**Part 5**

**Glioma Model and Culture Systems** 

342 Glioma – Exploring Its Biology and Practical Relevance

Panner, A., James, C., Berger, M. & Piepe, R. (2005). mTOR controls FLIPS translation and

Parney, I., Hao, C. & Petruk, K. (2000). Glioma immunology and immunotherapy.

Riboldi, E., Daniele, R., Cassatella, M., Sozzani, S. & Bosisio D. (2009). Engagement of

Röhn, T., Wagenknecht, B., Roth, W, Naumann, U., Gulbins, E., Krammer, P., Walczak, H. &

Sawada, M., Kiyono, T., Nakashima, S., Shinoda, J., Naganawa. T., Hara, S., Iwama, T. &

Siegelin, M., Reuss, D., Habel, A., Rami, A. & von Deimling, A. (2009) Quercetin promotes

Wesa, A. & Storkus, W. (2008). Killer dendritic cells: mechanisms of action and therapeutic implications for cancer. *Cell Death & Differentiation,* Vol.15, pp. 51-57 Weller, M., Frei, K., Groscurth, P., Krammer, P., Yonekawa, Y. & Fontana A. (1994). Anti-

Yang, R., Xu, D. Zhang, A. &. Gruber, A. (2001). Immature dendritic cells kill ovarian

in glioma cells. *Neuro-Oncology*, Vol.11, No.2 (April 2009), pp.122-131 Vanderheyde, N., Vandenabeele, P., Goldman M. & Willems F. (2004). Distinct mechanisms

cytochrome c release. *Oncogene*, Vol.20, No.31, pp. 4128-4137

pathways. *Cell Death and Differentiation*, Vol.11, pp.997–1008

*Investigation*, Vol. 94, No.3 ( September 1994), pp. 954–964

CTLs. *International Journal of Cancer*, Vol.94, No.3, pp.407-413

dendritic cells*. Immunology Letters*, Vol.91, No. 2

Vol.283, No.10, (March 2008), pp. 6438–6448

No.20, pp. 8809–8823

*Neurosurgery*, Vol.46, pp.778-791

*Immunobiology*, Vol.214, pp.868–876

Supports Infection with Chlamydia trachomatis. *The Journal of Biological Chemistry*,

TRAIL sensitivity in glioblastoma multiforme cells*. Molecular Cell Biology*, Vol.25,

BDCA-2 blocks TRAIL-mediated cytotoxic activity of plasmacytoid dendritic cells.

Weller, M. (2001). CCNU-dependent potentiation of TRAIL/Apo2L-induced apoptosis in human glioma cells is p53-independent but may involve enhanced

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carcinoma cells by a FAS/FASL pathway, enabling them to sensitize tumor-specific

**16** 

Lijun Sun

*P.R. China* 

**Animal Models of Glioma** 

*Department of Neurosurgery, Tianjin Huanhu Hospital* 

Gliomas are the most common primary tumors that arise from glial cells and their precursors in the central nervous system. Animal models always are important tool in the study of tumorigenesis, various therapy or preclinical trials for gliomas. It has been known since the 1970's that repetitive intravenous administration of nitrosourea compounds such as methynitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) produces glial-type neoplasms in immunocompetent rats. However, the long time required to induce neoplasms, and inconsistency of tumor development, led to a shift towards implantation of neoplastic cells propagated in vitro. Implantation of rodent glioma cells has proven an excellent intracranial brain tumor model due to their efficient tumorigenesis, reproducible and fast growth rates and accurate knowledge of the tumor location. Over the past few decades, several mouse glioma models have been generated based on human genetic abnormalities and the induced gliomas exhibit histological similarities to their human counterparts. More accurate animal models are required for research on the molecular and genetic bases of this disease. Here we expand on the existing animal models for gliomas

While there exist a multitude of methods for introducing glial-type neoplasms into the rodent central nervous system (CNS), which histologically mimic human primary tumors, these methods can be described as belonging to one of two groups: 1) Tumors created by methods which do not target a specific gene, and 2) Tumors created by targeted mutation of

While the majority of these models involve the use of rodent glioma cells injected in syngeneic hosts, it is also possible to use human glioma cells in vivo via their implantation in athymic mice. We will describe both of the two classes of glioma animal models, and eight commonly used rat brain tumor models and their application for the development of

The rat has been one of the most widely used experimental animals, and rat brain tumor models have been used extensively since the mid 1970s. Here we will focus on rat brain tumor models and their utility in evaluating the efficacy of various therapeutic modalities. Until recently, murine models (Fomchenko & Holland, 2006) were used less frequently than

**1. Introduction** 

with different strategies.

genes known to be mutated in human tumors.

**2.1 Traditional animal models of glioma** 

novel therapeutic and diagnostic modalities.

**2. Classification** 

## **Animal Models of Glioma**

Lijun Sun

*Department of Neurosurgery, Tianjin Huanhu Hospital P.R. China* 

#### **1. Introduction**

Gliomas are the most common primary tumors that arise from glial cells and their precursors in the central nervous system. Animal models always are important tool in the study of tumorigenesis, various therapy or preclinical trials for gliomas. It has been known since the 1970's that repetitive intravenous administration of nitrosourea compounds such as methynitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) produces glial-type neoplasms in immunocompetent rats. However, the long time required to induce neoplasms, and inconsistency of tumor development, led to a shift towards implantation of neoplastic cells propagated in vitro. Implantation of rodent glioma cells has proven an excellent intracranial brain tumor model due to their efficient tumorigenesis, reproducible and fast growth rates and accurate knowledge of the tumor location. Over the past few decades, several mouse glioma models have been generated based on human genetic abnormalities and the induced gliomas exhibit histological similarities to their human counterparts. More accurate animal models are required for research on the molecular and genetic bases of this disease. Here we expand on the existing animal models for gliomas with different strategies.

#### **2. Classification**

While there exist a multitude of methods for introducing glial-type neoplasms into the rodent central nervous system (CNS), which histologically mimic human primary tumors, these methods can be described as belonging to one of two groups: 1) Tumors created by methods which do not target a specific gene, and 2) Tumors created by targeted mutation of genes known to be mutated in human tumors.

#### **2.1 Traditional animal models of glioma**

While the majority of these models involve the use of rodent glioma cells injected in syngeneic hosts, it is also possible to use human glioma cells in vivo via their implantation in athymic mice. We will describe both of the two classes of glioma animal models, and eight commonly used rat brain tumor models and their application for the development of novel therapeutic and diagnostic modalities.

The rat has been one of the most widely used experimental animals, and rat brain tumor models have been used extensively since the mid 1970s. Here we will focus on rat brain tumor models and their utility in evaluating the efficacy of various therapeutic modalities. Until recently, murine models (Fomchenko & Holland, 2006) were used less frequently than

Animal Models of Glioma 347

tumor cell implantation, and relatively larger volumes (~20 μl) that can be injected versus mice (5 μl; Table 1). Mouse models, on the other hand, have allowed researchers to rigorously test hypotheses developed from examining human tumors by genetically manipulating them and controlling specific variables such as environmental influences, in order to better understand the roles of different pathways, cell types, stromal factors and genetic variation (Reilly et al, 2008). Mouse tumor models (Table 1) also have allowed researchers to test hypotheses derived from examining human tumors, in a controlled environment with specific genetic alterations and controlled environmental influences (Reilly et al, 2008). There is a general consensus that valid brain tumor models should fulfill the following criteria: (1) they should be derived from glial cells; (2) it should be possible to grow and clone them in vitro as continuous cell lines and propagate them in vivo by serial transplantation; (3) tumor growth rates should be predictable and reproducible; (4) the tumors should have glioma-like growth characteristics within the brain including neovascularization, alteration of the bloodbrain barrier (BBB), an invasive pattern of growth, and lack of encapsulation; (5) host survival time following i.c. tumor implantation should be of sufficient duration to permit therapy and determination of efficacy; (6) for therapy studies, the tumors should be either non or weakly immunogenic in syngeneic hosts; (7) they should not grow into the epidural space or extend beyond the brain and finally (8) their response or lack thereof to conventional treatment should be predictive of the response in

In studies carried out prior to the 1970s, either cells or tumor fragments were injected i.c. using a free hand approach, which generally lacked reproducibility and precision. A stereotactic implantation procedure using suspensions of tissue-culture-derived brain tumor cells was more successful (Barker et al, 1973). This procedure was further improved by the use of concentrated cell suspensions in small volumes, improved injection needles, better stereotactic localization to structures deeper in the white matter such as the caudate nucleus, the use of slower injection rates (Landen et al, 2004), 0.5–1.0% low gelling agarose to prevent backflow of tumor cells through the injection track (Kobayashi et al, 1980) and cleansing of the operative field with a solution of Betadine. Finally, rinsing the surface of the brain with sterile water destroys extravasated tumor cells by osmosis prior to closure of the skull with bone wax has also been recommended. This implantation procedure resulted in high success rates of i.c. tumor growth with the elimination of spinal and extracranial dissemination. The implantation of plastic (Kobayashi et al, 1980)or metallic screws (Lal et al, 2000) with an entry port, which are permanently implanted in the skull to inject tumor cells, have been very useful (Saini et al, 2004). Such devices can be left in place either at the time of or after tumor cell implantation in order to facilitate future administration of therapeutic agents at the same location without further stereotactic surgery. These are well tolerated and nonirritating in rats, but they cannot be as easily used in mice due to the thinness of their skulls. Keeping these general principles of tumor cell implantation in mind, we will now discuss the currently available rat glioma models that have been used in immunocompetent

**The C6 glioma** was produced by Benda et al. (Benda et al, 1968)and Schmidek et al. (Schmidek et al, 1971), in Sweet's laboratory at the Massachusetts General Hospital (MGH) by repetitively administering MNU to outbred Wistar rats over a period of approximately 8 months. When animals developed neurological signs, they were euthanized, and the tumors were excised and explanted into tissue culture. Among these was a tumor designated as "#6", which was subsequently cloned by Benda et al. and was shown to produce S-100

human brain tumors.

animals.

rat models, but the ability to produce genetically engineered cell lines (Lampson, 2001) has increased the use of murine models over the past five years. The relative advantages of rat and murine tumor models are summarized in Table 1. Feline and canine models have been used less frequently (Kimmelman & Nalbantoglu, 2007), but nevertheless, still provide an intermediate between rodent models and humans.

#### **2.1.1 Rat brain tumor models**

It was first reported in the early 1970s that CNS tumors could be induced reproducibly and selectively in adult rats that had been given repeated, weekly intravenous injections of MNU or a single dose of ENU. These studies led to the development of a number of rat brain tumor models that were highly reproducible and did not require the toxic application of a chemical carcinogen to the brain (Candolfi et al, 2007).


Table 1. Advantages and disadvantages of rat brain tumor models compared to mouse models.

The cellular signaling pathways important for the genesis of brain tumor are multiple, with feedback mechanisms that can dramatically affect the efficacy of molecularly targeted therapeutic strategies. The heterogeneous composition of human high grade gliomas, which consists of tumor stem cells and differentiated tumor cells with varying characteristics, further complicates their susceptibility to treatment. Brain tumors also can evolve within their microenvironment, adapting to changes that produce epigenetic effects thereby altering their biology, but concomitantly providing additional targets for therapeutic intervention. Finally, genetic variations between individuals can dictate how tumors initiate, progress, and respond to treatment. Rat brain tumor models have provided a wealth of information on the in vitro and in vivo responses to various therapeutic modalities. The larger rat brain (~1200 mg) compared to that of the mouse (~400 mg) allows for more precise

rat models, but the ability to produce genetically engineered cell lines (Lampson, 2001) has increased the use of murine models over the past five years. The relative advantages of rat and murine tumor models are summarized in Table 1. Feline and canine models have been used less frequently (Kimmelman & Nalbantoglu, 2007), but nevertheless, still provide an

It was first reported in the early 1970s that CNS tumors could be induced reproducibly and selectively in adult rats that had been given repeated, weekly intravenous injections of MNU or a single dose of ENU. These studies led to the development of a number of rat brain tumor models that were highly reproducible and did not require the toxic application

> Rat brain tumor models cannot be as easily genetically engineered and manipulated as mouse models in order to elucidate the importance of genetic factors, signaling pathways, cell types and stroma in tumor

> The potential to produce genetically engineered tumor cell lines is less in the rat

There are a smaller number of mAbs directed against rat surface antigens and chemokines compared to the mouse.

Rats are more expensive to purchase and

growth and invasion.

than in the mouse.

maintain than mice.

Table 1. Advantages and disadvantages of rat brain tumor models compared to mouse

The cellular signaling pathways important for the genesis of brain tumor are multiple, with feedback mechanisms that can dramatically affect the efficacy of molecularly targeted therapeutic strategies. The heterogeneous composition of human high grade gliomas, which consists of tumor stem cells and differentiated tumor cells with varying characteristics, further complicates their susceptibility to treatment. Brain tumors also can evolve within their microenvironment, adapting to changes that produce epigenetic effects thereby altering their biology, but concomitantly providing additional targets for therapeutic intervention. Finally, genetic variations between individuals can dictate how tumors initiate, progress, and respond to treatment. Rat brain tumor models have provided a wealth of information on the in vitro and in vivo responses to various therapeutic modalities. The larger rat brain (~1200 mg) compared to that of the mouse (~400 mg) allows for more precise

intermediate between rodent models and humans.

of a chemical carcinogen to the brain (Candolfi et al, 2007).

1. Larger size of the rat brain (compared to the mouse brain ~1200 mg vs ~ 400 mg) permits more precise stereotactic implantation than in mice, a longer interval of time until death and a thicker skull essentially eliminates osseous

2. Larger tumor size prior to death permits better in vivo localization and imaging by a variety of diagnostic modalities in

3. Larger tumor size prior to death permits the administration of larger amounts of various therapeutic agents, especially if administered i.c. by CED and more critical evaluation of their effectiveness.

4. More extensive literature on in vitro and in vivo studies of rat brain tumors compared to mouse tumors.

invasion and s.c. growth.

the rat.

models.

**Advantages Disadvantages** 

**2.1.1 Rat brain tumor models** 

tumor cell implantation, and relatively larger volumes (~20 μl) that can be injected versus mice (5 μl; Table 1). Mouse models, on the other hand, have allowed researchers to rigorously test hypotheses developed from examining human tumors by genetically manipulating them and controlling specific variables such as environmental influences, in order to better understand the roles of different pathways, cell types, stromal factors and genetic variation (Reilly et al, 2008). Mouse tumor models (Table 1) also have allowed researchers to test hypotheses derived from examining human tumors, in a controlled environment with specific genetic alterations and controlled environmental influences (Reilly et al, 2008). There is a general consensus that valid brain tumor models should fulfill the following criteria: (1) they should be derived from glial cells; (2) it should be possible to grow and clone them in vitro as continuous cell lines and propagate them in vivo by serial transplantation; (3) tumor growth rates should be predictable and reproducible; (4) the tumors should have glioma-like growth characteristics within the brain including neovascularization, alteration of the bloodbrain barrier (BBB), an invasive pattern of growth, and lack of encapsulation; (5) host survival time following i.c. tumor implantation should be of sufficient duration to permit therapy and determination of efficacy; (6) for therapy studies, the tumors should be either non or weakly immunogenic in syngeneic hosts; (7) they should not grow into the epidural space or extend beyond the brain and finally (8) their response or lack thereof to conventional treatment should be predictive of the response in human brain tumors.

In studies carried out prior to the 1970s, either cells or tumor fragments were injected i.c. using a free hand approach, which generally lacked reproducibility and precision. A stereotactic implantation procedure using suspensions of tissue-culture-derived brain tumor cells was more successful (Barker et al, 1973). This procedure was further improved by the use of concentrated cell suspensions in small volumes, improved injection needles, better stereotactic localization to structures deeper in the white matter such as the caudate nucleus, the use of slower injection rates (Landen et al, 2004), 0.5–1.0% low gelling agarose to prevent backflow of tumor cells through the injection track (Kobayashi et al, 1980) and cleansing of the operative field with a solution of Betadine. Finally, rinsing the surface of the brain with sterile water destroys extravasated tumor cells by osmosis prior to closure of the skull with bone wax has also been recommended. This implantation procedure resulted in high success rates of i.c. tumor growth with the elimination of spinal and extracranial dissemination. The implantation of plastic (Kobayashi et al, 1980)or metallic screws (Lal et al, 2000) with an entry port, which are permanently implanted in the skull to inject tumor cells, have been very useful (Saini et al, 2004). Such devices can be left in place either at the time of or after tumor cell implantation in order to facilitate future administration of therapeutic agents at the same location without further stereotactic surgery. These are well tolerated and nonirritating in rats, but they cannot be as easily used in mice due to the thinness of their skulls. Keeping these general principles of tumor cell implantation in mind, we will now discuss the currently available rat glioma models that have been used in immunocompetent animals.

**The C6 glioma** was produced by Benda et al. (Benda et al, 1968)and Schmidek et al. (Schmidek et al, 1971), in Sweet's laboratory at the Massachusetts General Hospital (MGH) by repetitively administering MNU to outbred Wistar rats over a period of approximately 8 months. When animals developed neurological signs, they were euthanized, and the tumors were excised and explanted into tissue culture. Among these was a tumor designated as "#6", which was subsequently cloned by Benda et al. and was shown to produce S-100

Animal Models of Glioma 349

Fig. 1. Histopathologic features of the C6, 9L, RG2, F98, CNS-1, and BT4C brain tumors. A The C6 glioma is composed of a pleomorphic population of cells with nuclei ranging from round to oblong. A herring-bone pattern of growth is seen in some areas and there is focal invasion of contiguous normal brain. There are scattered foci of necrosis with pseudo-palisading of tumor

protein. Following cloning, it was redesignated "C6" (Pfeiffer et al, 1970). The C6 glioma is composed of a pleomorphic population of cells with variably shaped nuclei. There is focal invasion into contiguous normal brain (Fig. 1a). Initially, the tumor was histopathologically classified as an astrocytoma, and eventually it was designated as a glial tumor following accession by the American Type Culture Collection, Rockville, MD (ATCC# CCL-107). The cells have been reported to have a mutant p16/Cdkn2a/ Ink4a locus (Schlegel et al, 1999) with no expression of p16 and p19ARF mRNAs, and a wildtype p53 (Asai et al, 1994). More recent molecular characterization, which compared changes in gene expression between the C6 glioma and rat stem cell-derived astrocytes, revealed that the changes in gene expression observed in the C6 cell line were the most similar to those reported in human brain tumors (Sibenaller et al, 2005). Compared to astrocytes, they also had increased expression of the PDGFβ, IGF-1, EGFR and Erb3/Her3 genes, which are frequently overexpressed in human gliomas (Guo et al, 2003; Heimberger et al,2005). In a recent study, the significance of PDGF in gliomagenesis in adult rats was established by infecting white matter with a retrovirus encoding for PDGF and GFP. Within 2 weeks 100% of the animals had tumors derived from both infected and uninfected glial progenitors, thereby implicating PDGF in both autocrine and paracrine stimulation of glial progenitor cells (Assanah et al, 2006). Although, IGF-1 was overexpressed in C6 glioma cells, there was reduced expression of IGF-2, FGF-9 and FGF-10 relative to astrocytes. Similar to the increased activity of the Ras pathway observed in human gliomas (Nakada et al, 2005), C6 cells also had an increase in both Ras expression and Ras guanine triphosphate activator protein (Sibenaller et al, 2005). However, contrary to what has been reported for human GBM, there was an increase in expression of Rb in these cells (Sibenaller et al, 2005). A subclone of C6 cells, stably expressing β-galactosidase, subsequently was described (Lampson et al, 1993) and this has permitted in vivo immunohistochemical analysis of these tumors in the brain. This clone is available through the ATCC (# CRL-2303). However, it must be noted that the β-galactosidase marker protein itself can serve as a tumor antigen, and immunization of rats against the reporter gene protected the animals against tumor growth.

The C6 rat glioma model has been widely used in experimental neuro-oncology to evaluate the therapeutic efficacy of a variety of modalities, including chemotherapy (Doblas et al, 2008), antiangiogenic therapy (Solly F et al, 2008), proteosome inhibitors (Ahmed et al, 2008), treatment with toxins (Zhao et al, 2008), radiation therapy (Sheehan et al, 2008), photodynamic therapy (Mannino et al, 2008), oncolytic viral therapy (Yang et al, 2004) and gene therapy (Tanriover et al, 2008). Since this tumor arose in an outbred Wistar rat, however, there is no syngeneic host in which it can be propagated. This is a very serious limitation that diminishes its usefulness for survival studies since the tumor is immunogenic, even in Wistar rats. The C6 glioma has been demonstrated to be immunogenic in Wistar and BDX rats (Parsa et al, 2000), and it therefore is not useful for evaluating the efficacy of immunotherapy. This problem is exemplified by prior studies in which C6 glioma cells were transfected with an antisense cDNA expression vector that downregulated the constitutive production of IGF-1 (Trojan et al, 1993). Not recognizing that the tumor was of Wistar origin, the authors unfortunately used BD IX rats, which they thought was the strain of origin, due to some ambiguity in the literature. Subsequently, it was reported that BD IX rats, which had been immunized with the C6 anti-sense IGF-1 transfected cells, were resistant to both s.c. and i.c. challenge of the C6 glioma. Similarly, Wistar rats, bearing C6 gliomas (s.c. or i.c.), developed potent humoral and cellular immune

protein. Following cloning, it was redesignated "C6" (Pfeiffer et al, 1970). The C6 glioma is composed of a pleomorphic population of cells with variably shaped nuclei. There is focal invasion into contiguous normal brain (Fig. 1a). Initially, the tumor was histopathologically classified as an astrocytoma, and eventually it was designated as a glial tumor following accession by the American Type Culture Collection, Rockville, MD (ATCC# CCL-107). The cells have been reported to have a mutant p16/Cdkn2a/ Ink4a locus (Schlegel et al, 1999) with no expression of p16 and p19ARF mRNAs, and a wildtype p53 (Asai et al, 1994). More recent molecular characterization, which compared changes in gene expression between the C6 glioma and rat stem cell-derived astrocytes, revealed that the changes in gene expression observed in the C6 cell line were the most similar to those reported in human brain tumors (Sibenaller et al, 2005). Compared to astrocytes, they also had increased expression of the PDGFβ, IGF-1, EGFR and Erb3/Her3 genes, which are frequently overexpressed in human gliomas (Guo et al, 2003; Heimberger et al,2005). In a recent study, the significance of PDGF in gliomagenesis in adult rats was established by infecting white matter with a retrovirus encoding for PDGF and GFP. Within 2 weeks 100% of the animals had tumors derived from both infected and uninfected glial progenitors, thereby implicating PDGF in both autocrine and paracrine stimulation of glial progenitor cells (Assanah et al, 2006). Although, IGF-1 was overexpressed in C6 glioma cells, there was reduced expression of IGF-2, FGF-9 and FGF-10 relative to astrocytes. Similar to the increased activity of the Ras pathway observed in human gliomas (Nakada et al, 2005), C6 cells also had an increase in both Ras expression and Ras guanine triphosphate activator protein (Sibenaller et al, 2005). However, contrary to what has been reported for human GBM, there was an increase in expression of Rb in these cells (Sibenaller et al, 2005). A subclone of C6 cells, stably expressing β-galactosidase, subsequently was described (Lampson et al, 1993) and this has permitted in vivo immunohistochemical analysis of these tumors in the brain. This clone is available through the ATCC (# CRL-2303). However, it must be noted that the β-galactosidase marker protein itself can serve as a tumor antigen, and immunization of rats against the reporter gene

The C6 rat glioma model has been widely used in experimental neuro-oncology to evaluate the therapeutic efficacy of a variety of modalities, including chemotherapy (Doblas et al, 2008), antiangiogenic therapy (Solly F et al, 2008), proteosome inhibitors (Ahmed et al, 2008), treatment with toxins (Zhao et al, 2008), radiation therapy (Sheehan et al, 2008), photodynamic therapy (Mannino et al, 2008), oncolytic viral therapy (Yang et al, 2004) and gene therapy (Tanriover et al, 2008). Since this tumor arose in an outbred Wistar rat, however, there is no syngeneic host in which it can be propagated. This is a very serious limitation that diminishes its usefulness for survival studies since the tumor is immunogenic, even in Wistar rats. The C6 glioma has been demonstrated to be immunogenic in Wistar and BDX rats (Parsa et al, 2000), and it therefore is not useful for evaluating the efficacy of immunotherapy. This problem is exemplified by prior studies in which C6 glioma cells were transfected with an antisense cDNA expression vector that downregulated the constitutive production of IGF-1 (Trojan et al, 1993). Not recognizing that the tumor was of Wistar origin, the authors unfortunately used BD IX rats, which they thought was the strain of origin, due to some ambiguity in the literature. Subsequently, it was reported that BD IX rats, which had been immunized with the C6 anti-sense IGF-1 transfected cells, were resistant to both s.c. and i.c. challenge of the C6 glioma. Similarly, Wistar rats, bearing C6 gliomas (s.c. or i.c.), developed potent humoral and cellular immune

protected the animals against tumor growth.

Fig. 1. Histopathologic features of the C6, 9L, RG2, F98, CNS-1, and BT4C brain tumors. A The C6 glioma is composed of a pleomorphic population of cells with nuclei ranging from round to oblong. A herring-bone pattern of growth is seen in some areas and there is focal invasion of contiguous normal brain. There are scattered foci of necrosis with pseudo-palisading of tumor

Animal Models of Glioma 351

(CSLCs) have been demonstrated in the 9L cell line. These CSLCs grow as neurospheres in chemically defined medium and express the neural stem cell markers Nestin and Sox2. They are self-renewable and differentiate in vitro into neuron- and glial-like cells (Ghods et al, 2007). The neurospheres have a lower proliferation rate and express several anti-apoptotic and drug related genes. Furthermore, these cells form tumors that are more aggressive than the parental 9L tumor (Ghods et al, 2007), which could be an important property in future studies. The 9L gliosarcoma model has been used extensively to investigate mechanisms and development of drug resistance (Barcellos-Hoff et al, 2006), transport of drugs across the blood-brain and bloodtumor barrier (Black et al, 2008), imaging of brain tumors including radiological techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) and imaging to evaluate tumor hypoxia and metabolism (Bansal et al, 2008), pharmacokinetic studies of nitrosourea (Warnke et al, 1987), mechanisms and effects of anti-angiogenic drugs (Yang et al, 2007), effects of radiation (Regnard et al, 2008), chemotherapy (Bencokova et al, 2008), gene therapy (Kumar et al, 2008), cancer stem cells (Ghods et al, 2007), immunotoxin treatment, immunotherapy and cytokine therapy (Liu et al, 2007) and oncolytic viral therapy (Madara et al, 2005). A number of these studies have yielded impressive therapeutic results, including apparent cures of tumor bearing animals. However, it must be emphasized that this tumor has been shown to be highly immunogenic. Animals immunized with X-irradiated 9L cells were resistant to both subcutaneous (s.c.) as well as i.c. tumor challenge, compared to 100% tumor takes in immunologically naïve animals (Blume et al, 1974). This report was first published in the proceedings of a meeting, which did not receive wide circulation, but subsequent studies have confirmed the immunogenicity of this model (Morantz et al, 1979). Expression of the s-Myc gene under the control of a CMV promotor resulted in complete suppression of 9L tumor growth, as well as rejection of subsequent challenges of tumor cells. Histological examination of the tumors after s-Myc therapy revealed massive mononuclear cell infiltration with CD8 + T lymphocytes, which accounted for >70% of these infiltrating cells. These observations suggested that tumor rejection was due to a potent T-cell mediated, antitumor immune response. This, and several more recent studies, have underscored the significance of the anti-tumor immune response following gene therapy induced tumor eradication observed with 9L model. It is now recognized that in vivo bystander cell killing (Chen et al, 1995), which has been observed with the 9L gliosarcoma following delivery of the Herpes simplex virus thymidine kinase gene (hsv-tk), (Moolten et al, 1986) followed by treatment with ganciclovir, was due in part to an anti-tumor immune response. The immunogenicity of the 9L glioma must be kept in mind when utilizing this model to evaluate the efficacy of novel therapeutic agents. Early studies employing radiation or chemotherapy alone were largely unsuccessful in curing the 9L tumor. However, the success obtained by boron neutron capture therapy and gene therapy highlights the importance of utilizing anti-tumor treatments that can destroy individual cancer cells and simultaneously spare host immune effector cells that can eradicate residual tumor cells (Moriuchi et al,

Despite the fact that the 9L arose in a Fischer rat, 9L gliosarcoma cells also can form i.c. tumors in allogeneic Wistar rats (Stojiljkovic et al, 2003). Histopathological evaluation revealed that these tumors formed circumscribed masses that were not infiltrative and did not spread into the subarachnoid space or ventricles. Immunostaining of the tumors revealed the presence of glial fibrillary acidic protein (GFAP)-positive, infiltrating astrocytic

2002).

cells at the periphery. B The 9L gliosarcoma is composed of spindle-shaped cells with a sarcomatoid appearance. A whorled pattern of growth is seen with sharp delineation of the margins of the tumor with little invasion of contiguous normal brain. C The RG2 glioma is very similar in appearance to the F98 glioma and also has a highly invasive pattern of growth. D The F98 glioma is composed of a mixed population of spindle-shaped cells with fusiform nuclei, frequently forming a whorled pattern of growth, and a smaller subpopulation of polygonal cells with round to oval nuclei. There is extensive invasion of contiguous normal brain with islands of tumor cells at varying distances from the main tumor mass, which form perivascular clusters. Usually, there is a central area of necrosis filled with tumor cell ghosts. E The CNS-1 glioma is composed of a pleomorphic population of cells that show great variation in size and shape. There is extensive invasion of contiguous normal brain with dense infiltrates in some areas and in others, more circumscribed clusters of tumor cells. Small foci of hemorrhage are scattered through the tumor. F The BT4C glioma is composed of a pleomorphic population of tumor cells with a sarcomatous pattern of growth. Scattered tumor giant cells are seen and mitotic figures are frequent. The tumor grows expansively and invades the surrounding normal brain along perivascular tracts and occasional tumor cell nests are seen in the surrounding normal brain. There is neo-vascularization, especially in the tumor periphery, where microhemorrhages are frequent. Central necrosis is usually not present but occasionally scattered areas of necrosis may be seen in larger tumors. All photomicrographs are at a magnification of 200×, except for F

responses to the tumor, and rats challenged simultaneously with s.c. and i.c. tumors, had a survival rate of 100% (Parsa et al, 2000). Since C6 glioma cells are allogeneic in all inbred strains, this should provide a strong cautionary note for studies employing this tumor model and they should not be used for immunotherapy studies. Despite this limitation, the C6 glioma model continues to be used for a variety of studies related to brain tumor biology (Karmakar et al, 2007). These have included studies on tumor growth, invasion, migration, BBB disruption, neovascularization, growth factor regulation and production, and biochemical studies (Assadian et al, 2008). Finally, single-cell clonal analysis has revealed that C6 cells also have cancer stem cell-like characteristics, including self-renewal, the potential for multi-lineage differentiation in vitro and tumor formation in vivo (Shen et al, 2008).

**The 9L gliosarcoma** has been the most widely used experimental rat brain tumor model. It was produced in Fisher 344 rats by the intravenous injection of 5 mg/kg of MNU for 26 weeks (Benda et al, 1971). The original tumor was designated as tumor #9, which subsequently was cloned at the Brain Tumor Research Center, University of California, San Francisco, and then was designated "9L". These tumor cells could be propagated in vitro, which made them very useful for in vivo studies to investigate the effects of various therapeutic modalities on brain tumors. 9L cells can be implanted i.c. into syngeneic Fischer rats, following which they give rise to rapidly growing tumors. These are composed of spindle-shaped cells with a sarcomatoid appearance. The tumor margins are sharply delineated with little obvious invasion into the contiguous normal brain (Fig. 1b). The 9L gliosarcoma has a mutant p53 gene (Asai et al, 1994), but there is normal expression of p16 and p19ARF mRNAs, indicating that there is a wild type p16/Cdkn2a/INK4α locus (Schlegel et al, 1999). Molecular characterization of the 9L relative to rat stem cell derived astrocytes revealed an increased expression of the genes encoding TGFα and its receptor, EGFR (Sibenaller et al, 2005). Interestingly, decreased expression of FGF-2, FGF-9, and FGFR-1 and PDGFRβ also was noted (Sibenaller et al, 2005). Recently, cancer stem-like cells

cells at the periphery. B The 9L gliosarcoma is composed of spindle-shaped cells with a sarcomatoid appearance. A whorled pattern of growth is seen with sharp delineation of the margins of the tumor with little invasion of contiguous normal brain. C The RG2 glioma is very similar in appearance to the F98 glioma and also has a highly invasive pattern of growth. D The F98 glioma is composed of a mixed population of spindle-shaped cells with fusiform nuclei, frequently forming a whorled pattern of growth, and a smaller subpopulation of polygonal cells with round to oval nuclei. There is extensive invasion of contiguous normal brain with islands of tumor cells at varying distances from the main tumor mass, which form perivascular clusters. Usually, there is a central area of necrosis filled with tumor cell ghosts. E The CNS-1 glioma is composed of a pleomorphic population of cells that show great variation in size and shape. There is extensive invasion of contiguous normal brain with dense infiltrates

in some areas and in others, more circumscribed clusters of tumor cells. Small foci of hemorrhage are scattered through the tumor. F The BT4C glioma is composed of a

are at a magnification of 200×, except for F

2008).

pleomorphic population of tumor cells with a sarcomatous pattern of growth. Scattered tumor giant cells are seen and mitotic figures are frequent. The tumor grows expansively and invades the surrounding normal brain along perivascular tracts and occasional tumor cell nests are seen in the surrounding normal brain. There is neo-vascularization, especially in the tumor periphery, where microhemorrhages are frequent. Central necrosis is usually not present but occasionally scattered areas of necrosis may be seen in larger tumors. All photomicrographs

responses to the tumor, and rats challenged simultaneously with s.c. and i.c. tumors, had a survival rate of 100% (Parsa et al, 2000). Since C6 glioma cells are allogeneic in all inbred strains, this should provide a strong cautionary note for studies employing this tumor model and they should not be used for immunotherapy studies. Despite this limitation, the C6 glioma model continues to be used for a variety of studies related to brain tumor biology (Karmakar et al, 2007). These have included studies on tumor growth, invasion, migration, BBB disruption, neovascularization, growth factor regulation and production, and biochemical studies (Assadian et al, 2008). Finally, single-cell clonal analysis has revealed that C6 cells also have cancer stem cell-like characteristics, including self-renewal, the potential for multi-lineage differentiation in vitro and tumor formation in vivo (Shen et al,

**The 9L gliosarcoma** has been the most widely used experimental rat brain tumor model. It was produced in Fisher 344 rats by the intravenous injection of 5 mg/kg of MNU for 26 weeks (Benda et al, 1971). The original tumor was designated as tumor #9, which subsequently was cloned at the Brain Tumor Research Center, University of California, San Francisco, and then was designated "9L". These tumor cells could be propagated in vitro, which made them very useful for in vivo studies to investigate the effects of various therapeutic modalities on brain tumors. 9L cells can be implanted i.c. into syngeneic Fischer rats, following which they give rise to rapidly growing tumors. These are composed of spindle-shaped cells with a sarcomatoid appearance. The tumor margins are sharply delineated with little obvious invasion into the contiguous normal brain (Fig. 1b). The 9L gliosarcoma has a mutant p53 gene (Asai et al, 1994), but there is normal expression of p16 and p19ARF mRNAs, indicating that there is a wild type p16/Cdkn2a/INK4α locus (Schlegel et al, 1999). Molecular characterization of the 9L relative to rat stem cell derived astrocytes revealed an increased expression of the genes encoding TGFα and its receptor, EGFR (Sibenaller et al, 2005). Interestingly, decreased expression of FGF-2, FGF-9, and FGFR-1 and PDGFRβ also was noted (Sibenaller et al, 2005). Recently, cancer stem-like cells (CSLCs) have been demonstrated in the 9L cell line. These CSLCs grow as neurospheres in chemically defined medium and express the neural stem cell markers Nestin and Sox2. They are self-renewable and differentiate in vitro into neuron- and glial-like cells (Ghods et al, 2007). The neurospheres have a lower proliferation rate and express several anti-apoptotic and drug related genes. Furthermore, these cells form tumors that are more aggressive than the parental 9L tumor (Ghods et al, 2007), which could be an important property in future studies. The 9L gliosarcoma model has been used extensively to investigate mechanisms and development of drug resistance (Barcellos-Hoff et al, 2006), transport of drugs across the blood-brain and bloodtumor barrier (Black et al, 2008), imaging of brain tumors including radiological techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) and imaging to evaluate tumor hypoxia and metabolism (Bansal et al, 2008), pharmacokinetic studies of nitrosourea (Warnke et al, 1987), mechanisms and effects of anti-angiogenic drugs (Yang et al, 2007), effects of radiation (Regnard et al, 2008), chemotherapy (Bencokova et al, 2008), gene therapy (Kumar et al, 2008), cancer stem cells (Ghods et al, 2007), immunotoxin treatment, immunotherapy and cytokine therapy (Liu et al, 2007) and oncolytic viral therapy (Madara et al, 2005). A number of these studies have yielded impressive therapeutic results, including apparent cures of tumor bearing animals. However, it must be emphasized that this tumor has been shown to be highly immunogenic. Animals immunized with X-irradiated 9L cells were resistant to both subcutaneous (s.c.) as well as i.c. tumor challenge, compared to 100% tumor takes in immunologically naïve animals (Blume et al, 1974). This report was first published in the proceedings of a meeting, which did not receive wide circulation, but subsequent studies have confirmed the immunogenicity of this model (Morantz et al, 1979). Expression of the s-Myc gene under the control of a CMV promotor resulted in complete suppression of 9L tumor growth, as well as rejection of subsequent challenges of tumor cells. Histological examination of the tumors after s-Myc therapy revealed massive mononuclear cell infiltration with CD8 + T lymphocytes, which accounted for >70% of these infiltrating cells. These observations suggested that tumor rejection was due to a potent T-cell mediated, antitumor immune response. This, and several more recent studies, have underscored the significance of the anti-tumor immune response following gene therapy induced tumor eradication observed with 9L model. It is now recognized that in vivo bystander cell killing (Chen et al, 1995), which has been observed with the 9L gliosarcoma following delivery of the Herpes simplex virus thymidine kinase gene (hsv-tk), (Moolten et al, 1986) followed by treatment with ganciclovir, was due in part to an anti-tumor immune response. The immunogenicity of the 9L glioma must be kept in mind when utilizing this model to evaluate the efficacy of novel therapeutic agents. Early studies employing radiation or chemotherapy alone were largely unsuccessful in curing the 9L tumor. However, the success obtained by boron neutron capture therapy and gene therapy highlights the importance of utilizing anti-tumor treatments that can destroy individual cancer cells and simultaneously spare host immune effector cells that can eradicate residual tumor cells (Moriuchi et al, 2002).

Despite the fact that the 9L arose in a Fischer rat, 9L gliosarcoma cells also can form i.c. tumors in allogeneic Wistar rats (Stojiljkovic et al, 2003). Histopathological evaluation revealed that these tumors formed circumscribed masses that were not infiltrative and did not spread into the subarachnoid space or ventricles. Immunostaining of the tumors revealed the presence of glial fibrillary acidic protein (GFAP)-positive, infiltrating astrocytic

Animal Models of Glioma 353

changes in vascular permeability (Ferrier et al, 2007), disruption of the BBB (Ningaraj et al, 2002), anti-angiogenic therapy (Zagorac et al, 2008), gene therapy, chemotherapy

The RG2 glioma is non-immunogenic in syngeneic Fischer rats and has low levels of MHC-1 expression compared to the C6 and 9L gliomas (Oshiro et al, 2001). However, in vitro treatment with IFN-γ upregulated MHC class I antigen expression and also resulted in a significant in vivo anti-tumor immune response with increased survival of treated animals. More recently, the RG2 glioma has been stably transfected with human Herpes virus Entry Mediator C (HveC) to facilitate HSV infection and has been used to study the therapeutic effects of oncolytic Herpes simplex virus-1 treatment (Kurozumi et al, 2007). The transfected cells retained their tumorigenicity following i.c. implantation in Fischer rats, and transfection of the HveC gene did not affect i.c. tumor growth (Wakimoto et al, 2004). However, it has not been determined if HveC can cause these cells to become immunogenic, and therefore, this must be taken into account when using the RG2 for immunotherapy

**The F98 glioma** (ATCC # CRL-2397) was produced by Wechsler in Koestner's laboratory at the same time as the RG2 glioma. It is composed of a mixed population of spindle-shaped cells, the majority of which have fusiform nuclei, and a smaller number of polygonal cells with round to oval nuclei. There is extensive invasion of contiguous normal brain with islands of tumor cells at varying distances from the tumor mass, many of which form perivascular clusters (Fig. 1d). Similar to human GBM, these cells overexpress PDGFβ, and Ras along with an increase in EGFR, cyclin D1 and cyclin D2 expression relative to rat astrocytes (Sibenaller et al, 2005). Like the C6 glioma, they also have increased expression of Rb relative to rat astrocytes. Immunofluorescence studies of F98 cells also revealed low expression of BRCA1, and a lack of radiation and cisplatin induced BRCA1 foci in these cells (Bencokova et al, 2008). Usually, there is a necrotic core, scattered mitotic cells and nonglomeruloid neovascular proliferation. The tumor is GFAP and vimentin positive with negligible staining for CD3 + T cells (Mathieu et al, 2007). Since it simulates the behavior of human GBMs in a number of important ways, such as its highly invasive pattern of growth and low immunogenicity, it has been used to evaluate the efficacy of a variety of experimental therapeutic agents. It is refractory to a number of therapeutic modalities, including systemic chemotherapy with paclitaxel, and carboplatin (von Eckardstein et al, 2005), and it is poorly responsive to photon-irradiation alone, which in part may be related to its functionally impaired BRCA1 status that can favor genomic instability and impaired DNA repair. Recently, it has been shown to be responsive to a combination of synchrotron radiation with cisplatin (Biston et al, 2004), and to convection enhanced delivery (CED) of carboplatin in combination with 6 MV photon-irradiation in rats bearing i.c. tumors (Rousseau et al, 2008). This model has been used extensively by Barth et al. to evaluate the efficacy of boron neutron capture therapy (BNCT) (Yang et al, 2008). Elleaume and her coworkers have evaluated cisplatin, carboplatin and iodine enhanced synchrotron stereotactic radiotherapy (Cho et al, 2002) in F98 glioma bearing rats (Adam et al, 2005). It has also been used to evaluate non-invasive MRI to visualize tumor growth, diffusion tensor imaging (Zhang et al, 2007), tumor angiogenesis and the tumor tropism of mesenchymal

The F98 glioma is very weakly immunogenic (Tzeng et al, 1991) and transfection with the gene encoding B7.1 co-stimulatory molecule (Paul et al, 2000), or syngeneic cellular vaccination combined with GMCSF, did not enhance its immunogenicity (Clavreul et al,

(Miknyoczki et al, 2007) and radionuclide therapy (Shen et al, 2004).

studies.

stem cells (Wu et al, 2008).

cells, and activated ED1 positive macrophages/ microglia. Higher numbers of K(ATP) and K(Ca) channels have been observed in 9L tumors grown in allogeneic Wistar rats compared to those grown in syngeneic Fischer rats. Furthermore, the allogeneic tumors showed a greater increase in brain tumor permeability upon treatment with potassium channel agonists, compared to those grown in syngeneic hosts. The 9L tumor model also has been used following treatment to study the effect of BBB disruption, implantation of devices for repeated intratumoral delivery and imaging (Bhattacharya et al, 2007).

The 9L gliosarcoma model also has been used to develop a model for brainstem tumors (Jallo et al, 2006). Progression to hemiparesis with the onset of symptoms occurred 17 days postimplantation into the brainstem. This model has been used to evaluate the efficacy of convection enhanced delivery (CED) of carboplatin to the brainstem, and to study the response of recurrent, chemo-resistant gliomas. Two bis-chloroethyl nitrosourea (BCNU) resistant cell lines were derived from 9L cells by treating them with BCNU in vitro or in vivo. Both of these cell lines formed tumors in a 100% of the animals following i.c. implantation, and were much more invasive than the parental 9L cells (Saito et al, 2004). The 9L gliosarcoma also has been used as a model to evaluate drug-resistant and invasive recurrent gliomas (Schepkin et al, 2006), but as previously indicated, caution must be used in evaluating results obtained with such a highly immunogenic tumor.

Although not fully appreciated, the **T9 glioma** was at one time, and still may be the same as the 9L gliosarcoma. The original stock of T9 cells was obtained from Sweet's laboratory at the MGH by Denlinger, and Koestner, and it was renamed T9 by them. Similar to the immunogenicity of the 9L gliosarcoma, the T9 glioma also was found to be highly immunogenic. Kida et al. found that rats immunized with irradiated T9 cells or T9 cells mixed with C. parvum rejected subsequent s.c. implants of T9 glioma cells (Kida et al, 1983). However, in order to immunize against intracranial tumors, rats initially had to reject intradermal T9 cells. As might have been predicted, these results indicated that, similar to the 9L gliosarcoma, the T9 glioma also was immunogenic. The T9 cell line subsequently has been shared among numerous investigators and has been used for many studies, including the evaluation of antiangiogenic (Jeffes et al, 2005), and chemotherapeutic agents (Pietronigro et al, 2003), immunotherapy (Shibuya et al, 1984), and gene therapy with interferon-β(Harada et al, 1995). Although tumor specific or tumor associated antigens have yet to be identified, for the 9L gliosarcoma and T9 glioma, it is only a matter of time before they are identified.

**The RG2 glioma** (ATCC #CRL-2433) was produced in Koestner's laboratory at The Ohio State University by the i.v. administration of ENU (50 mg/kg body weight) to a pregnant Fischer 344 rat on the 20th day of gestation. Subsequently, the in vitro growth and morphology of the F98 glioma was described in detail (Ko et al, 1980), and based on its histopathology it was classified as an anaplastic or undifferentiated glioma. The progeny of ENU-injected rats subsequently developed tumors, and following cloning by Wechsler in Germany, one of these clones was designated as "RG2" (rat glioma 2). The same clone was called the "D74-RG2" or "D74" in Koestner's laboratory at The Ohio State University. The RG2 glioma (Fig. 1c) is similar in microscopic appearance to the F98 glioma (Fig. 1d), and also has a highly invasive pattern of growth, which has made it a good representative model for GBM (Weizsacker et al, 1982). Gene expression profiling of these cells established that they had increased gene expression of PDGFβ, IGF-1, Ras, Erb3/HER3 precursor mRNA and cyclin D2. They express a wildtype p53 and a concurrent loss in the expression of the p16/Cdkn2a/Ink4 gene locus. It has been used for a variety of preclinical studies to evaluate

cells, and activated ED1 positive macrophages/ microglia. Higher numbers of K(ATP) and K(Ca) channels have been observed in 9L tumors grown in allogeneic Wistar rats compared to those grown in syngeneic Fischer rats. Furthermore, the allogeneic tumors showed a greater increase in brain tumor permeability upon treatment with potassium channel agonists, compared to those grown in syngeneic hosts. The 9L tumor model also has been used following treatment to study the effect of BBB disruption, implantation of devices for

The 9L gliosarcoma model also has been used to develop a model for brainstem tumors (Jallo et al, 2006). Progression to hemiparesis with the onset of symptoms occurred 17 days postimplantation into the brainstem. This model has been used to evaluate the efficacy of convection enhanced delivery (CED) of carboplatin to the brainstem, and to study the response of recurrent, chemo-resistant gliomas. Two bis-chloroethyl nitrosourea (BCNU) resistant cell lines were derived from 9L cells by treating them with BCNU in vitro or in vivo. Both of these cell lines formed tumors in a 100% of the animals following i.c. implantation, and were much more invasive than the parental 9L cells (Saito et al, 2004). The 9L gliosarcoma also has been used as a model to evaluate drug-resistant and invasive recurrent gliomas (Schepkin et al, 2006), but as previously indicated, caution must be used

Although not fully appreciated, the **T9 glioma** was at one time, and still may be the same as the 9L gliosarcoma. The original stock of T9 cells was obtained from Sweet's laboratory at the MGH by Denlinger, and Koestner, and it was renamed T9 by them. Similar to the immunogenicity of the 9L gliosarcoma, the T9 glioma also was found to be highly immunogenic. Kida et al. found that rats immunized with irradiated T9 cells or T9 cells mixed with C. parvum rejected subsequent s.c. implants of T9 glioma cells (Kida et al, 1983). However, in order to immunize against intracranial tumors, rats initially had to reject intradermal T9 cells. As might have been predicted, these results indicated that, similar to the 9L gliosarcoma, the T9 glioma also was immunogenic. The T9 cell line subsequently has been shared among numerous investigators and has been used for many studies, including the evaluation of antiangiogenic (Jeffes et al, 2005), and chemotherapeutic agents (Pietronigro et al, 2003), immunotherapy (Shibuya et al, 1984), and gene therapy with interferon-β(Harada et al, 1995). Although tumor specific or tumor associated antigens have yet to be identified, for the 9L gliosarcoma and T9 glioma, it is only a matter of time before

**The RG2 glioma** (ATCC #CRL-2433) was produced in Koestner's laboratory at The Ohio State University by the i.v. administration of ENU (50 mg/kg body weight) to a pregnant Fischer 344 rat on the 20th day of gestation. Subsequently, the in vitro growth and morphology of the F98 glioma was described in detail (Ko et al, 1980), and based on its histopathology it was classified as an anaplastic or undifferentiated glioma. The progeny of ENU-injected rats subsequently developed tumors, and following cloning by Wechsler in Germany, one of these clones was designated as "RG2" (rat glioma 2). The same clone was called the "D74-RG2" or "D74" in Koestner's laboratory at The Ohio State University. The RG2 glioma (Fig. 1c) is similar in microscopic appearance to the F98 glioma (Fig. 1d), and also has a highly invasive pattern of growth, which has made it a good representative model for GBM (Weizsacker et al, 1982). Gene expression profiling of these cells established that they had increased gene expression of PDGFβ, IGF-1, Ras, Erb3/HER3 precursor mRNA and cyclin D2. They express a wildtype p53 and a concurrent loss in the expression of the p16/Cdkn2a/Ink4 gene locus. It has been used for a variety of preclinical studies to evaluate

repeated intratumoral delivery and imaging (Bhattacharya et al, 2007).

in evaluating results obtained with such a highly immunogenic tumor.

they are identified.

changes in vascular permeability (Ferrier et al, 2007), disruption of the BBB (Ningaraj et al, 2002), anti-angiogenic therapy (Zagorac et al, 2008), gene therapy, chemotherapy (Miknyoczki et al, 2007) and radionuclide therapy (Shen et al, 2004).

The RG2 glioma is non-immunogenic in syngeneic Fischer rats and has low levels of MHC-1 expression compared to the C6 and 9L gliomas (Oshiro et al, 2001). However, in vitro treatment with IFN-γ upregulated MHC class I antigen expression and also resulted in a significant in vivo anti-tumor immune response with increased survival of treated animals. More recently, the RG2 glioma has been stably transfected with human Herpes virus Entry Mediator C (HveC) to facilitate HSV infection and has been used to study the therapeutic effects of oncolytic Herpes simplex virus-1 treatment (Kurozumi et al, 2007). The transfected cells retained their tumorigenicity following i.c. implantation in Fischer rats, and transfection of the HveC gene did not affect i.c. tumor growth (Wakimoto et al, 2004). However, it has not been determined if HveC can cause these cells to become immunogenic, and therefore, this must be taken into account when using the RG2 for immunotherapy studies.

**The F98 glioma** (ATCC # CRL-2397) was produced by Wechsler in Koestner's laboratory at the same time as the RG2 glioma. It is composed of a mixed population of spindle-shaped cells, the majority of which have fusiform nuclei, and a smaller number of polygonal cells with round to oval nuclei. There is extensive invasion of contiguous normal brain with islands of tumor cells at varying distances from the tumor mass, many of which form perivascular clusters (Fig. 1d). Similar to human GBM, these cells overexpress PDGFβ, and Ras along with an increase in EGFR, cyclin D1 and cyclin D2 expression relative to rat astrocytes (Sibenaller et al, 2005). Like the C6 glioma, they also have increased expression of Rb relative to rat astrocytes. Immunofluorescence studies of F98 cells also revealed low expression of BRCA1, and a lack of radiation and cisplatin induced BRCA1 foci in these cells (Bencokova et al, 2008). Usually, there is a necrotic core, scattered mitotic cells and nonglomeruloid neovascular proliferation. The tumor is GFAP and vimentin positive with negligible staining for CD3 + T cells (Mathieu et al, 2007). Since it simulates the behavior of human GBMs in a number of important ways, such as its highly invasive pattern of growth and low immunogenicity, it has been used to evaluate the efficacy of a variety of experimental therapeutic agents. It is refractory to a number of therapeutic modalities, including systemic chemotherapy with paclitaxel, and carboplatin (von Eckardstein et al, 2005), and it is poorly responsive to photon-irradiation alone, which in part may be related to its functionally impaired BRCA1 status that can favor genomic instability and impaired DNA repair. Recently, it has been shown to be responsive to a combination of synchrotron radiation with cisplatin (Biston et al, 2004), and to convection enhanced delivery (CED) of carboplatin in combination with 6 MV photon-irradiation in rats bearing i.c. tumors (Rousseau et al, 2008). This model has been used extensively by Barth et al. to evaluate the efficacy of boron neutron capture therapy (BNCT) (Yang et al, 2008). Elleaume and her coworkers have evaluated cisplatin, carboplatin and iodine enhanced synchrotron stereotactic radiotherapy (Cho et al, 2002) in F98 glioma bearing rats (Adam et al, 2005). It has also been used to evaluate non-invasive MRI to visualize tumor growth, diffusion tensor imaging (Zhang et al, 2007), tumor angiogenesis and the tumor tropism of mesenchymal stem cells (Wu et al, 2008).

The F98 glioma is very weakly immunogenic (Tzeng et al, 1991) and transfection with the gene encoding B7.1 co-stimulatory molecule (Paul et al, 2000), or syngeneic cellular vaccination combined with GMCSF, did not enhance its immunogenicity (Clavreul et al,

Animal Models of Glioma 355

animals were propagated in vitro and after 200 days in culture they became tumorigenic. The cells subsequently were implanted s.c. into BD IX rats and the resulting tumors contained a mixture of multipolar glia-like cells and flattened cells with fewer and shorter cytoplasmic processes and occasional giant cells (Laerum et al, 1977). BT4C glioma-derived tumors show high cellularity and have pleomorphic nuclei and numerous mitotic figures and the tumor blood vessels are irregular, dilated and show areas of proliferation (Fig. 1f) (Stuhr et al, 2007). At the molecular level, BT4C cells express VEGF, tPA, uPA and MVD in the periphery of the growing tumor and are S100 positive by immunohistochemistry (M. Johansson, Personal communication). This model has been useful to test novel chemotherapeutic targeting strategies (Pulkkinen et al, 2008), antitumor effects of gene therapy (Raty JK et al, 2004), anti-angiogenic agents alone (Huszthy et al, 2006) and in combination with radiation and temozolomide (Sandstrom et al, 2008). BT4C gliomas also have been used to investigate the impact of hyperoxia on tumor bearing rats. This resulted in slower growth accompanied by increased apoptosis of tumor cells and reduced microvessel density (MVD). Apart from studies to evaluate therapeutic efficacy, the BT4C glioma model also has been used to study the molecular and biological changes induced by chemotherapy (Vallbo et al, 2002), radiation therapy (Andersson et al, 2002) and suicide gene therapy (Griffin et al, 2003). BT4C cells, stably transfected with cDNA encoding βgalactosidase, have been used to evaluate the migration of single migrating tumor cell glioma spheroids and fetal brain aggregate coculture systems in vitro and in rat brains in

**Avian sarcoma virus induced and RT-2 glioma**. The induction of experimental brain tumors by the injection of Rous sarcoma virus has been described in canines, rats, and monkeys. Tumors were induced by inoculating neonatal Fischer rats i.c. with purified avian sarcoma virus (ASV) suspensions (Copeland et al, 1976). All of the animals developed tumors within 2 weeks following ASV injection, 94% of which were anaplastic astrocytomas, and the remainder were low grade gliomas or sarcomas (Prabhu et al, 2000). This model has been used to study the effects of chemo- and radiotherapy, BBB disruption, tumor permeability, and if de novo tumor induction is an important requirement. The response to immunotherapy indicated that these tumors were immunogenic, and expressed a variety of virally encoded tumor specific antigens. A continuous cell line, designated "RT-2", was derived from an ASV-induced Fischer rat tumor, and this has been used to study tumor growth, photochemotherapy, cytotoxic gene therapy (Valerie et al, 2001) and radiosensitization (Valerie et al, 2000). The RT2 tumor appears to be immunogenic, as evidenced by its ability to evoke a CD8 + T cell-mediated anti-tumor immune response (Shah et al, 2003), and this must be taken into account if it is used for immunotherapy studies. RT-2 cells expressing GFP have been used for quantitative assessment of glioma invasion in the rat brain (Mourad et al, 2003). The RT-2 glioma model also has been used to evaluate the therapeutic efficacy of oncolytic adenoviruses. Although they can be efficiently infected they do not permit efficient replication of E1- attenuated adenoviruses. These cells also have been transfected with cDNA encoding heat shock protein 72 (HSP72), which was thought to be necessary for replication of E1 deleted adenoviruses. These transfectants have been found to be permissive for replication of E1- deleted, conditionally replication-competent adenoviruses. The inherent immunogenicity of the RT-2 glioma may limit its usefulness for survival studies, but nevertheless it still may be a useful model for other types of studies. Rat brain tumor models have provided a wealth of information on the biology, biochemistry, imaging and experimental therapeutics of brain tumors in experimental

vivo (Garcia-Cabrera et al, 1996;).

2006). This makes it a very attractive model to investigate the mechanisms underlying glioma resistance to immunotherapy. It has also been used to study the molecular genetic alterations in GBMs (Hanissian et al, 2005), effects of infusion rates on drug distribution in i.c. tumors, and for suicide gene therapy with Herpes simplex virus-1 thymidine kinase (HSV-TK) (von Eckardstein et al, 2001). Like the 9L gliosarcoma, F98 cells also have been injected into the pontine tegmentum of the brainstem of Fischer rats to produce a model for brainstem tumors. The histopathological and radiobiological characteristics of these tumors were comparable to aggressive, primary human brainstem tumors, which could facilitate preclinical testing of therapeutics to treat these lethal tumors.

F98 cells have been stably transfected with expression vectors encoding for wildtype EGFR and EGFRvIII, and the resulting cell lines have been designated F98EGFR (ATCC# CRL-2948) and F98npEGFRvIII (ATCC# CRL-2949). They each express ~105 non-functional (i.e. nonphosphorylatable) receptor sites per cell. This is below the threshold number of 106 sites per cell that can evoke a xeno-immune response against human EGFR in rats. These cell lines have been used in Fischer rats for studies on molecular targeting (Yang et al, 2005) to evaluate the therapeutic efficacy of boronated mAbs and EGF for neutron capture therapy (NCT) (Wu et al, 2007). The boronated mAbs, L8A4, which is specific for EGFRvIII, and cetuximab, which recognizes wild type EGFR, specifically targeted their respective receptor positive i.c. tumors after CED and they were therapeutically effective following NCT.

A bioluminescent F98 cell line recently was constructed by stably transfecting F98 cells with the luciferase gene. When implanted i.c. into the brains of Fischer rats, tumor size could be monitored by measuring luminescence. This model should permit rapid, non-invasive imaging of i.c. tumor growth to evaluate novel therapeutic modalities (Bryant et al, 2008). Finally, F98 cells also are capable of growing as i.c. xenografts in cats (Ernestus et al, 1992), but since these cells can evoke a xenoimmune response, this model is of limited usefulness. It is important to note that, what may be therapeutically effective in the rat, may not be in the human. However, it probably is safe to say that if a particular therapeutic approach is ineffective in a rat model, it is even more unlikely to be so in humans.

**The CNS-1 glioma** was derived from an inbred Lewis rat that had received weekly i.v. injections of MNU for 6 months (Kruse et al, 1994). Following i.c. implantation into Lewis rats, it demonstrated an infiltrative pattern of growth with leptomeningeal, perivascular, and periventricular spread and extension of the tumor into the choroid plexus. Histologically, these tumors exhibited hypercellularity, nuclear atypia and pleomorphism, and had necrotic foci. These were surrounded by glioma cells arranged in a pseudopalisading pattern (Fig. 1e), although to a lesser extent than that seen in human GBM. These tumors are weakly immunogenic. Like human GBMs, these also were infiltrated with macrophages and T-cells, but did not have extensive glomeruloid endothelial/microvascular proliferation. Kielian et al. identified the constitutive expression of monocyte chemotactic factor 1 (MCP-1) by CNS-1 cells (Kielian et al, 2002). In vivo, CNS-1 tumors also showed extensive infiltration by macrophages, which might confer a growth advantage (Platten et al, 2003). This model has been useful to study glioma invasion (Owens et al, 1998), changes in the biology of glioma cells and their extracellular matrix (Lapointe et al, 2004), and gene therapy (Biglari et al, 2004). It also has been used to study the efficacy of immunotherapy as a potential treatment for human GBM (Ali et al, 2004) although its immunogenicity has not been studied in great detail.

**The BT4C glioma** was derived by giving a single transplacental administration of N-ethyl-Nnitrosourea (ENU) to pregnant BD IX rats. Dissociated brain tumor cells from one of these

2006). This makes it a very attractive model to investigate the mechanisms underlying glioma resistance to immunotherapy. It has also been used to study the molecular genetic alterations in GBMs (Hanissian et al, 2005), effects of infusion rates on drug distribution in i.c. tumors, and for suicide gene therapy with Herpes simplex virus-1 thymidine kinase (HSV-TK) (von Eckardstein et al, 2001). Like the 9L gliosarcoma, F98 cells also have been injected into the pontine tegmentum of the brainstem of Fischer rats to produce a model for brainstem tumors. The histopathological and radiobiological characteristics of these tumors were comparable to aggressive, primary human brainstem tumors, which could facilitate

F98 cells have been stably transfected with expression vectors encoding for wildtype EGFR and EGFRvIII, and the resulting cell lines have been designated F98EGFR (ATCC# CRL-2948) and F98npEGFRvIII (ATCC# CRL-2949). They each express ~105 non-functional (i.e. nonphosphorylatable) receptor sites per cell. This is below the threshold number of 106 sites per cell that can evoke a xeno-immune response against human EGFR in rats. These cell lines have been used in Fischer rats for studies on molecular targeting (Yang et al, 2005) to evaluate the therapeutic efficacy of boronated mAbs and EGF for neutron capture therapy (NCT) (Wu et al, 2007). The boronated mAbs, L8A4, which is specific for EGFRvIII, and cetuximab, which recognizes wild type EGFR, specifically targeted their respective receptor positive i.c. tumors after CED and they were therapeutically effective following NCT. A bioluminescent F98 cell line recently was constructed by stably transfecting F98 cells with the luciferase gene. When implanted i.c. into the brains of Fischer rats, tumor size could be monitored by measuring luminescence. This model should permit rapid, non-invasive imaging of i.c. tumor growth to evaluate novel therapeutic modalities (Bryant et al, 2008). Finally, F98 cells also are capable of growing as i.c. xenografts in cats (Ernestus et al, 1992), but since these cells can evoke a xenoimmune response, this model is of limited usefulness. It is important to note that, what may be therapeutically effective in the rat, may not be in the human. However, it probably is safe to say that if a particular therapeutic approach is

**The CNS-1 glioma** was derived from an inbred Lewis rat that had received weekly i.v. injections of MNU for 6 months (Kruse et al, 1994). Following i.c. implantation into Lewis rats, it demonstrated an infiltrative pattern of growth with leptomeningeal, perivascular, and periventricular spread and extension of the tumor into the choroid plexus. Histologically, these tumors exhibited hypercellularity, nuclear atypia and pleomorphism, and had necrotic foci. These were surrounded by glioma cells arranged in a pseudopalisading pattern (Fig. 1e), although to a lesser extent than that seen in human GBM. These tumors are weakly immunogenic. Like human GBMs, these also were infiltrated with macrophages and T-cells, but did not have extensive glomeruloid endothelial/microvascular proliferation. Kielian et al. identified the constitutive expression of monocyte chemotactic factor 1 (MCP-1) by CNS-1 cells (Kielian et al, 2002). In vivo, CNS-1 tumors also showed extensive infiltration by macrophages, which might confer a growth advantage (Platten et al, 2003). This model has been useful to study glioma invasion (Owens et al, 1998), changes in the biology of glioma cells and their extracellular matrix (Lapointe et al, 2004), and gene therapy (Biglari et al, 2004). It also has been used to study the efficacy of immunotherapy as a potential treatment for human GBM (Ali et al, 2004) although its

**The BT4C glioma** was derived by giving a single transplacental administration of N-ethyl-Nnitrosourea (ENU) to pregnant BD IX rats. Dissociated brain tumor cells from one of these

preclinical testing of therapeutics to treat these lethal tumors.

ineffective in a rat model, it is even more unlikely to be so in humans.

immunogenicity has not been studied in great detail.

animals were propagated in vitro and after 200 days in culture they became tumorigenic. The cells subsequently were implanted s.c. into BD IX rats and the resulting tumors contained a mixture of multipolar glia-like cells and flattened cells with fewer and shorter cytoplasmic processes and occasional giant cells (Laerum et al, 1977). BT4C glioma-derived tumors show high cellularity and have pleomorphic nuclei and numerous mitotic figures and the tumor blood vessels are irregular, dilated and show areas of proliferation (Fig. 1f) (Stuhr et al, 2007). At the molecular level, BT4C cells express VEGF, tPA, uPA and MVD in the periphery of the growing tumor and are S100 positive by immunohistochemistry (M. Johansson, Personal communication). This model has been useful to test novel chemotherapeutic targeting strategies (Pulkkinen et al, 2008), antitumor effects of gene therapy (Raty JK et al, 2004), anti-angiogenic agents alone (Huszthy et al, 2006) and in combination with radiation and temozolomide (Sandstrom et al, 2008). BT4C gliomas also have been used to investigate the impact of hyperoxia on tumor bearing rats. This resulted in slower growth accompanied by increased apoptosis of tumor cells and reduced microvessel density (MVD). Apart from studies to evaluate therapeutic efficacy, the BT4C glioma model also has been used to study the molecular and biological changes induced by chemotherapy (Vallbo et al, 2002), radiation therapy (Andersson et al, 2002) and suicide gene therapy (Griffin et al, 2003). BT4C cells, stably transfected with cDNA encoding βgalactosidase, have been used to evaluate the migration of single migrating tumor cell glioma spheroids and fetal brain aggregate coculture systems in vitro and in rat brains in vivo (Garcia-Cabrera et al, 1996;).

**Avian sarcoma virus induced and RT-2 glioma**. The induction of experimental brain tumors by the injection of Rous sarcoma virus has been described in canines, rats, and monkeys. Tumors were induced by inoculating neonatal Fischer rats i.c. with purified avian sarcoma virus (ASV) suspensions (Copeland et al, 1976). All of the animals developed tumors within 2 weeks following ASV injection, 94% of which were anaplastic astrocytomas, and the remainder were low grade gliomas or sarcomas (Prabhu et al, 2000). This model has been used to study the effects of chemo- and radiotherapy, BBB disruption, tumor permeability, and if de novo tumor induction is an important requirement. The response to immunotherapy indicated that these tumors were immunogenic, and expressed a variety of virally encoded tumor specific antigens. A continuous cell line, designated "RT-2", was derived from an ASV-induced Fischer rat tumor, and this has been used to study tumor growth, photochemotherapy, cytotoxic gene therapy (Valerie et al, 2001) and radiosensitization (Valerie et al, 2000). The RT2 tumor appears to be immunogenic, as evidenced by its ability to evoke a CD8 + T cell-mediated anti-tumor immune response (Shah et al, 2003), and this must be taken into account if it is used for immunotherapy studies. RT-2 cells expressing GFP have been used for quantitative assessment of glioma invasion in the rat brain (Mourad et al, 2003). The RT-2 glioma model also has been used to evaluate the therapeutic efficacy of oncolytic adenoviruses. Although they can be efficiently infected they do not permit efficient replication of E1- attenuated adenoviruses. These cells also have been transfected with cDNA encoding heat shock protein 72 (HSP72), which was thought to be necessary for replication of E1 deleted adenoviruses. These transfectants have been found to be permissive for replication of E1- deleted, conditionally replication-competent adenoviruses. The inherent immunogenicity of the RT-2 glioma may limit its usefulness for survival studies, but nevertheless it still may be a useful model for other types of studies.

Rat brain tumor models have provided a wealth of information on the biology, biochemistry, imaging and experimental therapeutics of brain tumors in experimental

Animal Models of Glioma 357

correct models of CNS cancers that result from activation and/or inactivation of

These models support the concept that the genetic alterations in human tumors, such as p53 loss and loss of PTEN function, are probably important in the development of astrocytomas (Grades II and III). Rodent models of GBM tumors are also available. In a somatic genetransfer model, simultaneous retroviral expression of constitutively active Ras and Akt gives rise to the formation of high-grade gliomas that are morphologically similar to human GBM tumors (Holland et al, 2000). Although Ras mutations are uncommon in GBM tumors, one study (Sharma et al, 2005) suggests that Ras activity is increased in human GBM biopsies due to a point mutation. In mice, the combination of EGFR amplification and either loss of p53 plus CDK4 overexpression or loss of INK4a-ARF is sufficient to induce glioma tumor formation that resembles that of human GBM tumors (Zhu et al, 2009). In an EGFR transgenic mouse model, LOH of p16INK4a, p19ARF, and PTEN cooperates with the amplification of EGFR to induce a highly infiltrative GBM tumor. Also, simultaneous deletion of p53 and PTEN in the mouse central nervous system generates an acute-onset, high-grade malignant glioma tumor that is histologically similar to human GBM tumors (Zheng et al, 2008). A new model of GBM tumor has been created by retroviral expression of PDGF-B in adult rat neural progenitor cells (Assanah et al, 2006). In this model, intracranial injection of retrovirus containing PDGF-B alone or in combination with PDGFRα results in the development of GBM-like tumors. To date, individual disruption or LOH of a single gene regulating the cell cycle, such as p53, INK4a, or ARF, has been insufficient to initiate gliomagenesis in vivo (Holland et al, 2001). Taken together, these studies suggest that alterations in neural progenitor cells probably give rise to at least some high-grade gliomas. There are limitations in the use of the above-discussed models. These include the facts that the tumor cells are not of human origin and that the rodents can in some instances require

Over the past two decades, scientists have developed a greater understanding of the molecular and genetic basis of brain tumorigenesis (Zhu et al, 2002). Evidence of the downregulation of tumor suppressor genes such as p53 and PTEN as well as elevated expression of growth factors, and their cognate tyrosine kinase receptors, such as PDGF and EGFR are found in a high percentage of human GBM tumors (Schwartzbaum et al, 2006). Researchers have exploited the role of these molecular pathways in brain tumor development to induce endogenous brain tumors in rodents. Thus, genetic engineering of mouse genes or intracranial delivery of oncogenic transgenes in adult mice and rats have been attempted in order to trigger the development of endogenous brain tumor in rodents. Germline deletion of the tumor suppressor genes p53 and NF1 increased the susceptibility of mice to develop astrocytomas (Reilly KM, 2009). These mice exhibit a wide range of astrocytoma stages, with tumor growth detected in 50-70% of the mice and median survival times of 6-8 months. This model is a valuable tool to study the development of secondary glioblastoma upon loss of p53. Germline deletion of other tumor suppressor genes, such as PTEN and Rb has also been attempted (Begemann et al, 2002). However, deletion of certain

endogenous genes in rodent genomes.

 Phosphatase and Tensin Homolog (PTEN), Epidermal Growth Factor Receptor (EGF-R), Platelet Derived Growth Factor (PDGF)

several months to reliably develop glioma tumors.

p53,

INK4a/ARF,

neuro-oncology, and there is every reason to believe that they will continue to do so. However, it is essential to recognize the limitations of each of the models that have been described, and depending on the nature of the study to be conducted, it is important that the appropriate model be selected. It now has become clear that immunogenic tumors such as the C6, 9L and T9 are not good choices for studies in immunocompetent rats, if the endpoint is prolongation of survival time or cure of the tumor. Destruction of tumor cells in these models, which have tumor infiltrating host immune effector cells within the tumor, can lead to significant amplification of an antitumor response. This may be the single most important in vivo contributor to the bystander effect that has been observed with gene therapy of the C6 and 9L gliomas following transfection with the HSV-tK gene and the lack of such immune amplification with the weakly immunogenic RG2 glioma. Anti-tumor immune response following transfection with suicide genes such as HSV-tK initially was unanticipated, but it is an important effect associated with both gene therapy and boron neutron capture therapy, but not with conventional chemo- and radiotherapy of the 9L gliosarcoma. Since human high grade brain tumors generally are regarded as being either non- or weakly immunogenic, therapeutic exploitation of this using modalities that spare tumor infiltrating host immune effector cells could have important therapeutic implications. Undoubtedly other rat brain tumor models will be developed, especially cell lines derived from genetically engineered rats that will expand the types of studies that can be carried out in this very important laboratory animal.

#### **2.1.2 Human glioma xenografts implanted in immunocompromised mice**

Xenograft models of malignant astrocytoma have been extensively used to assess the function of various signaling molecules or matrix proteins in glioma growth and invasion (Hingtgen et al, 2008). Xenograft models that transplant human malignant astrocytoma/glioma cells into the brains of immunocompromised mice (athymic nude or SCID) have the advantage of being relatively rapid models with which to assess the role of a particular molecule in positively or negatively regulating proliferation and/or regulating invasion in vivo. Also, these models are very useful for the initial evaluation of novel imaging techniques as well as new therapies for GBM, including antiangiogenic therapy chemotherapy, radiotherapy, targeted toxins, cytotoxic or conditionally replicative oncolytic viruses. One disadvantage of human xenograft models is that most human glioma cell lines are not invasive when propagated in vivo (Curtin et al, 2008). Another disadvantage is that the propagation of human malignant astrocytoma/glioma cell lines in culture can result in their loss of key genetic alterations, such as expression of the mutant EGFR (Tsurushima et al, 2007), that are the most likely to be important in gliomagenesis. This limitation has been overcome by propagating primary human GBM tumors in the nude mouse (either subcutaneously or intracerebrally) instead of in culture; when these tumors are propagated in vivo, the genetic alterations found in the patients biopsy are retained (Ozawa et al, 2005). For xenograft models it is also important to propagate the tumors for experimental analysis in an orthotopic environment (the brain) because the microenvironment in the brain (i.e., the extracellular matrix, growth factors, and stromal cells) is different from that found in the subcutaneous tissue.

#### **2.2 Gene trgeted animal models**

Recently, transgenic technology has allowed investigators to alter the function of specific genes of interest and thus exploit defined genetic lesions to produce more biologically correct models of CNS cancers that result from activation and/or inactivation of endogenous genes in rodent genomes.

p53,

356 Glioma – Exploring Its Biology and Practical Relevance

neuro-oncology, and there is every reason to believe that they will continue to do so. However, it is essential to recognize the limitations of each of the models that have been described, and depending on the nature of the study to be conducted, it is important that the appropriate model be selected. It now has become clear that immunogenic tumors such as the C6, 9L and T9 are not good choices for studies in immunocompetent rats, if the endpoint is prolongation of survival time or cure of the tumor. Destruction of tumor cells in these models, which have tumor infiltrating host immune effector cells within the tumor, can lead to significant amplification of an antitumor response. This may be the single most important in vivo contributor to the bystander effect that has been observed with gene therapy of the C6 and 9L gliomas following transfection with the HSV-tK gene and the lack of such immune amplification with the weakly immunogenic RG2 glioma. Anti-tumor immune response following transfection with suicide genes such as HSV-tK initially was unanticipated, but it is an important effect associated with both gene therapy and boron neutron capture therapy, but not with conventional chemo- and radiotherapy of the 9L gliosarcoma. Since human high grade brain tumors generally are regarded as being either non- or weakly immunogenic, therapeutic exploitation of this using modalities that spare tumor infiltrating host immune effector cells could have important therapeutic implications. Undoubtedly other rat brain tumor models will be developed, especially cell lines derived from genetically engineered rats that will expand the types of studies that can be carried out

in this very important laboratory animal.

**2.1.2 Human glioma xenografts implanted in immunocompromised mice** 

stromal cells) is different from that found in the subcutaneous tissue.

**2.2 Gene trgeted animal models** 

Xenograft models of malignant astrocytoma have been extensively used to assess the function of various signaling molecules or matrix proteins in glioma growth and invasion (Hingtgen et al, 2008). Xenograft models that transplant human malignant astrocytoma/glioma cells into the brains of immunocompromised mice (athymic nude or SCID) have the advantage of being relatively rapid models with which to assess the role of a particular molecule in positively or negatively regulating proliferation and/or regulating invasion in vivo. Also, these models are very useful for the initial evaluation of novel imaging techniques as well as new therapies for GBM, including antiangiogenic therapy chemotherapy, radiotherapy, targeted toxins, cytotoxic or conditionally replicative oncolytic viruses. One disadvantage of human xenograft models is that most human glioma cell lines are not invasive when propagated in vivo (Curtin et al, 2008). Another disadvantage is that the propagation of human malignant astrocytoma/glioma cell lines in culture can result in their loss of key genetic alterations, such as expression of the mutant EGFR (Tsurushima et al, 2007), that are the most likely to be important in gliomagenesis. This limitation has been overcome by propagating primary human GBM tumors in the nude mouse (either subcutaneously or intracerebrally) instead of in culture; when these tumors are propagated in vivo, the genetic alterations found in the patients biopsy are retained (Ozawa et al, 2005). For xenograft models it is also important to propagate the tumors for experimental analysis in an orthotopic environment (the brain) because the microenvironment in the brain (i.e., the extracellular matrix, growth factors, and

Recently, transgenic technology has allowed investigators to alter the function of specific genes of interest and thus exploit defined genetic lesions to produce more biologically


These models support the concept that the genetic alterations in human tumors, such as p53 loss and loss of PTEN function, are probably important in the development of astrocytomas (Grades II and III). Rodent models of GBM tumors are also available. In a somatic genetransfer model, simultaneous retroviral expression of constitutively active Ras and Akt gives rise to the formation of high-grade gliomas that are morphologically similar to human GBM tumors (Holland et al, 2000). Although Ras mutations are uncommon in GBM tumors, one study (Sharma et al, 2005) suggests that Ras activity is increased in human GBM biopsies due to a point mutation. In mice, the combination of EGFR amplification and either loss of p53 plus CDK4 overexpression or loss of INK4a-ARF is sufficient to induce glioma tumor formation that resembles that of human GBM tumors (Zhu et al, 2009). In an EGFR transgenic mouse model, LOH of p16INK4a, p19ARF, and PTEN cooperates with the amplification of EGFR to induce a highly infiltrative GBM tumor. Also, simultaneous deletion of p53 and PTEN in the mouse central nervous system generates an acute-onset, high-grade malignant glioma tumor that is histologically similar to human GBM tumors (Zheng et al, 2008). A new model of GBM tumor has been created by retroviral expression of PDGF-B in adult rat neural progenitor cells (Assanah et al, 2006). In this model, intracranial injection of retrovirus containing PDGF-B alone or in combination with PDGFRα results in the development of GBM-like tumors. To date, individual disruption or LOH of a single gene regulating the cell cycle, such as p53, INK4a, or ARF, has been insufficient to initiate gliomagenesis in vivo (Holland et al, 2001). Taken together, these studies suggest that alterations in neural progenitor cells probably give rise to at least some high-grade gliomas. There are limitations in the use of the above-discussed models. These include the facts that the tumor cells are not of human origin and that the rodents can in some instances require several months to reliably develop glioma tumors.

Over the past two decades, scientists have developed a greater understanding of the molecular and genetic basis of brain tumorigenesis (Zhu et al, 2002). Evidence of the downregulation of tumor suppressor genes such as p53 and PTEN as well as elevated expression of growth factors, and their cognate tyrosine kinase receptors, such as PDGF and EGFR are found in a high percentage of human GBM tumors (Schwartzbaum et al, 2006). Researchers have exploited the role of these molecular pathways in brain tumor development to induce endogenous brain tumors in rodents. Thus, genetic engineering of mouse genes or intracranial delivery of oncogenic transgenes in adult mice and rats have been attempted in order to trigger the development of endogenous brain tumor in rodents. Germline deletion of the tumor suppressor genes p53 and NF1 increased the susceptibility of mice to develop astrocytomas (Reilly KM, 2009). These mice exhibit a wide range of astrocytoma stages, with tumor growth detected in 50-70% of the mice and median survival times of 6-8 months. This model is a valuable tool to study the development of secondary glioblastoma upon loss of p53. Germline deletion of other tumor suppressor genes, such as PTEN and Rb has also been attempted (Begemann et al, 2002). However, deletion of certain

Animal Models of Glioma 359

mice developed invasive glioblastoma that exhibited neo-vascularization and tumor cell

In order to mimic the multiple genetic lesions encountered in human GBM, retroviral vectors that encode growth factors and a cycline-dependent kinase (cdk) were injected in the brain of neo-natal mice harboring additional mutations in tumor suppressor genes. Delivery of a constitutively active form of epidermal growth factor receptor gene (EGFR) in combination with basic fibroblast growth factor (bFGF) or ckd4 into the brain of neo-natal mice that are deficient in INK4a–ARF or p53 tumor suppressor genes led to formation of GBM in ~50% of the animals, while single mutations were unable of generating tumors (Holland et al, 1998). These findings support the notion that combination of genetic lesions is required for the induction of endogenous GBM in mice. Additionally, combined genetic aberrations can be targeted to specific cell populations by the development of transgenic mice that express the retroviral receptor under the control of cell-type specific promoters, such as the progenitor nestin promoter or the astrocyte GFAP promoter (Dai et al, 2001). This system is very functional because it allows cell-type-specific transfer of oncogenes

Lentiviral vectors have recently been employed to deliver oncogenes into the mouse brain. Considering that lentiviral vectors can transduce both dividing and non-dividing cells, these vectors constitute an attractive vehicle to deliver oncogenes to the brain of adult rodents (Singer O, Verma IM, 2008). In order to recapitulate the initiation of GBM, which is thought to arise upon genetic mutations in a few cells, oncogenic transgenes were delivered in a small population of cells in adult mouse brain by region-specific injection of lentiviral vectors encoding H-Ras or AKT. To target astrocytes the Cre-LoxP-controlled lentiviruses were injected in the cortex, hippocampus and subventricular zone of GFAP-Cre mice. Again, administration of single oncogenes did not induce formation of tumors for up to 10 months. However, when Ras and AKT were delivered together in the hippocampal area ~30% of mice exhibited brain tumors that exhibit a high degree of invasiveness within 3-5 months post injection. Only one mouse developed a tumor following transduction in the sub-ventricular zone, and no animals had tumors following transduction into the cortex. Combined delivery of H-Ras and AKT into p53 KO mice greatly increased the tumorigenesis of these vectors leading to 75 and 100% of the mice injected in the subventricular zone and hippocampus, respectively. These tumors also exhibited a much shorter tumor latency with many histopathological characteristics found in human GBM (Marumoto et al, 2009). These findings indicate that lentiviral vectors are useful tools to induce endogenous GBM in adult mice when several genetical abnormalities are induced in combination in the appropriate area of the brain. Another recent approach to induce endogenous GBM in mice is the use of the Sleeping Beauty (SB) transposable element to achieve integration of oncogenes in the genome of brain cells of neo-natal immune competent mice (Ohlfest et al, 2005). SB is a synthetic transposable element composed of a transposon DNA substrate and a transposase enzyme. SB transposase mediates excision and insertion of transposon DNA into the host genome, leading to long term expression (Ohlfest et al, 2004). Spontaneous brain tumors were induced by injecting SB-dependent plasmid harboring up to three genetic alterations (AKT, N-RAS, EGRFvIII, and/or shRNA specific for p53) into the lateral cerebral ventricle of neonatal mice of three different strains (Wiesner et al, 2009). The histological characteristics of the tumors were dependant of the combination of genetic lesions introduced to the mice, although most resembled human astrocytoma or GBM. In some mice, multifocal tumors, another hallmark of human GBM, was observed. The combination of N-RAs, EGFRvIII, and

infiltration throughout the brain parenchyma (Shih et al, 2004).

expressed within retroviral vectors under any type of promoter.

genes can lead to embryonic lethality or to the generation of tumors in other organs, limiting the utility of these models.

Tissue specific overexpression of putative oncogenes of interest, using methods which link the gene of interest to a glial specific promoter such as GFAP, S100β, or Nestin, provides an appealing approach towards the creation of spontaneously occurring brain tumors in animals seen in many germline knockout animals. Tissue targeted models involving deletion of tumor suppressor genes is more difficult. Conditional knockout models represent a promising new attempt to eliminate tumor suppressor function in a cell specific manner. These techniques have recently been utilized to create of variety of transgenic brain tumor models using targeted conditional knockouts of p53, PTEN, Ptc, and Rb. Frequently, conditional knockouts used in combination with oncogenes overexpressed on tissue specific promoters or introduced using viral vectors can create a localized tumor genetically similar to human cancer in an immune competent animal.

Transgenic mice that display cell type-specific overexpression of oncogenes have been employed to study genetic abnormalities in astrocytes and neural progenitors. This has proven useful to establish the role of oncogenes in the tumorgenesis and progression of GBM (Ding et al, 2001). Overexpression of the transcription factor E2F1 under the transcriptional control of the GFAP promoter led to the formation of astrocytomas in p53 KO mice, suggesting a role for E2F1 as an oncogene in the formation of brain tumors (Olson et al, 2007). Considering that cell typespecific expression of certain genes is lethal during early development, oncogene overexpression has also been approached by delivery of gene therapy vectors into the brain of pre-natal or adult rodents, leading to the formation of endogenous brain tumors. These tumors harbor the genetic abnormalities found in human GBM, as well as the histopathological hallmarks of human GBM, including the aggressive invasive behavior. The use of viral or plasmid based vectors to introduce genetic aberrations permits the tight anatomical restriction of tumor-forming genetic events to specific areas of the brain. Furthermore, viral and plasmid vectors allow for the delivery of multiple tumorigenic genes in any combination, thereby reducing the amount of time required to generate germline transgenic mouse models. Thus, endogenous rodent GBM models constitute a very promising and stringent animal model of GBM which recapitulates the most salient histopathological features, molecular attributes, and heterogeneity of human GBM in a syngeneic rodent background. However, the applicability of the endogenous brain tumor models to assess the pre-clinical efficacy of experimental therapeutics is still limited due to the long latency and the variable reproducibility of these models.

Extensive evidence from across this developing field suggests that formation of endogenous brain tumors using viral vectors or plasmid systems to deliver oncogenes is somewhat variable. The degree of penetrance, tumor latency, and histopathological characteristics are dependant on the species and age of animals, the identity of specific genetic alterations and the vector system used to deliver them, and the anatomical location of genetic alterations. Retroviral- mediated delivery of PDGF into the adult rat white matter leads to formation of brain tumors with histopathological features that resemble human GBM; 100% of the animals succumb due to tumor burden 14-20 days after injection (Assanah et al, 2006). However, when retro-PDGF is delivered into the brain of newborn mice brain tumor formation only occurred in ~40% of the animals within 14-29 weeks. The incidence and grade of brain tumor formation in mice has been suggested to be dependant on the levels of expression of PDGF. Newborn mice were administered with retroviral vectors encoding a PDGF gene that lacks its regulatory sequences, which leads to higher levels of PDGF expression. Within 4-12 weeks, 100% of these

genes can lead to embryonic lethality or to the generation of tumors in other organs, limiting

Tissue specific overexpression of putative oncogenes of interest, using methods which link the gene of interest to a glial specific promoter such as GFAP, S100β, or Nestin, provides an appealing approach towards the creation of spontaneously occurring brain tumors in animals seen in many germline knockout animals. Tissue targeted models involving deletion of tumor suppressor genes is more difficult. Conditional knockout models represent a promising new attempt to eliminate tumor suppressor function in a cell specific manner. These techniques have recently been utilized to create of variety of transgenic brain tumor models using targeted conditional knockouts of p53, PTEN, Ptc, and Rb. Frequently, conditional knockouts used in combination with oncogenes overexpressed on tissue specific promoters or introduced using viral vectors can create a localized tumor genetically similar

Transgenic mice that display cell type-specific overexpression of oncogenes have been employed to study genetic abnormalities in astrocytes and neural progenitors. This has proven useful to establish the role of oncogenes in the tumorgenesis and progression of GBM (Ding et al, 2001). Overexpression of the transcription factor E2F1 under the transcriptional control of the GFAP promoter led to the formation of astrocytomas in p53 KO mice, suggesting a role for E2F1 as an oncogene in the formation of brain tumors (Olson et al, 2007). Considering that cell typespecific expression of certain genes is lethal during early development, oncogene overexpression has also been approached by delivery of gene therapy vectors into the brain of pre-natal or adult rodents, leading to the formation of endogenous brain tumors. These tumors harbor the genetic abnormalities found in human GBM, as well as the histopathological hallmarks of human GBM, including the aggressive invasive behavior. The use of viral or plasmid based vectors to introduce genetic aberrations permits the tight anatomical restriction of tumor-forming genetic events to specific areas of the brain. Furthermore, viral and plasmid vectors allow for the delivery of multiple tumorigenic genes in any combination, thereby reducing the amount of time required to generate germline transgenic mouse models. Thus, endogenous rodent GBM models constitute a very promising and stringent animal model of GBM which recapitulates the most salient histopathological features, molecular attributes, and heterogeneity of human GBM in a syngeneic rodent background. However, the applicability of the endogenous brain tumor models to assess the pre-clinical efficacy of experimental therapeutics is still limited

due to the long latency and the variable reproducibility of these models.

Extensive evidence from across this developing field suggests that formation of endogenous brain tumors using viral vectors or plasmid systems to deliver oncogenes is somewhat variable. The degree of penetrance, tumor latency, and histopathological characteristics are dependant on the species and age of animals, the identity of specific genetic alterations and the vector system used to deliver them, and the anatomical location of genetic alterations. Retroviral- mediated delivery of PDGF into the adult rat white matter leads to formation of brain tumors with histopathological features that resemble human GBM; 100% of the animals succumb due to tumor burden 14-20 days after injection (Assanah et al, 2006). However, when retro-PDGF is delivered into the brain of newborn mice brain tumor formation only occurred in ~40% of the animals within 14-29 weeks. The incidence and grade of brain tumor formation in mice has been suggested to be dependant on the levels of expression of PDGF. Newborn mice were administered with retroviral vectors encoding a PDGF gene that lacks its regulatory sequences, which leads to higher levels of PDGF expression. Within 4-12 weeks, 100% of these

the utility of these models.

to human cancer in an immune competent animal.

mice developed invasive glioblastoma that exhibited neo-vascularization and tumor cell infiltration throughout the brain parenchyma (Shih et al, 2004).

In order to mimic the multiple genetic lesions encountered in human GBM, retroviral vectors that encode growth factors and a cycline-dependent kinase (cdk) were injected in the brain of neo-natal mice harboring additional mutations in tumor suppressor genes. Delivery of a constitutively active form of epidermal growth factor receptor gene (EGFR) in combination with basic fibroblast growth factor (bFGF) or ckd4 into the brain of neo-natal mice that are deficient in INK4a–ARF or p53 tumor suppressor genes led to formation of GBM in ~50% of the animals, while single mutations were unable of generating tumors (Holland et al, 1998). These findings support the notion that combination of genetic lesions is required for the induction of endogenous GBM in mice. Additionally, combined genetic aberrations can be targeted to specific cell populations by the development of transgenic mice that express the retroviral receptor under the control of cell-type specific promoters, such as the progenitor nestin promoter or the astrocyte GFAP promoter (Dai et al, 2001). This system is very functional because it allows cell-type-specific transfer of oncogenes expressed within retroviral vectors under any type of promoter.

Lentiviral vectors have recently been employed to deliver oncogenes into the mouse brain. Considering that lentiviral vectors can transduce both dividing and non-dividing cells, these vectors constitute an attractive vehicle to deliver oncogenes to the brain of adult rodents (Singer O, Verma IM, 2008). In order to recapitulate the initiation of GBM, which is thought to arise upon genetic mutations in a few cells, oncogenic transgenes were delivered in a small population of cells in adult mouse brain by region-specific injection of lentiviral vectors encoding H-Ras or AKT. To target astrocytes the Cre-LoxP-controlled lentiviruses were injected in the cortex, hippocampus and subventricular zone of GFAP-Cre mice. Again, administration of single oncogenes did not induce formation of tumors for up to 10 months. However, when Ras and AKT were delivered together in the hippocampal area ~30% of mice exhibited brain tumors that exhibit a high degree of invasiveness within 3-5 months post injection. Only one mouse developed a tumor following transduction in the sub-ventricular zone, and no animals had tumors following transduction into the cortex. Combined delivery of H-Ras and AKT into p53 KO mice greatly increased the tumorigenesis of these vectors leading to 75 and 100% of the mice injected in the subventricular zone and hippocampus, respectively. These tumors also exhibited a much shorter tumor latency with many histopathological characteristics found in human GBM (Marumoto et al, 2009). These findings indicate that lentiviral vectors are useful tools to induce endogenous GBM in adult mice when several genetical abnormalities are induced in combination in the appropriate area of the brain.

Another recent approach to induce endogenous GBM in mice is the use of the Sleeping Beauty (SB) transposable element to achieve integration of oncogenes in the genome of brain cells of neo-natal immune competent mice (Ohlfest et al, 2005). SB is a synthetic transposable element composed of a transposon DNA substrate and a transposase enzyme. SB transposase mediates excision and insertion of transposon DNA into the host genome, leading to long term expression (Ohlfest et al, 2004). Spontaneous brain tumors were induced by injecting SB-dependent plasmid harboring up to three genetic alterations (AKT, N-RAS, EGRFvIII, and/or shRNA specific for p53) into the lateral cerebral ventricle of neonatal mice of three different strains (Wiesner et al, 2009). The histological characteristics of the tumors were dependant of the combination of genetic lesions introduced to the mice, although most resembled human astrocytoma or GBM. In some mice, multifocal tumors, another hallmark of human GBM, was observed. The combination of N-RAs, EGFRvIII, and

Animal Models of Glioma 361

and have highly proliferative rate, and ability of self-renewal and differentiation. In vitro they form neurospheres and in vivo they growth intracranially in the brain of nude mice,

In summary, canine GBM emerges as an attractive animal model for testing novel therapies in a spontaneous tumor in the context of a large brain. The features of dog GBM make it a unique large animal model for preclinical cancer research with therapeutic outcomes which could better predict their efficacy in human trials. In spite of these attractive features, dogs are very expensive to treat and scarce, therefore the routine testing on novel therapeutics in

As a prelude to the implementation of gene therapy clinical trials for glioblastoma multiforme, it is critical to test potential novel therapies in relevant animal models of this disease. The ideal brain tumor model should exhibit predictable and reproducible intracranial growth patterns, have histopathological and biochemical resemblance to human GBMs and be nonimmunogenic. There are several models available in which it is feasible to study the efficacy and toxicity of different therapeutic approaches for this disease, i.e., antiangiogenic agents, proapoptotic molecules, immunotherapy, etc. these glioma models help unravel the biology of tumorigenesis and etiology of human central nervous system tumors. These mouse-modeling experiments may identify essential targets for therapy and provide

Main glioma animal models are murine implantation models traditionally, in this chapter, 9 rat models are described in detail. The most widely used model C6 rat glioma arose in an outbred Wistar rat, is non-syngeneic. Since the tumor is immunogenic, even in Wistar rats, the C6 glioma is not suitable for study of immunotherapy. Syngeneic murine models, i.e. CNS-1 cells in Lewis rats, F98 and RG-2 cells in Fisher rats, GL26 cells in C57BL6 mice, SMA-560 cells in VMDK mice, are non-immunogenic, constituting an excellent tool. Human glioma xenografts implanted in immunocompromised mice have been extensively employed in preclinical brain cancer research. Although their xenogeneic nature impairs the study of immune-mediated anti-tumor strategies, they allow assessing the efficacy of therapeutic approaches in human GBM cells in the context of normal brain tissue. In fact, human xenografts exhibit histopatological features that resemble the human GBM and

Recently, transgenic technique and gene knock-out technique rapidly developed, new animal models of gliomas were created. Central nervous system–specific inactivation of the genes encoding the tumor suppressors p53 and Nf1 leads to the spontaneous onset of Grade II and III astrocytoma tumors, as well as to GBM tumors in mice. This gliomagenesis can be accelerated by haploinsufficiency of the PTEN gene, and in neural progenitor cells conditional inactivation of p53 coordinates with a haploinsufficiency of PTEN and Nf1 to induce tumor formation. p53, INK4a/ARF, PTEN, EGFR, PDGF are the most popular genes in glioma research. Viral vectors or plasmid systems are used to deliver oncogenes. By means of linking the gene of interest to a glial specific promoter such as GFAP, S100β, or Nestin, transgenic mice that display cell type-specific overexpression of oncogenes. When

large animal models are necessary, dog glioma models are available for alternative.

forming GBMs that exhibit histopathological features of dog GBM.

test animals for preclinical trials of mechanistically designed therapeutics.

retain gene amplifications detected in the in situ tumors.

these animals would be unfeasible.

**4. Conclusion** 

**3. Application and future projection** 

p53 silencing was the most robust combination of genes with a 100% penetrance and a median survival of 83 days. These tumors were highly invasive and immunoreactive for nestin and GFAP indicating heterogeneity in the tumor mass. The SB is a very attractive and versatile system to induce endogenous brain tumors, allowing integration of large transposons (<10 kb) into the genome of many strains of mice.

In summary, endogenous rodent brain tumor models that recapitulate the genetic aberrations found in human GBM are very useful for the study of gliomagenesis; however, their variable tumor formation rate and long latency limits their use for testing preclinical treatments. Nevertheless, the use of imaging techniques to confirm tumor formation before the treatment would allow rigorous evaluation of novel therapies in these models, which resemble hystologically and genetically the human disease.

#### **2.3 Other models**

Dogs bearing spontaneous GBM constitute a valuable tool in preclinical brain cancer research. GBM is the most common primary brain tumor in dogs, and brachycephalic breeds such as Boston terriers and Boxers (Heidner et al, 1991) are predisposed to develop spontaneous GBM (Stoica et al, 2004). Dog GBM exhibits the same histopathological characteristics of the human disease, including necrosis with pseudopalizading, neovascularization and endothelial proliferation. The presence of pseudopalisading necroses and endothelial proliferation that closely resemble those found in human GBMs suggest the presence of a hypoxic environment in dog GBM, as described in human patients (Rong et al, 2006). Importantly, canine GBM is highly invasive and exhibits the classical patterns of human GBM invasion, which makes it a very valuable tool to test not only the efficacy of novel therapies, but also their toxicity to the normal brain. The large size of the dog brain would be useful for preclinical assessment of doses and volumes in order to optimize treatment protocols before the translation into the clinic. Also, the detection of therapy-induced toxicity and side effects, as well as behavioral abnormalities are technically very well developed in dogs and constitutes a routine assessment in clinical veterinary practice. Moreover, the individual variability of outbreed dogs could help to better predict the clinical outcomes in human patients. Clinical signs and prognosis of dogs with spontaneous GBM are very similar to those in human, and there is a high correlation of neuro-imaging features seen with MRI in canine and human GBM, which is also used as a diagnostic tool for canine GBM (Lipsitz et al, 2003). The standard care of treatment in dogs with GBM is very similar to that used in human patients, consisting of surgical resection followed by radiation therapy and chemotherapy which leads to a median survival of 8.5-10 months. This allows performing preclinical trials that will mimic more closely the clinical scenario, in which new therapies are applied in patients that simultaneously undergo traditional treatment. Candolfi and others have previously demonstrated the feasibility of delivering therapeutic transgenes to dog GBM cells in vitro and dog brain cells in vivo upon intracranial injection of gene therapy vectors, such as type 5 adenoviral vectors (Candolfi et al, 2007), adeno-associated viral vectors (Ciron et al, 2006), plasmid DNA/polyethylenimine (PEI) complexes (Oh et al, 2007), which suggests that dogs bearing spontaneous GBM would be a suitable model to test novel gene therapy approaches. Importantly, the availability of canine GBM J3T (Rainov et al, 2000) and W&W (Garcia-Escudero et al, 2008) cell lines allows in vitro screening of novel therapeutic agents before moving to preclinical trials in dogs bearing spontaneous GBM. Also, the characterization of cancer stem cells from a GBM in a Boxer has been recently reported (Stoica et al, 2009). These cells exhibit cancer stem markers

p53 silencing was the most robust combination of genes with a 100% penetrance and a median survival of 83 days. These tumors were highly invasive and immunoreactive for nestin and GFAP indicating heterogeneity in the tumor mass. The SB is a very attractive and versatile system to induce endogenous brain tumors, allowing integration of large

In summary, endogenous rodent brain tumor models that recapitulate the genetic aberrations found in human GBM are very useful for the study of gliomagenesis; however, their variable tumor formation rate and long latency limits their use for testing preclinical treatments. Nevertheless, the use of imaging techniques to confirm tumor formation before the treatment would allow rigorous evaluation of novel therapies in these models, which

Dogs bearing spontaneous GBM constitute a valuable tool in preclinical brain cancer research. GBM is the most common primary brain tumor in dogs, and brachycephalic breeds such as Boston terriers and Boxers (Heidner et al, 1991) are predisposed to develop spontaneous GBM (Stoica et al, 2004). Dog GBM exhibits the same histopathological characteristics of the human disease, including necrosis with pseudopalizading, neovascularization and endothelial proliferation. The presence of pseudopalisading necroses and endothelial proliferation that closely resemble those found in human GBMs suggest the presence of a hypoxic environment in dog GBM, as described in human patients (Rong et al, 2006). Importantly, canine GBM is highly invasive and exhibits the classical patterns of human GBM invasion, which makes it a very valuable tool to test not only the efficacy of novel therapies, but also their toxicity to the normal brain. The large size of the dog brain would be useful for preclinical assessment of doses and volumes in order to optimize treatment protocols before the translation into the clinic. Also, the detection of therapy-induced toxicity and side effects, as well as behavioral abnormalities are technically very well developed in dogs and constitutes a routine assessment in clinical veterinary practice. Moreover, the individual variability of outbreed dogs could help to better predict the clinical outcomes in human patients. Clinical signs and prognosis of dogs with spontaneous GBM are very similar to those in human, and there is a high correlation of neuro-imaging features seen with MRI in canine and human GBM, which is also used as a diagnostic tool for canine GBM (Lipsitz et al, 2003). The standard care of treatment in dogs with GBM is very similar to that used in human patients, consisting of surgical resection followed by radiation therapy and chemotherapy which leads to a median survival of 8.5-10 months. This allows performing preclinical trials that will mimic more closely the clinical scenario, in which new therapies are applied in patients that simultaneously undergo traditional treatment. Candolfi and others have previously demonstrated the feasibility of delivering therapeutic transgenes to dog GBM cells in vitro and dog brain cells in vivo upon intracranial injection of gene therapy vectors, such as type 5 adenoviral vectors (Candolfi et al, 2007), adeno-associated viral vectors (Ciron et al, 2006), plasmid DNA/polyethylenimine (PEI) complexes (Oh et al, 2007), which suggests that dogs bearing spontaneous GBM would be a suitable model to test novel gene therapy approaches. Importantly, the availability of canine GBM J3T (Rainov et al, 2000) and W&W (Garcia-Escudero et al, 2008) cell lines allows in vitro screening of novel therapeutic agents before moving to preclinical trials in dogs bearing spontaneous GBM. Also, the characterization of cancer stem cells from a GBM in a Boxer has been recently reported (Stoica et al, 2009). These cells exhibit cancer stem markers

transposons (<10 kb) into the genome of many strains of mice.

resemble hystologically and genetically the human disease.

**2.3 Other models** 

and have highly proliferative rate, and ability of self-renewal and differentiation. In vitro they form neurospheres and in vivo they growth intracranially in the brain of nude mice, forming GBMs that exhibit histopathological features of dog GBM.

In summary, canine GBM emerges as an attractive animal model for testing novel therapies in a spontaneous tumor in the context of a large brain. The features of dog GBM make it a unique large animal model for preclinical cancer research with therapeutic outcomes which could better predict their efficacy in human trials. In spite of these attractive features, dogs are very expensive to treat and scarce, therefore the routine testing on novel therapeutics in these animals would be unfeasible.

#### **3. Application and future projection**

As a prelude to the implementation of gene therapy clinical trials for glioblastoma multiforme, it is critical to test potential novel therapies in relevant animal models of this disease. The ideal brain tumor model should exhibit predictable and reproducible intracranial growth patterns, have histopathological and biochemical resemblance to human GBMs and be nonimmunogenic. There are several models available in which it is feasible to study the efficacy and toxicity of different therapeutic approaches for this disease, i.e., antiangiogenic agents, proapoptotic molecules, immunotherapy, etc. these glioma models help unravel the biology of tumorigenesis and etiology of human central nervous system tumors. These mouse-modeling experiments may identify essential targets for therapy and provide test animals for preclinical trials of mechanistically designed therapeutics.

#### **4. Conclusion**

Main glioma animal models are murine implantation models traditionally, in this chapter, 9 rat models are described in detail. The most widely used model C6 rat glioma arose in an outbred Wistar rat, is non-syngeneic. Since the tumor is immunogenic, even in Wistar rats, the C6 glioma is not suitable for study of immunotherapy. Syngeneic murine models, i.e. CNS-1 cells in Lewis rats, F98 and RG-2 cells in Fisher rats, GL26 cells in C57BL6 mice, SMA-560 cells in VMDK mice, are non-immunogenic, constituting an excellent tool. Human glioma xenografts implanted in immunocompromised mice have been extensively employed in preclinical brain cancer research. Although their xenogeneic nature impairs the study of immune-mediated anti-tumor strategies, they allow assessing the efficacy of therapeutic approaches in human GBM cells in the context of normal brain tissue. In fact, human xenografts exhibit histopatological features that resemble the human GBM and retain gene amplifications detected in the in situ tumors.

Recently, transgenic technique and gene knock-out technique rapidly developed, new animal models of gliomas were created. Central nervous system–specific inactivation of the genes encoding the tumor suppressors p53 and Nf1 leads to the spontaneous onset of Grade II and III astrocytoma tumors, as well as to GBM tumors in mice. This gliomagenesis can be accelerated by haploinsufficiency of the PTEN gene, and in neural progenitor cells conditional inactivation of p53 coordinates with a haploinsufficiency of PTEN and Nf1 to induce tumor formation. p53, INK4a/ARF, PTEN, EGFR, PDGF are the most popular genes in glioma research. Viral vectors or plasmid systems are used to deliver oncogenes. By means of linking the gene of interest to a glial specific promoter such as GFAP, S100β, or Nestin, transgenic mice that display cell type-specific overexpression of oncogenes. When large animal models are necessary, dog glioma models are available for alternative.

Animal Models of Glioma 363

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8517

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Abbreviations (GS-Gliosarcoma, GBM-glioblastoma multiforme, Astro-astrocytoma, ODGoligodendroglioma, MB-Medulloblastoma, KO-knockout)

Table 2. A summary of existing animal models of brain tumors

#### **5. References**

362 Glioma – Exploring Its Biology and Practical Relevance

 **Tumorigenesis Method Technique Tumor Animal Implantation** 9 L Gliosarcoma Syngeneic Graft GS Rat

**Genetic** p53 +/-, NF-1 +/- Germline mutations Astro Mouse

Human Tumor Cells (U87,

GFAP- p53 +/-, NF-1 +/-,

INK4a/ARF -/-, PDGF Overexpression

INK4a/ARF -/-, EGF-R overexpression

INK4a/ARF -/-, Ras, Akt

Ras, Akt overexpression,

RAS, EGF-R targeted overexpression

INK4a/ARF -/-, PDGF overexp., PTEN -/-

P53 +/-, S100β promoter

INK4a-ARF +/-, S100β promoter v-erbB

oligodendroglioma, MB-Medulloblastoma, KO-knockout)

Table 2. A summary of existing animal models of brain tumors

driven-v-erbB

p53 +/-, EGF-R overexpression

overexpression

PTEN -/-

U251)

PTEN-/-

C6 Syngeneic Graft GBM Rat T9 Syngeneic Graft GS Rat RG2 Syngeneic Graft GBM Rat F98 Syngeneic Graft GBM Rat RT-2 Syngeneic Graft GBM Rat CNS-1 Syngeneic Graft GBM Rat GL261 Syngeneic Graft GBM Mouse

GFAP- p53 +/-, NF-1 +/- Conditional KO Astr o Mouse

GFAP- p53 +/-, PTEN-/- Conditional KO Astro Mouse

Ras, Akt overexpression RCAS Astro Mouse

GFAP-V12 Ras, EGFRvIII Astrocyte targeted mutation, Adenovirus Astro Mouse

PDGF-B overexpression MMLV retrovirus ODG Mouse PDGF-B overexpression RCAS ODG Mouse

Ptc +/- Germline mutation or Conditional KO MB Mouse Ptc +/-, p53 -/- Germline mutations MB Mouse Shh, n-Myc RCAS MB Mouse Rb +/-, p53 +/- GFAP-conditional KO MB Mouse BRCA2 -/-, p53 +/- Nestin-conditional KO MB Mouse Xrcc4 -/-, p53 -/- Nestin-conditional KO MB Mouse SmoM2 GFAP-conditional KO MB Mouse

Germline mutation

Rb inactivation, PTEN -/- GFAP-Cre targeted conditional KO ODG

Conditional KO

Germline mutation, Oligodendrocyte mutation

Germline mutation, Oligodendrocyte mutation

Germline mutation, Oligodendrocyte mutation

Abbreviations (GS-Gliosarcoma, GBM-glioblastoma multiforme, Astro-astrocytoma, ODG-

Germline mutation, RCAS,

GFAP-V12 Ras, PTEN -/- Astrocyte targeted mutation,

Xenograft GBM Mouse

Conditional KO Astro Mouse

Germline mutation, RCAS Astro Mouse

Germline mutation, RCAS Astro Mouse

Germline mutation, RCAS Astro Mouse

RCAS, Conditional KO Astro Mouse

Astrocyte targeted mutations Astro Mouse

Astro Mouse

ODG Mouse

ODG Mouse

ODG Mouse

ODG Mouse


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capture therapy and immunoprophylaxis for advanced intracerebral gliosarcomas


**17** 

*Denmark* 

**Three-Dimensional In Vitro Models** 

Søren Kabell Nissen and Bjarne Winther Kristensen

Stine Skov Jensen, Charlotte Aaberg-Jessen, Ida Pind Jakobsen, Simon Kjær Hermansen,

*University of Southern Denmark* 

**in Glioma Research – Focus on Spheroids** 

*Department of Pathology, Odense University Hospital, Institute of Clinical Research* 

In the field of glioma research, in vitro models are widely used to investigate tumor biology as well as tumor response to chemotherapy and radiation. There is an increasing need to improve these in vitro models in order to meet the new challenges arising in drug discovery. It is thus important that development of new drugs is based on the latest knowledge about glioma biology such as for example the recent discovery of tumor stem cells (Reya et al., 2001). When investigating glioblastomas in vitro – and especially the supposed tumor stem cells – three dimensional multicellular spheroid models have recently come into focus. The aim of this chapter is to review the development as well as the most recent aspects of the three-dimensional glioma in vitro models focusing on glioma spheroids. The implementation of these models in current and in future in vitro glioma research will be

Cell lines cultured as monolayers have been the in vitro model of choice for many years (Ponten & Macintyre, 1968). However, the three-dimensional aspect came into focus in the 1970's, where scientists started to grow tumor cells from cell lines as multicellular spheroids (Yuhas et al., 1977). Over the years the spheroid model has been improved by deriving spheroids from cells obtained from dissociated primary glioblastoma tissue (Mackillop et al., 1985) as well as by using organotypic primary spheroids derived from small tumor

In general, most in vitro studies are performed with cells cultured in conventional serumcontaining medium. Recently – as the tumor stem cell theory has evolved – the culturing medium has come into focus. It has thus been demonstrated that the use of serum-free medium for culturing of cell line-derived spheroids preserved the in vivo-like features as well as the tumor stem cell-like phenotype suggesting crucial importance of the use of

Identification of the glioma stem cells is still a matter of discussion. The most used marker in the field has been the cell surface marker CD133. Expression of this putative tumor stem cell marker in gliomas has been studied in several papers demonstrating clusters or niches of CD133 positive tumor cells as well as CD133 positive single cells dispersed in the tumor

discussed putting emphasis on the themes described below.

serum-free medium in tumor stem cell research (Lee et al., 2006).

fragments (Bjerkvig et al., 1990).

**1. Introduction** 


## **Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids**

Stine Skov Jensen, Charlotte Aaberg-Jessen, Ida Pind Jakobsen, Simon Kjær Hermansen, Søren Kabell Nissen and Bjarne Winther Kristensen *Department of Pathology, Odense University Hospital, Institute of Clinical Research University of Southern Denmark Denmark* 

#### **1. Introduction**

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brain tumors revealed by high-resolution diffusion tensor MRI. *Magnetic resonance* 

encapsulated in liposomes inhibits the growth of C6 gliomas in rat brains. *Journal of* 

neural and glioma stem/progenitor cell renewal and differentiation. *Nature*,

EGFR signaling cooperates with loss of tumor suppressor gene functions in gliomagenesis. *Proceedings of the National Academy of Sciences of the United States of*  In the field of glioma research, in vitro models are widely used to investigate tumor biology as well as tumor response to chemotherapy and radiation. There is an increasing need to improve these in vitro models in order to meet the new challenges arising in drug discovery. It is thus important that development of new drugs is based on the latest knowledge about glioma biology such as for example the recent discovery of tumor stem cells (Reya et al., 2001). When investigating glioblastomas in vitro – and especially the supposed tumor stem cells – three dimensional multicellular spheroid models have recently come into focus.

The aim of this chapter is to review the development as well as the most recent aspects of the three-dimensional glioma in vitro models focusing on glioma spheroids. The implementation of these models in current and in future in vitro glioma research will be discussed putting emphasis on the themes described below.

Cell lines cultured as monolayers have been the in vitro model of choice for many years (Ponten & Macintyre, 1968). However, the three-dimensional aspect came into focus in the 1970's, where scientists started to grow tumor cells from cell lines as multicellular spheroids (Yuhas et al., 1977). Over the years the spheroid model has been improved by deriving spheroids from cells obtained from dissociated primary glioblastoma tissue (Mackillop et al., 1985) as well as by using organotypic primary spheroids derived from small tumor fragments (Bjerkvig et al., 1990).

In general, most in vitro studies are performed with cells cultured in conventional serumcontaining medium. Recently – as the tumor stem cell theory has evolved – the culturing medium has come into focus. It has thus been demonstrated that the use of serum-free medium for culturing of cell line-derived spheroids preserved the in vivo-like features as well as the tumor stem cell-like phenotype suggesting crucial importance of the use of serum-free medium in tumor stem cell research (Lee et al., 2006).

Identification of the glioma stem cells is still a matter of discussion. The most used marker in the field has been the cell surface marker CD133. Expression of this putative tumor stem cell marker in gliomas has been studied in several papers demonstrating clusters or niches of CD133 positive tumor cells as well as CD133 positive single cells dispersed in the tumor

Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 375

over 40 years new genomic mutations have arisen capable of giving rise to different phenotypic subpopulations in different laboratories. It is also well known that the phenotypic characteristics and genetic aberrations found within in vitro cells passaged repeatedly for about 10 times in serum containing medium often show only little resemblance with the original primary tumor (Lee et al., 2006). It is therefore not surprising that the U87MG cell line is known to have a highly aberrant genomic structure as visualized by karyotyping (Galli et al., 2004). Galli et al. (Galli et al., 2004) demonstrated loss of chromosome 1, 9, 10, 11, 12, 13, 14, 16, 19, 20, 22 and X. Furthermore, 11 unidentified abnormal chromosomes were found, whereas no gains of chromosomes were seen. In addition to this, Clark et al (Clark et al., 2010) have sequenced the genome of U87MG in order to further characterize it. They identified 35 interchromosomal translocation events, 1,315 structural variations (>100 bp), 191,743 small (< 21 bp) insertions and deletions as well as 2,384,470 single nucleotide variations. Protein coding sequences were disrupted predominantly by small insertions and deletions as well as larger deletions and translocations and 512 genes were homozygously mutated. Surprisingly, the study by Clark et al. also indicated that although this U87MG cell line has been cultured for more than 40 years, the cell line has now been relatively stable for years and is not rapidly changing anymore. This relative stability could be an advantage when using the U87MG cell line. In addition, the use of this cell line for four decades has resulted in a very well characterized

In general, there are many advantages when using cell lines. When first established, they are easy to handle in the laboratory and a large number of cells can be obtained in a short period of time, making it feasible to conduct large scale studies. In addition, cell lines are relatively easy to manipulate genetically by transfection and knock-down etc. establishing subpopulations with specific gene expression. These cell lines are important tools in the field of basic research investigating cellular pathways involved in tumor biology and response to different drugs. In addition to the obvious advantages, there will always be challenges when working with cell lines. As mentioned above, tumor cell lines are very likely to acquire new mutations and chromosome damage when undergoing cell division, because of the unstable genome in the tumor cells. The longer cultures are maintained and passaged the more changes accumulate (Lee et al., 2006) leading to changes in tumor cell behaviour. This is one of the main problems by culturing cell lines for many years. There will always be a possibility that the cells further mutate and several subpopulations will arise. This is important to keep in mind, when comparing results obtained with the same commercial cell

Another obstacle to overcome when using cell lines is the heterogeneity seen in tumors like glioblastomas. It is not possible to maintain the high degree of heterogeneity in long term cell cultures. In order to improve models using cell lines, short term cultures prepared from fresh tumor biopsies can be an alternative (Kolenda et al., 2010; Potter et al., 2009). The use of short term cultures may reduce differences between the tumor of origin and the cultured cells. In a study by Potter et al. (Potter et al., 2009) short term cultures from 6 pediatric pilocytic astrocytomas and 3 adult glioblastomas were established and cultured in conventional medium containing fetal calf serum. Gene expression profiles of the derived short term cell cultures harvested below passage 8 and their respective original biopsies were performed. They demonstrated that although short term cultures more resemble in situ gliomas than homogenous long term cultures, significant changes in gene expression were found between the biopsies and the derived short term cultures. The most significant

line but in different laboratories at different time points.

cell line.

(Christensen et al., 2008; Hermansen et al., 2011). These important tumor stem cell niches are preserved in primary glioma spheroids in contrast to cell line spheroids (Christensen et al., 2010).

Another important aspect in culturing of glioma stem cells is hypoxia, since several studies have shown that hypoxia influences radiation resistance. This may be explained by effects of hypoxia in vitro on proliferation of the tumor cells, spheroid formation and expression of stem cell markers, suggesting that also this aspect should be taken into consideration (Heddleston et al., 2009; Kolenda et al., 2010; McCord et al., 2009; Soeda et al., 2009).

Several studies have used the different types of spheroids mentioned above for investigating the effects of chemotherapy and radiation on the tumor cells and in particular on the tumor stem-like cells (Bao et al., 2006; Bauman et al., 1999; Fehlauer et al., 2005; Fehlauer et al., 2006; Fehlauer et al., 2007; Genc et al., 2004; Gliemroth et al., 2003; Haas-Kogan et al., 1996; Johannessen et al., 2009; Kaaijk et al., 1997; Khaitan et al., 2009; Sunayama et al., 2010; Terzis et al., 1997; Terzis et al., 1998; Wakimoto et al., 2009; Wang et al., 2010). After such treatments cell viability and cell proliferation assays as well as secondary spheroid formation assays have been used to evaluate induced effects. Moreover, expression of apoptosis and proliferation markers and for example stem cell markers have been investigated immunohistochemically in paraffin embedded spheroids (Christensen et al., 2010). The advantages using this panel of methods will be an important part of this chapter.

High grade gliomas are known to be highly invasive and new knowledge concerning tumor cell invasion also incorporating the tumor stem cell aspect is urgently needed. In our laboratory we have worked to improve in vitro models when investigating the invasive features of gliomas. This led to establishment of an in vivo-like model of invasion, where spheroids are implanted into organotypic brain slice cultures.

Taken together the three-dimensional multicellular spheroid model is the three dimensional model of choice for in vivo-like glioma in vitro studies. However, at the same time this model is under ongoing development to become an even more in vivo-like model in order to meet the new challenges of glioma research and drug development. The use of spheroids in especially tumor stem cell research has been fast increasing in recent years making spheroids an important tool also in future glioma research.

#### **2. Establishment and development of the spheroid model**

In the field of cancer research it is important to continuously develop in vitro models mimicking in vivo conditions as much as possible. By isolating cells from tumor tissue, tumor cell lines can be established from almost any kind of tumor including brain tumors such as glioblastomas. Glioblastoma cell lines are traditionally cultured as adherent monolayers but can also be cultured as single cell suspensions or spheroids. One of the most used cell lines in glioblastoma research is the cell line U87MG established by Pontén and Macintyre in 1968 (Ponten & Macintyre, 1968). This cell line was established from an astrocytic tumor with necrosis, which corresponds to this tumor being a glioblastoma multiforme according to WHO (World Health Organization) guidelines 2007 (Louis et al. 2007). U87MG was originally established in a traditional serum containing medium and the cells were described as large, extremely bizarre and very slowly growing. Today U87MG can be described as having only limited pleomorphism as well as being fast growing, clearly indicating that such cell lines change over time. U87MG has been used in a variety of glioma studies and is still used. Since the cell line has been cultured in different laboratories for

(Christensen et al., 2008; Hermansen et al., 2011). These important tumor stem cell niches are preserved in primary glioma spheroids in contrast to cell line spheroids (Christensen et al.,

Another important aspect in culturing of glioma stem cells is hypoxia, since several studies have shown that hypoxia influences radiation resistance. This may be explained by effects of hypoxia in vitro on proliferation of the tumor cells, spheroid formation and expression of stem cell markers, suggesting that also this aspect should be taken into consideration

Several studies have used the different types of spheroids mentioned above for investigating the effects of chemotherapy and radiation on the tumor cells and in particular on the tumor stem-like cells (Bao et al., 2006; Bauman et al., 1999; Fehlauer et al., 2005; Fehlauer et al., 2006; Fehlauer et al., 2007; Genc et al., 2004; Gliemroth et al., 2003; Haas-Kogan et al., 1996; Johannessen et al., 2009; Kaaijk et al., 1997; Khaitan et al., 2009; Sunayama et al., 2010; Terzis et al., 1997; Terzis et al., 1998; Wakimoto et al., 2009; Wang et al., 2010). After such treatments cell viability and cell proliferation assays as well as secondary spheroid formation assays have been used to evaluate induced effects. Moreover, expression of apoptosis and proliferation markers and for example stem cell markers have been investigated immunohistochemically in paraffin embedded spheroids (Christensen et al., 2010). The advantages using this panel of methods will be an important part of this chapter. High grade gliomas are known to be highly invasive and new knowledge concerning tumor cell invasion also incorporating the tumor stem cell aspect is urgently needed. In our laboratory we have worked to improve in vitro models when investigating the invasive features of gliomas. This led to establishment of an in vivo-like model of invasion, where

Taken together the three-dimensional multicellular spheroid model is the three dimensional model of choice for in vivo-like glioma in vitro studies. However, at the same time this model is under ongoing development to become an even more in vivo-like model in order to meet the new challenges of glioma research and drug development. The use of spheroids in especially tumor stem cell research has been fast increasing in recent years making

In the field of cancer research it is important to continuously develop in vitro models mimicking in vivo conditions as much as possible. By isolating cells from tumor tissue, tumor cell lines can be established from almost any kind of tumor including brain tumors such as glioblastomas. Glioblastoma cell lines are traditionally cultured as adherent monolayers but can also be cultured as single cell suspensions or spheroids. One of the most used cell lines in glioblastoma research is the cell line U87MG established by Pontén and Macintyre in 1968 (Ponten & Macintyre, 1968). This cell line was established from an astrocytic tumor with necrosis, which corresponds to this tumor being a glioblastoma multiforme according to WHO (World Health Organization) guidelines 2007 (Louis et al. 2007). U87MG was originally established in a traditional serum containing medium and the cells were described as large, extremely bizarre and very slowly growing. Today U87MG can be described as having only limited pleomorphism as well as being fast growing, clearly indicating that such cell lines change over time. U87MG has been used in a variety of glioma studies and is still used. Since the cell line has been cultured in different laboratories for

(Heddleston et al., 2009; Kolenda et al., 2010; McCord et al., 2009; Soeda et al., 2009).

spheroids are implanted into organotypic brain slice cultures.

spheroids an important tool also in future glioma research.

**2. Establishment and development of the spheroid model** 

2010).

over 40 years new genomic mutations have arisen capable of giving rise to different phenotypic subpopulations in different laboratories. It is also well known that the phenotypic characteristics and genetic aberrations found within in vitro cells passaged repeatedly for about 10 times in serum containing medium often show only little resemblance with the original primary tumor (Lee et al., 2006). It is therefore not surprising that the U87MG cell line is known to have a highly aberrant genomic structure as visualized by karyotyping (Galli et al., 2004). Galli et al. (Galli et al., 2004) demonstrated loss of chromosome 1, 9, 10, 11, 12, 13, 14, 16, 19, 20, 22 and X. Furthermore, 11 unidentified abnormal chromosomes were found, whereas no gains of chromosomes were seen. In addition to this, Clark et al (Clark et al., 2010) have sequenced the genome of U87MG in order to further characterize it. They identified 35 interchromosomal translocation events, 1,315 structural variations (>100 bp), 191,743 small (< 21 bp) insertions and deletions as well as 2,384,470 single nucleotide variations. Protein coding sequences were disrupted predominantly by small insertions and deletions as well as larger deletions and translocations and 512 genes were homozygously mutated. Surprisingly, the study by Clark et al. also indicated that although this U87MG cell line has been cultured for more than 40 years, the cell line has now been relatively stable for years and is not rapidly changing anymore. This relative stability could be an advantage when using the U87MG cell line. In addition, the use of this cell line for four decades has resulted in a very well characterized cell line.

In general, there are many advantages when using cell lines. When first established, they are easy to handle in the laboratory and a large number of cells can be obtained in a short period of time, making it feasible to conduct large scale studies. In addition, cell lines are relatively easy to manipulate genetically by transfection and knock-down etc. establishing subpopulations with specific gene expression. These cell lines are important tools in the field of basic research investigating cellular pathways involved in tumor biology and response to different drugs. In addition to the obvious advantages, there will always be challenges when working with cell lines. As mentioned above, tumor cell lines are very likely to acquire new mutations and chromosome damage when undergoing cell division, because of the unstable genome in the tumor cells. The longer cultures are maintained and passaged the more changes accumulate (Lee et al., 2006) leading to changes in tumor cell behaviour. This is one of the main problems by culturing cell lines for many years. There will always be a possibility that the cells further mutate and several subpopulations will arise. This is important to keep in mind, when comparing results obtained with the same commercial cell line but in different laboratories at different time points.

Another obstacle to overcome when using cell lines is the heterogeneity seen in tumors like glioblastomas. It is not possible to maintain the high degree of heterogeneity in long term cell cultures. In order to improve models using cell lines, short term cultures prepared from fresh tumor biopsies can be an alternative (Kolenda et al., 2010; Potter et al., 2009). The use of short term cultures may reduce differences between the tumor of origin and the cultured cells. In a study by Potter et al. (Potter et al., 2009) short term cultures from 6 pediatric pilocytic astrocytomas and 3 adult glioblastomas were established and cultured in conventional medium containing fetal calf serum. Gene expression profiles of the derived short term cell cultures harvested below passage 8 and their respective original biopsies were performed. They demonstrated that although short term cultures more resemble in situ gliomas than homogenous long term cultures, significant changes in gene expression were found between the biopsies and the derived short term cultures. The most significant

Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 377

al., 1990) it was shown that when culturing small tumor fragments from astrocytic brain tumors of increasing grade, small primary spheroids were formed for the majority of the tumors within 3-5 days. The spheroids were analyzed by light microscopy as well as transmission- and scanning electron microscopy (TEM and SEM, respectively) after 3 and 10 weeks of culture, showing the unique preservation of cell- to cell interaction, blood vessels, extracellular matrix, and macrophages. It was moreover demonstrated that the primary spheroids could be cultured for 70 days with preservation of the histology of the spheroids. In a similar study in our laboratory, glioma tissue was collected from 11 patients. The tissue pieces from 7 of these patients formed vital spheroids within a week. Thereafter, the spheroids were fixed, paraffin embedded and investigated immunohistochemically as described later in this chapter. Areas of necrosis were seen in some of the spheroids,

whereas blood vessels were present in the majority of the glioma-derived spheroids.

Fig. 1. Adherent monolayer cells and free floating spheroids. U87MG grows as an adherent monolayer when the cells are cultured in serum containing medium (A). However, in serum-free medium U87MG grows as spheroids (B). Tissue derived from freshly removed glioblastoma (C) and cells from a glioblastoma short term culture (D) also grow as spheroids when cultured in a serum-free medium. The organotypic spheroids (C) can be grown in serum containing medium as well and preserves in both media some of the characteristics found in the primary tumor such as tumor necrosis and blood vessels, whereas the short

term culture spheroid in (D) has lost these characteristics.

functions differing for the glioblastomas were associated with cell structure, shape, motility, proliferation, cellular development, cell death, cellular assembly and organization, cell-tocell signaling and interaction, as well as cell cycle.

As our knowledge of tumor cells expands, there is a need to establish more advanced models to mimic the tumor in situ. One of the main problems is the fact, that the cells are removed from their natural environment, dissociated and cultured as single cells. It may therefore be important to prevent this in order to be able to mimic the natural environment as much as possible. Moreover in the recent years, the use of serum containing cell culturing medium has come into focus. The composition of serum has not been fully understood for many years, but it is known that it supplies the cells with nutrients, vitamins, hormones, and growth-, differentiation-, and attachment-factors. These factors may affect the cells in ways we are not fully aware of. It is also well known that there are differences between batches of serum (Fisher & Wieser, 1983). Another important issue is the fact that only a small fraction of cells in the organism is in direct contact with serum. This may be a problem in glioma research, since the brain and brain tumor tissues are not among these cells. In this context, it is also worth highlighting that neural stem cells should be cultured under serumfree conditions similar to what has been found for the so called glioblastoma tumor stem cells.

The three-dimensional glioma spheroid model came into focus in the 1970's, where scientists started to grow tumor cells as multicellular spheroids using tumor cells from conventional monolayer cultures (Yuhas et al., 1977). Such spheroids are usually formed by aggregation of cells growing into the larger three-dimensional spheroids. They are believed to be a better model than monolayer cultures due to a three-dimensional structure with more in vivo-like intercellular contacts. This model was later on further improved by deriving spheroids from single cells obtained from dissociated primary glioblastoma tissue (Mackillop et al., 1985). In order to obtain an even more in vivo-like model the primary organotypic spheroids were introduced (Bjerkvig et al., 1990). Organotypic means that the properties characteristic of the tissue of origin is maintained. These spheroids are derived from freshly removed glioma tissue and have been shown to be a valid tumor model providing a biological system that mimics the original glioma in vivo.

When deriving primary spheroids from glioma biopsies, it is important to process the tumor tissue as soon as it is removed. As we have published earlier (Christensen et al., 2010) the glioma tissue should be collected directly in the operation theatre, where the tissue is placed in a tube with Hanks' Balanced Salt Solution supplemented with 0.9 % glucose and transported to the laboratory. The tumor tissue can then be processed according to the study by Bjerkvig et al. (Bjerkvig et al., 1990), where small tumor fragments of approximately 200- 400 µm in diameter are obtained after sectioning the tumor tissue manually using scalpels. These fragments are then transferred to 0.75% agar-coated culture flasks of 75 cm2 with prewarmed medium. The cultures should be kept in a standard tissue culture incubator (95% humidity, 95% air, and 5% CO2) and the following day the culturing medium should be changed in order to remove dead blood cells and cellular debris. The tumor fragments should then be examined under a light microscope every day, until they round up to form spheroids within 5-15 days.

The main advantages by primary organotypic spheroids are the preservation of the original intercellular contacts and the tumor heterogeneity. However, because of this heterogeneity it is important to include a larger number of spheroids in in vitro studies using primary spheroids in order to obtain reproducible results. In a study by Bjerkvig et al., (Bjerkvig et

functions differing for the glioblastomas were associated with cell structure, shape, motility, proliferation, cellular development, cell death, cellular assembly and organization, cell-to-

As our knowledge of tumor cells expands, there is a need to establish more advanced models to mimic the tumor in situ. One of the main problems is the fact, that the cells are removed from their natural environment, dissociated and cultured as single cells. It may therefore be important to prevent this in order to be able to mimic the natural environment as much as possible. Moreover in the recent years, the use of serum containing cell culturing medium has come into focus. The composition of serum has not been fully understood for many years, but it is known that it supplies the cells with nutrients, vitamins, hormones, and growth-, differentiation-, and attachment-factors. These factors may affect the cells in ways we are not fully aware of. It is also well known that there are differences between batches of serum (Fisher & Wieser, 1983). Another important issue is the fact that only a small fraction of cells in the organism is in direct contact with serum. This may be a problem in glioma research, since the brain and brain tumor tissues are not among these cells. In this context, it is also worth highlighting that neural stem cells should be cultured under serumfree conditions similar to what has been found for the so called glioblastoma tumor stem

The three-dimensional glioma spheroid model came into focus in the 1970's, where scientists started to grow tumor cells as multicellular spheroids using tumor cells from conventional monolayer cultures (Yuhas et al., 1977). Such spheroids are usually formed by aggregation of cells growing into the larger three-dimensional spheroids. They are believed to be a better model than monolayer cultures due to a three-dimensional structure with more in vivo-like intercellular contacts. This model was later on further improved by deriving spheroids from single cells obtained from dissociated primary glioblastoma tissue (Mackillop et al., 1985). In order to obtain an even more in vivo-like model the primary organotypic spheroids were introduced (Bjerkvig et al., 1990). Organotypic means that the properties characteristic of the tissue of origin is maintained. These spheroids are derived from freshly removed glioma tissue and have been shown to be a valid tumor model providing a biological system that

When deriving primary spheroids from glioma biopsies, it is important to process the tumor tissue as soon as it is removed. As we have published earlier (Christensen et al., 2010) the glioma tissue should be collected directly in the operation theatre, where the tissue is placed in a tube with Hanks' Balanced Salt Solution supplemented with 0.9 % glucose and transported to the laboratory. The tumor tissue can then be processed according to the study by Bjerkvig et al. (Bjerkvig et al., 1990), where small tumor fragments of approximately 200- 400 µm in diameter are obtained after sectioning the tumor tissue manually using scalpels. These fragments are then transferred to 0.75% agar-coated culture flasks of 75 cm2 with prewarmed medium. The cultures should be kept in a standard tissue culture incubator (95% humidity, 95% air, and 5% CO2) and the following day the culturing medium should be changed in order to remove dead blood cells and cellular debris. The tumor fragments should then be examined under a light microscope every day, until they round up to form

The main advantages by primary organotypic spheroids are the preservation of the original intercellular contacts and the tumor heterogeneity. However, because of this heterogeneity it is important to include a larger number of spheroids in in vitro studies using primary spheroids in order to obtain reproducible results. In a study by Bjerkvig et al., (Bjerkvig et

cell signaling and interaction, as well as cell cycle.

cells.

mimics the original glioma in vivo.

spheroids within 5-15 days.

al., 1990) it was shown that when culturing small tumor fragments from astrocytic brain tumors of increasing grade, small primary spheroids were formed for the majority of the tumors within 3-5 days. The spheroids were analyzed by light microscopy as well as transmission- and scanning electron microscopy (TEM and SEM, respectively) after 3 and 10 weeks of culture, showing the unique preservation of cell- to cell interaction, blood vessels, extracellular matrix, and macrophages. It was moreover demonstrated that the primary spheroids could be cultured for 70 days with preservation of the histology of the spheroids. In a similar study in our laboratory, glioma tissue was collected from 11 patients. The tissue pieces from 7 of these patients formed vital spheroids within a week. Thereafter, the spheroids were fixed, paraffin embedded and investigated immunohistochemically as described later in this chapter. Areas of necrosis were seen in some of the spheroids, whereas blood vessels were present in the majority of the glioma-derived spheroids.

Fig. 1. Adherent monolayer cells and free floating spheroids. U87MG grows as an adherent monolayer when the cells are cultured in serum containing medium (A). However, in serum-free medium U87MG grows as spheroids (B). Tissue derived from freshly removed glioblastoma (C) and cells from a glioblastoma short term culture (D) also grow as spheroids when cultured in a serum-free medium. The organotypic spheroids (C) can be grown in serum containing medium as well and preserves in both media some of the characteristics found in the primary tumor such as tumor necrosis and blood vessels, whereas the short term culture spheroid in (D) has lost these characteristics.

Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 379

mentioned earlier, normal neural stem cells are cultured under serum-free conditions, since it is well known that serum causes irreversible differentiation of neural stem cells (Gage et al., 1995). Lee et al. (Lee et al., 2006) cultured glioblastoma short term cultures in a serumfree medium similar to the medium used for culturing neural stem cells in order to preserve and select for tumor stem-like cells. This serum-free medium consisted of neurobasal medium supplemented with EGF and bFGF, because EGF and bFGF earlier seemed to select for tumor stem-like cells by inducing proliferation of multipotent, self-renewing, and expandable tumor stem cells (Galli et al., 2004; Ignatova et al., 2002; Lee et al., 2006). In the study by Lee et al., (Lee et al., 2006) dissociated glioblastoma cells, cultured as short term cultures, formed spheroids expressing putative tumor stem cell markers but when culturing the selected cells in serum containing medium, they irreversibly differentiated into neural and glial cell lineages. Interestingly, this is in line with the irreversible differentiation of

In the search for improvement of in vitro models we performed a study in our laboratory (Christensen et al., 2010), where organotypic primary spheroids were cultured in serum-free medium. In terms of the tumor stem cell concept, culturing of these organotypic primary spheroids in serum-free conditions may be closer to the in vivo situation than using tumor stem cell line-derived spheroids, especially regarding studies of radiation and chemosensitivity. We investigated the influence of serum-containing medium and serum-free medium on the phenotype of primary glioma spheroids. The aim was to elucidate whether serum-free medium also favors the presence of tumor cells expressing stem cell markers in these spheroids, when investigated immunohistochemically. The results based on seven malignant astrocytomas WHO Grade III–IV, supported the hypothesis that putative brain tumor stem cells are better preserved in serum-free culture medium with EGF and bFGF. When comparing spheroids from both media, we found increased CD133 expression when culturing primary glioma spheroids in serum-free medium compared to serum-containing medium, which is in line with the study by Lee et al. (Lee et al., 2006) using short term cultures. In contrast to Lee, who found a drastic decrease in Sox2, Bmi-1, and Nestin when culturing short term cultures in serum, we only found a slightly decreased expression of Sox2, whereas Bmi-1 and Nestin were equally expressed in both media. This better preservation of stem cell marker expression in serum-containing medium in primary glioma spheroids (Christensen et al., 2010) may be explained by primary spheroids preserving an intact microenvironment, whereas Lee et al. (Lee et al., 2006) repeatedly dissociated the spheroids. Another interesting observation was that primary glioma spheroids cultured in serum-free medium contained more blood vessels than in serum-containing medium, and furthermore, many blood vessels were hyperplastic. The immunohistochemical comparison showed more CD34 and VWF, but less CD31 in serum-free medium compared to serumcontaining medium. This increase was accompanied with more CD133 positive cells, thus suggesting that the close relationship between blood vessels and tumor stem-like cells may

As it is clear from the tumor stem cell research field, markers specific for tumor stem cells are of crucial importance. This has resulted in the development of a great number of antibodies against tumor stem cell-related proteins. Some of the most important markers in the field of brain tumors have been (table 1) CD133 (Bandopadhyay et al. 2010; Bidlingmaier et al., 2008; Christensen et al., 2008; Dell'albani, 2008; Fargeas et al., 2007; Griguer et al., 2008; Jaszai et al., 2007; Mizrak et al., 2008; Pfenninger et al., 2007; Wang et al., 2008; Zeppernick et al., 2008), A2B5 (Balik et al., 2009; Merzak et al., 1994; Ogden et al., 2008; Piepmeier et al.,

neural stem cells under the same conditions (Gage et al., 1995).

be better preserved in serum-free medium.


Box 1. Preparation of organotypic primary spheroids

#### **3. The tumor stem cell paradigm and the spheroid model**

The tumor stem cell paradigm proposes that only a small subset of cells – the so-called tumor stem cells - within the tumor cell population is able to initiate and sustain tumor growth (Ward & Dirks, 2007). These tumor stem cells have been found in a variety of different cancers such as leukaemia (Bonnet & Dick, 1997), colon cancer (Daidone et al., 2004), breast cancer (Al-Hajj et al., 2003) and brain cancer (Singh et al., 2004). With the discovery of the neural stem cells (Reynolds & Weiss, 1992) it also became plausible that brain tumors could be derived from the transformation of neural stem cells or progenitor cells (Singh et al., 2004). The neural stem cells were first isolated by Reynolds and Weiss in 1992 (Reynolds & Weiss, 1992). They found a small population of cells isolated from the adult striatum in mouse brain that were able to proliferate and differentiate. They cultured these cells in a serum-free environment supplemented with the growth factors EGF (epidermal growth factor) and bFGF (basic fibroblast growth factor), and the cells grew as neurospheres. When dissociating the neurospheres and re-plating them as single cells new neurospheres developed. Under these serum-free conditions most differentiating and differentiated cells died, whereas the neural stem cells responded to the growth factors and proliferated to form neurospheres (Vescovi et al., 2006). By applying the same conditions to human glioblastoma cells, it was possible to isolate a population of cells that formed tumorspheres. These cells were capable of differentiation and self-renewal (Galli et al., 2004; Ignatova et al., 2002; Lee et al., 2006; Singh et al., 2003). Furthermore, the cells from the tumorspheres gave rise to tumors resembling the primary tumor when injected into the brains of immunodeficient mice, suggesting that a population of the cells isolated were brain tumor-initiating stem-like cells.

When culturing putative tumor stem cells, spheroid models are often used. Especially the clonogenic neurosphere assay is used for preserving tumor stem-like cells in serum-free medium (Lee et al., 2006). In this assay, primary human brain tumor tissue form spheroids after repeated dissociation into single cells. However, cell-to-cell interactions are interrupted and this might affect experimental results obtained with the tumor stem cell line-derived spheroids. This could be particularly important in tumor stem cell research, since the suggested close relationship between brain tumor stem cells and adjacent endothelial cells (Bao et al., 2006; Calabrese et al., 2007) is lost in these spheroids. In addition, the culturing medium has come into focus when performing studies focusing on tumor stem cells. As

1. Collect tissue and transport the freshly removed tumor tissue in Hanks' Balanced Salt

3. Section the tumor tissue manually using two scalpels until tumor fragments

7. Change the medium twice a week in 10-15 days until the fragments round up and

The tumor stem cell paradigm proposes that only a small subset of cells – the so-called tumor stem cells - within the tumor cell population is able to initiate and sustain tumor growth (Ward & Dirks, 2007). These tumor stem cells have been found in a variety of different cancers such as leukaemia (Bonnet & Dick, 1997), colon cancer (Daidone et al., 2004), breast cancer (Al-Hajj et al., 2003) and brain cancer (Singh et al., 2004). With the discovery of the neural stem cells (Reynolds & Weiss, 1992) it also became plausible that brain tumors could be derived from the transformation of neural stem cells or progenitor cells (Singh et al., 2004). The neural stem cells were first isolated by Reynolds and Weiss in 1992 (Reynolds & Weiss, 1992). They found a small population of cells isolated from the adult striatum in mouse brain that were able to proliferate and differentiate. They cultured these cells in a serum-free environment supplemented with the growth factors EGF (epidermal growth factor) and bFGF (basic fibroblast growth factor), and the cells grew as neurospheres. When dissociating the neurospheres and re-plating them as single cells new neurospheres developed. Under these serum-free conditions most differentiating and differentiated cells died, whereas the neural stem cells responded to the growth factors and proliferated to form neurospheres (Vescovi et al., 2006). By applying the same conditions to human glioblastoma cells, it was possible to isolate a population of cells that formed tumorspheres. These cells were capable of differentiation and self-renewal (Galli et al., 2004; Ignatova et al., 2002; Lee et al., 2006; Singh et al., 2003). Furthermore, the cells from the tumorspheres gave rise to tumors resembling the primary tumor when injected into the brains of immunodeficient mice, suggesting that a population of the cells isolated were brain

When culturing putative tumor stem cells, spheroid models are often used. Especially the clonogenic neurosphere assay is used for preserving tumor stem-like cells in serum-free medium (Lee et al., 2006). In this assay, primary human brain tumor tissue form spheroids after repeated dissociation into single cells. However, cell-to-cell interactions are interrupted and this might affect experimental results obtained with the tumor stem cell line-derived spheroids. This could be particularly important in tumor stem cell research, since the suggested close relationship between brain tumor stem cells and adjacent endothelial cells (Bao et al., 2006; Calabrese et al., 2007) is lost in these spheroids. In addition, the culturing medium has come into focus when performing studies focusing on tumor stem cells. As

4. Culture fragments in 0.75% agar coated culture flasks containing 20 ml medium 5. Incubate the cultures in 36°C humidified air containing 5% CO2 and 95% atmospheric

Solution supplemented with 0.9% glucose

Box 1. Preparation of organotypic primary spheroids

**3. The tumor stem cell paradigm and the spheroid model** 

of 50-400 µm in diameter are obtained

2. Place the tissue in a sterile petri dish

6. Change the medium the next day

form spheroids

tumor-initiating stem-like cells.

air

mentioned earlier, normal neural stem cells are cultured under serum-free conditions, since it is well known that serum causes irreversible differentiation of neural stem cells (Gage et al., 1995). Lee et al. (Lee et al., 2006) cultured glioblastoma short term cultures in a serumfree medium similar to the medium used for culturing neural stem cells in order to preserve and select for tumor stem-like cells. This serum-free medium consisted of neurobasal medium supplemented with EGF and bFGF, because EGF and bFGF earlier seemed to select for tumor stem-like cells by inducing proliferation of multipotent, self-renewing, and expandable tumor stem cells (Galli et al., 2004; Ignatova et al., 2002; Lee et al., 2006). In the study by Lee et al., (Lee et al., 2006) dissociated glioblastoma cells, cultured as short term cultures, formed spheroids expressing putative tumor stem cell markers but when culturing the selected cells in serum containing medium, they irreversibly differentiated into neural and glial cell lineages. Interestingly, this is in line with the irreversible differentiation of neural stem cells under the same conditions (Gage et al., 1995).

In the search for improvement of in vitro models we performed a study in our laboratory (Christensen et al., 2010), where organotypic primary spheroids were cultured in serum-free medium. In terms of the tumor stem cell concept, culturing of these organotypic primary spheroids in serum-free conditions may be closer to the in vivo situation than using tumor stem cell line-derived spheroids, especially regarding studies of radiation and chemosensitivity. We investigated the influence of serum-containing medium and serum-free medium on the phenotype of primary glioma spheroids. The aim was to elucidate whether serum-free medium also favors the presence of tumor cells expressing stem cell markers in these spheroids, when investigated immunohistochemically. The results based on seven malignant astrocytomas WHO Grade III–IV, supported the hypothesis that putative brain tumor stem cells are better preserved in serum-free culture medium with EGF and bFGF. When comparing spheroids from both media, we found increased CD133 expression when culturing primary glioma spheroids in serum-free medium compared to serum-containing medium, which is in line with the study by Lee et al. (Lee et al., 2006) using short term cultures. In contrast to Lee, who found a drastic decrease in Sox2, Bmi-1, and Nestin when culturing short term cultures in serum, we only found a slightly decreased expression of Sox2, whereas Bmi-1 and Nestin were equally expressed in both media. This better preservation of stem cell marker expression in serum-containing medium in primary glioma spheroids (Christensen et al., 2010) may be explained by primary spheroids preserving an intact microenvironment, whereas Lee et al. (Lee et al., 2006) repeatedly dissociated the spheroids. Another interesting observation was that primary glioma spheroids cultured in serum-free medium contained more blood vessels than in serum-containing medium, and furthermore, many blood vessels were hyperplastic. The immunohistochemical comparison showed more CD34 and VWF, but less CD31 in serum-free medium compared to serumcontaining medium. This increase was accompanied with more CD133 positive cells, thus suggesting that the close relationship between blood vessels and tumor stem-like cells may be better preserved in serum-free medium.

As it is clear from the tumor stem cell research field, markers specific for tumor stem cells are of crucial importance. This has resulted in the development of a great number of antibodies against tumor stem cell-related proteins. Some of the most important markers in the field of brain tumors have been (table 1) CD133 (Bandopadhyay et al. 2010; Bidlingmaier et al., 2008; Christensen et al., 2008; Dell'albani, 2008; Fargeas et al., 2007; Griguer et al., 2008; Jaszai et al., 2007; Mizrak et al., 2008; Pfenninger et al., 2007; Wang et al., 2008; Zeppernick et al., 2008), A2B5 (Balik et al., 2009; Merzak et al., 1994; Ogden et al., 2008; Piepmeier et al.,

Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 381

Ogasawara et al., 2008; Ordonez, 2006; Shibahara et

Dahlstrand et al., 1992a; Dahlstrand et al., 1992b; Dell'albani, 2008; Ehrmann et al., 2005; Ma et al., 2008; Maderna et al., 2007; Strojnik et al., 2007; Wan et al., 2011

Kanemura et al., 2001; Ma et al., 2008; Okano et al., 2005; Sakakibara & Okano, 1997; Thon et al., 2010; Toda et al.,

Bruggeman et al., 2007; Hayry et al., 2008; Park et al., 2004; Zencak et al., 2005

Gangemi et al., 2009; Ma et al., 2008; Phi et al., 2008

2001

al., 2006

**Markers Short introduction References**  or neurons. A high expression of Podoplanin has been found in high grade astrocytomas. However, no protein has been detected in diffuse astrocytomas or in normal brain tissue. Podoplanin has been suggested to be expressed in human glioma stem cells. This podoplanin positive cell population formed neurospheres in vitro and tumors in vivo. Moreover, these cells showed increased

resistance to radiation.

Nestin Nestin is a protein belonging to the class VI of

Musashi-1 Musashi-1 belongs to a family of evolutionary

tumors including gliomas.

including glioblastomas.

Sox2 Sox2 (SRY (sex determining region Y)-box 2) is

Bmi-1 Bmi-1 (B lymphoma Mo-MLV insertion region)

intermediate filaments and it appears after neurulation in the CNS stem cells. In the normal adult brain, nestin is only expressed in the neural stem cells lining the ventricular wall and the central canal. It is believed to be a marker of proliferating and migrating cells. Nestin has been found in several tumor types including gliomas. The expression of nestin may be related to a dedifferentiated tumor stem cell status, enhanced cell motility, invasive potential and increased malignancy.

well conserved neural RNA-binding proteins. Musashi-1 is found in neural stem cells and progenitor cells in the adult human brain and plays important roles in cell fate decision, including the maintenance of the stem cell state, differentiation, and tumorigenesis. Musashi-1 has been found in a variety of

is a *Polycomb* group transcription repressor, thought to be essential for self-renewal of neural stem cells and maintenance of the stem cell population by preventing premature senescence. Bmi-1 is found mainly around the ventricles in the subventricular zone and *in vitro* in cortical neural stem cells as well as in progenitor cells. Bmi-1 has been found to be highly expressed in human brain tumors

a transcription factor that plays a role in sustaining self-renewal and maintaining

1993; Tchoghandjian et al., 2009), Podoplanin (Goodman et al. 2009; Grau et al., 2008; Mishima et al., 2006; Nakamura et al., 2006; Ogasawara et al., 2008; Ordonez, 2006; Shibahara et al., 2006), Nestin (Dahlstrand et al., 1992a; Dahlstrand et al., 1992b; Dell'albani, 2008; Ehrmann et al., 2005; Ma et al., 2008; Maderna et al., 2007; Strojnik et al., 2007; Wan et al., 2011), Mushashi-1 (Kanemura et al., 2001; Ma et al., 2008; Okano et al., 2005; Sakakibara & Okano, 1997; Thon et al., 2010; Toda et al., 2001), Bmi-1 (Bruggeman et al., 2007; Hayry et al., 2008; Park et al., 2004; Zencak et al., 2005) and Sox2 (Gangemi et al., 2009; Ma et al., 2008; Phi et al., 2008) and new upcoming markers such as ID1 (Kamalian et al., 2008; Maw et al., 2009; Nam & Benezra, 2009; Schindl et al., 2001; Schindl et al., 2003; Schoppmann et al., 2003; Tang et al., 2009), NG2 (Brekke et al., 2006; Chekenya et al., 1999; Chekenya et al., 2002a; Chekenya et al., 2002b; Chekenya et al., 2008; Chekenya & Immervoll, 2007; Chekenya & Pilkington, 2002; Joo et al., 2008; Petrovici et al., 2010; Stallcup & Huang, 2008) and CD15 (Capela & Temple, 2002; Capela & Temple, 2006; Read et al., 2009; Ward et al., 2009). Until now, the most widely used marker in brain tumors has been CD133 (Bidlingmaier et al., 2008; Christensen et al., 2008; Dell'albani, 2008; Fargeas et al., 2007; Griguer et al., 2008; Jaszai et al., 2007; Mizrak et al., 2008; Pfenninger et al., 2007; Wang et al., 2008; Zeppernick et al., 2008).


1993; Tchoghandjian et al., 2009), Podoplanin (Goodman et al. 2009; Grau et al., 2008; Mishima et al., 2006; Nakamura et al., 2006; Ogasawara et al., 2008; Ordonez, 2006; Shibahara et al., 2006), Nestin (Dahlstrand et al., 1992a; Dahlstrand et al., 1992b; Dell'albani, 2008; Ehrmann et al., 2005; Ma et al., 2008; Maderna et al., 2007; Strojnik et al., 2007; Wan et al., 2011), Mushashi-1 (Kanemura et al., 2001; Ma et al., 2008; Okano et al., 2005; Sakakibara & Okano, 1997; Thon et al., 2010; Toda et al., 2001), Bmi-1 (Bruggeman et al., 2007; Hayry et al., 2008; Park et al., 2004; Zencak et al., 2005) and Sox2 (Gangemi et al., 2009; Ma et al., 2008; Phi et al., 2008) and new upcoming markers such as ID1 (Kamalian et al., 2008; Maw et al., 2009; Nam & Benezra, 2009; Schindl et al., 2001; Schindl et al., 2003; Schoppmann et al., 2003; Tang et al., 2009), NG2 (Brekke et al., 2006; Chekenya et al., 1999; Chekenya et al., 2002a; Chekenya et al., 2002b; Chekenya et al., 2008; Chekenya & Immervoll, 2007; Chekenya & Pilkington, 2002; Joo et al., 2008; Petrovici et al., 2010; Stallcup & Huang, 2008) and CD15 (Capela & Temple, 2002; Capela & Temple, 2006; Read et al., 2009; Ward et al., 2009). Until now, the most widely used marker in brain tumors has been CD133 (Bidlingmaier et al., 2008; Christensen et al., 2008; Dell'albani, 2008; Fargeas et al., 2007; Griguer et al., 2008; Jaszai et al., 2007; Mizrak et al., 2008; Pfenninger et al., 2007; Wang et al., 2008; Zeppernick et

**Markers Short introduction References** 

five transmembrane domains. CD133 has been found in a variety of non-pathogen human tissues including the brain. However, the function remains unknown. It was identified as a marker of hematopoietic stem cells in 1997 and later as a marker of human neural stem cells. Since 2004, CD133 has been widely used for identifying tumor stem cells in brain tumors. However, some results suggest that CD133 is not specific for tumor stem cells. Downregulation of CD133 in glioma cell lines has been suggested to influence migration, spheroid formation and resistance to

Bandopadhyay et al. 2010; Bidlingmaier et al., 2008; Christensen et al., 2008; Dell'albani, 2008; Fargeas et al., 2007; Griguer et al., 2008; Jaszai et al., 2007; Mizrak et al., 2008; Pfenninger et al., 2007; Wang et al., 2008; Zeppernick et al., 2008

Balik et al., 2009; Merzak et al., 1994; Ogden et al., 2008; Piepmeier et al., 1993; Tchoghandjian et al., 2009

Goodman et al. 2009; Grau et al., 2008; Mishima et al., 2006; Nakamura et al., 2006;

CD133 CD133 is a cell membrane glycoprotein with

chemotherapeutics.

A2B5 A2B5 is a cell surface ganglioside found on

to basement membrane components.

Podoplanin Podoplanin is a mucin-type transmembrane

white matter progenitors of the oligodendrocyte lineage. A2B5 has been found in gliomas in a population of cells, which are distinct from the CD133+ population but have the capacity to initiate tumors. A2B5 might be involved in glioma cell invasion in vitro, probably because of adhesion of the molecule

glycoprotein found in several normal tissues but not in mature astrocytes, oligodendrocytes

al., 2008).


Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 383

Fig. 2. Spheroids derived from a glioblastoma short term culture were stained

CD133 (B), Podoplanin (C), Nestin (D), Bmi-1 (E) and Sox2 (F).

immunohistochemically with a panel of stem cell markers. After culturing, the spheroids were formalin fixed, paraffin embedded and sectioned in 3 µm thin sections followed by immunohistochemical staining. The section in (A) was stained with hematoxylin and eosin (HE) which is widely used in histology to identify cell nucleus and cytoplasm. Moreover, sections were immunohistochemically stained with the stem or tumor stem cell markers


Table 1. Some of the most used stem cell markers in the field of brain tumors.

Kamalian et al., 2008; Maw et al., 2009; Nam & Benezra, 2009; Schindl et al., 2001; Schindl et al., 2003; Schoppmann et al., 2003;

Brekke et al., 2006; Chekenya et al., 1999; Chekenya et al., 2002a; Chekenya et al., 2002b; Chekenya et al., 2008; Chekenya & Immervoll, 2007; Chekenya & Pilkington, 2002; Joo et al., 2008; Petrovici et al., 2010; Stallcup & Huang, 2008

Capela & Temple, 2002; Capela & Temple, 2006; Read et al., 2009; Ward et al., 2009

Tang et al., 2009

**Markers Short introduction References**  neuronal stem cell fate. It is found in the ventricular and sub-ventricular zone in fetal brains, but only in the ependymal cells in the human adult brain. It has been found to be highly expressed in glioblastoma cells compared to normal human brain and is believed to be involved in proliferation and

> class of transcription factors known as helixloop-helix (HLH) proteins. The Id gene family is involved in regulation of cell-cycle status and differentiation during embryogenesis and has been found in a rare type of neural stem cells, the B1 type, where it is necessary for selfrenewal. Expression of Id proteins has been demonstrated in a variety of human tumors including gliomas and has been investigated as a potential proto-oncogene. Overexpression of Id1 in human tumor cells induces cell proliferation and invasion, and also protects

tumorigenesis.

ID1 ID1 (inhibitor of DNA binding 1) belongs to a

cells against drug-induced apoptosis.

interacts with the ECM to mediate cell adhesion and proliferation. It is expressed on oligodendrocyte precursor cells in the adult CNS. It has been found in human acute myeloid leukemia and in gliomas, where it in the latter seems to increase tumor cell proliferation in vitro and promote

also known as LeX or stage-specific embryonic antigen 1, SSEA-1, is an extracellular matrixassociated carbohydrate. CD15 is secreted by neural progenitor cells including stem cells into the stem cell niche, where it binds factors such as WNT-1 that are important for progenitor proliferation and self-renewal. It is highly expressed on pluripotent stem cells and has been found in CNS germinal zones. It has been found in various normal tissues but also

NG2 NG2 is a transmembrane proteoglycan that

CD15 CD15 (leukocyte cluster of differentiation 15)

in different cancers including gliomas.

Table 1. Some of the most used stem cell markers in the field of brain tumors.

angiogenesis in vivo.

Fig. 2. Spheroids derived from a glioblastoma short term culture were stained immunohistochemically with a panel of stem cell markers. After culturing, the spheroids were formalin fixed, paraffin embedded and sectioned in 3 µm thin sections followed by immunohistochemical staining. The section in (A) was stained with hematoxylin and eosin (HE) which is widely used in histology to identify cell nucleus and cytoplasm. Moreover, sections were immunohistochemically stained with the stem or tumor stem cell markers CD133 (B), Podoplanin (C), Nestin (D), Bmi-1 (E) and Sox2 (F).

Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 385

Low oxygen levels in different tumor types are believed to increase the population of tumor stem cells and to promote a stem-like state (Bar et al., 2010; Heddleston et al., 2009; Saigusa et al., 2011; Soeda et al., 2009; Wang et al., 2011; Xing et al., 2011; Yeung et al., 2011). This is similar to results obtained for embryonic stem cells showing that low oxygen levels promote maintenance of pluripotent potential, and maintenance of the cells in an undifferentiated stem cell state (Ezashi et al., 2005; Heddleston et al., 2009). The existence of tumor stem cells has been suggested to be restricted to perivascular niches and hypoxic areas within the tumor (Heddleston et al., 2009) explaining the poor outcome and therapeutic resistance seen in these hypoxic tumors. In addition to obtaining a more in vivo like metabolic milieu when culturing cells in hypoxic conditions, hypoxia also seems to promote the existence and propagation of tumor stem cells (Heddleston et al., 2009; McCord et al., 2009; Seno et al., 2009; Soeda et al., 2009). Several studies thus reported an increase in spheroid diameter, cell proliferation and number of spheroids (Heddleston et al., 2009; McCord et al., 2009; Soeda et al., 2009) when culturing spheroids in hypoxic compared to normoxic conditions. In a study from our group (Kolenda et al., 2010), spheroids obtained from a glioblastoma short term culture and the commercial glioblastoma cell line U87MG were cultured in both normoxia and hypoxia. Interestingly, a significant increase in the expression of the proposed stem cell markers CD133, Podoplanin and Bmi-1 was found in both types of spheroids when cultured in hypoxia. Furthermore, a study by Heddelston et al. (Heddleston et al., 2009) proposed that a phenotypic shift from non-stem to stem-like cells was obtained when culturing tumor cells in hypoxia. On the more mechanistic level, the spheroid formation in hypoxia has been shown to be affected by the hypoxia inducible factors as shown in studies by Li et al. (Li et al., 2009) and Méndez et al. (Mendez et al., 2010). Knockdown of HIF altered spheroid formation in glioma spheroids, resulting in smaller and fewer spheroids. Overall these findings suggest that culturing of cells in hypoxia as spheroids provides important in vivolike conditions that are optimal when studying the stem cell biology of brain tumors.

In the last three decades radiotherapy has been the standard treatment or part of the standard treatment for newly diagnosed glioblastoma patients (Stupp et al., 2009) providing a significant survival benefit (Laperriere et al., 2002). However, due to resistance to the current treatment of a subset of cells, it remains palliative. Primary spheroids obtained from glioma tissue have for years been reported to be a useful model for investigating in vitro radiobiology due to the preserved cellular organization. Features existing in these spheroids such as cell-cell contact, variation in the cell cycle distribution, diffusion effects, altered metabolism and hypoxia may influence the outcome of treatment, contributing to a better resemblance of the in vivo situation than obtained with a monolayer model (Olive & Durand, 1994; Sutherland & Durand, 1972). One feature of particular importance in these spheroids is possibly the low oxygen status being partly responsible for the increased radioresistance of the spheroid tumor cells (Blazek et al., 2007; Hsieh et al., 2010; Sutherland, 1998). Ionising radiation causes the formation of reactive oxygen species (ROS) (Brahme & Lind, 2010; 2008) and oxygen has therefore long been known to be a potent radiosensitizer (Vlashi et al., 2009). ROS causes damage to cellular components including DNA damage (Nishikawa, 2008) and are critical for irradiation-induced killing of tumor cells (Diehn et al., 2009). However, there have also been reports of no evident correlation between hypoxia and radioresistance (Buffa et al., 2001; Gorlach & Acker, 1994; Sminia et al., 2003) A study by

**5. Primary spheroids and radiotherapy** 

CD133 was initially identified as a marker of hematopoietic stem cells in 1997 (Miraglia et al., 1997; Yin et al., 1997) and later as a marker of human neural stem cells (Uchida et al., 2000). In 2003 a CD133+ subpopulation of cells with stem cell properties were isolated from medulloblastomas and pilocytic astrocytomas by flow cytometry (Singh et al., 2003). The isolated CD133+ cells formed primary neurospheres in vitro, whereas the CD133- cells did not. As previously mentioned, the sphere forming capability is believed to be a stem cell hallmark. In 2004 the same group isolated CD133+ subpopulations from medulloblastomas and glioblastomas and showed that they also exhibited stem cell properties in vivo. The CD133+ population could initiate phenotypically similar tumors, when injected intracranially into NOD/SCID mice in numbers as few as 100 CD133+ cells. This was not the case for CD133- cells, where up to 100,000 cells could not initiate new tumor formation (Singh et al., 2004). Although these results had a great impact on the field of glioma research, it should be mentioned that today controversies exist in this area. An important paper contributing to this controversy was a paper showing that also CD133- cells were tumorigenic and could give rise to CD133+ cells (Wang et al., 2008).

In accordance with the general idea of neural stem cells residing in discrete stem cell niches in the adult subventricular zone (Riquelme et al., 2008; Zhu et al., 2005), we have found CD133+ cells in this particular zone (Hermansen et al., 2011). In line with this, immunohistochemical studies performed by different groups (Calabrese et al., 2007; Thon et al., 2010; Zeppernick et al., 2008) including our group (Christensen et al., 2008; Hermansen et al., 2011) have shown that CD133 is located in clusters or niches in brain tumors, some of which are perivascular. The size of the niches varies from large positive areas to small perivascular niches comprising only a few cells. Several studies, including studies from our group (Christensen et al., 2008; Hermansen et al., 2011; Immervoll et al., 2008) have, however, also reported a widespread CD133 expression pattern in areas of various normal tissues, which is not normally associated with stem cells. This suggests that CD133 is not specific for stem cells and should be used in combination with other stem or progenitor cell markers to isolate tumor stem cells.

### **4. Hypoxia and tumor stem cells**

Several studies associate tumor hypoxia with poor patient outcome and resistance to therapies (Bar, 2011; Li et al., 2009; Mashiko et al., 2011). In line with this, one of the hallmarks of glioblastomas is the presence of necrosis, occurring as a consequence of poor oxygenation and nutrition because of rapid tumor growth and formation of vessel thrombosis (Hulleman & Helin, 2005; Louis et al., 2007; Preusser et al., 2006). The hypoxiainducible factors (HIFs) are transcription factors upregulated at low oxygen levels. These factors mediate the cellular hypoxia response influencing angiogenesis, cell survival, chemotherapy and radiation resistance, invasion and metastasis (Bar, 2011).

Usually culturing of cells is performed at 21 % O2, but with the knowledge that the physiological oxygen concentration in the healthy brain ranges between 2.5 % and 12.5 % O2 and in glioblastomas is even lower (Bar, 2011), it is worth considering culturing cells at lower oxygen concentrations. Spheroids of large sizes become hypoxic even if cultured in normoxia because of a diffusion gradient. However, a study by Glicklis et al. (Glicklis et al., 2004) has described that hepatocyte spheroids with diameters up to 100 μm have a good oxygenation status. Other studies by Fehlauer et al. (Fehlauer et al., 2005; Fehlauer et al., 2006; Fehlauer et al., 2007) have reported that by using glioma spheroids with diameters of 200-250 μm, there are only few hypoxic cells and no central necrosis present.

CD133 was initially identified as a marker of hematopoietic stem cells in 1997 (Miraglia et al., 1997; Yin et al., 1997) and later as a marker of human neural stem cells (Uchida et al., 2000). In 2003 a CD133+ subpopulation of cells with stem cell properties were isolated from medulloblastomas and pilocytic astrocytomas by flow cytometry (Singh et al., 2003). The isolated CD133+ cells formed primary neurospheres in vitro, whereas the CD133- cells did not. As previously mentioned, the sphere forming capability is believed to be a stem cell hallmark. In 2004 the same group isolated CD133+ subpopulations from medulloblastomas and glioblastomas and showed that they also exhibited stem cell properties in vivo. The CD133+ population could initiate phenotypically similar tumors, when injected intracranially into NOD/SCID mice in numbers as few as 100 CD133+ cells. This was not the case for CD133- cells, where up to 100,000 cells could not initiate new tumor formation (Singh et al., 2004). Although these results had a great impact on the field of glioma research, it should be mentioned that today controversies exist in this area. An important paper contributing to this controversy was a paper showing that also CD133- cells were

In accordance with the general idea of neural stem cells residing in discrete stem cell niches in the adult subventricular zone (Riquelme et al., 2008; Zhu et al., 2005), we have found CD133+ cells in this particular zone (Hermansen et al., 2011). In line with this, immunohistochemical studies performed by different groups (Calabrese et al., 2007; Thon et al., 2010; Zeppernick et al., 2008) including our group (Christensen et al., 2008; Hermansen et al., 2011) have shown that CD133 is located in clusters or niches in brain tumors, some of which are perivascular. The size of the niches varies from large positive areas to small perivascular niches comprising only a few cells. Several studies, including studies from our group (Christensen et al., 2008; Hermansen et al., 2011; Immervoll et al., 2008) have, however, also reported a widespread CD133 expression pattern in areas of various normal tissues, which is not normally associated with stem cells. This suggests that CD133 is not specific for stem cells and should be used in combination with other stem or progenitor cell

Several studies associate tumor hypoxia with poor patient outcome and resistance to therapies (Bar, 2011; Li et al., 2009; Mashiko et al., 2011). In line with this, one of the hallmarks of glioblastomas is the presence of necrosis, occurring as a consequence of poor oxygenation and nutrition because of rapid tumor growth and formation of vessel thrombosis (Hulleman & Helin, 2005; Louis et al., 2007; Preusser et al., 2006). The hypoxiainducible factors (HIFs) are transcription factors upregulated at low oxygen levels. These factors mediate the cellular hypoxia response influencing angiogenesis, cell survival,

Usually culturing of cells is performed at 21 % O2, but with the knowledge that the physiological oxygen concentration in the healthy brain ranges between 2.5 % and 12.5 % O2 and in glioblastomas is even lower (Bar, 2011), it is worth considering culturing cells at lower oxygen concentrations. Spheroids of large sizes become hypoxic even if cultured in normoxia because of a diffusion gradient. However, a study by Glicklis et al. (Glicklis et al., 2004) has described that hepatocyte spheroids with diameters up to 100 μm have a good oxygenation status. Other studies by Fehlauer et al. (Fehlauer et al., 2005; Fehlauer et al., 2006; Fehlauer et al., 2007) have reported that by using glioma spheroids with diameters of

chemotherapy and radiation resistance, invasion and metastasis (Bar, 2011).

200-250 μm, there are only few hypoxic cells and no central necrosis present.

tumorigenic and could give rise to CD133+ cells (Wang et al., 2008).

markers to isolate tumor stem cells.

**4. Hypoxia and tumor stem cells** 

Low oxygen levels in different tumor types are believed to increase the population of tumor stem cells and to promote a stem-like state (Bar et al., 2010; Heddleston et al., 2009; Saigusa et al., 2011; Soeda et al., 2009; Wang et al., 2011; Xing et al., 2011; Yeung et al., 2011). This is similar to results obtained for embryonic stem cells showing that low oxygen levels promote maintenance of pluripotent potential, and maintenance of the cells in an undifferentiated stem cell state (Ezashi et al., 2005; Heddleston et al., 2009). The existence of tumor stem cells has been suggested to be restricted to perivascular niches and hypoxic areas within the tumor (Heddleston et al., 2009) explaining the poor outcome and therapeutic resistance seen in these hypoxic tumors. In addition to obtaining a more in vivo like metabolic milieu when culturing cells in hypoxic conditions, hypoxia also seems to promote the existence and propagation of tumor stem cells (Heddleston et al., 2009; McCord et al., 2009; Seno et al., 2009; Soeda et al., 2009). Several studies thus reported an increase in spheroid diameter, cell proliferation and number of spheroids (Heddleston et al., 2009; McCord et al., 2009; Soeda et al., 2009) when culturing spheroids in hypoxic compared to normoxic conditions. In a study from our group (Kolenda et al., 2010), spheroids obtained from a glioblastoma short term culture and the commercial glioblastoma cell line U87MG were cultured in both normoxia and hypoxia. Interestingly, a significant increase in the expression of the proposed stem cell markers CD133, Podoplanin and Bmi-1 was found in both types of spheroids when cultured in hypoxia. Furthermore, a study by Heddelston et al. (Heddleston et al., 2009) proposed that a phenotypic shift from non-stem to stem-like cells was obtained when culturing tumor cells in hypoxia. On the more mechanistic level, the spheroid formation in hypoxia has been shown to be affected by the hypoxia inducible factors as shown in studies by Li et al. (Li et al., 2009) and Méndez et al. (Mendez et al., 2010). Knockdown of HIF altered spheroid formation in glioma spheroids, resulting in smaller and fewer spheroids. Overall these findings suggest that culturing of cells in hypoxia as spheroids provides important in vivolike conditions that are optimal when studying the stem cell biology of brain tumors.

#### **5. Primary spheroids and radiotherapy**

In the last three decades radiotherapy has been the standard treatment or part of the standard treatment for newly diagnosed glioblastoma patients (Stupp et al., 2009) providing a significant survival benefit (Laperriere et al., 2002). However, due to resistance to the current treatment of a subset of cells, it remains palliative. Primary spheroids obtained from glioma tissue have for years been reported to be a useful model for investigating in vitro radiobiology due to the preserved cellular organization. Features existing in these spheroids such as cell-cell contact, variation in the cell cycle distribution, diffusion effects, altered metabolism and hypoxia may influence the outcome of treatment, contributing to a better resemblance of the in vivo situation than obtained with a monolayer model (Olive & Durand, 1994; Sutherland & Durand, 1972). One feature of particular importance in these spheroids is possibly the low oxygen status being partly responsible for the increased radioresistance of the spheroid tumor cells (Blazek et al., 2007; Hsieh et al., 2010; Sutherland, 1998). Ionising radiation causes the formation of reactive oxygen species (ROS) (Brahme & Lind, 2010; 2008) and oxygen has therefore long been known to be a potent radiosensitizer (Vlashi et al., 2009). ROS causes damage to cellular components including DNA damage (Nishikawa, 2008) and are critical for irradiation-induced killing of tumor cells (Diehn et al., 2009). However, there have also been reports of no evident correlation between hypoxia and radioresistance (Buffa et al., 2001; Gorlach & Acker, 1994; Sminia et al., 2003) A study by

Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 387

viability and the proliferation of cells in the spheroids, including the ability of the cells to

Frequently used assays for measuring the viability of the cells after treatment is tetrazoliumbased cell proliferation assays. Several variations of this assay exists (XTT, MTT, MTS or WST-1) (Berridge et al., 2005), but all utilize the conversion of tetrazolium salts by active mitochondria into dark red formazan that can be monitored by absorbance measurements (Berridge et al., 2005). Usually these assays are used on adherent monolayer cultures, which consist of uniform cell populations. However, these assays have also been used on spheroids consisting of more heterogenous cell populations. In one study (Johannessen et al., 2009) the doxorubicin sensitivity was determined in high and low passage spheroids by a MTS-assay. This was done by placing one spheroid per well in a 96 well plate, measuring viability relative to size after incubation with doxorubicin for 96 hours. After the viability measurements, the spheroids were allowed to adhere to the bottom of the plastic plates resulting in cell migration from the spheroids. Immunostaining of the migratory cells were

performed using the neural stem cell markers Nestin, Vimentin and Musashi-1.

Another widely used assay is the lactate dehydrogenase assay (LDH-assay), measuring cell death. The LDH-assay indirectly measures plasma membrane damage, which is related to cell death. Due to membrane damage, LDH leaks to the culture medium, where it participates in the conversion of tetrazolium salts to formazan. The amount of formazan produced is directly proportional to the amount of LDH in the culture medium, which in turn is directly proportional to the number of dead or damaged cells (Korzeniewski &

The size of spheroids after drug treatment has also been used as a measure of cell viability (Fehlauer et al., 2007; Johannessen et al., 2009; Khaitan et al., 2009; Yamaguchi et al., 2010). Khaitan et al. (Khaitan et al., 2009) investigated the effect of the glycolytic inhibitor 2-deoxy-D-glucose on spheroids derived from a human glioma cell line by measuring the size of the spheroids after drug exposure. In a stem cell context, number and size of primary and secondary spheroids have also been widely used as measures of the self-renewal potential, which is one of the hallmarks of stem cells. Especially the traits that are attributable to tumor stem cells are of interest, as the tumor stem cell hypothesis states that the tumor stem cells need to be targeted specifically in order to improve cancer treatment. In the so called spheroid formation assays or clonogenic assays, the ability of the cells to form spheroids is investigated. In many studies (Sunayama et al., 2010; Wakimoto et al., 2009; Wang et al., 2010; Zhu et al., 2010) this is primarily done after treatment of the cells, thereby investigating the effect of a given drug on the ability of the cells to self-renew. Different experimental setups have been employed using different cell densities, probably resulting in two different assays - one assay with high cell densities for evaluation of proliferation and another assay with small so-called clonal cell densities for evaluation of self-renewal or clonogenic capabilities of the cells. High cell densities often result in cell and spheroid fusion due to a high motility of the spheroids (Singec et al., 2006) and it is therefore not possible to investigate the self-renewal mechanism in this assay. A plating density of 20 cells/µl has been considered as clonal conditions in terms of neurosphere formation (Singec et al., 2006). In glioma studies cell densities ranging between 0.15 cells/µl to 300 cells/µl have been used (Kolenda et al., 2010; Sunayama et al., 2010; Wakimoto et al., 2009; Wang et al., 2010; Zhu et

form secondary spheroid.

Callewaert, 1983).

al., 2010) when studying spheroid formation.

Sminia et al. (Sminia et al., 2003) found that both hypoxic and well-oxygenated organotypic multicellular spheroids derived from glioblastoma specimens showed high resistance to irradiation.

A study by Kaaijk et al. (Kaaijk et al., 1997) described the observation of only minor histological changes including a few shrunken nuclei, but no major histological damage in normoxic organotypic multicellular glioblastoma spheroids after a single dose of 50 Gy. This is in line with a study by Bauman et al. (Bauman et al., 1999), where C6 astrocytoma spheroids were implanted into a collagen type I gel. Following irradiation with 12 and 25 Gy, neither the hypoxic core nor the rim of the spheroids experienced a significant increase in the fraction of apoptotic cells. Similar to this, U87MG monolayer cultures irradiated with 8 and 20 Gy showed no considerable apoptosis five days after treatment and remained viable ten weeks after a 40 Gy dose was administered. However, in fact Kaaijk et al. reported that proliferation in three investigated organotypic multicellular spheroids was decreased 7-20 fold relative to untreated controls one week after hypofractionated radiation with a total of 40 Gy. Moreover, Fehlauer et al. (Fehlauer et al., 2005; Fehlauer et al., 2006) described in two studies a decrease in the percentage of MIB-1 positive proliferative cells in organotypic multicellular spheroids following irradiation with 20 Gy.

It has also been suggested that tumor stem cells might show increased radioresistance compared to more differentiated cells (Bao et al., 2006; Phillips et al., 2006; Rich, 2007). Bao et al. (Bao et al., 2006) thus showed that CD133+ cells survived ionizing radiation better than CD133- cells and that the fraction of CD133+ cells was enriched in gliomas after radiotherapy, suggesting that the CD133+ cellular population of gliomas is contributing to glioma radioresistance and could be the source of tumor repopulation after radiation. Liu et al. (Liu et al., 2006) investigated mRNA levels of various markers including BCRP1 (breast cancer resistance protein), MGMT (O-6-methylguanine-DNA methyltransferase), anti-apoptosis proteins and inhibitors of apoptosis protein families in CD133+ cells isolated by FACS. These markers are involved in treatment resistance and elevated mRNA levels were shown in CD133+ cells compared to CD133- cells. A significant degree of resistance towards chemotherapeutics such as temozolomide, carboplatin, paclitaxel and etoposide were demonstrated in the CD133+ cells (Liu et al., 2006). In line with these results Liu et al. showed enrichment of CD133+ cells in five recurrent gliomas when compared to the respective newly diagnosed tumors. Furthermore, results obtained in our laboratory have shown a much more pronounced reduction in the secondary spheroid formation capacity of irradiated spheroids derived from recently established glioma spheroids with stem cell characteristics compared to U87MG derived spheroids without these characteristics (Jakobsen et al. 2011).

#### **6. Spheroids and chemotherapy**

Besides irradiation and surgery, the treatment of glioblastomas consists of chemotherapy. Although the introduction of temozolomide as standard chemotherapeutic in 2005 (Stupp et al., 2005) has increased the overall patient survival, new and more efficient chemotherapeutics or targeted therapies are urgently needed. Here spheroids also have an important role to play.

Investigations of the specific effects of chemotherapeutics and other drugs on glioma spheroids are often done by investigating the size and number of spheroids as well as the

Sminia et al. (Sminia et al., 2003) found that both hypoxic and well-oxygenated organotypic multicellular spheroids derived from glioblastoma specimens showed high resistance to

A study by Kaaijk et al. (Kaaijk et al., 1997) described the observation of only minor histological changes including a few shrunken nuclei, but no major histological damage in normoxic organotypic multicellular glioblastoma spheroids after a single dose of 50 Gy. This is in line with a study by Bauman et al. (Bauman et al., 1999), where C6 astrocytoma spheroids were implanted into a collagen type I gel. Following irradiation with 12 and 25 Gy, neither the hypoxic core nor the rim of the spheroids experienced a significant increase in the fraction of apoptotic cells. Similar to this, U87MG monolayer cultures irradiated with 8 and 20 Gy showed no considerable apoptosis five days after treatment and remained viable ten weeks after a 40 Gy dose was administered. However, in fact Kaaijk et al. reported that proliferation in three investigated organotypic multicellular spheroids was decreased 7-20 fold relative to untreated controls one week after hypofractionated radiation with a total of 40 Gy. Moreover, Fehlauer et al. (Fehlauer et al., 2005; Fehlauer et al., 2006) described in two studies a decrease in the percentage of MIB-1 positive proliferative cells in

It has also been suggested that tumor stem cells might show increased radioresistance compared to more differentiated cells (Bao et al., 2006; Phillips et al., 2006; Rich, 2007). Bao et al. (Bao et al., 2006) thus showed that CD133+ cells survived ionizing radiation better than CD133- cells and that the fraction of CD133+ cells was enriched in gliomas after radiotherapy, suggesting that the CD133+ cellular population of gliomas is contributing to glioma radioresistance and could be the source of tumor repopulation after radiation. Liu et al. (Liu et al., 2006) investigated mRNA levels of various markers including BCRP1 (breast cancer resistance protein), MGMT (O-6-methylguanine-DNA methyltransferase), anti-apoptosis proteins and inhibitors of apoptosis protein families in CD133+ cells isolated by FACS. These markers are involved in treatment resistance and elevated mRNA levels were shown in CD133+ cells compared to CD133- cells. A significant degree of resistance towards chemotherapeutics such as temozolomide, carboplatin, paclitaxel and etoposide were demonstrated in the CD133+ cells (Liu et al., 2006). In line with these results Liu et al. showed enrichment of CD133+ cells in five recurrent gliomas when compared to the respective newly diagnosed tumors. Furthermore, results obtained in our laboratory have shown a much more pronounced reduction in the secondary spheroid formation capacity of irradiated spheroids derived from recently established glioma spheroids with stem cell characteristics compared to U87MG derived spheroids without these characteristics

Besides irradiation and surgery, the treatment of glioblastomas consists of chemotherapy. Although the introduction of temozolomide as standard chemotherapeutic in 2005 (Stupp et al., 2005) has increased the overall patient survival, new and more efficient chemotherapeutics or targeted therapies are urgently needed. Here spheroids also have an

Investigations of the specific effects of chemotherapeutics and other drugs on glioma spheroids are often done by investigating the size and number of spheroids as well as the

organotypic multicellular spheroids following irradiation with 20 Gy.

irradiation.

(Jakobsen et al. 2011).

important role to play.

**6. Spheroids and chemotherapy** 

viability and the proliferation of cells in the spheroids, including the ability of the cells to form secondary spheroid.

Frequently used assays for measuring the viability of the cells after treatment is tetrazoliumbased cell proliferation assays. Several variations of this assay exists (XTT, MTT, MTS or WST-1) (Berridge et al., 2005), but all utilize the conversion of tetrazolium salts by active mitochondria into dark red formazan that can be monitored by absorbance measurements (Berridge et al., 2005). Usually these assays are used on adherent monolayer cultures, which consist of uniform cell populations. However, these assays have also been used on spheroids consisting of more heterogenous cell populations. In one study (Johannessen et al., 2009) the doxorubicin sensitivity was determined in high and low passage spheroids by a MTS-assay. This was done by placing one spheroid per well in a 96 well plate, measuring viability relative to size after incubation with doxorubicin for 96 hours. After the viability measurements, the spheroids were allowed to adhere to the bottom of the plastic plates resulting in cell migration from the spheroids. Immunostaining of the migratory cells were performed using the neural stem cell markers Nestin, Vimentin and Musashi-1.

Another widely used assay is the lactate dehydrogenase assay (LDH-assay), measuring cell death. The LDH-assay indirectly measures plasma membrane damage, which is related to cell death. Due to membrane damage, LDH leaks to the culture medium, where it participates in the conversion of tetrazolium salts to formazan. The amount of formazan produced is directly proportional to the amount of LDH in the culture medium, which in turn is directly proportional to the number of dead or damaged cells (Korzeniewski & Callewaert, 1983).

The size of spheroids after drug treatment has also been used as a measure of cell viability (Fehlauer et al., 2007; Johannessen et al., 2009; Khaitan et al., 2009; Yamaguchi et al., 2010). Khaitan et al. (Khaitan et al., 2009) investigated the effect of the glycolytic inhibitor 2-deoxy-D-glucose on spheroids derived from a human glioma cell line by measuring the size of the spheroids after drug exposure. In a stem cell context, number and size of primary and secondary spheroids have also been widely used as measures of the self-renewal potential, which is one of the hallmarks of stem cells. Especially the traits that are attributable to tumor stem cells are of interest, as the tumor stem cell hypothesis states that the tumor stem cells need to be targeted specifically in order to improve cancer treatment. In the so called spheroid formation assays or clonogenic assays, the ability of the cells to form spheroids is investigated. In many studies (Sunayama et al., 2010; Wakimoto et al., 2009; Wang et al., 2010; Zhu et al., 2010) this is primarily done after treatment of the cells, thereby investigating the effect of a given drug on the ability of the cells to self-renew. Different experimental setups have been employed using different cell densities, probably resulting in two different assays - one assay with high cell densities for evaluation of proliferation and another assay with small so-called clonal cell densities for evaluation of self-renewal or clonogenic capabilities of the cells. High cell densities often result in cell and spheroid fusion due to a high motility of the spheroids (Singec et al., 2006) and it is therefore not possible to investigate the self-renewal mechanism in this assay. A plating density of 20 cells/µl has been considered as clonal conditions in terms of neurosphere formation (Singec et al., 2006). In glioma studies cell densities ranging between 0.15 cells/µl to 300 cells/µl have been used (Kolenda et al., 2010; Sunayama et al., 2010; Wakimoto et al., 2009; Wang et al., 2010; Zhu et al., 2010) when studying spheroid formation.

Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 389

Besides investigating tumor invasion into the brain tissue, the model offers several other applications. Guillamo et al. (Guillamo et al., 2009) investigated the invasion, proliferation and angiogenesis of six human malignant glioma spheroids implanted into organotypic brain slice cultures, when these co-cultures were treated with gefinitib. Some of the tumors implanted had EGFR amplifications resulting in more pronounced invasion than tumors without EGFR amplification. Upon treatment with gefinitib only tumor cell invasion from tumors with EGFR amplification was inhibited, whereas vascular density was decreased in all tumors. In another study, Caspani et al. (Caspani et al., 2006) used the co-culture model to investigate the re-organization of the cytoskeleton in migrating glioblastoma cells. Cells transfected with green fluorescent protein were introduced into collagen gels, brain slice cultures and in vivo into mice brains and the re-organization and motility of the

glioblastoma cells in the different models were monitored by confocal microscopy.

details.

Organotypic brain slice cultures from rodents have been used in a variety of different studies using tissue obtained from different areas in the brain. The organotypic brain slice cultures used in the invasion studies varied from explants from spinal cord (Caspani et al., 2006), brain slices cut in the sagittal plane (Caspani et al., 2006) and the coronal plane (De et al., 2002; Guillamo et al., 2009; Matsumura et al., 2000; Ohnishi et al., 1998; Palfi et al., 2004) as well as entorhinohippocampal slice cultures (Eyupoglu et al., 2005). Since glioblastomas are often located in the subcortical white matter of the cerebral hemisphere and tumor infiltration often extends into the adjacent cortex and through corpus callosum (Louis et al., 2007) we have used organotypic corticostriatal brain slice cultures with cortex, striatum and corpus callosum for our studies of the invasive features of the glioblastoma cells. Such corticostriatal slice cultures should be cultured by the interface method placing the brain slices at the interface between air and culturing medium on a porous, transparent and lowprotein binding membrane. This allows the cultures to be oxygenated on one site, while receiving nutrients from the other site. In the following text our approach is described in

The corticostriatal slice cultures are prepared from newborn Wistar rat pups by a method slightly modified from Kristensen et al. (Kristensen et al., 1999). The brain is aseptically removed from the pup and placed in a petri dish under a stereomicroscope, where the meninges are carefully removed. Hereafter the brain is sectioned coronally in 400 μm slices on a McIIwain tissue Chopper and the slices are transferred to a petri dish containing Hanks' Balanced Salt Solution supplemented with 0.9% glucose. The brain slices are separated from each other and the sections containing cortex and striatum are divided into the two hemispheric parts resulting in the final brain slices. These slices are randomly moved to the insert membranes with four cultures on each membrane. Finally, the membrane inserts are placed in a 6-well plate in 1 ml preheated medium, and incubated in

Cell line spheroids or organotypic primary spheroids are implanted into organotypic brain slice cultures in the corpus callosum area between cortex and striatum, whereby a co-culture is established. In order to identify and follow tumor cells when they invade the slice, the spheroids can be labeled with the fluorescent dye, DiI (1,1′-Dioctadecyl-3,3,3′,3′ tetramethylindocarbocyanine perchlorate) for 24 h before implantation, enabling confocal microscopy. The labeled spheroids are prepared by adding a DiI solution to the medium with spheroids, achieving a concentration of 25 μg/ml. Before implantation the spheroids are washed in medium to avoid bringing excess dye onto the brain slice cultures. Spheroids around 200-400 μm in diameter are captured using a denudation pipette and

36°C humidified air containing 5% CO2 and 95% atmospheric air.

#### **7. An in vivo-like in vitro model of glioma invasion**

Gliomas are known to be highly invasive and new knowledge concerning tumor cell invasion also incorporating the tumor stem cell aspect is urgently needed. In our laboratory we have worked to improve in vitro models when investigating the invasive features of glioblastoma cells. This led to establishment of an in vivo-like model of invasion, where spheroids are implanted into organotypic brain slice cultures.

The organotypic brain slice cultures became very popular research tools especially with the development of the roller-tube technique by Gähwiler in 1981 (Gahwiler, 1981) and the inter-face culturing method developed by Stoppini in 1991 (Stoppini et al., 1991). These cultures preserve many of the basic structural and connective tissue structures present in the tissue, when it is localized in the brain (Gahwiler, 1988). By implanting the organotypic spheroids into the organotypic brain slice cultures, it is possible to establish an organotypic spheroid-based slice-culture invasion assay, suitable for following tumor cell invasion into the brain tissue in vitro.

The investigation of glioma invasion has been performed since the 1980's but the models used have improved during the years. Different assays have been used to address the invasive capacities of the tumor cells. A frequently used migration assay allows spheroids to adhere to the bottom of coated plastic plates, and after a period of time the distance of migrating cells from the spheroid can be measured (Gliemroth et al., 2003; Narla et al., 1998; Terzis et al., 1997; Terzis et al., 1998). Another extensively used invasion assay is the Boyden or Boyden-like chamber-based assays. The Boyden chamber was first introduced by Boyden in order to investigate the chemotactic effect of mixtures of antibody and antigen on leukocytes (Boyden, 1962). The principle in the Boyden chambers is cell migration into a microporous membrane, often made of matrigel (Deryugina et al., 1997; Paulus & Tonn, 1994; Schichor et al., 2005). The membrane is placed in between two medium-filled compartments; the upper compartment containing cells whereas the lower compartment may contain a chemotactic agent. After an incubation period, the cells migrating through the microporous membrane can be stained and counted (Chen, 2005). In a variation of the Boyden Chamber assay, slices of porcine white and gray matter were placed on top of a filter between the two compartments facilitating the cells to migrate through the porcine brain slice, making this assay a combination of the Boyden Chamber and the organotypic coculture system (Schichor et al., 2005).

In the first real invasion studies, tissue aggregates from rat brain or chick heart were (Lund-Johansen et al., 1990) placed next to the tumor tissue, but first with the development of the organotypic brain slice culture, it was possible to preserve the brain architecture and organization in an optimal way, thereby creating the conditions necessary for a more in vivo-like model of glioma invasion. Several groups have been using this model to investigate the invasion of glioma cell into organotypic brain slice cultures (Aaberg-Jessen et al. 2011; Caspani et al., 2006; De et al., 2002; Eyupoglu et al., 2005; Guillamo et al., 2009; Jensen et al. 2010; Matsumura et al., 2000; Ohnishi et al., 1998; Palfi et al., 2004; Stoppini et al., 1991). In one study using this model, invasion was shown to be associated with the histological type and grade of the tumor (Palfi et al., 2004) and in another study invasion and tumor-induced neurotoxicity was shown to be associated (Eyupoglu et al., 2005). Most interestingly, quantitative analysis of invasion has also been performed (De et al., 2002) using confocal laser scanning microscopy and a three-dimensional visualization after having followed invasion over several weeks (Matsumura et al., 2000).

Gliomas are known to be highly invasive and new knowledge concerning tumor cell invasion also incorporating the tumor stem cell aspect is urgently needed. In our laboratory we have worked to improve in vitro models when investigating the invasive features of glioblastoma cells. This led to establishment of an in vivo-like model of invasion, where spheroids are

The organotypic brain slice cultures became very popular research tools especially with the development of the roller-tube technique by Gähwiler in 1981 (Gahwiler, 1981) and the inter-face culturing method developed by Stoppini in 1991 (Stoppini et al., 1991). These cultures preserve many of the basic structural and connective tissue structures present in the tissue, when it is localized in the brain (Gahwiler, 1988). By implanting the organotypic spheroids into the organotypic brain slice cultures, it is possible to establish an organotypic spheroid-based slice-culture invasion assay, suitable for following tumor cell invasion into

The investigation of glioma invasion has been performed since the 1980's but the models used have improved during the years. Different assays have been used to address the invasive capacities of the tumor cells. A frequently used migration assay allows spheroids to adhere to the bottom of coated plastic plates, and after a period of time the distance of migrating cells from the spheroid can be measured (Gliemroth et al., 2003; Narla et al., 1998; Terzis et al., 1997; Terzis et al., 1998). Another extensively used invasion assay is the Boyden or Boyden-like chamber-based assays. The Boyden chamber was first introduced by Boyden in order to investigate the chemotactic effect of mixtures of antibody and antigen on leukocytes (Boyden, 1962). The principle in the Boyden chambers is cell migration into a microporous membrane, often made of matrigel (Deryugina et al., 1997; Paulus & Tonn, 1994; Schichor et al., 2005). The membrane is placed in between two medium-filled compartments; the upper compartment containing cells whereas the lower compartment may contain a chemotactic agent. After an incubation period, the cells migrating through the microporous membrane can be stained and counted (Chen, 2005). In a variation of the Boyden Chamber assay, slices of porcine white and gray matter were placed on top of a filter between the two compartments facilitating the cells to migrate through the porcine brain slice, making this assay a combination of the Boyden Chamber and the organotypic co-

In the first real invasion studies, tissue aggregates from rat brain or chick heart were (Lund-Johansen et al., 1990) placed next to the tumor tissue, but first with the development of the organotypic brain slice culture, it was possible to preserve the brain architecture and organization in an optimal way, thereby creating the conditions necessary for a more in vivo-like model of glioma invasion. Several groups have been using this model to investigate the invasion of glioma cell into organotypic brain slice cultures (Aaberg-Jessen et al. 2011; Caspani et al., 2006; De et al., 2002; Eyupoglu et al., 2005; Guillamo et al., 2009; Jensen et al. 2010; Matsumura et al., 2000; Ohnishi et al., 1998; Palfi et al., 2004; Stoppini et al., 1991). In one study using this model, invasion was shown to be associated with the histological type and grade of the tumor (Palfi et al., 2004) and in another study invasion and tumor-induced neurotoxicity was shown to be associated (Eyupoglu et al., 2005). Most interestingly, quantitative analysis of invasion has also been performed (De et al., 2002) using confocal laser scanning microscopy and a three-dimensional visualization after having

**7. An in vivo-like in vitro model of glioma invasion** 

implanted into organotypic brain slice cultures.

the brain tissue in vitro.

culture system (Schichor et al., 2005).

followed invasion over several weeks (Matsumura et al., 2000).

Besides investigating tumor invasion into the brain tissue, the model offers several other applications. Guillamo et al. (Guillamo et al., 2009) investigated the invasion, proliferation and angiogenesis of six human malignant glioma spheroids implanted into organotypic brain slice cultures, when these co-cultures were treated with gefinitib. Some of the tumors implanted had EGFR amplifications resulting in more pronounced invasion than tumors without EGFR amplification. Upon treatment with gefinitib only tumor cell invasion from tumors with EGFR amplification was inhibited, whereas vascular density was decreased in all tumors. In another study, Caspani et al. (Caspani et al., 2006) used the co-culture model to investigate the re-organization of the cytoskeleton in migrating glioblastoma cells. Cells transfected with green fluorescent protein were introduced into collagen gels, brain slice cultures and in vivo into mice brains and the re-organization and motility of the glioblastoma cells in the different models were monitored by confocal microscopy.

Organotypic brain slice cultures from rodents have been used in a variety of different studies using tissue obtained from different areas in the brain. The organotypic brain slice cultures used in the invasion studies varied from explants from spinal cord (Caspani et al., 2006), brain slices cut in the sagittal plane (Caspani et al., 2006) and the coronal plane (De et al., 2002; Guillamo et al., 2009; Matsumura et al., 2000; Ohnishi et al., 1998; Palfi et al., 2004) as well as entorhinohippocampal slice cultures (Eyupoglu et al., 2005). Since glioblastomas are often located in the subcortical white matter of the cerebral hemisphere and tumor infiltration often extends into the adjacent cortex and through corpus callosum (Louis et al., 2007) we have used organotypic corticostriatal brain slice cultures with cortex, striatum and corpus callosum for our studies of the invasive features of the glioblastoma cells. Such corticostriatal slice cultures should be cultured by the interface method placing the brain slices at the interface between air and culturing medium on a porous, transparent and lowprotein binding membrane. This allows the cultures to be oxygenated on one site, while receiving nutrients from the other site. In the following text our approach is described in details.

The corticostriatal slice cultures are prepared from newborn Wistar rat pups by a method slightly modified from Kristensen et al. (Kristensen et al., 1999). The brain is aseptically removed from the pup and placed in a petri dish under a stereomicroscope, where the meninges are carefully removed. Hereafter the brain is sectioned coronally in 400 μm slices on a McIIwain tissue Chopper and the slices are transferred to a petri dish containing Hanks' Balanced Salt Solution supplemented with 0.9% glucose. The brain slices are separated from each other and the sections containing cortex and striatum are divided into the two hemispheric parts resulting in the final brain slices. These slices are randomly moved to the insert membranes with four cultures on each membrane. Finally, the membrane inserts are placed in a 6-well plate in 1 ml preheated medium, and incubated in 36°C humidified air containing 5% CO2 and 95% atmospheric air.

Cell line spheroids or organotypic primary spheroids are implanted into organotypic brain slice cultures in the corpus callosum area between cortex and striatum, whereby a co-culture is established. In order to identify and follow tumor cells when they invade the slice, the spheroids can be labeled with the fluorescent dye, DiI (1,1′-Dioctadecyl-3,3,3′,3′ tetramethylindocarbocyanine perchlorate) for 24 h before implantation, enabling confocal microscopy. The labeled spheroids are prepared by adding a DiI solution to the medium with spheroids, achieving a concentration of 25 μg/ml. Before implantation the spheroids are washed in medium to avoid bringing excess dye onto the brain slice cultures. Spheroids around 200-400 μm in diameter are captured using a denudation pipette and

Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 391

Fig. 3. Schematic overview of the implantation of glioma spheroids into organotypic rat corticostriatal brain slice cultures. Glioma tissue is obtained from patients and collected in the operation theatre. Thereafter, it is processed and cultured in the laboratory until spheroids are formed. Alternatively, spheroids from established short term cultures or cell lines can be used. Simultaneously, brain slice cultures from newborn rats are prepared and cultured by the interface method. The spheroids are labeled with the fluorescent dye DiI and implanted into the brain slice cultures in the corpus callosum area between cortex and striatum, here illustrated by a phase contrast image of a spheroid immediately after implantation and after 14 days of culturing. Note the less marked edge of the spheroid as

the cells migrate into the surrounding brain tissue (Cx- cortex and Str-striatum).

thereafter carefully placed next to corpus callosum between cortex and striatum in the brain slice cultures. The culturing plates are placed in the incubator in 36°C humidified air containing 5% CO2 and 95% atmospheric air, whereafter the medium is changed twice a week. By monitoring the co-culture with confocal time-lapse microscopy, tumor cell invasion into the surrounding rat brain tissue can be visualized. Using a confocal microscope, a z-stack can be made consisting of thin sections at different levels of the culture. This makes it possible to follow invasion from all parts of the spheroid in different layers of the brain slice culture, whereby a three-dimentional movie and image can be constructed or alternatively an accumulated two-dimensional image based on overlay of all the images from one z-stack.


Box 2. Preparation of organotypic corticostriatal brain slice cultures and implantation of spheroids.

thereafter carefully placed next to corpus callosum between cortex and striatum in the brain slice cultures. The culturing plates are placed in the incubator in 36°C humidified air containing 5% CO2 and 95% atmospheric air, whereafter the medium is changed twice a week. By monitoring the co-culture with confocal time-lapse microscopy, tumor cell invasion into the surrounding rat brain tissue can be visualized. Using a confocal microscope, a z-stack can be made consisting of thin sections at different levels of the culture. This makes it possible to follow invasion from all parts of the spheroid in different layers of the brain slice culture, whereby a three-dimentional movie and image can be constructed or alternatively an accumulated two-dimensional image based on

4. Section the brain coronally in 400 µm slices on a McIIwain tissue Chopper 5. Separate the brain slices and choose the slices containing cortex and striatum

7. Move slices randomly to a transparent insert membrane

12. Wash the spheroids 3 times with medium before implantation

16. Monitor the DiI-labeled spheroids using confocal microscopy

6. Divide these slices into the two identical halves for obtaining the final brain slice

8. Place the insert membranes in a 6-well culturing plate with 1 ml preheated medium 9. Incubate the cultures in 36°C humidified air containing 5% CO2 and 95% atmospheric

11. Label organotypic spheroids or spheroids derived from short term cultures with

14. Place the spheroids in the area between cortex and striatum next to corpus callosum

15. Incubate the co-cultures in 36°C humidified air containing 5% CO2 and 95%

Box 2. Preparation of organotypic corticostriatal brain slice cultures and implantation of

overlay of all the images from one z-stack.

1. Decapitate a newborn rat pup 2. Aseptically remove the brain 3. Remove the meninges

10. Change the medium twice a week

by a denudation pipette

atmospheric air

25µg/ml DiI for 24 h before implantation

13. Use spheroids in the size range from 200-400 µm

cultures

air

spheroids.

Fig. 3. Schematic overview of the implantation of glioma spheroids into organotypic rat corticostriatal brain slice cultures. Glioma tissue is obtained from patients and collected in the operation theatre. Thereafter, it is processed and cultured in the laboratory until spheroids are formed. Alternatively, spheroids from established short term cultures or cell lines can be used. Simultaneously, brain slice cultures from newborn rats are prepared and cultured by the interface method. The spheroids are labeled with the fluorescent dye DiI and implanted into the brain slice cultures in the corpus callosum area between cortex and striatum, here illustrated by a phase contrast image of a spheroid immediately after implantation and after 14 days of culturing. Note the less marked edge of the spheroid as the cells migrate into the surrounding brain tissue (Cx- cortex and Str-striatum).

Three-Dimensional In Vitro Models in Glioma Research – Focus on Spheroids 393

environment. As discussed in the chapter, spheroids have been used in a wide range of experiments investigating radiation responses, effects of chemotherapy and effects of different types of experimental drugs as well as in migration and invasion studies. The experimental setups may be somewhat more difficult than by using monolayer cultures, but since the spheroid models are supposed to be closer to the in vivo situation, the results and answers obtained are also supposed to be closer to what it true for the corresponding tumors in the human brain. However, efforts should be made to develop these threedimensional models to become even more in vivo-like, in order to meet new challenges in glioma research and drug development. The use of spheroids in especially tumor stem cell research has been fast increasing in recent years making spheroids an important tool also in

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Fig. 4. Confocal images of an invasive DiI-labeled glioma spheroid implanted into an organotypic rat brain slice culture. The spheroid is followed by confocal timelapse microscopy for a period of 72 hours. The images are accumulated images based on overlay of all the images from one z-stack. A z-stack consists of several images obtained at different levels of the co-culture.

#### **8. Conclusion**

We conclude that the three-dimensional spheroid models offers advantages in glioma research taking tumor biology and microenvironment into account. Especially, when using the organotypic models, where the structure and organization of the tissue is preserved, features close to the in vivo situation are supposed to be obtained. In tumor stem cell research, the spheroids are a necessary tool as this culture method seems to promote the existence of these cells. This is especially the case when culturing the spheroids in a hypoxic environment. As discussed in the chapter, spheroids have been used in a wide range of experiments investigating radiation responses, effects of chemotherapy and effects of different types of experimental drugs as well as in migration and invasion studies. The experimental setups may be somewhat more difficult than by using monolayer cultures, but since the spheroid models are supposed to be closer to the in vivo situation, the results and answers obtained are also supposed to be closer to what it true for the corresponding tumors in the human brain. However, efforts should be made to develop these threedimensional models to become even more in vivo-like, in order to meet new challenges in glioma research and drug development. The use of spheroids in especially tumor stem cell research has been fast increasing in recent years making spheroids an important tool also in future glioma research.

#### **9. References**

392 Glioma – Exploring Its Biology and Practical Relevance

Fig. 4. Confocal images of an invasive DiI-labeled glioma spheroid implanted into an organotypic rat brain slice culture. The spheroid is followed by confocal timelapse

levels of the co-culture.

**8. Conclusion** 

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We conclude that the three-dimensional spheroid models offers advantages in glioma research taking tumor biology and microenvironment into account. Especially, when using the organotypic models, where the structure and organization of the tissue is preserved, features close to the in vivo situation are supposed to be obtained. In tumor stem cell research, the spheroids are a necessary tool as this culture method seems to promote the existence of these cells. This is especially the case when culturing the spheroids in a hypoxic


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**18** 

*1,2Spain 3Switzerland* 

**Endogenous Experimental Glioma Model, Links** 

**Between Glioma Stem Cells and Angiogenesis** 

*3Unit of Anatomy, Department of Medicine, University of Fribourg, Fribourg,* 

Glioblastomas (GBM) are the most malignant solid tumours (grade IV) of CNS. They are glial lineage neoplasias with a high proliferative and invasive capacity, reaching to occupy an entire lobe of the brain (Kleihues et al., 2007). According with their genesis, they can be differentiated between primary and secondary glioblastoma. The primary is the most common glioblastoma. This is a new generated tumour after a brief medical history (three months), with no evidence of a less malignant lesion. On the other hand, secondary glioblastoma develops from diffuse astrocytoma, anaplastic astrocytoma or oligodendroglioma and malignant progression. Its development time is about five years. It is thought that both types of glioblastomas may be generated from neoplastic cells with characteristic of stem cells (Ohgaki & Kleihues, 2009). In addition, these cancer stem cells called "glioma stem cells" (GSCs) may be the responsible for glioma recurrences due to chemo-and radio resistance (Bao et al., 2006; Rich, 2007). Glioma stem cells (GSCs) are a subpopulation of neoplastic cells identified in glioma sharing properties with neural stem cells (self-renewal, high proliferation rate, undifferentiating, and neurospheres conformation) and the capacity for leading the tumourigenesis and tumour malignancy. The proliferation and the invasion into adjacent normal parenchyma have been attributed to glioma stem cells as well. Indeed, they were related to the angiogenesis process

 The microvascular network in gliomas has to get adapt to metabolic tissue requirements (Folkman, 2000). When the vascular network cannot satisfy cell requirements (Oxygen pressure of 5-10 mm Hg) tissue hypoxia occurs. This situation triggers the synthesis of proangiogenic factors as matrix metalloprotease (MMP-2), angiopoietin-1, phosphoglycerate kinase (PGK), erythropoietin (EPO), and vascular endothelial growth factor (VEGF)-A

Vascular endothelial growth factor (VEGF) is a major regulator of tumour angiogenesis (Bulnes & Lafuente, 2007; Lafuente et al., 1999; Machein & Plate, 2004; Marti et al., 2000).

**1. Introduction** 

(Fong, 2008).

needed for the growth and survival of the neoplasia.

Susana Bulnes1, Harkaitz Bengoetxea2, Naiara Ortuzar2,

Enrike G. Argandoña3 and José Vicente Lafuente2 *1Laboratory of Clinical and Experimental Neuroscience (LaNCE), Department of Nursing I, University of the Basque Country, Leioa 2Laboratory of Clinical and Experimental Neuroscience (LaNCE), Department of Neuroscience, University of the Basque Country, Leioa,* 

Enger, P. O. (2008). CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells. *Int.J.Cancer*. Vol. 122, No. 4, pp. 761-768


### **Endogenous Experimental Glioma Model, Links Between Glioma Stem Cells and Angiogenesis**

Susana Bulnes1, Harkaitz Bengoetxea2, Naiara Ortuzar2, Enrike G. Argandoña3 and José Vicente Lafuente2

*1Laboratory of Clinical and Experimental Neuroscience (LaNCE), Department of Nursing I, University of the Basque Country, Leioa 2Laboratory of Clinical and Experimental Neuroscience (LaNCE), Department of Neuroscience, University of the Basque Country, Leioa, 3Unit of Anatomy, Department of Medicine, University of Fribourg, Fribourg, 1,2Spain 3Switzerland* 

#### **1. Introduction**

404 Glioma – Exploring Its Biology and Practical Relevance

Wang, J., Wakeman, T. P., Lathia, J. D., Hjelmeland, A. B., Wang, X. F., White, R. R., Rich, J.

Wang, Y., Liu, Y., Malek, S. N., Zheng, P., & Liu, Y. (2011). Targeting HIF1alpha Eliminates

Ward, R. J. & Dirks, P. B. (2007). Cancer stem cells: at the headwaters of tumor development.

Ward, R. J., Lee, L., Graham, K., Satkunendran, T., Yoshikawa, K., Ling, E., Harper, L.,

Xing, F., Okuda, H., Watabe, M., Kobayashi, A., Pai, S. K., Liu, W., Pandey, P. R., Fukuda,

Yamaguchi, S., Kobayashi, H., Narita, T., Kanehira, K., Sonezaki, S., Kubota, Y., Terasaka, S.,

Yeung, T. M., Gandhi, S. C., & Bodmer, W. F. (2011). Hypoxia and lineage specification of

Yin, A. H., Miraglia, S., Zanjani, E. D., Almeida-Porada, G., Ogawa, M., Leary, A. G.,

Zencak, D., Lingbeek, M., Kostic, C., Tekaya, M., Tanger, E., Hornfeld, D., Jaquet, M.,

population and proliferation. *J.Neurosci.* Vol. 25, No. 24, pp. 5774-5783 Zeppernick, F., Ahmadi, R., Campos, B., Dictus, C., Helmke, B. M., Becker, N., Lichter, P.,

sphere cells. *Mol.Cancer Ther.* Vol. 9, No. 7, pp. 2131-2141

spheroids in vitro. *Photochem.Photobiol.* Vol. 86, No. 4, pp. 964-971

medulloblastoma. *Cancer Res.* Vol. 69, No. 11, pp. 4682-4690

rise to CD133 positive cells. *Int.J.Cancer*. Vol. 122, No. 4, pp. 761-768

*Stem Cells*. Vol. 28, No. 1, pp. 17-28

*Annu.Rev.Pathol.* Vol. 2, No. 175-189

*Oncogene*. April 18, Epub ahead of print.

399-411

11, pp. 4382-4387

10, pp. 3639-3643

Enger, P. O. (2008). CD133 negative glioma cells form tumors in nude rats and give

N., & Sullenger, B. A. (2010). Notch promotes radioresistance of glioma stem cells.

Cancer Stem Cells in Hematological Malignancies. *Cell Stem Cell*. Vol. 8, No. 4, pp.

Austin, R., Nieuwenhuis, E., Clarke, I. D., Hui, C. C., & Dirks, P. B. (2009). Multipotent CD15+ cancer stem cells in patched-1-deficient mouse

K., Hirota, S., Sugai, T., Wakabayshi, G., Koeda, K., Kashiwaba, M., Suzuki, K., Chiba, T., Endo, M., Mo, Y. Y., & Watabe, K. (2011). Hypoxia-induced Jagged2 promotes breast cancer metastasis and self-renewal of cancer stem-like cells.

& Iwasaki, Y. (2010). Novel photodynamic therapy using water-dispersed TiO2 polyethylene glycol compound: evaluation of antitumor effect on glioma cells and

cell line-derived colorectal cancer stem cells. *Proc.Natl.Acad.Sci.U.S.A*. Vol. 108, No.

Olweus, J., Kearney, J., & Buck, D. W. (1997). AC133, a novel marker for human hematopoietic stem and progenitor cells. *Blood*. Vol. 90, No. 12, pp. 5002-5012 Yuhas, J. M., Li, A. P., Martinez, A. O., & Ladman, A. J. (1977). A simplified method for

production and growth of multicellular tumor spheroids. *Cancer Res.* Vol. 37, No.

Munier, F. L., Schorderet, D. F., van, L. M., & Arsenijevic, Y. (2005). Bmi1 loss produces an increase in astroglial cells and a decrease in neural stem cell

Unterberg, A., Radlwimmer, B., & Herold-Mende, C. C. (2008). Stem cell marker CD133 affects clinical outcome in glioma patients. *Clin.Cancer Res.* Vol. 14, No. 1, pp. 123-129 Zhu, X., Bidlingmaier, S., Hashizume, R., James, C. D., Berger, M. S., & Liu, B. (2010).

Identification of internalizing human single-chain antibodies targeting brain tumor

F. (2005). Early inactivation of p53 tumor suppressor gene cooperating with NF1

Zhu, Y., Guignard, F., Zhao, D., Liu, L., Burns, D. K., Mason, R. P., Messing, A., & Parada, L.

loss induces malignant astrocytoma. *Cancer Cell*. Vol. 8, No. 2, pp. 119-130

Glioblastomas (GBM) are the most malignant solid tumours (grade IV) of CNS. They are glial lineage neoplasias with a high proliferative and invasive capacity, reaching to occupy an entire lobe of the brain (Kleihues et al., 2007). According with their genesis, they can be differentiated between primary and secondary glioblastoma. The primary is the most common glioblastoma. This is a new generated tumour after a brief medical history (three months), with no evidence of a less malignant lesion. On the other hand, secondary glioblastoma develops from diffuse astrocytoma, anaplastic astrocytoma or oligodendroglioma and malignant progression. Its development time is about five years. It is thought that both types of glioblastomas may be generated from neoplastic cells with characteristic of stem cells (Ohgaki & Kleihues, 2009). In addition, these cancer stem cells called "glioma stem cells" (GSCs) may be the responsible for glioma recurrences due to chemo-and radio resistance (Bao et al., 2006; Rich, 2007). Glioma stem cells (GSCs) are a subpopulation of neoplastic cells identified in glioma sharing properties with neural stem cells (self-renewal, high proliferation rate, undifferentiating, and neurospheres conformation) and the capacity for leading the tumourigenesis and tumour malignancy. The proliferation and the invasion into adjacent normal parenchyma have been attributed to glioma stem cells as well. Indeed, they were related to the angiogenesis process needed for the growth and survival of the neoplasia.

 The microvascular network in gliomas has to get adapt to metabolic tissue requirements (Folkman, 2000). When the vascular network cannot satisfy cell requirements (Oxygen pressure of 5-10 mm Hg) tissue hypoxia occurs. This situation triggers the synthesis of proangiogenic factors as matrix metalloprotease (MMP-2), angiopoietin-1, phosphoglycerate kinase (PGK), erythropoietin (EPO), and vascular endothelial growth factor (VEGF)-A (Fong, 2008).

Vascular endothelial growth factor (VEGF) is a major regulator of tumour angiogenesis (Bulnes & Lafuente, 2007; Lafuente et al., 1999; Machein & Plate, 2004; Marti et al., 2000).

Endogenous Experimental Glioma Model,

carry out in human brain.

**3. Stem cells and cancer stem cells** 

al., 2009) or experimental therapeutic agents (Kish et al., 2001).

Links Between Glioma Stem Cells and Angiogenesis 407

substances as nitroso compounds is one of the most commonly-used methods to induce experimental CNS neoplasm. There is strong experimental data showing that nitrosamides (R1NNO-COR2), a type of N-nitroso compounds (NOC), are potent neuro-carcinogens when administered transplacentally. N-nitrosoureas MNU and ENU (a class of nitrosamides) have been demonstrated to be carcinogenic in animals, and particularly related to the development of CNS tumours. N-ethyl-N-nitrosourea (ENU) acts alkylating the O6 in the guanine (G:C---T:A transition) and the O2 in the thymine (T:A---A:T transversion). The accumulation of these successive DNA mutations seems to be responsible of the neurooncogenic effect of ENU (Bulnes-Sesma et al., 2006; O'Neill, 2000). Recently it has been reported that ENU exposure affects primitive neuroepithelial cells of the subventricular plate (SVZ) and germinative zone (VZ). ENU prenatal exposure affects the differentiation of these cells generating glial lineage tumours (Burger, 1988; Vaquero et al., 1994; Yoshimura et al., 1998) and its exposure in adult affects the neurogenesis of the SVZ (Capilla-Gonzalez et al., 2010). In previous studies we found that gliomas induced in offspring were similar to the human gliomas (Kokkinakis et al., 2004). Therefore, ENU brain induced tumours have allowed the study of several aspects of glioma behaviour, for example, microvascular organization (Schlageter et al., 1999; Yoshimura et al., 1998); neoplastic cell dedifferentiation (Jang et al., 2004); gene mutations (Bielas & Heddle, 2000; O'Neil, 2000); microcirculation and angiogenesis process (Bulnes & Lafuente, 2007; Bulnes et

In our model, the glioma induction was performed by prenatal exposure of Sprague Dawley rats to ENU. Briefly, pregnancy rats, on the 15th day of gestation, were given a single i.p. injection of 80 mg of ENU/kg body weight (Bulnes et al., 2009; Bulnes et al., 2010). Offspring rats exposed to ENU were reared in standard laboratory conditions and the study was performed from 5 months to one year of age. The identification of ENU-Gliomas was performed by T2-w and postcontrast T1-weighted NMR images and by histopathology diagnosis from H&E staining and immunophenotypic study as previously described (Bulnes & Lafuente 2007) (Figure 1, 2). Following our results, ENU-glioma starts from the fifth month of offspring rat age and becomes GBM at 10 months of age (Bulnes-Sesma et al., 2006). ENU-glioma starts as cellular proliferation growing near ventricles in association with subcortical white matter. Over 6 months of extrauterine life, this tumour proliferation become nodular and rats display neurological signs (Figure 1). Around one year they grow as a GBM toward the contralateral hemisphere (Figure 2). Following our findings, we have identified three stages of ENU-glioma development: initial, intermediate and advanced. The advanced stage corresponds to anaplastic oligodendroglioma or glioblastoma (GBM) similar to the human. ENU-GBM may reach to infiltrate whole cerebral hemisphere, showing malignant histopathological features such as: high tissue heterogeneity, aberrant angioarchitecture, macro-haemorrhages, macrocysts or palisade necrosis (Klehiues et al., 2007). Thanks to this model we could isolate early glioma stages, which is impossible to

Stem cells are functionally defined as self-renewing and multipotent cells that exhibit multilineage differentiation (Till & McCulloch, 2011). Nowadays they have been proposed to be an important tool in regenerative therapy being used to regenerate tissue in many diseases like heart stroke, neurodegenerative diseases, etc (Nadig, 2009). However, in oncology and especially in cerebral gliomas, the presence of the stem cells has been related

VEGF acts as mitogen, survival, antiapoptotic and vascular permeability factor (VPF) for the endothelial cells (Dvorak, 2006). The increase of this pro-angiogenic factor, secreted either by neoplastic cells or by cells of the tumour microenvironment, induces the start of angiogenesis, the called "angiogenic switch" (Bergers & Benjamin, 2003). This event results in the transition from avascularised hyperplasia to outgrowing vascularised tumour and eventually to malignant progression. It has been shown in human glioma biopsies that VEGF overexpression correlates directly to proliferation, vascularization and degree of malignancy, and therefore inversely to prognosis (Ke et al., 2000; Lafuente et al., 1999; Plate, 1999). The synthesis of VEGF is mediated by the Hypoxia-Inducible Factor (HIF-1), a critical step for the formation of new blood vessels and for the adaptation of microenvironment to the growth of gliomas (Jin et al., 2000; Marti et al., 2000; Semenza, 2003). Recent researches have reported that glioma stem cells play a pivotal role inducing the angiogenesis via HIF-1/VEGF (Bao et al., 2006). By the other hand, hypoxia has been related to clones selection of tumour cells. These clones adapted to the tumour microenvironment have acquired the phenotype of tumour stem cell with increased proliferative and infiltrative capacity (Heddleston et al., 2009; Li et al., 2009). Invasion of adjacent normal parenchyma has been attributed to glioma stem cells as well.

Due to these evidences, GSCs are currently being considered as a potential therapeutic target of the tumours. Recent studies have been focused on the identification of GSCs. In human glioblastomas they have been identified using CD133 marker (Ignatova et al., 2002). However, little is known about their genesis during glioma progression, especially during the early stages.

Some authors have previously reported the induction of glial tumour in rats by transplacentary administration of the carcinogen ethylnitrosourea (ENU) as a suitable method for studying the natural development of glioma (Bulnes-Sesma et al., 2006; Zook et al., 2000). In addition to this, it has been reported that ENU glioma model is a representative model for human glioma due to its location and also to its similar cellular, molecular and genetic alterations (Kokkinakis et al., 2004). Our experience with this model has proven to be useful to study many aspects of tumourigenesis and neoangiogenesis. In previous researches we reported the progression of tumour malignancy associated with vascular structural alterations and blood brain barrier (BBB) disturbances (Bulnes & Lafuente, 2007; Bulnes et al., 2009). ENU induced glioma permitted us to identify tumour development stages following microvascular changes. In addition, it was possible to study the angiogenesis process. Recently, we have used this model to study the relationship between glioma stem cells and angiogenesis process during the neoplasia development.

Many evidences corroborate the hypothesis that "glioma stem cells" have a close relationship with angiogenesis process, intratumour hypoxia and neoplastic microvascular network. In this chapter we centred to show this relationship from early to advanced stages of glioma using ENU-model.

#### **2. Endogenous glioma model**

Over the years, different methods have been employed to induce experimental tumours in the Central Nervous System of animals. Exposure to radiation, inoculation of carcinogenic virus, xenografts of tumour cell lines or tumour fragments in nude rats or mice, administration of chemical substances (Bulnes-Sesma et al., 2006) and genetically engineered mouse models have been used to replicate CNS tumours. The administration of chemical

VEGF acts as mitogen, survival, antiapoptotic and vascular permeability factor (VPF) for the endothelial cells (Dvorak, 2006). The increase of this pro-angiogenic factor, secreted either by neoplastic cells or by cells of the tumour microenvironment, induces the start of angiogenesis, the called "angiogenic switch" (Bergers & Benjamin, 2003). This event results in the transition from avascularised hyperplasia to outgrowing vascularised tumour and eventually to malignant progression. It has been shown in human glioma biopsies that VEGF overexpression correlates directly to proliferation, vascularization and degree of malignancy, and therefore inversely to prognosis (Ke et al., 2000; Lafuente et al., 1999; Plate, 1999). The synthesis of VEGF is mediated by the Hypoxia-Inducible Factor (HIF-1), a critical step for the formation of new blood vessels and for the adaptation of microenvironment to the growth of gliomas (Jin et al., 2000; Marti et al., 2000; Semenza, 2003). Recent researches have reported that glioma stem cells play a pivotal role inducing the angiogenesis via HIF-1/VEGF (Bao et al., 2006). By the other hand, hypoxia has been related to clones selection of tumour cells. These clones adapted to the tumour microenvironment have acquired the phenotype of tumour stem cell with increased proliferative and infiltrative capacity (Heddleston et al., 2009; Li et al., 2009). Invasion of adjacent normal parenchyma has been

Due to these evidences, GSCs are currently being considered as a potential therapeutic target of the tumours. Recent studies have been focused on the identification of GSCs. In human glioblastomas they have been identified using CD133 marker (Ignatova et al., 2002). However, little is known about their genesis during glioma progression, especially during

Some authors have previously reported the induction of glial tumour in rats by transplacentary administration of the carcinogen ethylnitrosourea (ENU) as a suitable method for studying the natural development of glioma (Bulnes-Sesma et al., 2006; Zook et al., 2000). In addition to this, it has been reported that ENU glioma model is a representative model for human glioma due to its location and also to its similar cellular, molecular and genetic alterations (Kokkinakis et al., 2004). Our experience with this model has proven to be useful to study many aspects of tumourigenesis and neoangiogenesis. In previous researches we reported the progression of tumour malignancy associated with vascular structural alterations and blood brain barrier (BBB) disturbances (Bulnes & Lafuente, 2007; Bulnes et al., 2009). ENU induced glioma permitted us to identify tumour development stages following microvascular changes. In addition, it was possible to study the angiogenesis process. Recently, we have used this model to study the relationship between

Many evidences corroborate the hypothesis that "glioma stem cells" have a close relationship with angiogenesis process, intratumour hypoxia and neoplastic microvascular network. In this chapter we centred to show this relationship from early to advanced stages

Over the years, different methods have been employed to induce experimental tumours in the Central Nervous System of animals. Exposure to radiation, inoculation of carcinogenic virus, xenografts of tumour cell lines or tumour fragments in nude rats or mice, administration of chemical substances (Bulnes-Sesma et al., 2006) and genetically engineered mouse models have been used to replicate CNS tumours. The administration of chemical

glioma stem cells and angiogenesis process during the neoplasia development.

attributed to glioma stem cells as well.

the early stages.

of glioma using ENU-model.

**2. Endogenous glioma model** 

substances as nitroso compounds is one of the most commonly-used methods to induce experimental CNS neoplasm. There is strong experimental data showing that nitrosamides (R1NNO-COR2), a type of N-nitroso compounds (NOC), are potent neuro-carcinogens when administered transplacentally. N-nitrosoureas MNU and ENU (a class of nitrosamides) have been demonstrated to be carcinogenic in animals, and particularly related to the development of CNS tumours. N-ethyl-N-nitrosourea (ENU) acts alkylating the O6 in the guanine (G:C---T:A transition) and the O2 in the thymine (T:A---A:T transversion). The accumulation of these successive DNA mutations seems to be responsible of the neurooncogenic effect of ENU (Bulnes-Sesma et al., 2006; O'Neill, 2000). Recently it has been reported that ENU exposure affects primitive neuroepithelial cells of the subventricular plate (SVZ) and germinative zone (VZ). ENU prenatal exposure affects the differentiation of these cells generating glial lineage tumours (Burger, 1988; Vaquero et al., 1994; Yoshimura et al., 1998) and its exposure in adult affects the neurogenesis of the SVZ (Capilla-Gonzalez et al., 2010). In previous studies we found that gliomas induced in offspring were similar to the human gliomas (Kokkinakis et al., 2004). Therefore, ENU brain induced tumours have allowed the study of several aspects of glioma behaviour, for example, microvascular organization (Schlageter et al., 1999; Yoshimura et al., 1998); neoplastic cell dedifferentiation (Jang et al., 2004); gene mutations (Bielas & Heddle, 2000; O'Neil, 2000); microcirculation and angiogenesis process (Bulnes & Lafuente, 2007; Bulnes et al., 2009) or experimental therapeutic agents (Kish et al., 2001).

In our model, the glioma induction was performed by prenatal exposure of Sprague Dawley rats to ENU. Briefly, pregnancy rats, on the 15th day of gestation, were given a single i.p. injection of 80 mg of ENU/kg body weight (Bulnes et al., 2009; Bulnes et al., 2010). Offspring rats exposed to ENU were reared in standard laboratory conditions and the study was performed from 5 months to one year of age. The identification of ENU-Gliomas was performed by T2-w and postcontrast T1-weighted NMR images and by histopathology diagnosis from H&E staining and immunophenotypic study as previously described (Bulnes & Lafuente 2007) (Figure 1, 2). Following our results, ENU-glioma starts from the fifth month of offspring rat age and becomes GBM at 10 months of age (Bulnes-Sesma et al., 2006). ENU-glioma starts as cellular proliferation growing near ventricles in association with subcortical white matter. Over 6 months of extrauterine life, this tumour proliferation become nodular and rats display neurological signs (Figure 1). Around one year they grow as a GBM toward the contralateral hemisphere (Figure 2). Following our findings, we have identified three stages of ENU-glioma development: initial, intermediate and advanced. The advanced stage corresponds to anaplastic oligodendroglioma or glioblastoma (GBM) similar to the human. ENU-GBM may reach to infiltrate whole cerebral hemisphere, showing malignant histopathological features such as: high tissue heterogeneity, aberrant angioarchitecture, macro-haemorrhages, macrocysts or palisade necrosis (Klehiues et al., 2007). Thanks to this model we could isolate early glioma stages, which is impossible to carry out in human brain.

#### **3. Stem cells and cancer stem cells**

Stem cells are functionally defined as self-renewing and multipotent cells that exhibit multilineage differentiation (Till & McCulloch, 2011). Nowadays they have been proposed to be an important tool in regenerative therapy being used to regenerate tissue in many diseases like heart stroke, neurodegenerative diseases, etc (Nadig, 2009). However, in oncology and especially in cerebral gliomas, the presence of the stem cells has been related

Endogenous Experimental Glioma Model,

white matter (Nunes et al., 2003).

**3.1 Glioma stem cells (GSCs)** 

al., 2009).

al., 2002) etc.

Links Between Glioma Stem Cells and Angiogenesis 409

In the middle of the 60s, Altman and Das reported the first evidences about stem cells in adult brain. They observed stem cells in the hippocampus and olfactory bulb of rats, and it supposed the first sign of division of stem cells. Later on they were called Neural Stem Cells (NSCs). NSCs were considered the unique population of Central Nervous System cells characterized by self-renewal and multilineage differentiation properties (Muller et al., 2006). They can form neurospheres (Reynolds & Weiss, 1992) and differentiate in vitro into the three neuroectodermal lineages astrocytes, oligodendrocytes and neurons (Alvarez-Buylla & Garcia-Verdugo, 2002). Furthermore, when they are transplanted in vivo in the cerebellum, they can generate neurons and glial cells (Lee et al., 2005). Also, after transplantation into nude mice they can differentiate into neuroblasts (Tamaki et al., 2002). NSCs reside in the germinal layers of the developing brain, initially in the early neuroepithelium, later in the ventricular (VZ) and subventricular zone (SVZ) during embryogenesis (Götz & Huttner, 2005). In adult brain, three areas are supposed to harbour neural stem cells: dentate gyrus of hippocampus, SVZ (Doetsch et al., 1999; Eriksson et al., 1998) and the fibbers connecting olfactory bulb to lateral ventricle (Lois & Alvarez-Buylla, 1994; Whitman & Greer, 2009). In recent times, they were also isolated in the subcortical

In the 1960s, evidence emerged supporting the presence of stem cells in tumours. Bergsagel and Valeriote (1968) showed that only certain cells within a tumour had the capacity to generate a new tumour; they termed these cells "tumour stem cells". After this, tumour stem cells were identified in breast tumour (Al-Hajj et al., 2003), pancreatic tumour (Esposito et

The first concept of cancer stem cell, later on also called tumour initiating cells, appeared in the beginning of the 90s. Bonnet and Dick (1977) describe how some cells, isolated from leukaemia patient´s blood, had proliferation and differentiation capacities in vivo. Fan et al. (2007) described cancer stem cells as the cellular subpopulation capable of tumour regeneration within a permissive environment. Rich and collaborators reported that cancer stem cells have tumourigenic, infiltration and angiogenesis properties as well as

The relation between stem cells and cancer stem cells was studied. The results explained that both cellular types share the previously mentioned characteristics, as well as many cell signalling pathways as oncogene bcl-2, Sonic hedgehog (Shh) and Wnt signalling cascade (Reya et al., 2001). Both types of stem cells also share common markers like CD133, Nestin (Dahlstrand et al., 1992) and transcription factor Sox2 (Gangemi et al., 2009). However, there are differences between stem cells and cancer stem cells, such as expression of different markers, chromosomal alterations and tumourigenic capacity. Holland et al. (2000) published that cancer stem cells could develop from modified neural stem cells. They have been described many pathways that can lead to cancer stem cell formation like Notch

(Takebe & Ivy 2010), Akt (Germano et al., 2010) activation or p53 pathway alteration.

Dahlstrand et al. (1992) identified a cancer stem cells subtype inside glial lineage brain tumours which were called Glioma Stem Cells (GSCs). These GSCs may be responsible for maintenance of the entire tumour and also they have the potential, when injected in immunodeficient mice, to generate gliomas similar to the original tumours (Heddleston et

radio/chemo-resistance (Rich, 2007; Hadjipanayis & Van Meir, 2009).

to a poor prognosis. Recent investigations in glioblastomas have reported that these cancer stem cells called glioma stem cells (GSCs) have tumourigenic capacities like tumour malignant process, peripheral tissue infiltration and angiogenesis induction (Hadjipanayis & Van Meir, 2009; Rich, 2007).

Fig. 1. Coronal sections of rat brains displaying ENU-glioma showed by MRI on T2-w and T1-w after injection of gadolinium. a, b) Small neoplastic mass growing on the cerebral cortex with an homogeneous hyperintense signal on T2-w images. These neoplastic masses correspond to initial stage of ENU-glioma. e, f) Both masses display an isointense signal on T1-w. c, d) ENU-glioma tumour with nodular shape showed on T2-w hyperintense signal that represents intermediate stage. g, h) At this stage there is a gadolinium contrast enhancement observed as homogeneous soft hyperintense signal on T1-w image.

to a poor prognosis. Recent investigations in glioblastomas have reported that these cancer stem cells called glioma stem cells (GSCs) have tumourigenic capacities like tumour malignant process, peripheral tissue infiltration and angiogenesis induction (Hadjipanayis

Fig. 1. Coronal sections of rat brains displaying ENU-glioma showed by MRI on T2-w and T1-w after injection of gadolinium. a, b) Small neoplastic mass growing on the cerebral cortex with an homogeneous hyperintense signal on T2-w images. These neoplastic masses correspond to initial stage of ENU-glioma. e, f) Both masses display an isointense signal on T1-w. c, d) ENU-glioma tumour with nodular shape showed on T2-w hyperintense signal that represents intermediate stage. g, h) At this stage there is a gadolinium contrast enhancement observed as homogeneous soft hyperintense signal on T1-w image.

Fig. 2. Coronal sections of rat brains with ENU-glioma of advanced stage showed by MRI on

enhancement of this T1-w image adopts a rim shape bordering the neoplastic mass. This rim

T2-w and T1-w after injection of gadolinium. All of these anaplastic gliomas display heterogeneous hyperintense signal on T2 (a-d) and on T1-w (e-h). This heterogeneity is due to the presence of histopathology features of malignity. c-d) ENU-GBMs high-proliferative covering a whole cerebral hemisphere. The T2-w images reveal an intratumour hyperintense

signal corresponding with intratumour oedema or macrocysts. g-h). Gadolinium

represents the microvascular proliferation with dysfunction of Blood Brain Barrier.

& Van Meir, 2009; Rich, 2007).

In the middle of the 60s, Altman and Das reported the first evidences about stem cells in adult brain. They observed stem cells in the hippocampus and olfactory bulb of rats, and it supposed the first sign of division of stem cells. Later on they were called Neural Stem Cells (NSCs). NSCs were considered the unique population of Central Nervous System cells characterized by self-renewal and multilineage differentiation properties (Muller et al., 2006). They can form neurospheres (Reynolds & Weiss, 1992) and differentiate in vitro into the three neuroectodermal lineages astrocytes, oligodendrocytes and neurons (Alvarez-Buylla & Garcia-Verdugo, 2002). Furthermore, when they are transplanted in vivo in the cerebellum, they can generate neurons and glial cells (Lee et al., 2005). Also, after transplantation into nude mice they can differentiate into neuroblasts (Tamaki et al., 2002). NSCs reside in the germinal layers of the developing brain, initially in the early neuroepithelium, later in the ventricular (VZ) and subventricular zone (SVZ) during embryogenesis (Götz & Huttner, 2005). In adult brain, three areas are supposed to harbour neural stem cells: dentate gyrus of hippocampus, SVZ (Doetsch et al., 1999; Eriksson et al., 1998) and the fibbers connecting olfactory bulb to lateral ventricle (Lois & Alvarez-Buylla, 1994; Whitman & Greer, 2009). In recent times, they were also isolated in the subcortical white matter (Nunes et al., 2003).

In the 1960s, evidence emerged supporting the presence of stem cells in tumours. Bergsagel and Valeriote (1968) showed that only certain cells within a tumour had the capacity to generate a new tumour; they termed these cells "tumour stem cells". After this, tumour stem cells were identified in breast tumour (Al-Hajj et al., 2003), pancreatic tumour (Esposito et al., 2002) etc.

The first concept of cancer stem cell, later on also called tumour initiating cells, appeared in the beginning of the 90s. Bonnet and Dick (1977) describe how some cells, isolated from leukaemia patient´s blood, had proliferation and differentiation capacities in vivo. Fan et al. (2007) described cancer stem cells as the cellular subpopulation capable of tumour regeneration within a permissive environment. Rich and collaborators reported that cancer stem cells have tumourigenic, infiltration and angiogenesis properties as well as radio/chemo-resistance (Rich, 2007; Hadjipanayis & Van Meir, 2009).

The relation between stem cells and cancer stem cells was studied. The results explained that both cellular types share the previously mentioned characteristics, as well as many cell signalling pathways as oncogene bcl-2, Sonic hedgehog (Shh) and Wnt signalling cascade (Reya et al., 2001). Both types of stem cells also share common markers like CD133, Nestin (Dahlstrand et al., 1992) and transcription factor Sox2 (Gangemi et al., 2009). However, there are differences between stem cells and cancer stem cells, such as expression of different markers, chromosomal alterations and tumourigenic capacity. Holland et al. (2000) published that cancer stem cells could develop from modified neural stem cells. They have been described many pathways that can lead to cancer stem cell formation like Notch (Takebe & Ivy 2010), Akt (Germano et al., 2010) activation or p53 pathway alteration.

#### **3.1 Glioma stem cells (GSCs)**

Dahlstrand et al. (1992) identified a cancer stem cells subtype inside glial lineage brain tumours which were called Glioma Stem Cells (GSCs). These GSCs may be responsible for maintenance of the entire tumour and also they have the potential, when injected in immunodeficient mice, to generate gliomas similar to the original tumours (Heddleston et al., 2009).

Endogenous Experimental Glioma Model,

tumour (Germano et al., 2010).

**4. Tumour angiogenesis** 

et al., 1993; Neufeld et al., 1999).

to the new microenvironment, are transformed to GSCs.

**4.1 Vascular endothelial growth factor (VEGF)** 

Links Between Glioma Stem Cells and Angiogenesis 411

colon cancer (Cheng et al., 2009; Collins et al., 2005; O'Brien et al., 2007). In human glioblastoma, CD133 expression has been associated to GSCs and bad prognosis of the

Gliomas proliferate in the brain, a privileged organ from the point of view of blood supply. The exchange of metabolites between blood and cerebral tissue occurs essentially in the brain capillaries. The diameter of brain capillaries in the adult human is between 5 and 7 microns. These microvessels feed to the cells that are 10-20 microns away. Although the distance between cells and microvessels is lesser than 20 µm, the growth and survival of the gliomas depend on vascular remodelling and angiogenesis (Folkman, 2006). Along the early stages of small gliomas the metabolic demand is supplied by the vast microvascular network but when the metabolic supply has been exceeded, new formation of vessels becomes necessary (Carmeliet & Jain, 2000; Yancopoulos et al., 2000). The genesis of the new vessels from preexisting ones is called angiogenesis in opposite to vasculogenesis refereed to the formation of

vessels from hemopoietic niches (Carmeliet, 2003; Risau & Falmme 1995; Risau, 1997).

Angiogenesis is a complex process that requires proteolytic and mitogenic activity of endothelial cells and interaction of these with the extracellular matrix molecules and cells of peri-endothelial support cells (pericytes and smooth muscle cells). Many molecules and pathways are involved in this process, such as VEGF, its receptors VEGFR-1 and VEGFR-2, the endothelial receptor tyrosine kinase tie-1 and tie-2 and the angiopoietin ligands 1 and 2. Many other molecules as PDGF and TGF-β, integrin receptors, are very important (Millauer

Angiogenesis requires some angiogenic stimulus, such as hypoxia, new metabolic requirements or tumour growth to start. Intratumour hypoxia occurs at the time when there is an imbalance between supply and demand oxygen due to the irregular and chaotic blood flow (Jensen, 2006). The relative tissue hypoxia triggers the production of hypoxia inducible factor-1α, upregulating the expression of VEGF. In addition to this, it was reported that hypoxia plays a fundamental role in the induction of cell phenotype neoplastic to the undifferentiated state of GSCs. According to recent research, hypoxia selects tumour cell clones that have adapted to the tumour microenvironment and have acquired the phenotype tumour stem cell, with its capabilities of proliferation and infiltration (Heddleston et al., 2009; Li et al., 2009). Heddleston et al. (2009) observed how in cultures of human glioma neoplastic cells exposed to hypoxia reverted to a state of tumour stem cells. Griguer et al. (2008) related the appearance of CD133 + cells with oxygen stress in gliomas. On the other hand, it was observed a decrease in the expression of CD133 when reverted to conditions of normoxia. Furthermore, studies of human GBM have described the relationship between the gradient of intratumour oxygen and the appearance of the phenotype tumour stem cell (Pistollato et al., 2010). As above, only a cluster of neoplastic cells resists to the conditions of hypoxia and intratumoural ischemia. This group of cells may be stem cell precursors, and after adapting

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis in development (Bengoetxea et al., 2008; Ferrara et al., 2003; Ment et al., 1997) and pathological

GSCs indeed of share properties of somatic or embryonic stem cells (high proliferation rate, undifferentiating, formation of neurospheres) are chemo-and radio resistant (Bao et al., 2006, Rich, 2007). Their radiotherapy resistance may be thanks to a more efficient DNA reparation mechanism and protein kinases phosphorilation Chk1 and Chk2 (Bao et al., 2006). The resistance to chemotherapeutic drugs is through membrane transporters that bomb the drugs outside the cell (Donnenberg & Donnenberg, 2005).

The first GSCs identification was found in the tumour advanced stage corresponding with human-GBM (Ignatova et al., 2000). However, the first moment of GSCs expression remains unknown, as well as their role in early stages of tumour development. It is very important to identify and explain GSCs apparition in early glioma stages to research about future tumour therapy.

The discovery of GSCs in gliomas involved the creation of a new glioma-genesis hypothesis called "hierarchical hypothesis". Before GSCs discovery, glioma development was explained by the "stochastic theory". Stochastic theory is based on all neoplastic cells are clones from a single undifferentiated cell and they have the same genetic alterations (Hadjipanayis & Van Meir, 2009). Nowadays the "hierarchical theory" explains that only a few neoplastic cells can adapt to the tumour environment and are able to start the tumourigenic process. Even though the low proliferation of GSCs, they guide the tumour growth giving raise to more mature cells with limited proliferation capacity (Shen et al., 2008).

After the glioma stem cells finding, the research about glioma development has been centred in the identification of them. So far markers as CD133/Promonin-1, presents in glioma stem cells (Dell'Albani, 2008), Nestin, a protein found in neural stem cells in SVZ and other markers of neuroepithelial stem cells (Jang et al., 2004) including Musashi-1, Sox-2, GFAP, Map-2, Neural-tubulin, Neurofilament O4 and Noggin were used in order to identify tumour stem cells. But the lack of a specific marker makes it very difficult to identify (Hadjipanayis & Van Meir, 2009; Li et al., 2009).

*Nestin* is an intermediate filament protein typical for neural precursor cells. It has been extensively used as a marker for neural stem cells. It is expressed in primitive neuroepithelial cells of all regions of CNS during the development. In adult its expression is restricted to the ventricular wall (SVZ) and the central canal. In pathological conditions like brain trauma, CNS ischemia, neurotoxicity, neoplastic transformation and in response to cellular stress, the nestin over-expression was showed (Holmin et al., 1997, Jang et al., 2004). In primary malignant tumours of CNS high amounts of cells positive for Nestin have been reported. Nestin has been described as a marker of GSCs in astroglial tumours (Singh et al., 2003), indicating undifferentiating and malignance degree (Schiffer et al., 2010), but it is not specific for glioma stem cells (Hadjipanayis & Van Meir, 2009). Indeed, Nestin expression has been described to appear since the first stages in glioma models (Jang et al., 2004).

*CD133 (prominin-1*) was the first identified member of the prominin family of pentaspan membrane proteins which acts as a marker of hematopoietic progenitor cells. It is a cell surface marker used for the identification and isolation of stem/progenitor cells in several tissues, for instance, endothelium, brain, bone narrow, liver, prostate, pancreas and foreskin (Mizrak et al., 2008). CD133 was originally described as an hematopoietic stem cell marker and was subsequently related to number of progenitor cells including neuroepithelium (Corbeil et al., 2000) as well as cancer stem cells in various tumours such as prostate and colon cancer (Cheng et al., 2009; Collins et al., 2005; O'Brien et al., 2007). In human glioblastoma, CD133 expression has been associated to GSCs and bad prognosis of the tumour (Germano et al., 2010).

### **4. Tumour angiogenesis**

410 Glioma – Exploring Its Biology and Practical Relevance

GSCs indeed of share properties of somatic or embryonic stem cells (high proliferation rate, undifferentiating, formation of neurospheres) are chemo-and radio resistant (Bao et al., 2006, Rich, 2007). Their radiotherapy resistance may be thanks to a more efficient DNA reparation mechanism and protein kinases phosphorilation Chk1 and Chk2 (Bao et al., 2006). The resistance to chemotherapeutic drugs is through membrane transporters that bomb the

The first GSCs identification was found in the tumour advanced stage corresponding with human-GBM (Ignatova et al., 2000). However, the first moment of GSCs expression remains unknown, as well as their role in early stages of tumour development. It is very important to identify and explain GSCs apparition in early glioma stages to research about future tumour

The discovery of GSCs in gliomas involved the creation of a new glioma-genesis hypothesis called "hierarchical hypothesis". Before GSCs discovery, glioma development was explained by the "stochastic theory". Stochastic theory is based on all neoplastic cells are clones from a single undifferentiated cell and they have the same genetic alterations (Hadjipanayis & Van Meir, 2009). Nowadays the "hierarchical theory" explains that only a few neoplastic cells can adapt to the tumour environment and are able to start the tumourigenic process. Even though the low proliferation of GSCs, they guide the tumour growth giving raise to more mature cells with limited proliferation capacity (Shen et al.,

After the glioma stem cells finding, the research about glioma development has been centred in the identification of them. So far markers as CD133/Promonin-1, presents in glioma stem cells (Dell'Albani, 2008), Nestin, a protein found in neural stem cells in SVZ and other markers of neuroepithelial stem cells (Jang et al., 2004) including Musashi-1, Sox-2, GFAP, Map-2, Neural-tubulin, Neurofilament O4 and Noggin were used in order to identify tumour stem cells. But the lack of a specific marker makes it very difficult to identify

*Nestin* is an intermediate filament protein typical for neural precursor cells. It has been extensively used as a marker for neural stem cells. It is expressed in primitive neuroepithelial cells of all regions of CNS during the development. In adult its expression is restricted to the ventricular wall (SVZ) and the central canal. In pathological conditions like brain trauma, CNS ischemia, neurotoxicity, neoplastic transformation and in response to cellular stress, the nestin over-expression was showed (Holmin et al., 1997, Jang et al., 2004). In primary malignant tumours of CNS high amounts of cells positive for Nestin have been reported. Nestin has been described as a marker of GSCs in astroglial tumours (Singh et al., 2003), indicating undifferentiating and malignance degree (Schiffer et al., 2010), but it is not specific for glioma stem cells (Hadjipanayis & Van Meir, 2009). Indeed, Nestin expression has been described to appear since the first stages in glioma models (Jang et al., 2004). *CD133 (prominin-1*) was the first identified member of the prominin family of pentaspan membrane proteins which acts as a marker of hematopoietic progenitor cells. It is a cell surface marker used for the identification and isolation of stem/progenitor cells in several tissues, for instance, endothelium, brain, bone narrow, liver, prostate, pancreas and foreskin (Mizrak et al., 2008). CD133 was originally described as an hematopoietic stem cell marker and was subsequently related to number of progenitor cells including neuroepithelium (Corbeil et al., 2000) as well as cancer stem cells in various tumours such as prostate and

drugs outside the cell (Donnenberg & Donnenberg, 2005).

(Hadjipanayis & Van Meir, 2009; Li et al., 2009).

therapy.

2008).

Gliomas proliferate in the brain, a privileged organ from the point of view of blood supply. The exchange of metabolites between blood and cerebral tissue occurs essentially in the brain capillaries. The diameter of brain capillaries in the adult human is between 5 and 7 microns. These microvessels feed to the cells that are 10-20 microns away. Although the distance between cells and microvessels is lesser than 20 µm, the growth and survival of the gliomas depend on vascular remodelling and angiogenesis (Folkman, 2006). Along the early stages of small gliomas the metabolic demand is supplied by the vast microvascular network but when the metabolic supply has been exceeded, new formation of vessels becomes necessary (Carmeliet & Jain, 2000; Yancopoulos et al., 2000). The genesis of the new vessels from preexisting ones is called angiogenesis in opposite to vasculogenesis refereed to the formation of vessels from hemopoietic niches (Carmeliet, 2003; Risau & Falmme 1995; Risau, 1997).

Angiogenesis is a complex process that requires proteolytic and mitogenic activity of endothelial cells and interaction of these with the extracellular matrix molecules and cells of peri-endothelial support cells (pericytes and smooth muscle cells). Many molecules and pathways are involved in this process, such as VEGF, its receptors VEGFR-1 and VEGFR-2, the endothelial receptor tyrosine kinase tie-1 and tie-2 and the angiopoietin ligands 1 and 2. Many other molecules as PDGF and TGF-β, integrin receptors, are very important (Millauer et al., 1993; Neufeld et al., 1999).

Angiogenesis requires some angiogenic stimulus, such as hypoxia, new metabolic requirements or tumour growth to start. Intratumour hypoxia occurs at the time when there is an imbalance between supply and demand oxygen due to the irregular and chaotic blood flow (Jensen, 2006). The relative tissue hypoxia triggers the production of hypoxia inducible factor-1α, upregulating the expression of VEGF. In addition to this, it was reported that hypoxia plays a fundamental role in the induction of cell phenotype neoplastic to the undifferentiated state of GSCs. According to recent research, hypoxia selects tumour cell clones that have adapted to the tumour microenvironment and have acquired the phenotype tumour stem cell, with its capabilities of proliferation and infiltration (Heddleston et al., 2009; Li et al., 2009).

Heddleston et al. (2009) observed how in cultures of human glioma neoplastic cells exposed to hypoxia reverted to a state of tumour stem cells. Griguer et al. (2008) related the appearance of CD133 + cells with oxygen stress in gliomas. On the other hand, it was observed a decrease in the expression of CD133 when reverted to conditions of normoxia. Furthermore, studies of human GBM have described the relationship between the gradient of intratumour oxygen and the appearance of the phenotype tumour stem cell (Pistollato et al., 2010). As above, only a cluster of neoplastic cells resists to the conditions of hypoxia and intratumoural ischemia. This group of cells may be stem cell precursors, and after adapting to the new microenvironment, are transformed to GSCs.

#### **4.1 Vascular endothelial growth factor (VEGF)**

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis in development (Bengoetxea et al., 2008; Ferrara et al., 2003; Ment et al., 1997) and pathological

Endogenous Experimental Glioma Model,

dilated vessels (Klehues et al., 2007).

Links Between Glioma Stem Cells and Angiogenesis 413

vessels such as: multilayered "glomeruloid tuft"; "garland" of proliferated vessels and huge

One result to take in consideration was the gradient from the well-oxygenated tumour periphery to the central hypoxic core of ENU-glioblastoma. Dilated intratumour vessels, expressing VEGF (Lafuente et al., 1999) increase their lumen on account of endothelial elongation but not of cell proliferation (Helmlinger et al., 2000). The intratumour area displays irregularly branching vessels, variable intravascular spaces and large avascular areas. It is also worth mentioning that perivascular cells of aberrant vessels of ENU-GBM often displayed a high activity for BChE, depicted by a strong brown staining (Bulnes et al., 2009). BChE activity is strongly related to neurogenesis and cellular proliferation (Mack & Robitzki, 2000), having a great role in tumourigenesis. These findings have led us to postulate that these perivascular cells might be stem cells proliferating around intratumour vessels (Anderson et al., 2005; Brat et al., 2004) and migrating through the vascular extracellular matrix (Ruoslahti, 2002). This could corroborate the hypothesis that stem cells adapted to hypoxic stress use the vascular extracellular matrix for migration and invasion. In addition to this, in previous work we have

shown that these cells co-expressed Ki-67 and VEGF (Bulnes & Lafuente, 2007).

Fig. 3. Angioarchitecture study of gliomas shown by butyrylcholinesterase histochemistry.

a) Angioarchitecture of the cerebral cortex of the rat brain. b) Periventricular small neoplastic mass (initial stage) showing some strongly-positive vessels for BChE. c) Intermediate ENU-glioma stage displaying a network of numerous tortuous capillaries of anarchic distribution. d) Malignant infiltrating macrotumour, with dilated vessels of the

intratumour area with strongly BChE positive cells. (Scale bar of 50µm).

disease (Bulnes & Lafuente, 2007; Lafuente et al., 1999; Marti et al., 2000; Plate, 1992). However, the role of VEGF in nervous tissue is even more extensive. Previous studies showed that VEGF also has strong neuroprotective, neurotrophic and neurogenic properties (Jin et al., 2002; Ortuzar et al., 2011; Rosenstein & Krum, 2004; Storkebaum et al., 2004).

Although the synthesis of this proangiogenic cytokine is associated to tumour cells and endothelial cells, it has been described in others, such as: neurons, astrocytes, pericytes, smooth muscle cells, macrophages, lymphoid cells, platelets and fibroblasts (Zagzag et al., 2000). The VEGF family consists of five different homologous factors, VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PIGF) (Ferrara et al., 2003). VEGF-A (VEGF) is the predominant form and is a hypoxia-inducible 45 KDa homodimeric glycoprotein.

VEGF-A acts as mitogen, survival and antiapoptotic factor for the endothelial cells from arteries, veins and lymphatics. Faced with increased secretion of VEGF and its binding to receptors on the surface of endothelial cells, VEGF is a signal transduction leading to production of molecules including enzymes for the degradation of extracellular matrix and increase of vascular permeability. This will facilitate cell proliferation, survival and migration of endothelial cells. It is also known as the vascular permeability factor (VPF) (Dvorak, 2006) on the basis of its ability to induce leakage through the blood brain barrier in some pathological situations (Ferrara, 2001; Lafuente et al., 1999; Lafuente et al., 2002). Helmlinger et al. (2000) stated that in the vasodilatation process the VEGF induced the elongation of endothelial cells but not their proliferation. In the angiogenesis process, VEGF works in line with other factors such as angiopoietin and ephrins (Tonini et al., 2003). It has been shown in human biopsies that VEGF overexpression in gliomas correlates directly to proliferation, vascularization and degree of malignancy, and therefore inversely to prognosis (Ke et al., 2000; Lafuente et al., 1999; Plate, 1999).

#### **5. ENU glioma microvascular adaptation**

Along the glioma progression, there is a transition from the homogeneous capillary network to an anarchic angioarchitecture. Microvessels have to adapt in order to maintain blood perfusion and metabolic support in adverse conditions, constituting a peculiar tissular microenvironment in response to hypoxia (Blouw et al., 2003). Glioma microvascular remodelling consists in a process of vascular aberration along the neoplasia development. Vascular development process led to microvascular proliferations that are a histopathological hallmark of glioblastoma (Kleihues et al., 2007). Some authors consider the core of a high-grade glioma as an avascular zone, since it has scarce capillaries with wide lumen and a fragmented basal membrane, being rather inefficient for metabolic exchange (Vajkoczy & Menger, 2004).

Tumour blood vessels have multiple abnormalities that result in a heterogeneous environment. They are disorganized, tortuous, sinusoidal, branchy and leaky, the diameter is irregular and the walls are thinner than those found in healthy brain tissue (Bigner et al., 1998). Following our results obtained by LEA and Butyrylcholinesterase (BChE) histochemistry (Bulnes et al., 2009) we showed a transition from the homogeneous capillary network of early stages to an anarchic angioarchitecture of advanced ENU-glioma stages (Figure 3). It was found that the vessel density decreased and the vascular size increased in order to glioma malignity (Bulnes et al., 2009). The initial stage of ENU-glioma was constituted by microvessels similar to the brain capillaries, the intermediate stage by tortuous, disorganized and dilated vessels and the advanced stage by anarchic and aberrant

disease (Bulnes & Lafuente, 2007; Lafuente et al., 1999; Marti et al., 2000; Plate, 1992). However, the role of VEGF in nervous tissue is even more extensive. Previous studies showed that VEGF also has strong neuroprotective, neurotrophic and neurogenic properties (Jin et al., 2002; Ortuzar et al., 2011; Rosenstein & Krum, 2004; Storkebaum et al., 2004). Although the synthesis of this proangiogenic cytokine is associated to tumour cells and endothelial cells, it has been described in others, such as: neurons, astrocytes, pericytes, smooth muscle cells, macrophages, lymphoid cells, platelets and fibroblasts (Zagzag et al., 2000). The VEGF family consists of five different homologous factors, VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PIGF) (Ferrara et al., 2003). VEGF-A (VEGF) is the predominant form and is a hypoxia-inducible 45 KDa homodimeric glycoprotein. VEGF-A acts as mitogen, survival and antiapoptotic factor for the endothelial cells from arteries, veins and lymphatics. Faced with increased secretion of VEGF and its binding to receptors on the surface of endothelial cells, VEGF is a signal transduction leading to production of molecules including enzymes for the degradation of extracellular matrix and increase of vascular permeability. This will facilitate cell proliferation, survival and migration of endothelial cells. It is also known as the vascular permeability factor (VPF) (Dvorak, 2006) on the basis of its ability to induce leakage through the blood brain barrier in some pathological situations (Ferrara, 2001; Lafuente et al., 1999; Lafuente et al., 2002). Helmlinger et al. (2000) stated that in the vasodilatation process the VEGF induced the elongation of endothelial cells but not their proliferation. In the angiogenesis process, VEGF works in line with other factors such as angiopoietin and ephrins (Tonini et al., 2003). It has been shown in human biopsies that VEGF overexpression in gliomas correlates directly to proliferation, vascularization and degree of malignancy, and therefore inversely to

Along the glioma progression, there is a transition from the homogeneous capillary network to an anarchic angioarchitecture. Microvessels have to adapt in order to maintain blood perfusion and metabolic support in adverse conditions, constituting a peculiar tissular microenvironment in response to hypoxia (Blouw et al., 2003). Glioma microvascular remodelling consists in a process of vascular aberration along the neoplasia development. Vascular development process led to microvascular proliferations that are a histopathological hallmark of glioblastoma (Kleihues et al., 2007). Some authors consider the core of a high-grade glioma as an avascular zone, since it has scarce capillaries with wide lumen and a fragmented basal membrane, being rather inefficient for metabolic exchange

Tumour blood vessels have multiple abnormalities that result in a heterogeneous environment. They are disorganized, tortuous, sinusoidal, branchy and leaky, the diameter is irregular and the walls are thinner than those found in healthy brain tissue (Bigner et al., 1998). Following our results obtained by LEA and Butyrylcholinesterase (BChE) histochemistry (Bulnes et al., 2009) we showed a transition from the homogeneous capillary network of early stages to an anarchic angioarchitecture of advanced ENU-glioma stages (Figure 3). It was found that the vessel density decreased and the vascular size increased in order to glioma malignity (Bulnes et al., 2009). The initial stage of ENU-glioma was constituted by microvessels similar to the brain capillaries, the intermediate stage by tortuous, disorganized and dilated vessels and the advanced stage by anarchic and aberrant

prognosis (Ke et al., 2000; Lafuente et al., 1999; Plate, 1999).

**5. ENU glioma microvascular adaptation** 

(Vajkoczy & Menger, 2004).

vessels such as: multilayered "glomeruloid tuft"; "garland" of proliferated vessels and huge dilated vessels (Klehues et al., 2007).

One result to take in consideration was the gradient from the well-oxygenated tumour periphery to the central hypoxic core of ENU-glioblastoma. Dilated intratumour vessels, expressing VEGF (Lafuente et al., 1999) increase their lumen on account of endothelial elongation but not of cell proliferation (Helmlinger et al., 2000). The intratumour area displays irregularly branching vessels, variable intravascular spaces and large avascular areas. It is also worth mentioning that perivascular cells of aberrant vessels of ENU-GBM often displayed a high activity for BChE, depicted by a strong brown staining (Bulnes et al., 2009). BChE activity is strongly related to neurogenesis and cellular proliferation (Mack & Robitzki, 2000), having a great role in tumourigenesis. These findings have led us to postulate that these perivascular cells might be stem cells proliferating around intratumour vessels (Anderson et al., 2005; Brat et al., 2004) and migrating through the vascular extracellular matrix (Ruoslahti, 2002). This could corroborate the hypothesis that stem cells adapted to hypoxic stress use the vascular extracellular matrix for migration and invasion. In addition to this, in previous work we have shown that these cells co-expressed Ki-67 and VEGF (Bulnes & Lafuente, 2007).

Fig. 3. Angioarchitecture study of gliomas shown by butyrylcholinesterase histochemistry. a) Angioarchitecture of the cerebral cortex of the rat brain. b) Periventricular small neoplastic mass (initial stage) showing some strongly-positive vessels for BChE. c) Intermediate ENU-glioma stage displaying a network of numerous tortuous capillaries of anarchic distribution. d) Malignant infiltrating macrotumour, with dilated vessels of the intratumour area with strongly BChE positive cells. (Scale bar of 50µm).

Endogenous Experimental Glioma Model,

Links Between Glioma Stem Cells and Angiogenesis 415

(Bulnes et al., 2010). Because stem cells have been associated with the synthesis of VEGF (Bao et al., 2006), we focused on the identification of GSC using antibodies against the antigens CD133 and Nestin. We showed three distribution patterns of these cells (Figure 5): 1- isolated in the tumour periphery areas; 2- numerous small cells forming intratumour niches and 3- cells around the tortuous and aberrant vessel (intermediate-advanced stages).

Fig. 4. Vascular endothelial growth factor and endothelial nitric oxide synthase expression during ENU-glioma development. Confocal microphotographs showing VEGF165 (a-c, red)

immunofluorescence for tomato lectin LEA (green). (a, d) Initial stages of gliomas display basal stain of VEGF165 (a) and overexpression of eNOS only in dilated vessels (d, white arrow). (b, e) Anaplastic ENU-glioma corresponding with the intermediate tumour stage

overexpression of eNOS (e, yellow) in dilated and tortuous vessels from intratumour area. (c, f) ENU-induced glioblastomas show an heterogeneous pattern of expression for both markers. VEGF distribution is mainly showed in the peritumour neoangiogenic area (c) while eNOS overexpress as patching in vascular sections of intratumour aberrant

According to human astrocitomas, in ENU-glioma the number of positive cells for CD133 and Nestin antibodies increases with malignant grades of the tumour (Ma et al., 2008). Nestin+ cells were found in every stage of tumour development. It corroborated that the expression of Nestin is linked to the glioma grade, as stated in previous researches

and eNOS (d-f, red) in different stages of glioma. Vascular network is showed by

shows overexpression of VEGF165 in the neoangiogenic tumour border (b) and

microvessels (f). (Bar scale of 200 µm).

(Ehrmann et al., 2005).

Glioma malignancy process is mediated by the vascular remodelling and the angiogenesis process where the blood brain barrier (BBB) function is implicated. The BBB is the set of mechanisms (physical and metabolic) that regulate the passage of elements from the blood plasma to neural tissue. This especial barrier is necessary for the cerebral homeostasis and it is associated with the hydrostatic and osmotic pressure gradients across the capillary (Hatashita & Hoff, 1986).

In pathological conditions, the increase of vascular permeability could be due to the blood brain barrier dysfunction, to a structural break-down or to its immaturity. Endothelial cells (ECs) of tumour vessels do not form a closed barrier, and pericytes are loosely attached (Baluk, et al., 2005). Defective tight junctions explain the tumour vessel leakiness which leads to blood brain barrier (BBB) breakdown and the oedema associated with brain tumours (Hashizume et al., 2000; Papadopoulos et al., 2004). Brain oedema in gliomas is an epiphenomenon related to BBB breakdown and is another cause of tumour mortality (Ballabh et al., 2004). The BBB distortion and permeability increase have been related to intravital dyes extravasation (Lafuente et al., 1994, 2004), Gd-DTPA contrast enhancement on T1-w images (Brasch & Turetschek, 2000; Cha et al., 2003; Claes et al., 2007) and to changes in the expression of BBB markers as glucose transporter-1 (GluT-1) (Dobrogowska & Vorbrodt, 1999) and structural rat specific antigen of BBB (EBA) (Argandona et al., 2005; Lafuente et al., 2006; Lin & Ginsberg, 2000; Krum et al., 2002; Sternberger et al., 1989; Zhu et al., 2001).

In our ENU model, vascular adaptations predominate over angiogenesis (Lafuente et al., 2000; Bian et al., 2006). Microvascular adaptations in early development stages are based on vasodilatation, endothelium elongation and permeability increase mediated by VEGF-A without BBB dysfunction. On the other hand, in malignant gliomas the microvascular adaptations vary according to blood flow perfusion. Permeability increase in intratumour vessels is not enough to supply the metabolic demand, and triggering of the angiogenesis process on the tumour border is necessary. When the blood flow inside and around the tumour becomes irregular and chaotic, partly due to the aberrant microvessels, the relative tissue hypoxia triggers the production of hypoxia inducible factor-1α (Chen et al., 2009; Jain et al., 2007), upregulating the expression of VEGF-A and endothelial nitric oxide synthase (eNOS). VEGF-A induces the synthesis of NO by phosphorylation of endothelial NO synthase via PI-3K/Akt kinase (Osuka et al., 2004, Ziche & Morbidelli, 2009), thus promoting BBB breakdown and increasing permeability. Although, the role of eNOS and VEGF-A in tumour induced brain oedema is still a matter of debate. Our previous studies demonstrates that eNOS overexpression in the microvasculature of intermediate and advanced ENU-gliomas correlates with the loss of immunostaining for primary BBB markers GluT-1 and EBA (Bulnes et al., 2010) (Figure 4).

Following the finding showed in human tissues, in ENU-malignant glioma astrocytic processes and pericytes were loosely attached to endothelial cells of tumour vessels without forming a continuous layer (Baluk et al., 2005) (result not published). In addition to this, defective tight junctions (TJs) without occludin protein expression, also lead to oedema associated with ENU induced brain tumours. We showed an intratumoural glioma oedema instead of peritumoural one by gadolinium contrast enhancement and intravital dyes extravasation (Bulnes et al., 2009, 2010).

#### **6. Glioma stem cells and angiogenesis in ENU model**

The moment named "angiogenenic switch", when the angiogenesis starts, is showed at ENU-glioma intermediate stage due to the presence of overexpression of VEGF and eNOS

Glioma malignancy process is mediated by the vascular remodelling and the angiogenesis process where the blood brain barrier (BBB) function is implicated. The BBB is the set of mechanisms (physical and metabolic) that regulate the passage of elements from the blood plasma to neural tissue. This especial barrier is necessary for the cerebral homeostasis and it is associated with the hydrostatic and osmotic pressure gradients across the capillary

In pathological conditions, the increase of vascular permeability could be due to the blood brain barrier dysfunction, to a structural break-down or to its immaturity. Endothelial cells (ECs) of tumour vessels do not form a closed barrier, and pericytes are loosely attached (Baluk, et al., 2005). Defective tight junctions explain the tumour vessel leakiness which leads to blood brain barrier (BBB) breakdown and the oedema associated with brain tumours (Hashizume et al., 2000; Papadopoulos et al., 2004). Brain oedema in gliomas is an epiphenomenon related to BBB breakdown and is another cause of tumour mortality (Ballabh et al., 2004). The BBB distortion and permeability increase have been related to intravital dyes extravasation (Lafuente et al., 1994, 2004), Gd-DTPA contrast enhancement on T1-w images (Brasch & Turetschek, 2000; Cha et al., 2003; Claes et al., 2007) and to changes in the expression of BBB markers as glucose transporter-1 (GluT-1) (Dobrogowska & Vorbrodt, 1999) and structural rat specific antigen of BBB (EBA) (Argandona et al., 2005; Lafuente et al., 2006; Lin & Ginsberg,

In our ENU model, vascular adaptations predominate over angiogenesis (Lafuente et al., 2000; Bian et al., 2006). Microvascular adaptations in early development stages are based on vasodilatation, endothelium elongation and permeability increase mediated by VEGF-A without BBB dysfunction. On the other hand, in malignant gliomas the microvascular adaptations vary according to blood flow perfusion. Permeability increase in intratumour vessels is not enough to supply the metabolic demand, and triggering of the angiogenesis process on the tumour border is necessary. When the blood flow inside and around the tumour becomes irregular and chaotic, partly due to the aberrant microvessels, the relative tissue hypoxia triggers the production of hypoxia inducible factor-1α (Chen et al., 2009; Jain et al., 2007), upregulating the expression of VEGF-A and endothelial nitric oxide synthase (eNOS). VEGF-A induces the synthesis of NO by phosphorylation of endothelial NO synthase via PI-3K/Akt kinase (Osuka et al., 2004, Ziche & Morbidelli, 2009), thus promoting BBB breakdown and increasing permeability. Although, the role of eNOS and VEGF-A in tumour induced brain oedema is still a matter of debate. Our previous studies demonstrates that eNOS overexpression in the microvasculature of intermediate and advanced ENU-gliomas correlates with the loss of immunostaining for primary BBB

Following the finding showed in human tissues, in ENU-malignant glioma astrocytic processes and pericytes were loosely attached to endothelial cells of tumour vessels without forming a continuous layer (Baluk et al., 2005) (result not published). In addition to this, defective tight junctions (TJs) without occludin protein expression, also lead to oedema associated with ENU induced brain tumours. We showed an intratumoural glioma oedema instead of peritumoural one by gadolinium contrast enhancement and intravital dyes

The moment named "angiogenenic switch", when the angiogenesis starts, is showed at ENU-glioma intermediate stage due to the presence of overexpression of VEGF and eNOS

2000; Krum et al., 2002; Sternberger et al., 1989; Zhu et al., 2001).

markers GluT-1 and EBA (Bulnes et al., 2010) (Figure 4).

**6. Glioma stem cells and angiogenesis in ENU model** 

extravasation (Bulnes et al., 2009, 2010).

(Hatashita & Hoff, 1986).

(Bulnes et al., 2010). Because stem cells have been associated with the synthesis of VEGF (Bao et al., 2006), we focused on the identification of GSC using antibodies against the antigens CD133 and Nestin. We showed three distribution patterns of these cells (Figure 5): 1- isolated in the tumour periphery areas; 2- numerous small cells forming intratumour niches and 3- cells around the tortuous and aberrant vessel (intermediate-advanced stages).

Fig. 4. Vascular endothelial growth factor and endothelial nitric oxide synthase expression during ENU-glioma development. Confocal microphotographs showing VEGF165 (a-c, red) and eNOS (d-f, red) in different stages of glioma. Vascular network is showed by immunofluorescence for tomato lectin LEA (green). (a, d) Initial stages of gliomas display basal stain of VEGF165 (a) and overexpression of eNOS only in dilated vessels (d, white arrow). (b, e) Anaplastic ENU-glioma corresponding with the intermediate tumour stage shows overexpression of VEGF165 in the neoangiogenic tumour border (b) and overexpression of eNOS (e, yellow) in dilated and tortuous vessels from intratumour area. (c, f) ENU-induced glioblastomas show an heterogeneous pattern of expression for both markers. VEGF distribution is mainly showed in the peritumour neoangiogenic area (c) while eNOS overexpress as patching in vascular sections of intratumour aberrant microvessels (f). (Bar scale of 200 µm).

According to human astrocitomas, in ENU-glioma the number of positive cells for CD133 and Nestin antibodies increases with malignant grades of the tumour (Ma et al., 2008). Nestin+ cells were found in every stage of tumour development. It corroborated that the expression of Nestin is linked to the glioma grade, as stated in previous researches (Ehrmann et al., 2005).

Endogenous Experimental Glioma Model,

Links Between Glioma Stem Cells and Angiogenesis 417

neoplasia proliferation and invasion. These cells may be use extracellular matrix of vessel

wall to migrate and infiltrate the brain parenchyma (Borovski et al., 2009).

Fig. 6. Relationship between stem cell markers and proangiogenic factor VEGF in intratumour niches of advanced ENU-glioma stage. Study performed by double

immunofluorescence, all tumours are counterstained with Hoechst. a-c) Microphotographs of Nestin+ cells (a, in green) and VEGF+ cells (b, in red) and colocalization (yellow, c). VEGF+ cells predominate over Nestin+ cells. Some cells with big cytoplasm are Nestin-VEGF+. Small Nestin+ cells form a cluster and lack the staining of VEGF (at the top). d-f) Colocalization (yellow) of glial fibrillary acidic protein (GFAP, green) and VEGF (red). All VEGF+ cells in this intratumour area are stained for GFAP and display the astrocyte shape. g-i) Relationship between the two markers of stem cells: Nestin (green) and CD133 (reed). This niche shows higher density of nestin+ cells (g) than CD133+ cells (h). Almost all of the CD133+ cells coexpress nestin antibody (i, yellow). j-l) Coexpression of GFAP (green) and CD133 (red). Some cells coexpress both antibodies (l, yellow). (x400 Amplification)

Fig. 5. Immunoexpression of Nestin antigen in 4 μm paraffin sections showed by DAB staining (Brown). a-b) Intratumour area of ENU-Glioma showing two kinds of isolated cells marked by Nestin antibody. a) Cells of big cytoplasm and nucleon distributed predominantly near the periphery of the tumour. They display an astrocyte shape and GFAP positivity. b) Small cells with scarce cytoplasm and prolongations. c-d) Two distribution of stem cells: Intratumour niches (c) and around the vascular endothelium of neoplastic microvessels (d). (Bar scale of 10µm).

By the other hand, CD133+ cells were only present since intermediate stages corresponding with "angiogenenic switch". The distribution of CD133+ cells corresponds mainly to overexpression of VEGF in neoangiogenic border and intratumour hypoxic areas of neoplasia (Bulnes & Lafuente, 2007). It has been reported that tumour stem cells overexpress VEGF factor, so this cell population could be involved in the process of angiogenesis. Our results agree with the staining of CD133 described in the advanced and medium stage of human gliomas. Therefore, CD133 expression has been related to poor prognosis (Zeppernick et al., 2008).

We showed that some cells coexpress the antibodies Nestin, CD133 and VEGF165. They were forming niches around microvessels or into hypoxic areas (Figure 6). Only cells distributed in the periphery of neoplasia were stained for GFAP and displayed astrocyte morphology.

The relationship between CD133+ cells and vessels wall was shown around the glomeruloid vessels, distributed in the neoangiogenic border of ENU-GBM, and delimiting huge dilated intratumour vessels (Figure 7). The presence of CD133+ cells near these aberrant vessels which display BBB disturbance may corroborate the pivotal role of stem cells in the

Fig. 5. Immunoexpression of Nestin antigen in 4 μm paraffin sections showed by DAB staining (Brown). a-b) Intratumour area of ENU-Glioma showing two kinds of isolated cells

predominantly near the periphery of the tumour. They display an astrocyte shape and GFAP positivity. b) Small cells with scarce cytoplasm and prolongations. c-d) Two distribution of stem cells: Intratumour niches (c) and around the vascular endothelium of

By the other hand, CD133+ cells were only present since intermediate stages corresponding with "angiogenenic switch". The distribution of CD133+ cells corresponds mainly to overexpression of VEGF in neoangiogenic border and intratumour hypoxic areas of neoplasia (Bulnes & Lafuente, 2007). It has been reported that tumour stem cells overexpress VEGF factor, so this cell population could be involved in the process of angiogenesis. Our results agree with the staining of CD133 described in the advanced and medium stage of human gliomas. Therefore, CD133 expression has been related to poor

We showed that some cells coexpress the antibodies Nestin, CD133 and VEGF165. They were forming niches around microvessels or into hypoxic areas (Figure 6). Only cells distributed in the periphery of neoplasia were stained for GFAP and displayed astrocyte morphology. The relationship between CD133+ cells and vessels wall was shown around the glomeruloid vessels, distributed in the neoangiogenic border of ENU-GBM, and delimiting huge dilated intratumour vessels (Figure 7). The presence of CD133+ cells near these aberrant vessels which display BBB disturbance may corroborate the pivotal role of stem cells in the

marked by Nestin antibody. a) Cells of big cytoplasm and nucleon distributed

neoplastic microvessels (d). (Bar scale of 10µm).

prognosis (Zeppernick et al., 2008).

neoplasia proliferation and invasion. These cells may be use extracellular matrix of vessel wall to migrate and infiltrate the brain parenchyma (Borovski et al., 2009).

Fig. 6. Relationship between stem cell markers and proangiogenic factor VEGF in intratumour niches of advanced ENU-glioma stage. Study performed by double immunofluorescence, all tumours are counterstained with Hoechst. a-c) Microphotographs of Nestin+ cells (a, in green) and VEGF+ cells (b, in red) and colocalization (yellow, c). VEGF+ cells predominate over Nestin+ cells. Some cells with big cytoplasm are Nestin-VEGF+. Small Nestin+ cells form a cluster and lack the staining of VEGF (at the top). d-f) Colocalization (yellow) of glial fibrillary acidic protein (GFAP, green) and VEGF (red). All VEGF+ cells in this intratumour area are stained for GFAP and display the astrocyte shape. g-i) Relationship between the two markers of stem cells: Nestin (green) and CD133 (reed). This niche shows higher density of nestin+ cells (g) than CD133+ cells (h). Almost all of the CD133+ cells coexpress nestin antibody (i, yellow). j-l) Coexpression of GFAP (green) and CD133 (red). Some cells coexpress both antibodies (l, yellow). (x400 Amplification)

Endogenous Experimental Glioma Model,

**7. Conclusion** 

endothelial differentiation.

**8. Acknowledgment** 

Basque Government.

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Links Between Glioma Stem Cells and Angiogenesis 419

Following evidences reported in the literature and our findings, the distribution of "glioma stem cells" close to microvascular wall during the glioma malignancy process suggests a synergistic role of both structures. Indeed, based on our results we corroborate the hypothesis that glioma stem cells may induce angiogenesis via VEGF synthesis or

This knowledge will contribute to the generation of new antitumour therapy treatment against glioma stem cells. ENU experimental model would be considered as an useful

This work has been partially supported by Gangoiti Foundation, SAIOTEK and GIC 491/10

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Fig. 7. Immunofluorescence confocal images of CD133 antibody (red) in ENU-glioblastoma. All sections are counterstained with Hoechst (blue). a) Intratumour niche displaying some CD133+ cells. b) Tortuous vessel of the periphery of the neoplasia with CD133+ structures attached to the vascular endothelium. c) Aberrant vessels sections demarcated by CD133+cells. d) Vessels with huge lumen display CD133+ cells around some vascular sections. (Scale bar of 20µm).

Although some authors proposed that CD133+ cells were selected cells with tumorigenic capacity (Schiffer et al., 2010), others postulated that a fraction of CD133+ cells might be related to the endothelial differentiation and could generate tumour vessels (Wang et al., 2010). Recently, Soda et al. (2011) reported that part of the vasculature of GBM was originated from tumour cells. Therefore, some researchers as Wang et al. (2010) and Ricci-Vitiani et al. (2010) were centred to describe the proportion of the stem cells that contributed to blood vessels in glioblastoma. After their results they postulated that glioblastoma microvessels were originated from tumour stem like cells.

### **7. Conclusion**

418 Glioma – Exploring Its Biology and Practical Relevance

Fig. 7. Immunofluorescence confocal images of CD133 antibody (red) in ENU-glioblastoma. All sections are counterstained with Hoechst (blue). a) Intratumour niche displaying some CD133+ cells. b) Tortuous vessel of the periphery of the neoplasia with CD133+ structures

Although some authors proposed that CD133+ cells were selected cells with tumorigenic capacity (Schiffer et al., 2010), others postulated that a fraction of CD133+ cells might be related to the endothelial differentiation and could generate tumour vessels (Wang et al., 2010). Recently, Soda et al. (2011) reported that part of the vasculature of GBM was originated from tumour cells. Therefore, some researchers as Wang et al. (2010) and Ricci-Vitiani et al. (2010) were centred to describe the proportion of the stem cells that contributed to blood vessels in glioblastoma. After their results they postulated that glioblastoma

attached to the vascular endothelium. c) Aberrant vessels sections demarcated by CD133+cells. d) Vessels with huge lumen display CD133+ cells around some vascular

microvessels were originated from tumour stem like cells.

sections. (Scale bar of 20µm).

Following evidences reported in the literature and our findings, the distribution of "glioma stem cells" close to microvascular wall during the glioma malignancy process suggests a synergistic role of both structures. Indeed, based on our results we corroborate the hypothesis that glioma stem cells may induce angiogenesis via VEGF synthesis or endothelial differentiation.

This knowledge will contribute to the generation of new antitumour therapy treatment against glioma stem cells. ENU experimental model would be considered as an useful option to check a design of treatment strategies against these cells.

### **8. Acknowledgment**

This work has been partially supported by Gangoiti Foundation, SAIOTEK and GIC 491/10 Basque Government.

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**19** 

*Spain* 

Bárbara Meléndez et al.\* *Virgen de la Salud Hospital, Toledo,* 

**Copy Number Alterations in Glioma Cell Lines** 

Established tumor-derived cell lines are widely and routinely used as *in vitro* cancer models for various kinds of biomedical research. The easy management of these cell cultures, in contrast to the inherent difficulty in establishing and mantaining primary tumoral cultures, has contributed to the wide use of these inmortalized cell lines in order to characterize the biological significance of specific genomic aberrations identified in primary tumors. Therefore, it has been assumed that the genomic and expression aberrations of long-term established cell lines resemble, and are representative, of the primary tumor from which the cell line was derived. Indeed, the cell line-based research has been performed, not only for the definition of the molecular biology of several cancer models, but also for the investigation of new targeted therapeutic agents in a prior step to clinical practice. The use of tumor-derived cell lines has been highly relevant for the testing and development of new therapeutical agents, with several cancer cell-line panels having been developed for drug

Controversial concerning the ability of tumor-derived cell lines to accurately reflect the phenotype and genotype of the parental histology has been documented. A previous report of Greshock and coworkers using array-based Comparative Genomic Hybridization (aCGH) data of seven diagnosis-specific matched tumors and cell lines showed that, on average, cell lines preserve *in vitro* the genetic aberrations that are unique to the parent histology from which they were derived, while acquiring additional locus-specific alterations in long-term cultures (Greshock et al, 2007). In contrast, a study on breast cancer cell lines and primary tumors highlight that cell lines do not always represent the genotypes of parental tumor tissues (Tsuji et al, 2010). Furthermore, a parallel genomic and expression study on glioma cell lines and primary tumors states that in this specific cancer type, cell lines are poor representative of the primary tumors (Li et al, 2008). Given the importance of the use of cell lines as models for the study of the biology and development of tumors, and for the testing of the mode of action of new therapeutical agents, the knowledge of which genomic alterations are tumor-specific or which are necessary for the maintenance of the cell line in

Ainoha García-Claver1, Yolanda Ruano1, Yolanda Campos-Martín1, Ángel Rodríguez de Lope1, Elisa Pérez-Magán1, Pilar Mur1, Sofía Torres2, Mar Lorente2, Guillermo Velasco2 and Manuela Mollejo1

sensitivity screening and new agents' discovery (Sharma et al, 2010).

**1. Introduction** 

culture, becomes essential.

*1Virgen de la Salud Hospital, Toledo, Spain 2Universidad Complutense, Madrid, Spain* 

 \*

neurocarcinogenesis. *Acta Neurochirurgica*, Vol. 131, No.3-4, pp. 294-301, ISSN 00016268


### **Copy Number Alterations in Glioma Cell Lines**

Bárbara Meléndez et al.\*

*Virgen de la Salud Hospital, Toledo, Spain* 

#### **1. Introduction**

428 Glioma – Exploring Its Biology and Practical Relevance

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Established tumor-derived cell lines are widely and routinely used as *in vitro* cancer models for various kinds of biomedical research. The easy management of these cell cultures, in contrast to the inherent difficulty in establishing and mantaining primary tumoral cultures, has contributed to the wide use of these inmortalized cell lines in order to characterize the biological significance of specific genomic aberrations identified in primary tumors. Therefore, it has been assumed that the genomic and expression aberrations of long-term established cell lines resemble, and are representative, of the primary tumor from which the cell line was derived. Indeed, the cell line-based research has been performed, not only for the definition of the molecular biology of several cancer models, but also for the investigation of new targeted therapeutic agents in a prior step to clinical practice. The use of tumor-derived cell lines has been highly relevant for the testing and development of new therapeutical agents, with several cancer cell-line panels having been developed for drug sensitivity screening and new agents' discovery (Sharma et al, 2010).

Controversial concerning the ability of tumor-derived cell lines to accurately reflect the phenotype and genotype of the parental histology has been documented. A previous report of Greshock and coworkers using array-based Comparative Genomic Hybridization (aCGH) data of seven diagnosis-specific matched tumors and cell lines showed that, on average, cell lines preserve *in vitro* the genetic aberrations that are unique to the parent histology from which they were derived, while acquiring additional locus-specific alterations in long-term cultures (Greshock et al, 2007). In contrast, a study on breast cancer cell lines and primary tumors highlight that cell lines do not always represent the genotypes of parental tumor tissues (Tsuji et al, 2010). Furthermore, a parallel genomic and expression study on glioma cell lines and primary tumors states that in this specific cancer type, cell lines are poor representative of the primary tumors (Li et al, 2008). Given the importance of the use of cell lines as models for the study of the biology and development of tumors, and for the testing of the mode of action of new therapeutical agents, the knowledge of which genomic alterations are tumor-specific or which are necessary for the maintenance of the cell line in culture, becomes essential.

<sup>\*</sup> Ainoha García-Claver1, Yolanda Ruano1, Yolanda Campos-Martín1, Ángel Rodríguez de Lope1,

Elisa Pérez-Magán1, Pilar Mur1, Sofía Torres2, Mar Lorente2, Guillermo Velasco2 and Manuela Mollejo1 *1Virgen de la Salud Hospital, Toledo, Spain* 

*<sup>2</sup>Universidad Complutense, Madrid, Spain* 

Copy Number Alterations in Glioma Cell Lines 431

**CHROMOSOME 4**

**CHROMOSOME 10**

Fig. 2. RT-PCR analysis for the detection of EGFR wild-type (EGFRwt) and EGFRvIII mutant receptor. The inset shows control gene GAPDH results. Line 1: GOS3, 2: A172, 3: U118, 4:

Analysis of the high-level copy number changes detected by aCGH in the eleven glioma cell lines revealed higher frequency of genomic losses than gains. A stringent filter was applied

SF767, 5: T98, 6: wt EGFR control, 7: EGFRvIII control; M: molecular marker.

Fig. 1. aCGH results of chromosomes 4 (a) and 10 (b) in SW1783 and SF767 cell lines, respectively. Moving average of log2-genomic ratios over five neighbouring genes are



**2.1.2 Homozygous deletions** 

in order to detect homozygous deletions.

plotted.

0

0,5

**b)**

0

0.5

**a)**

Sometimes cell line studies are interpreted in the context of artifacts introduced by selection and establishment of cell lines in vitro, given the prevalence of documented cell line-specific cytogenetic changes acquired with multiple growth passages which is associated with random genomic instability. Therefore, the ability of glioma cell-line models to accurately reflect the phenotype and genotype of the parental glioma tumors remains unstudied. The aim of this study is to compare the genomic aberrations of the most commonly used glioma cell lines for *in vitro* analysis with those alterations more prevalent in primary glioma tumors.

#### **2. Copy number alterations in glioma cell lines**

#### **2.1 High-level DNA copy number alterations in glioma cell lines 2.1.1 Amplifications**

Genomic high-level DNA copy number gains (regions of amplification, or amplicons, i.e. chromosome regions that show more than 5- to 10-fold copy number increases) were detected at 4q, 10q and 19q in two of the cell lines: SW1783 (4q12) and SF767 (10q21.2-q23.1 and 19p12) (table 1, figure 1). The MLPA analysis confirmed some of the genomic alterations observed by aCGH, such as the amplification of *PDGFRA* (4q12) which was observed in SW1783 cell line (see below table 3).


Table 1. Summary of high-level gains (amplifications) detected by aCGH

Amplification of the EGFR gene, located on chromosome 7, and subsequent over-expression of EGFR protein, is the most common genetic alteration found in primary glioblastoma (GBM), the most aggressive high-grade glioma. This amplification is detected in about 40% of these tumors, and is present as double-minute extrachromosomal elements (Louis et al, 2007). Amplification of the EGFR gene is often associated with structural rearrangements, resulting in tumors expressing both wild-type EGFR as well as the mutated EGFR. The most common truncated EGFR variant is the EGFRvIII one, consisting of 801-bp in-frame deletion comprising exons 2-7 of the gene.

Among the cell lines analyzed in this study, some of them derived from primary GBMs, none of them carried either amplification of the EGFR gene, nor the EGFRvIII mutant form of the receptor (Figure 2). Besides, EGFR sequence analysis of exons 18-21, coding for the tyrosine kinase domain, revealed not a mutation in this region, unlike what is found in nonsmall lung cancer tumors.

Sometimes cell line studies are interpreted in the context of artifacts introduced by selection and establishment of cell lines in vitro, given the prevalence of documented cell line-specific cytogenetic changes acquired with multiple growth passages which is associated with random genomic instability. Therefore, the ability of glioma cell-line models to accurately reflect the phenotype and genotype of the parental glioma tumors remains unstudied. The aim of this study is to compare the genomic aberrations of the most commonly used glioma cell lines for

Genomic high-level DNA copy number gains (regions of amplification, or amplicons, i.e. chromosome regions that show more than 5- to 10-fold copy number increases) were detected at 4q, 10q and 19q in two of the cell lines: SW1783 (4q12) and SF767 (10q21.2-q23.1 and 19p12) (table 1, figure 1). The MLPA analysis confirmed some of the genomic alterations observed by aCGH, such as the amplification of *PDGFRA* (4q12) which was observed in

**CHROMOSOME GENES CELL LINE (Region**

**19p12 ZNF43,SINE-R ,ZNF208,ZNF257 SF767 (0.28)**

Amplification of the EGFR gene, located on chromosome 7, and subsequent over-expression of EGFR protein, is the most common genetic alteration found in primary glioblastoma (GBM), the most aggressive high-grade glioma. This amplification is detected in about 40% of these tumors, and is present as double-minute extrachromosomal elements (Louis et al, 2007). Amplification of the EGFR gene is often associated with structural rearrangements, resulting in tumors expressing both wild-type EGFR as well as the mutated EGFR. The most common truncated EGFR variant is the EGFRvIII one, consisting of 801-bp in-frame deletion

Among the cell lines analyzed in this study, some of them derived from primary GBMs, none of them carried either amplification of the EGFR gene, nor the EGFRvIII mutant form of the receptor (Figure 2). Besides, EGFR sequence analysis of exons 18-21, coding for the tyrosine kinase domain, revealed not a mutation in this region, unlike what is found in non-

**CHIC2, GSH2, PDGFRA, KIT, KDR, SRD5A2L, TMEM165, CLOCK, PDCL2, NMU, EXOC1, CEP135, AASDH, PPAT, PAICS, SRP72, HOP, SPINK2, REST,** 

**COL13A1, H2AFY2, AIFM2, TYSND1, SAR1A, PPA1, NPFFR1, LRRC20, EIF4EBP2, NODAL, PRF1, ADAMTS14, SGPL1, PCBD1, UNC5B, SLC29A3, CDH23, PSAP, CHST3, SPOCK2, ASCC1, DNAJB12, CBARA1, CCDC109A, OIT3, PLA2G12B, P4HA1, NUDT13, ECD, DNAJC9, MRPS16, TTC18, ANXA7, ZMYND17, PPP3CB, , USP54, MYOZ1, SYNPO2L, SEC24C, FUT11, NDST2, CAMK2G, PLAU, VCL, AP3M1, ADK, MYST4, DUSP13, SAMD8, VDAC2, KCNMA1, DLG5, NAG13, POLR3A, RPS24, ZMIZ1, PPIF, SFTPD,** 

**ANXA11, MAT1A, DYDC1, DYDC2, TSPAN14, NRG3**

Table 1. Summary of high-level gains (amplifications) detected by aCGH

**Size Mb)**

**SW1783 (3.57)**

**SF767 (13.37)**

*in vitro* analysis with those alterations more prevalent in primary glioma tumors.

**2. Copy number alterations in glioma cell lines** 

**2.1.1 Amplifications** 

**4q12**

**10q21.2 - q23.1**

SW1783 cell line (see below table 3).

**OLR2B, IGFBP7**

comprising exons 2-7 of the gene.

small lung cancer tumors.

**2.1 High-level DNA copy number alterations in glioma cell lines** 

Fig. 1. aCGH results of chromosomes 4 (a) and 10 (b) in SW1783 and SF767 cell lines, respectively. Moving average of log2-genomic ratios over five neighbouring genes are plotted.

Fig. 2. RT-PCR analysis for the detection of EGFR wild-type (EGFRwt) and EGFRvIII mutant receptor. The inset shows control gene GAPDH results. Line 1: GOS3, 2: A172, 3: U118, 4: SF767, 5: T98, 6: wt EGFR control, 7: EGFRvIII control; M: molecular marker.

#### **2.1.2 Homozygous deletions**

Analysis of the high-level copy number changes detected by aCGH in the eleven glioma cell lines revealed higher frequency of genomic losses than gains. A stringent filter was applied in order to detect homozygous deletions.

Copy Number Alterations in Glioma Cell Lines 433

showed a substitution in exon 7 (c.691C>T) which results in a stop codon (CGA>TGA). The latter mutation was confirmed with the database from the Cancer Genome Project (CGP, Sanger Institute). The CGP report the same mutation that we found in cell line U373, for the U251 glioma cell line, which is derived from the same tumour as U373, and thus contains

**SF767 (5.09)**

**CDKN2B U118, U87,LN18, H4, SW1088, U373, A172, GOS3**

**MLLT3, IFNB1 U118 (10.86), U87 (3.52), LN18 IFNW1 U118, U87,LN18, H4 (1.22)**

**ELAVL2 U118, LN18, SW1088**

**hel-N1 U118, LN18**

**MOBKL2B, LRRN6C U118, SW1088**

**ACTA2, FAS,CH25H,LIPA SW1088 (1.50)**

**BAGE H4 A172, U118, GOS3 (0.04)**

**PLAA, IFT74, LNG01784, TEK,** 

**10p11.21 PARD3 T98 (0.11)**

**10q23.2 - q23.31 PAPSS2,ATAD1,PTEN H4,SW1088 LIPF,ANKRD22,STAMBPL1,** 

**10q25.2 TCF7L2 T98 (0.16) 12q21.2 PAWR GOS3 (0.14)**

**21p11.1 BAGE4,BAGE5,BAGE3,BAGE2,** 

**LINGO2 U118**

Table 2. Homozygous losses detected in glioma cell lines by aCGH

**MINPP1 H4 (0.73)**

**KLHL9,IFNA2,IFNA8 U118, U87,LN18, H4, SW1088 (7.22) IFNE1,MTAP U118, U87,LN18, H4, SW1088,A172 (0.71) 9p21.3-p21.1 CDKN2A U118,U87,LN18,H4,SW1088,T98,U373,A172,GOS3 (0.18)**

the same TT insertion mutation in PTEN.

**1p31.1 LRRC44, FPGT, TNNI3K,** 

**3p24.3 TBC1D5,SATB1,KCNH8,**

**3p12.2-p11.2 IGSF4D,VGLL3,CHMP2B,**

**HDAC9**

**7p21.2-p21.1**

**CHROMOSOME GENES CELL LINE (Mb lost)**

**CRYZ, TYW3 U118 1q42.2 DISC1,SIPA1L2,PCNXL2 GOS3 (1.33) 2q42.2 BAZ2B GOS3 (0.12)**

**EFHB,RAB5A, SGOL1, PCAF H4 (4.63)**

**POU1F1,HTR1F, CGGBP1 LN18 (6.32)**

**3p24.1 TGFBR2 SF767 (0.23)**

**4q34.1 FBXO8,HPGD,GLRA3 U118 (0.40) 5q14.1 THBS4, SERINC5 SF767 (0.13) 6q22.2 ROS1,DCBLD1 U118 (0.17)**

> **ETV1, DGKB, MEOX2, OSTDC1, ANKMY2, BZW2, TSPAN13, AGR2, BCMP11, AHR, SNX13,**

**9p22.1-p21.1 SLC24A2 LN18 (6.37)**

**1p33 FAF1, CDKN2C U87 (0.17), T98 (0.07), U373 (0.23)**

Genomic homozygous losses were detected at 1p, 1q, 2q, 3p, 4q, 5q, 6q, 7p, 9p, 10p, 10q and 21p (Table 2).

Homozygous losses affecting two or more cell lines were detected at 1p33, 9p21.3-21.1, 10q23.2-23.3 and 21p11.1 (Table 2). Main target genes of these regions were: *CDKN2C* (p18INK4c) on chromosome 1, *CDKN2A* (p16INK4a) and CDKN2B (p15INK4b) on chromosome 9, and *PTEN* on chromosome 10. The most frequent homozygous gene loss was the loss of *CDKN2A* (p16INK4a) and *CDKN2B* (p15INK4b), affecting nine (82%) and eight (73%) of 11 glioma cell lines, respectively.

#### 2.1.2.1 Loss of *CDKN2C*

The Cancer Cell Line project (CCL) database from the Genome Cancer Project of the Sanger Institute (http://www.sanger.ac.uk/genetics/CGP/CellLines/) was used to confirm these alterations when possible. Homozygous deletion of CDKN2C (1p33) was described in this project for T98 and U87 cell lines. Homozygous deletion of CDKN2C on U373 cell line was not reported in this project. By contrast, this deletion was not reported in the study of Li and coworkers for T98 and U87 cell lines (Li et al, 2008).

#### 2.1.2.2 Loss of *CDKN2A* and *CDKN2B*

*CDKN2A* (9p21.3) loss of cell lines A172, H4, SW1088, T98, U118 and U87 was reported by the CCL project. Similarly, this gene was described as not mutated in SW1783, therefore confirming our results. Data from GOS3, LN18 and U373 were not provided in this database. Deletion of the 9p21 region was also reported in A172 and U87 cell lines by Li and coworkers, again validating our findings. Strikingly, T98 cell line was not deleted in that study (Li et al, 2008). Furthermore, the MLPA analyses performed on the cell lines confirmed the homozygous deletions observed by aCGH (Table 2). Therefore homozygous deletion of the *CDKN2A* gene was present in 9 of the 11 glioma cell lines (Table 2, Figure 3). Remarkably, there were two cell lines that lack any alteration at the *CDNK2A* locus, either by homozygous or hemizygous loss of the region.

#### 2.1.2.3 Loss of *PTEN*

The aCGH analysis revealed homozygous deletion of PTEN in SW1088 and H4 cell lines (Table 2), which was confirmed by the MLPA assay (Figure 4). In addition, homozygous deletion of PTEN in these cell lines was also reported by the CCL project. PTEN hemizygous deletion was detected in SF767 and GOS3 cell lines by aCGH and MLPA. Surprisingly, A172 cell line had homozygous deletion of all the PTEN probes of the MLPA assay except those of exons 1 and 2. This loss could not be detected by the aCGH analysis, probably because only two of the three probes included in the microarray were in the deleted part of PTEN (homozygous losses were considered as present when three consecutive clones were under the threshold 1.0) (Figure 4).

Further analyses of PTEN sequence were performed attending to PTEN expression (see Table 5 in section 3). Western-blot analysis showed PTEN expression in T98, LN18, GOS3 and SF767 (the two latter carring hemizygous deletion of the gene). Lack of protein expression was found in 7 of the eleven cell lines, three of them having homozygous deletion of PTEN. Therefore, we carried out exon-sequencing analysis of the other four PTEN deficient cell lines (U118, U87, U373 and SW1783) in order to detect putative mutations of the genomic sequence that could explain the observed suppression of protein expression. U118 and U87 presented a substitution mutation (G>T) in the splicing site of exons 8 (c.1026+1G>T) and 3 (c.209+1G>T), respectively; U373 showed an homozygous TT insertion in exon 7 causing a shift in the reading frame (c.723\_724insTT); and SW1783

Genomic homozygous losses were detected at 1p, 1q, 2q, 3p, 4q, 5q, 6q, 7p, 9p, 10p, 10q and

Homozygous losses affecting two or more cell lines were detected at 1p33, 9p21.3-21.1, 10q23.2-23.3 and 21p11.1 (Table 2). Main target genes of these regions were: *CDKN2C* (p18INK4c) on chromosome 1, *CDKN2A* (p16INK4a) and CDKN2B (p15INK4b) on chromosome 9, and *PTEN* on chromosome 10. The most frequent homozygous gene loss was the loss of *CDKN2A* (p16INK4a) and *CDKN2B* (p15INK4b), affecting nine (82%) and eight (73%) of 11

The Cancer Cell Line project (CCL) database from the Genome Cancer Project of the Sanger Institute (http://www.sanger.ac.uk/genetics/CGP/CellLines/) was used to confirm these alterations when possible. Homozygous deletion of CDKN2C (1p33) was described in this project for T98 and U87 cell lines. Homozygous deletion of CDKN2C on U373 cell line was not reported in this project. By contrast, this deletion was not reported in the study of Li and

*CDKN2A* (9p21.3) loss of cell lines A172, H4, SW1088, T98, U118 and U87 was reported by the CCL project. Similarly, this gene was described as not mutated in SW1783, therefore confirming our results. Data from GOS3, LN18 and U373 were not provided in this database. Deletion of the 9p21 region was also reported in A172 and U87 cell lines by Li and coworkers, again validating our findings. Strikingly, T98 cell line was not deleted in that study (Li et al, 2008). Furthermore, the MLPA analyses performed on the cell lines confirmed the homozygous deletions observed by aCGH (Table 2). Therefore homozygous deletion of the *CDKN2A* gene was present in 9 of the 11 glioma cell lines (Table 2, Figure 3). Remarkably, there were two cell lines that lack any alteration at the *CDNK2A* locus, either

The aCGH analysis revealed homozygous deletion of PTEN in SW1088 and H4 cell lines (Table 2), which was confirmed by the MLPA assay (Figure 4). In addition, homozygous deletion of PTEN in these cell lines was also reported by the CCL project. PTEN hemizygous deletion was detected in SF767 and GOS3 cell lines by aCGH and MLPA. Surprisingly, A172 cell line had homozygous deletion of all the PTEN probes of the MLPA assay except those of exons 1 and 2. This loss could not be detected by the aCGH analysis, probably because only two of the three probes included in the microarray were in the deleted part of PTEN (homozygous losses were considered as present when three consecutive clones were under

Further analyses of PTEN sequence were performed attending to PTEN expression (see Table 5 in section 3). Western-blot analysis showed PTEN expression in T98, LN18, GOS3 and SF767 (the two latter carring hemizygous deletion of the gene). Lack of protein expression was found in 7 of the eleven cell lines, three of them having homozygous deletion of PTEN. Therefore, we carried out exon-sequencing analysis of the other four PTEN deficient cell lines (U118, U87, U373 and SW1783) in order to detect putative mutations of the genomic sequence that could explain the observed suppression of protein expression. U118 and U87 presented a substitution mutation (G>T) in the splicing site of exons 8 (c.1026+1G>T) and 3 (c.209+1G>T), respectively; U373 showed an homozygous TT insertion in exon 7 causing a shift in the reading frame (c.723\_724insTT); and SW1783

21p (Table 2).

glioma cell lines, respectively.

coworkers for T98 and U87 cell lines (Li et al, 2008).

by homozygous or hemizygous loss of the region.

2.1.2.2 Loss of *CDKN2A* and *CDKN2B* 

2.1.2.1 Loss of *CDKN2C* 

2.1.2.3 Loss of *PTEN* 

the threshold 1.0) (Figure 4).

showed a substitution in exon 7 (c.691C>T) which results in a stop codon (CGA>TGA). The latter mutation was confirmed with the database from the Cancer Genome Project (CGP, Sanger Institute). The CGP report the same mutation that we found in cell line U373, for the U251 glioma cell line, which is derived from the same tumour as U373, and thus contains the same TT insertion mutation in PTEN.


Table 2. Homozygous losses detected in glioma cell lines by aCGH

Copy Number Alterations in Glioma Cell Lines 435

**CHROMOSOME 9**

**0.5**

**b)**

**0**

**-0.5**

**-1.0**

**-1.5**

**1.0**

**0.5**

**0**

represents a probe of the MLPA assay).

**CDKN2A EGFR ERRB2 PTEN P53**

Fig. 3. Homozygous loss detected on chromosome 9 (including *CDKN2A*locus) in two representative cell lines: U118 (a) and LN18 (b). Upper panel: aCGH plot (moving average of log2-genomic ratios over five neighbouring genes); Lower panel: MLPA graph (each bar

**U118**

**CHROMOSOME 9**

**CDKN2A EGFR ERRB2 PTEN P53**

0

5

5

**1.5**

**1.0**

**0.5**

**0**

**0.5**

**a)**

**0**

**-0.5**

**-1.0**

**-1.5**

Fig. 3. Homozygous loss detected on chromosome 9 (including *CDKN2A*locus) in two representative cell lines: U118 (a) and LN18 (b). Upper panel: aCGH plot (moving average of log2-genomic ratios over five neighbouring genes); Lower panel: MLPA graph (each bar represents a probe of the MLPA assay).

Copy Number Alterations in Glioma Cell Lines 437

Analyses of the DNA copy number changes in 11 of the most commonly used glioma cell lines revealed higher frecquency of genomic losses than gains. While 22.15% of the analyzed probes were lost, only 12.35% of them presented gains. Chromosomes containing frequently gained probes among all the cell lines included chromosomes 7, 16, 17, 19 and 20. Similarly, chromosomes containing frequently lost probes included chromosomes 4, 6, 10, 13, 14 and 18 (Figure 5). Surprisingly, chromosome 9, presenting loss of the *CDKN2A*/*CDKN2B* locus in most of the cell lines (9 out of 11 cell lines) presented a similar percentage of gained and loss probes. This result may be explained due to this loss is relatively small in most of the cell lines, and to the low-level DNA copy number gain of most of chromosome 9 in SF767

**2.2 Low-level DNA copy number alterations in glioma cell lines** 

cell line (data not shown).

0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00

**Arbitrary units (Y-axis)** 

**Chr.1**

cell lines (table 3).

grade gliomas.

**chr2**

**chr3**

**chr4**

**chr5**

**chr6**

the analyzed probes in the microarray per chromosome.

**chr7**

**chr8**

**chr9**

**chr10**

**chr11**

Fig. 5. Percentage of low-level DNA copy number gains (black) and losses (grey) relative to

Chromosome 7 was one of the most gained chromosomes, with complete or almost complete chromosome 7 gain in SW1088 and GOS3 cell lines, or with relative wide regions of gain in H4, U373, U118 or A172 cell lines. Gain of the *EGFR* gene (located at 7p12) was evaluated by MLPA assays, showing EGFR low-level copy number gain in 8 out of the 11

Other gains detected by MLPA analysis were *PI3KCA*, *BRAF* and *BIRC5*. Three of the cell lines presented a *PI3KCA* gain (3q). *PIK3CA* is one of the three genes encoding components of PI3K which is involved in activation of AKT signaling. Amplification of *PIK3CA* has been observed in various types of cancer, including gliomas (Karakas, 2006; Kita, 2007; Vogt, 2006). *BRAF* oncogene (7q34) was gained in five of the cell lines. BRAF is a serine/threonine kinase that is frequently activated in many types of cancer by a specific mutation (V600E). In pilocytic astrocytomas, BRAF is frequently activated by tandem duplication and rearrangement of part of the gene, resulting in fusion proteins containing the kinase domain (exons 9-18). Activation of BRAF through these mechanisms of duplication or fusion is infrequent in diffusely infiltrating astrocytic gliomas (Bar et al, 2008; Riemenscheneider et al, 2010). All the cell lines analyzed in this study were obtained from adult patients with high

**chr12**

**chr13**

**chr14**

**chr15**

**chr16**

**chr17**

**chr18**

**chr19**

**chr20**

**chr21**

**chr22**

Fig. 4. Homozygous loss detected on chromosome 10 (including *PTEN*) in two representative cell lines: SW1088 (a) and H4 (b). Upper panel: aCGH plot (moving average of log2-genomic ratios over five neighbouring genes); Lower panel: MLPA graph (each bar represents a probe of the MLPA assay).

**CHROMOSOME 10**

**CHROMOSOME 10**

**CDKN2A EGFR ERRB2 PTEN P53**

**CDKN2A EGFR ERRB2 PTEN P53**

representative cell lines: SW1088 (a) and H4 (b). Upper panel: aCGH plot (moving average of log2-genomic ratios over five neighbouring genes); Lower panel: MLPA graph (each bar

Fig. 4. Homozygous loss detected on chromosome 10 (including *PTEN*) in two

**H4**

5

**1.5**

**1.0**

**0.5**

**0**

**0.5**

**b)**

**0**

**-0.5**

**-1.0**

0

**0**

represents a probe of the MLPA assay).

0

**0.5**

0

**1.0**

0

**1.5**

0

**-1.5**

5

**-0.5**

**-1.0**

5

**a)**

**0.5**

**0**

#### **2.2 Low-level DNA copy number alterations in glioma cell lines**

Analyses of the DNA copy number changes in 11 of the most commonly used glioma cell lines revealed higher frecquency of genomic losses than gains. While 22.15% of the analyzed probes were lost, only 12.35% of them presented gains. Chromosomes containing frequently gained probes among all the cell lines included chromosomes 7, 16, 17, 19 and 20. Similarly, chromosomes containing frequently lost probes included chromosomes 4, 6, 10, 13, 14 and 18 (Figure 5). Surprisingly, chromosome 9, presenting loss of the *CDKN2A*/*CDKN2B* locus in most of the cell lines (9 out of 11 cell lines) presented a similar percentage of gained and loss probes. This result may be explained due to this loss is relatively small in most of the cell lines, and to the low-level DNA copy number gain of most of chromosome 9 in SF767 cell line (data not shown).

Fig. 5. Percentage of low-level DNA copy number gains (black) and losses (grey) relative to the analyzed probes in the microarray per chromosome.

Chromosome 7 was one of the most gained chromosomes, with complete or almost complete chromosome 7 gain in SW1088 and GOS3 cell lines, or with relative wide regions of gain in H4, U373, U118 or A172 cell lines. Gain of the *EGFR* gene (located at 7p12) was evaluated by MLPA assays, showing EGFR low-level copy number gain in 8 out of the 11 cell lines (table 3).

Other gains detected by MLPA analysis were *PI3KCA*, *BRAF* and *BIRC5*. Three of the cell lines presented a *PI3KCA* gain (3q). *PIK3CA* is one of the three genes encoding components of PI3K which is involved in activation of AKT signaling. Amplification of *PIK3CA* has been observed in various types of cancer, including gliomas (Karakas, 2006; Kita, 2007; Vogt, 2006). *BRAF* oncogene (7q34) was gained in five of the cell lines. BRAF is a serine/threonine kinase that is frequently activated in many types of cancer by a specific mutation (V600E). In pilocytic astrocytomas, BRAF is frequently activated by tandem duplication and rearrangement of part of the gene, resulting in fusion proteins containing the kinase domain (exons 9-18). Activation of BRAF through these mechanisms of duplication or fusion is infrequent in diffusely infiltrating astrocytic gliomas (Bar et al, 2008; Riemenscheneider et al, 2010). All the cell lines analyzed in this study were obtained from adult patients with high grade gliomas.

Copy Number Alterations in Glioma Cell Lines 439

**A172** 

**CDKN2A EGFR ERRB2 PTEN P53**

**A172**

Chromosome 7 Chromosome 9 Chromosome 10

**SW1088**

**CDKN2A EGFR ERRB2 PTEN P53**

Chromosome 7 Chromosome 9 Chromosome 10

Fig. 6. Genomic analysis of A172 (a, b) and SW1088 cell lines (c, d). a) MLPA analysis (each bar represents a probe of the MLPA assay) showing *EGFR* gain, *CDKN2A* homozygous deletion, and *PTEN* homozygous deletions except for exons 1 and 2. b) aCGH analysis (moving average of log2-genomic ratios over five neighbouring genes) of chromosomes 7, 9 and 10. c) MLPA analysis showing *EGFR* gain, and homozygous deletions of *CDKN2A* and *PTEN* d) aCGH analysis of chromosomes 7, 9 and 10. Observe that no *PTEN* deletions

0

**b)**



(10q23.2) were detected in A172 cell line compared to SW1088.

0.00

**d)**

0.50

1.00

1.50

2.00

**c)**

0.5

1

1.5

2

**a)**

BIRC5 or survivin (17q) was gained in five of the cell lines. Survivin, which promotes cell proliferation, angiogenes and inhibits apoptosis, is frequently overexpressed in proliferating tissues and tumors (Zhen et al, 2005). In gliomas, survivin overexpression is significantly associated with tumorigenesis and progression, and with a worse prognosis of patients (Shirai et al, 2009). Previous studies revealed, as well, BIRC5 gain and overexpression in oligodendroglial tumors (Blesa et al, 2009). High expression of BIRC5 in nervous system tumors have been already reported (Das, 2002; Hogdson, 2009; Sasaki, 2002).

As a summary, at the gene-level, the most represented gains and losses in the 11 analyzed cell lines are shown in table 4.


Table 3. Summary of gene-specific MLPA-validated copy number alterations (HOM LOSS: homozygous loss; HEMI LOSS: one copy loss; GAIN: low-level copy number gains; A: amplifications).


Table 4. Summary of the alterations most represented on the eleven glioma cell lines studied. (Total: number of cell lines presenting the alteration described)

BIRC5 or survivin (17q) was gained in five of the cell lines. Survivin, which promotes cell proliferation, angiogenes and inhibits apoptosis, is frequently overexpressed in proliferating tissues and tumors (Zhen et al, 2005). In gliomas, survivin overexpression is significantly associated with tumorigenesis and progression, and with a worse prognosis of patients (Shirai et al, 2009). Previous studies revealed, as well, BIRC5 gain and overexpression in oligodendroglial tumors (Blesa et al, 2009). High expression of BIRC5 in nervous system

As a summary, at the gene-level, the most represented gains and losses in the 11 analyzed

9p21 *CDKN2A* U373, U118, SW1088, GOS3, A172, H4, T98, U87, LN18

7p12 *EGFR* U373, U118, SW1088, GOS3, A172, H4, T98, SF767

tumors have been already reported (Das, 2002; Hogdson, 2009; Sasaki, 2002).

**CHROMOSOME GENE NAME CELL LINE** 

1p13.2 *NRAS* A172, H4 10q23 *PTEN* SF767, GOS3

1p13.2 *NRAS* U373 1q32 *PI3KC2B* A172 2q35 *IGFBP2* SW1088

17p11.2 *TOM1L2* LN18

**A** 4q11 *PDGFRA* SW1783

21q22.3 *RUNX1* H4, A172, T98

10q23 *PTEN* A172, SW1088, H4

3q26.3 *PIK3CA* A172, SW1783, H4

7q34 *BRAF* U87, U373, SW1088, GOS3, T98

17q25 *BIRC5* H4, LN18, T98, U373, SW1783

Table 3. Summary of gene-specific MLPA-validated copy number alterations (HOM LOSS: homozygous loss; HEMI LOSS: one copy loss; GAIN: low-level copy number gains; A:

> **Gene (location) Total Gene (location) Total**  EGFR (7p12) 8 CDKN2A (9p21) 9 BRAF (7q34) 5 CDKN2B (9p21) 8 BIRC5 (17q25) 5 MTAP (& others; 9p21) 6 PI3KCA (3q26.3) 3 BAGE (21p11.1) 4

Table 4. Summary of the alterations most represented on the eleven glioma cell lines

studied. (Total: number of cell lines presenting the alteration described)

**GAIN HOMOZYGOUS DELETION** 

PTEN (10q23) 3

CDKN2C (1p33) 3

cell lines are shown in table 4.

**HOM LOSS** 

**HEMI LOSS** 

**GAIN** 

amplifications).

Fig. 6. Genomic analysis of A172 (a, b) and SW1088 cell lines (c, d). a) MLPA analysis (each bar represents a probe of the MLPA assay) showing *EGFR* gain, *CDKN2A* homozygous deletion, and *PTEN* homozygous deletions except for exons 1 and 2. b) aCGH analysis (moving average of log2-genomic ratios over five neighbouring genes) of chromosomes 7, 9 and 10. c) MLPA analysis showing *EGFR* gain, and homozygous deletions of *CDKN2A* and *PTEN* d) aCGH analysis of chromosomes 7, 9 and 10. Observe that no *PTEN* deletions (10q23.2) were detected in A172 cell line compared to SW1088.

Copy Number Alterations in Glioma Cell Lines 441

minutes, i.e. small and circular fragments of extrachromosomal DNA that are replicated in the nucleus of the cell during cell division but that, unlike actual chromosomes, lack centromere or telomere. This EGFR amplification has not been detected in any of the analyzed glioma cell lines, probably due to the difficulty in maintaining a highly unstable extrachromosomal fragment that lacks centromere, in long-term cultures. A recent report, however, describes another type of EGFR gain in which extra copies (in small numbers) of EGFR are inserted in different loci of chromosome 7 (Lopez-Gines et al, 2010). The presence

**T98G** del HOMO N - G No N p.M237I **LN18** del HOMO N - N No N\* nd **SF767** N del HEMI - G No N\* nd **U373** del HOMO N\* c.723\_724insTT G No N nd **U87MG** del HOMO N\* c.209+1G>T N No N\* nd **SW1088** del HOMO del HOMO\* - G No N p.R273C **H4** del HOMO del HOMO\* - G No N\* nd **SW1783** N N\* c.691C>T N No N p.R273C **U118** del HOMO N\* c.1026+1G>T G No N p.R213Q **GOS3** del HOMO del HEMI - G No N nd **A172** del HOMO del HOMO\*,# - G No N\* nd

\*Protein expression not detected (Western-blot or Immunohistochemistry, data not shown) #deletion except for exons 1 and 2. del HOMO: homozygous deletion; del Hemi: hemizygous deletion; G: Gain; N: No copy number change; No: EGFRvIII mutation not detected; p53 mut: data from the Sanger database;

Thus, at least for what concerns to the EGFR amplification, glioma cell lines seem not to resemble primary tumors. This result contrast to what is found in breast cancer cell lines, where amplification of *ERBB2* (17q12) is detected indeed more frequently in cell lines that in primary tumors (Tsuji et al, 2010). Of note, amplification of *ERBB2* takes place within homogeneously staining regions, where the extra copies of the gene are integrated within

Similarly, other amplifications reported in primary GBM tumors have not been found in these cell lines, such as those of 1q (*MDM4*, *PIK3C2B*), 7q (*MET*, *PEX1*, *CDK6*), 12p (*CCDN2*) 12q (*MDM2*, *GLI*, *CDK4*) or 13q (Rao et al, 2010; Ruano et al, 2006). The only common amplification detected in glioma cell lines and tumors was that of 4q (*PDGFRA*) which was detected in SW1783 cell line. *PDGFRA* encodes for a cell surface tyrosine kinase receptor of the members of the platelet-derived growth factor family. These growth factors are mitogens for cells of mesenchymal origin and activate intracellular signaling through the MAPK, PI3K and PKCgamma pathways with roles in the regulation of many biological processes including embryonic development, angiogenesis, cell proliferation and differentiation. On the other hand, to our knowledge, amplifications of 10q and 19p detected in SF767 cell line

Digital karyotyping for eight tumor-derived cultured samples and one bulk tumor was used by Rao and coworkers (2010) to describe genomic alterations in GBM. This group described

*CDKN2A PTEN PTEN seq EGFR EGFRvIII Tp53 p53 mut*

*PI3K* pathway *TP53* pathway

of this type of gain in glioma cell lines remains to be studied.

Table 5. Alterations of the RB, TP53 and PI3K pathways.

have not been reported before in glioma tumors.

the chromosome, thus allowing its maintenance in established cell lines.

*RB* pathway

nd: no data from available.

#### **3. Comparison between copy number alterations in glioma cell lines and primary tumors**

Gliomas are the most frequent primary brain tumors, and include a variety of different histological tumor types and malignancy grades. High-grade gliomas are those graded as III or IV according to the criteria of the World Health Organization (WHO) classification system (Louis et al, 2007), including anaplasic astrocytoma (WHO grade III) and GBM (WHO grade IV). High-grade gliomas may arise from diffuse astrocytoma WHO grade II or III, or *de novo*, i.e. without evidence of a less malignant precursor lesion. GBM is the most frequent primary brain tumor. Primary GBM manifest rapidly de novo, while secondary GBM develops slowly from diffuse or anaplastic astrocytomas.

It is important to note that most of the cell lines used in this study derived from astrocytoma tumors of high-grade (8 cell lines: T98, LN18, U373, SW1088, H4, SW1783, U118, and A172), with the exception of GOS3 cell line that was derived from a high-grade mixed tumor with oligodendroglial component.

From a genetic point of view, progression to malignancy in gliomas is a multistep process, driven by the sequential acquisition and accumulation of genetic alterations. Distinctions between the genetic alterations identified in primary and secondary GBM have been made, with *TP53* mutations occurring more commonly in secondary GBMs and *EGFR* amplifications, and *PTEN* mutations occurring more frequently in primary GBMs. However, none of these alterations sufficiently distinguishes between primary and secondary GBM.

Recently, a comprehensive sequencing and genomic copy number analysis of GBM tumors showed that the majority of the tumors analyzed had alterations in genes encoding components of each of the *TP53*, *RB1*, and *PI3K* pathways, previously known to be altered in GBMs (Parsons et al, 2008). In these tumors, all but one of the cancers with mutations in members of a pathway did not have alterations in other members of the same family, suggesting that such alterations are functionally equivalent in tumorigenesis. Opposite to what is found in primary and secondary GBMs, glioma cell lines usually harbor functional alterations of the three pathways simultaneously (e.g. SW1088, SW1783 or U118, table 5).

Alteration mutations of the tumor suppressor gene *TP53* (located at 17p13.1) and loss of heterozygosity on chromosome arm 17p are frequent in secondary GBM. While *TP53* copy number analysis showed nor gains or losses in the cell lines tested, neither by CGH nor by MLPA, point mutations have been reported by the Sanger database in some of the analyzed cell lines (Table 5).

Primary GBM, on another hand, characterises by *EGFR* amplification or overexpression, *PTEN* mutation, trisomy of chromosome 7, monosomy of 10 and genomic gains of 12p, 19q and 20q (Riemenschneider et al, 2010).

Regarding alterations of *PTEN* gen (*PI3K* pathway), loss of chromosome 10 is one of the most frequent alteration in primary GBM tumors (60-80% of cases). While many tumors show loss of one entire copy of chromosome 10, loss of heterozygosity (LOH) studies have reported the involvement of several regions of LOH, suggesting several potential tumor suppressor genes in addition to *PTEN*. The cell lines analyzed in our study frequently presented alteration of *PTEN* gene (nine out of 11 cell lines), either by mutation or genomic loss. Absence of PTEN protein expression in these cell lines was confirmed in seven of these cell lines by western blot (data not shown).

Concerning amplifications, EGFR high-level copy number gain is the most frequent alteration found in primary GBM. As mentioned before, this alteration is present as double-

Gliomas are the most frequent primary brain tumors, and include a variety of different histological tumor types and malignancy grades. High-grade gliomas are those graded as III or IV according to the criteria of the World Health Organization (WHO) classification system (Louis et al, 2007), including anaplasic astrocytoma (WHO grade III) and GBM (WHO grade IV). High-grade gliomas may arise from diffuse astrocytoma WHO grade II or III, or *de novo*, i.e. without evidence of a less malignant precursor lesion. GBM is the most frequent primary brain tumor. Primary GBM manifest rapidly de novo, while secondary

It is important to note that most of the cell lines used in this study derived from astrocytoma tumors of high-grade (8 cell lines: T98, LN18, U373, SW1088, H4, SW1783, U118, and A172), with the exception of GOS3 cell line that was derived from a high-grade mixed tumor with

From a genetic point of view, progression to malignancy in gliomas is a multistep process, driven by the sequential acquisition and accumulation of genetic alterations. Distinctions between the genetic alterations identified in primary and secondary GBM have been made, with *TP53* mutations occurring more commonly in secondary GBMs and *EGFR* amplifications, and *PTEN* mutations occurring more frequently in primary GBMs. However, none of these alterations sufficiently distinguishes between primary and secondary GBM. Recently, a comprehensive sequencing and genomic copy number analysis of GBM tumors showed that the majority of the tumors analyzed had alterations in genes encoding components of each of the *TP53*, *RB1*, and *PI3K* pathways, previously known to be altered in GBMs (Parsons et al, 2008). In these tumors, all but one of the cancers with mutations in members of a pathway did not have alterations in other members of the same family, suggesting that such alterations are functionally equivalent in tumorigenesis. Opposite to what is found in primary and secondary GBMs, glioma cell lines usually harbor functional alterations of the three pathways simultaneously (e.g. SW1088, SW1783 or U118, table 5). Alteration mutations of the tumor suppressor gene *TP53* (located at 17p13.1) and loss of heterozygosity on chromosome arm 17p are frequent in secondary GBM. While *TP53* copy number analysis showed nor gains or losses in the cell lines tested, neither by CGH nor by MLPA, point mutations have been reported by the Sanger database in some of the analyzed

Primary GBM, on another hand, characterises by *EGFR* amplification or overexpression, *PTEN* mutation, trisomy of chromosome 7, monosomy of 10 and genomic gains of 12p, 19q

Regarding alterations of *PTEN* gen (*PI3K* pathway), loss of chromosome 10 is one of the most frequent alteration in primary GBM tumors (60-80% of cases). While many tumors show loss of one entire copy of chromosome 10, loss of heterozygosity (LOH) studies have reported the involvement of several regions of LOH, suggesting several potential tumor suppressor genes in addition to *PTEN*. The cell lines analyzed in our study frequently presented alteration of *PTEN* gene (nine out of 11 cell lines), either by mutation or genomic loss. Absence of PTEN protein expression in these cell lines was confirmed in seven of these

Concerning amplifications, EGFR high-level copy number gain is the most frequent alteration found in primary GBM. As mentioned before, this alteration is present as double-

**3. Comparison between copy number alterations in glioma cell lines and** 

GBM develops slowly from diffuse or anaplastic astrocytomas.

**primary tumors** 

oligodendroglial component.

cell lines (Table 5).

and 20q (Riemenschneider et al, 2010).

cell lines by western blot (data not shown).

minutes, i.e. small and circular fragments of extrachromosomal DNA that are replicated in the nucleus of the cell during cell division but that, unlike actual chromosomes, lack centromere or telomere. This EGFR amplification has not been detected in any of the analyzed glioma cell lines, probably due to the difficulty in maintaining a highly unstable extrachromosomal fragment that lacks centromere, in long-term cultures. A recent report, however, describes another type of EGFR gain in which extra copies (in small numbers) of EGFR are inserted in different loci of chromosome 7 (Lopez-Gines et al, 2010). The presence of this type of gain in glioma cell lines remains to be studied.


\*Protein expression not detected (Western-blot or Immunohistochemistry, data not shown) #deletion except for exons 1 and 2. del HOMO: homozygous deletion; del Hemi: hemizygous deletion; G: Gain; N: No copy number change; No: EGFRvIII mutation not detected; p53 mut: data from the Sanger database; nd: no data from available.

Table 5. Alterations of the RB, TP53 and PI3K pathways.

Thus, at least for what concerns to the EGFR amplification, glioma cell lines seem not to resemble primary tumors. This result contrast to what is found in breast cancer cell lines, where amplification of *ERBB2* (17q12) is detected indeed more frequently in cell lines that in primary tumors (Tsuji et al, 2010). Of note, amplification of *ERBB2* takes place within homogeneously staining regions, where the extra copies of the gene are integrated within the chromosome, thus allowing its maintenance in established cell lines.

Similarly, other amplifications reported in primary GBM tumors have not been found in these cell lines, such as those of 1q (*MDM4*, *PIK3C2B*), 7q (*MET*, *PEX1*, *CDK6*), 12p (*CCDN2*) 12q (*MDM2*, *GLI*, *CDK4*) or 13q (Rao et al, 2010; Ruano et al, 2006). The only common amplification detected in glioma cell lines and tumors was that of 4q (*PDGFRA*) which was detected in SW1783 cell line. *PDGFRA* encodes for a cell surface tyrosine kinase receptor of the members of the platelet-derived growth factor family. These growth factors are mitogens for cells of mesenchymal origin and activate intracellular signaling through the MAPK, PI3K and PKCgamma pathways with roles in the regulation of many biological processes including embryonic development, angiogenesis, cell proliferation and differentiation. On the other hand, to our knowledge, amplifications of 10q and 19p detected in SF767 cell line have not been reported before in glioma tumors.

Digital karyotyping for eight tumor-derived cultured samples and one bulk tumor was used by Rao and coworkers (2010) to describe genomic alterations in GBM. This group described

Copy Number Alterations in Glioma Cell Lines 443

Several of the frequent genomic alterations detected in glioma cell lines are not found in primary tumors, suggesting that some of the commonly seen alterations *in vitro* could be artifacts secondary to the selection pressure for optimal cell growth *in vitro* following years of passage. This observation has been reported previously in gliomas (Li et al, 2008), but the presence of acquired locus-specific alterations in culture has also been recognized in tumors and cell lines of other histologies (Greshock et al, 2007). For example, genome-specific copy number alterations of chromosomes 5 (gained), 8, 11 and 18 (lost) in glioma cell lines have been attributed exclusively to the phenotype of established cell lines. Furthermore, other copy number alterations not commonly found in cell lines, such as those of specific areas of

Our findings (Figure 5) have identified areas of low-level gain on chromosomes 5, 16 and 17 affecting between 5 and 7 cell lines, which do not feature GBM tumors. In addition, areas of loss of chromosomes 6, 8, 11, and, most importantly, loss of chromosome 18 have been identified in most of cell lines. These alterations seem to be culture-associated changes present in cell lines and suggest a genomic instability phenotype in established cell lines that

Absence of chromosome 13 deletions in glioma cell lines, which were commonly found in primary GBMs, was reported by Li and coworkers (2008) as a striking discrepancy between cell lines and tumors. Our study, however, did detected chromosome 13 losses (Figure 5). In the present study, complete loss of chromosome 13 was identified by aCGH in H4, LN18, U373, SW1088 and U118 cell lines, while partial loss was detected in U87, SF767, SW1783 and A172 cell lines. No loss was observed in T98 and GOS3 cell lines. Curiously, cell lines analyzed in common by our study and that of Li, had partial chromosome 13 loss in our study and partial chromosome 13 LOH in the study of Li and coworkers (U87 and A172), or

The human glioma cell lines GOS3, U87MG (U87), A172, SW1783, U118 MG (U118), T98G (T98), SW1088, H4, LN18, U373MG (U373) and SF767WL (SF767) were kindly provided by Dr. Velasco (Complutense University of Madrid, Spain) or Dr. Setién (Catalan Institute of Oncology, Spain). These cell lines were maintained in RPMI medium containing 10% FBS (Gibco, Grand Island, NY) in standard culture conditions. Total DNA and RNA were extracted from cell cultures according to standard phenol-chloroform and Trizol (Invitrogen, Carlsbad, CA) techniques, respectively. Nucleic acids obtained were quantified using

Copy number analyses of the 11 glioma cell lines were screened by array-based Comparative Genomic Hybridization (aCGH) in the Microarrays Analysis Service of the CIPF (Centro de Investigación Principe Felipe, Valencia). "Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis" protocol Version 4.0 (Agilent Technologies, Palo Alto, California USA. p/n G4410-90010) was followed to obtain labeled DNA. 2000 ng of DNA from samples and reference DNA (pool of sex-matched normal brain DNA) was

chromosomes 2, 3, 6 and 8 have been rarely observed in primary tumors.

**4. Cell culture specific aberrations** 

is not present in primary tumor tissues.

no chosmosome 13 loss in both studies (T98).

**5.2 Comparative genomic hybridization** 

NanoDrop-1000 (NanoDrop Technologies, Inc., Wilmington, DE).

**5. Material and methods 5.1 Cell lines and cell culture** 

amplifications in 1q, 7p, 8q and 12q, and homozygous deletions in 1p, 9p and 9q. However 7p11.2-12.1 (*EGFR*), 8q24.21 (*MYC*) and 12q15 (*MDM2*) amplifications were found just in case of the tumor sample, consistent with previous observations that adherent GBM cells tend to lose EGFR amplification during culturing. The most frequent amplifications found by this group was 12q13.3-q14.1, which targeted *GLI1* and *CDK4* oncogenes, affecting 3 samples. Two of the samples showed amplification of *PI3KC2B* and *MDM4* in 1q32.1. Table 6 shows comparison of our results with those published by Rao and coworkers (2010). Lowlevel copy number gains (e.g. *PI3KC2B*: A172 cell lines; *EGFR:* 8/11 cell lines) but not amplifications were detected in the cell lines.

The p16ink4a/CDK4/RB1 pathway is important for the control of progression through G1 into the S phase of the cell cycle. In GBM tumors, alterations affecting this pathway are found at an overall frequency of 40-50% (Louis et al, 2007). Homozygous deletions affecting *CDKN2A* locus (9p21) were described by digital karyotyping in 44% of cultured samples (four out of nine) (Rao et al, 2010). Our study reveals 82% (9/11) and 73% (8/11) of cell lines carrying homozygous deletions for *CDKN2A* and *CDKN2B* genes, respectively (Table 6).


Table 6. Comparison of results obtained by Digital Karyotyping (Rao et al, 2010) with aCGH alterations observed in glioma cell lines. Only amplification data from Rao´s study was available.

Finally, regarding the number of DNA copy number alterations in cell lines, the lost probes almost doubled the gained ones, with an average of losses and gains per cell line of 9,908 and 5,072 probes, respectively. This result contrast to what is observed in primary GBM tumors, having similar numbers of gains and losses (Ruano et al, 2006). Accordingly, similar results were obtained in tumor-derived cell lines from other histologies (Greshock et al, 2007) and specifically in breast cancer cell lines (Tsuji et al, 2010; Naylor et al, 2005), with more alterations found in cell lines than in tissue specimens, as a general trend. In fact, genomic losses in breast cancer cell lines almost doubled the gains (Tsuji et al, 2010). These observations may suggest the accumulation of genomic alterations in long-term cultures that are not present in primary tissues.

amplifications in 1q, 7p, 8q and 12q, and homozygous deletions in 1p, 9p and 9q. However 7p11.2-12.1 (*EGFR*), 8q24.21 (*MYC*) and 12q15 (*MDM2*) amplifications were found just in case of the tumor sample, consistent with previous observations that adherent GBM cells tend to lose EGFR amplification during culturing. The most frequent amplifications found by this group was 12q13.3-q14.1, which targeted *GLI1* and *CDK4* oncogenes, affecting 3 samples. Two of the samples showed amplification of *PI3KC2B* and *MDM4* in 1q32.1. Table 6 shows comparison of our results with those published by Rao and coworkers (2010). Lowlevel copy number gains (e.g. *PI3KC2B*: A172 cell lines; *EGFR:* 8/11 cell lines) but not

The p16ink4a/CDK4/RB1 pathway is important for the control of progression through G1 into the S phase of the cell cycle. In GBM tumors, alterations affecting this pathway are found at an overall frequency of 40-50% (Louis et al, 2007). Homozygous deletions affecting *CDKN2A* locus (9p21) were described by digital karyotyping in 44% of cultured samples (four out of nine) (Rao et al, 2010). Our study reveals 82% (9/11) and 73% (8/11) of cell lines carrying homozygous deletions for *CDKN2A* and *CDKN2B* genes, respectively (Table 6).

**(n=9)** 

22 22 **Our group % (n=11)** 

> 9 (G) 0

**Chromosome band Target oncogene Rao %** 

PIK3C2B MDM4

7p11.2-12.1 EGFR 11 73 (G) 8q24.21 MYC 11 45 (G) 12q13.3-q14.1 GLI1,CD4 33 18 (G) 12q14.1 Unknown 22 9 (G) 12q15 MDM2 11 0

1p36.31-p36.23 TP73, LRRC47, DFFB 33 18 9p21.3-22.3 CDKN2A, CDKN2B 44 82, 73 9q34.3 CACNA1B 44 0

Table 6. Comparison of results obtained by Digital Karyotyping (Rao et al, 2010) with aCGH alterations observed in glioma cell lines. Only amplification data from Rao´s study was

Finally, regarding the number of DNA copy number alterations in cell lines, the lost probes almost doubled the gained ones, with an average of losses and gains per cell line of 9,908 and 5,072 probes, respectively. This result contrast to what is observed in primary GBM tumors, having similar numbers of gains and losses (Ruano et al, 2006). Accordingly, similar results were obtained in tumor-derived cell lines from other histologies (Greshock et al, 2007) and specifically in breast cancer cell lines (Tsuji et al, 2010; Naylor et al, 2005), with more alterations found in cell lines than in tissue specimens, as a general trend. In fact, genomic losses in breast cancer cell lines almost doubled the gains (Tsuji et al, 2010). These observations may suggest the accumulation of genomic alterations in long-term cultures

amplifications were detected in the cell lines.

**Amplifications/Gains (G)** 

**Homozygous deletions** 

that are not present in primary tissues.

1q32.1 1q32.1

available.

#### **4. Cell culture specific aberrations**

Several of the frequent genomic alterations detected in glioma cell lines are not found in primary tumors, suggesting that some of the commonly seen alterations *in vitro* could be artifacts secondary to the selection pressure for optimal cell growth *in vitro* following years of passage. This observation has been reported previously in gliomas (Li et al, 2008), but the presence of acquired locus-specific alterations in culture has also been recognized in tumors and cell lines of other histologies (Greshock et al, 2007). For example, genome-specific copy number alterations of chromosomes 5 (gained), 8, 11 and 18 (lost) in glioma cell lines have been attributed exclusively to the phenotype of established cell lines. Furthermore, other copy number alterations not commonly found in cell lines, such as those of specific areas of chromosomes 2, 3, 6 and 8 have been rarely observed in primary tumors.

Our findings (Figure 5) have identified areas of low-level gain on chromosomes 5, 16 and 17 affecting between 5 and 7 cell lines, which do not feature GBM tumors. In addition, areas of loss of chromosomes 6, 8, 11, and, most importantly, loss of chromosome 18 have been identified in most of cell lines. These alterations seem to be culture-associated changes present in cell lines and suggest a genomic instability phenotype in established cell lines that is not present in primary tumor tissues.

Absence of chromosome 13 deletions in glioma cell lines, which were commonly found in primary GBMs, was reported by Li and coworkers (2008) as a striking discrepancy between cell lines and tumors. Our study, however, did detected chromosome 13 losses (Figure 5). In the present study, complete loss of chromosome 13 was identified by aCGH in H4, LN18, U373, SW1088 and U118 cell lines, while partial loss was detected in U87, SF767, SW1783 and A172 cell lines. No loss was observed in T98 and GOS3 cell lines. Curiously, cell lines analyzed in common by our study and that of Li, had partial chromosome 13 loss in our study and partial chromosome 13 LOH in the study of Li and coworkers (U87 and A172), or no chosmosome 13 loss in both studies (T98).

#### **5. Material and methods**

#### **5.1 Cell lines and cell culture**

The human glioma cell lines GOS3, U87MG (U87), A172, SW1783, U118 MG (U118), T98G (T98), SW1088, H4, LN18, U373MG (U373) and SF767WL (SF767) were kindly provided by Dr. Velasco (Complutense University of Madrid, Spain) or Dr. Setién (Catalan Institute of Oncology, Spain). These cell lines were maintained in RPMI medium containing 10% FBS (Gibco, Grand Island, NY) in standard culture conditions. Total DNA and RNA were extracted from cell cultures according to standard phenol-chloroform and Trizol (Invitrogen, Carlsbad, CA) techniques, respectively. Nucleic acids obtained were quantified using NanoDrop-1000 (NanoDrop Technologies, Inc., Wilmington, DE).

#### **5.2 Comparative genomic hybridization**

Copy number analyses of the 11 glioma cell lines were screened by array-based Comparative Genomic Hybridization (aCGH) in the Microarrays Analysis Service of the CIPF (Centro de Investigación Principe Felipe, Valencia). "Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis" protocol Version 4.0 (Agilent Technologies, Palo Alto, California USA. p/n G4410-90010) was followed to obtain labeled DNA. 2000 ng of DNA from samples and reference DNA (pool of sex-matched normal brain DNA) was

Copy Number Alterations in Glioma Cell Lines 445

High-level copy number alterations have been observed in cell lines of different sources such as breast, melanoma or lung tumors. Some authors suggest that some of the commonly seen alterations in the glioma cell lines can be due to the *in vitro* cell growth process following long term passage cultures. These observations are based on (i) the comparison of the genomic alterations of glioma and other non glioma cancer cell lines: some of these alterations are common between established cancer cell lines from different origin and uncommon in glioma tumors (Li et al, 2008). ii) Differential expression analyses suggest that established cancer cell lines share an underlying molecular similarity more closely related to their *in vitro* culture conditions than to their original tumor type of origin. Although some functional signalling pathways are up-regulated both in glioma tumors and glioma cell lines (epidermal growth factor receptor, vascular endothelial growth factor receptor, p53, PI3K pathway), there are some others gene expression sets whose up-regulation is just seen in cancer cell lines (cell cycle, proteasome activity, purine metabolism, mitochondrial activity). Our findings show that established glioma cell lines and glioma tumours have differences in genomic alterations, concluding that glioma cell lines may not be such an accurate representation or model system for primary gliomas as would be desirable. As opposed to primary tumors, glioma cell lines did not present either *EGFR* amplification, or presence of EGFRvIII variant, events that are frequent in high-grade gliomas. Homozygous *CDKN2A* deletion was frequently observed in glioma cell lines, as occur in cell lines derived from other histologies and in glioma tumors. Chromosome 7 gain and *PTEN* deletions represent

The easy of management of glioma cell lines make these cell lines as good candidate models for exploring basic glioma biology and for the use and discovery of therapeutic agents in preclinical screens. However, it is of interest that cell clycle-related alterations of gene expression are importantly affected in these cell lines, and that most drugs have been tested for cytotoxicity against rapidly dividing cells. Therefore, selection bias toward the identification of therapeutic agents involved in molecular functions more related to the long

On the other hand, many efforts are being done to create adequate culture conditions that allow the maintenance of the genomic profiles of the original tumor, such as glioma stemlike cell cultures, which may be more representative of their parent tumors. Several reports have demonstrated that glioma cultures under serum free conditions and stimulated with mitogens, epidermal growth factor and fibroblast growth factor, grow as neurospheres and maintain a phenotype and genotype closer to that typical of primary tumours compared to traditional serum-derived cell lines and culture techniques (Fael Al-Mayhani et al, 2009; Ernst et al, 2009). Perhaps, the standardization of this culture method could enhance and

We gratefully acknowledge Drs. G. Velasco and F. Setien for kindly provinding cell lines. This work was partially supported by grants G-2009\_E/04 from Fundación Sociosanitaria de Castilla-La Mancha and the Consejería de Salud y Bienestar Social, Junta de Comunidades de Castilla-La Mancha; and FIS PI07/0662, FIS10/01974 from the Fondo de Investigaciones

the most specific glioma alterations present in these cell lines.

term culture than to glioma biology could occur.

improve the research with cell lines in brain tumors.

Sanitarias (FIS) of the Instituto de Salud Carlos III (Spain).

**7. Acknowledgment** 

**6. Conclusion** 

fragmented and labeled (Cyanine 3-dUTP for the cell lines DNA and cyanine 5-dUTP for the reference DNA) according to the "Agilent Genomic DNA labeling kit plus" protocol. Labeled DNA was hybridized with Human Genome CGH Microarray 44 k (Agilent p/n G4426B-014950) containing 45,214 probes with 42,494 distinct biological features. Arrays were scanned in an Agilent Microarray Scanner (Agilent G2565BA). Data was analyzed using DNA Analytics 4.0 CGH Module (Agilent Technologies). Genomic alterations were detected using an ADM-2 algorithm with two different filters: one, used to detect low level alterations, , detects those alterations affecting to three consecutive probes with a ratio above or below to 0.25; the other, used to obtain amplifications or homozygous deletion, included in 2.1 section, detects only three consecutive probes above or below a ratio of 1.0.

#### **5.3 MLPA analysis**

Specific gene alterations were validated by Multiple ligation probe assay experiments (MLPA®, Mrc-Holland, The Netherlands) with SALSA MLPA kit P105 Glioma-2 for EGFR,

PTEN, CDKN2A and p53. Besides, SALSA® MLPA® kit P173 was used to detect copy number alteration of several genes which are frequently altered in several tumors, such as: BCL2L11, BIRC5, BRAF, ERBB4, JAK2, NRAS, PDGFRA, PIK3C2B, PIK3CA. MLPA assays were carried in total DNA from the eleven cell lines, obtained by standard methods, following manufacturers' conditions. Polymerase chain reaction products were separated and quantified on an ABI PRISM 310 DNA sequencer (Applied Biosystems), and electropherograms were analyzed using GeneMapper v.3.7 software (Applied Biosystems). Three nontumor reference samples were included in each run.

#### **5.4 EGFRvIII analysis**

Presence of EGFR vIII variant was determined by RT-PCR from total RNA of the cell cultures. cDNA was obtained from lµg of total RNA using the Superscript System (Gibco®). Primers and PCR conditions used were previously described (Lee et al, 2006). Amplifications products were visualized in bromure ethydium 2% agarose gel.

#### **5.5 EGFR and PTEN sequence analysis**

Mutations in exons 1 to 9 of PTEN gene and 18 to 21 of the EGFR gene were screened by direct sequencing in an ABI PRISM 310 DNA Analyser (Applied Biosystems) according to the manufacturer's instructions. PCR primers and conditions for EGFR amplification were previously described (Hsieh et al, 2006).


Table 7. PTEN sequence and annealing temperature used for PCR reactions of nine exon primers

#### **6. Conclusion**

444 Glioma – Exploring Its Biology and Practical Relevance

fragmented and labeled (Cyanine 3-dUTP for the cell lines DNA and cyanine 5-dUTP for the reference DNA) according to the "Agilent Genomic DNA labeling kit plus" protocol. Labeled DNA was hybridized with Human Genome CGH Microarray 44 k (Agilent p/n G4426B-014950) containing 45,214 probes with 42,494 distinct biological features. Arrays were scanned in an Agilent Microarray Scanner (Agilent G2565BA). Data was analyzed using DNA Analytics 4.0 CGH Module (Agilent Technologies). Genomic alterations were detected using an ADM-2 algorithm with two different filters: one, used to detect low level alterations, , detects those alterations affecting to three consecutive probes with a ratio above or below to 0.25; the other, used to obtain amplifications or homozygous deletion, included

Specific gene alterations were validated by Multiple ligation probe assay experiments (MLPA®, Mrc-Holland, The Netherlands) with SALSA MLPA kit P105 Glioma-2 for EGFR, PTEN, CDKN2A and p53. Besides, SALSA® MLPA® kit P173 was used to detect copy number alteration of several genes which are frequently altered in several tumors, such as: BCL2L11, BIRC5, BRAF, ERBB4, JAK2, NRAS, PDGFRA, PIK3C2B, PIK3CA. MLPA assays were carried in total DNA from the eleven cell lines, obtained by standard methods, following manufacturers' conditions. Polymerase chain reaction products were separated and quantified on an ABI PRISM 310 DNA sequencer (Applied Biosystems), and electropherograms were analyzed using GeneMapper v.3.7 software (Applied Biosystems).

Presence of EGFR vIII variant was determined by RT-PCR from total RNA of the cell cultures. cDNA was obtained from lµg of total RNA using the Superscript System (Gibco®). Primers and PCR conditions used were previously described (Lee et al, 2006).

Mutations in exons 1 to 9 of PTEN gene and 18 to 21 of the EGFR gene were screened by direct sequencing in an ABI PRISM 310 DNA Analyser (Applied Biosystems) according to the manufacturer's instructions. PCR primers and conditions for EGFR amplification were

Exon Upstream primer 5'-3' Downstream primer 5'-3' Annealing T (°C) 1 TCCTCCTTTTTCTTCAGCCAC GAAAGGTAAAGAGGAGCAGCC 56 2 GCTGCATATTTCAATCAAACTAA ACATCAATATTTGAAATAGAAAATC 54 3 TGTTAATGGTGGCTTTTTG GCAAGCATACAAATAAGAAAAC 56 4 TTCCTAAGTGCAAAAGATAAC TACAGTCTATCGGGTTTAAGT 56 5 TTTTTTTTTCTTATTCTGAGGTTAT GAAGAGGAAAGGAAAAACATC 51 6 AGTGAAATAACTATAATGGAACA GAAGGATGAGAATTTCAAGC 54 7 AATACTGGTATGTATTTAACCAT TCTCCCAATGAAAGTAAAGTA 56 8 TTTTTAGGACAAAATGTTTCAC CCCACAAAATGTTTAATTTAAC 54 9 GTTTTCATTTTAAATTTTCTTTC TGGTGTTTTATCCCTCTTG 54 Table 7. PTEN sequence and annealing temperature used for PCR reactions of nine exon

Amplifications products were visualized in bromure ethydium 2% agarose gel.

in 2.1 section, detects only three consecutive probes above or below a ratio of 1.0.

Three nontumor reference samples were included in each run.

**5.3 MLPA analysis** 

**5.4 EGFRvIII analysis** 

primers

**5.5 EGFR and PTEN sequence analysis** 

previously described (Hsieh et al, 2006).

High-level copy number alterations have been observed in cell lines of different sources such as breast, melanoma or lung tumors. Some authors suggest that some of the commonly seen alterations in the glioma cell lines can be due to the *in vitro* cell growth process following long term passage cultures. These observations are based on (i) the comparison of the genomic alterations of glioma and other non glioma cancer cell lines: some of these alterations are common between established cancer cell lines from different origin and uncommon in glioma tumors (Li et al, 2008). ii) Differential expression analyses suggest that established cancer cell lines share an underlying molecular similarity more closely related to their *in vitro* culture conditions than to their original tumor type of origin. Although some functional signalling pathways are up-regulated both in glioma tumors and glioma cell lines (epidermal growth factor receptor, vascular endothelial growth factor receptor, p53, PI3K pathway), there are some others gene expression sets whose up-regulation is just seen in cancer cell lines (cell cycle, proteasome activity, purine metabolism, mitochondrial activity).

Our findings show that established glioma cell lines and glioma tumours have differences in genomic alterations, concluding that glioma cell lines may not be such an accurate representation or model system for primary gliomas as would be desirable. As opposed to primary tumors, glioma cell lines did not present either *EGFR* amplification, or presence of EGFRvIII variant, events that are frequent in high-grade gliomas. Homozygous *CDKN2A* deletion was frequently observed in glioma cell lines, as occur in cell lines derived from other histologies and in glioma tumors. Chromosome 7 gain and *PTEN* deletions represent the most specific glioma alterations present in these cell lines.

The easy of management of glioma cell lines make these cell lines as good candidate models for exploring basic glioma biology and for the use and discovery of therapeutic agents in preclinical screens. However, it is of interest that cell clycle-related alterations of gene expression are importantly affected in these cell lines, and that most drugs have been tested for cytotoxicity against rapidly dividing cells. Therefore, selection bias toward the identification of therapeutic agents involved in molecular functions more related to the long term culture than to glioma biology could occur.

On the other hand, many efforts are being done to create adequate culture conditions that allow the maintenance of the genomic profiles of the original tumor, such as glioma stemlike cell cultures, which may be more representative of their parent tumors. Several reports have demonstrated that glioma cultures under serum free conditions and stimulated with mitogens, epidermal growth factor and fibroblast growth factor, grow as neurospheres and maintain a phenotype and genotype closer to that typical of primary tumours compared to traditional serum-derived cell lines and culture techniques (Fael Al-Mayhani et al, 2009; Ernst et al, 2009). Perhaps, the standardization of this culture method could enhance and improve the research with cell lines in brain tumors.

#### **7. Acknowledgment**

We gratefully acknowledge Drs. G. Velasco and F. Setien for kindly provinding cell lines. This work was partially supported by grants G-2009\_E/04 from Fundación Sociosanitaria de Castilla-La Mancha and the Consejería de Salud y Bienestar Social, Junta de Comunidades de Castilla-La Mancha; and FIS PI07/0662, FIS10/01974 from the Fondo de Investigaciones Sanitarias (FIS) of the Instituto de Salud Carlos III (Spain).

Copy Number Alterations in Glioma Cell Lines 447

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**Part 6** 

**Miscellaneous** 

448 Glioma – Exploring Its Biology and Practical Relevance

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2005) Vol. 104, pp 2775-2783, ISSN: 1097-0142

with proliferation, apoptosis, and angiogenesis in brain gliomas. *Cancer* (April

**20** 

*Canada* 


**Oxidative Stress and Glutamate** 

Glioma are a family of glial cell tumours of the central nervous system (CNS) wellcharacterized as aggressive cancers with dismally limited treatment options. The relatively recent discoveries of mechanisms surrounding glioma cell antioxidant protection and neuronal and glial cell destruction have opened the gates to a new therapeutic avenue whose implications to the field of cancer biology extend far beyond the treatment of glioma alone. Stemming from the discovery of significant glutamate release and glutathione production by glioma cells, the mechanisms through which glioma mediate oxidative stress

and influence their extracellular microenvironment are now being unravelled.

antioxidant increases are driven by an upregulation of system xc

far as clinical trials. Glutamate receptor antagonists and system xc

pathways are great, and the discovery of system xc

This chapter will discuss the upregulation of the cystine/glutamate antiporter, system xc

glioma and the far ranging consequences that stem from this compensatory action. Specifically, a characteristic shift of cancer cell metabolism away from the tricarboxylic acid (TCA) cycle and towards increased rates of glycolysis, a process termed the Warburg effect, produces a high amount of reactive oxygen species (ROS) that would prove cytotoxic without adequate cellular antioxidant defences. In response to this metabolic abnormality, glioma have demonstrated increased synthesis of the primary cellular antioxidant glutathione and an increased circulation of the cystine/cysteine redox cycle. These

with the rate limiting substrate for glutathione synthesis, cysteine, and acts as one half of the transport machinery for the cystine/cysteine cycle. The increased tolerance to oxidative stress that is conferred by these mechanisms allows glioma survival and growth advantages and mediate chemo- and radiation-resistance to treatment. The corollary effect of cystine

molecule, glutamate. This release has destructive consequences for the peritumoral brain. Glutamate induces neuronal and glial excitotoxic cell death, and acts in an autocrine and paracrine signalling manner to stimulate glioma cell growth and migration. Treatments based on these mechanisms are currently under development and some have progressed as

primary avenues of investigation. The potential treatment benefits of targeting these

glioma suggests that study of this pathway may produce wide-ranging cancer treatment


**1. Introduction** 

import *via* system xc

options.

**Release in Glioma** 

*McMaster University* 




Robert Ungard and Gurmit Singh

## **Oxidative Stress and Glutamate Release in Glioma**

Robert Ungard and Gurmit Singh *McMaster University Canada* 

#### **1. Introduction**

Glioma are a family of glial cell tumours of the central nervous system (CNS) wellcharacterized as aggressive cancers with dismally limited treatment options. The relatively recent discoveries of mechanisms surrounding glioma cell antioxidant protection and neuronal and glial cell destruction have opened the gates to a new therapeutic avenue whose implications to the field of cancer biology extend far beyond the treatment of glioma alone. Stemming from the discovery of significant glutamate release and glutathione production by glioma cells, the mechanisms through which glioma mediate oxidative stress and influence their extracellular microenvironment are now being unravelled.

This chapter will discuss the upregulation of the cystine/glutamate antiporter, system xc -, in glioma and the far ranging consequences that stem from this compensatory action. Specifically, a characteristic shift of cancer cell metabolism away from the tricarboxylic acid (TCA) cycle and towards increased rates of glycolysis, a process termed the Warburg effect, produces a high amount of reactive oxygen species (ROS) that would prove cytotoxic without adequate cellular antioxidant defences. In response to this metabolic abnormality, glioma have demonstrated increased synthesis of the primary cellular antioxidant glutathione and an increased circulation of the cystine/cysteine redox cycle. These antioxidant increases are driven by an upregulation of system xc -, which supplies the cell with the rate limiting substrate for glutathione synthesis, cysteine, and acts as one half of the transport machinery for the cystine/cysteine cycle. The increased tolerance to oxidative stress that is conferred by these mechanisms allows glioma survival and growth advantages and mediate chemo- and radiation-resistance to treatment. The corollary effect of cystine import *via* system xc - is the export of the neurotransmitter and ubiquitous cell-signalling molecule, glutamate. This release has destructive consequences for the peritumoral brain. Glutamate induces neuronal and glial excitotoxic cell death, and acts in an autocrine and paracrine signalling manner to stimulate glioma cell growth and migration. Treatments based on these mechanisms are currently under development and some have progressed as far as clinical trials. Glutamate receptor antagonists and system xc - inhibitors are as of yet the primary avenues of investigation. The potential treatment benefits of targeting these pathways are great, and the discovery of system xc - prevalence in other cancers beyond glioma suggests that study of this pathway may produce wide-ranging cancer treatment options.

Oxidative Stress and Glutamate Release in Glioma 453

In several cancers including glioma, cysteine must be obtained through the import of cystine from the extracellular environment. Cystine is imported into the cell *via* the system xc

cystine/glutamate antiporter; a transporter that is a feature of many cancer cell lines and endogenous to many tissues in the body. Cystine is the oxidized form of the amino acid, comprised of two cysteine molecules joined by a covalent double bond, and more prevalent in the oxidizing extracellular space. In the reducing environment of the cell, imported cystine is rapidly reduced to cysteine which is then incorporated as a substrate in GSH biosynthesis or serves to propel the cystine/cysteine redox cycle that plays a large role in maintaining extracellular redox balance (Ishii et al., 1992). The exported glutamate can have a number of deleterious effects upon the surrounding host tissue, many of which favour




the heavy subunit is 4F2hc



glutamate first described in human fibroblasts by Bannai & Kitamura (1980), and later named by Makowske & Christensen (1982). It is classified within the family of heteromeric amino acid transporters, all of which are comprised of a single heavy polypeptide subunit (SLC3 family) and a single light subunit (SLC7 family) coupled *via* a disulfide bridge (Chillarón et al., 2001). These transporters are essential for the import of amino acids to the

(SLC3A2), a type II membrane glycoprotein common to many amino acid transporters (Verrey et al., 2004). It plays a regulatory role, functioning to traffic and adhere the transporter complex to the cell membrane. It features one transmembrane domain, and has a molecular weight of ~85 kDa (Lim & Donaldson, 2010). 4F2hc is not essential to the

with similar transport and adherence capabilities, (ex. rBAT) without losing antiporter

entirely responsible for the amino acid exchange function of the transporter and unique to

(Lim & Donaldson, 2010). Cystine and glutamate are exchanged with a 1:1 stoichiometry that does not require an ionic gradient, rather it is thought that glutamate, which must be eliminated from the cytosol to prevent toxicity, provides the concentration gradient

brain, it is a feature of both neurons and glial cells. Specifically, xCT was found to be expressed in neurons of the cerebral cortex, GFAP positive glial cells, vascular endothelial

thought to be related to the organ's relatively high rate of glucose metabolism and the need for antioxidants to protect highly sensitive neurons from the resulting ROS production (Conrad & Sato, 2011). The expression of xCT fluctuates greatly, and can be readily induced under a number of stimuli including low levels of extracellular cystine (Bannai & Kitamura, 1982), and oxidative stress (Bannai et al., 1991). In astrocytes, the induced upregulation of xCT increased GSH synthesis and release and conferred antioxidant protection on immature

cells and the leptomeninges (Burdo et al., 2006). The prominence of system xc


is expressed endogenously in a number of tissues in the body. In the human


**- antiporter and glioma** 

cancer cell survival and progression (Ishii et al., 1992).

cell that cannot be intracellularly synthesized. In system xc

function (Wang et al., 2003). The light subunit of system xc

necessary for transporter function (Bannai & Ishii, 1988).

**3. The System xc**

**3.1 System xc**

System xc

system xc

**3.1.1 System xc**


System xc

**-**

transport action of system xc

 **in glia** 

#### **2. Glioma metabolism and oxidative stress**

Cancer cells exist under self-induced conditions of abnormally elevated oxidative stress resulting from a characteristic shift in glucose metabolism away from the TCA cycle and towards a high rate of aerobic glycolysis (Kroemer & Pouyssegur, 2008). This metabolic shift results in less efficient ATP production from glucose by the cell, but serves to confer unique benefits upon the cancer cell allowing survival in conditions of high proliferation, high oxidative stress, and varying access to blood vasculature. One adaptive characteristic of cancer cells is the upregulation of antioxidant defence mechanisms, necessary for protection from the high level of ROS generated by escalated glycolysis.

#### **2.1 Cancer cell metabolism**

In normal cells, the metabolism of glucose generates ATP through glycolysis followed by a high rate of pyruvate metabolism through the TCA cycle. The final electron acceptor in the TCA cycle is oxygen, without which, the cycle ceases to function, and pyruvate is converted to lactate *via* anaerobic glycolysis (Kim & Dang, 2006). Anaerobic glycolysis is prevalent in hypoxic environments when the TCA cycle has no access to oxygen, however, glycolysis is also predominant in cancer cells even during aerobic conditions (Kim & Dang, 2006). This phenomenon of cancer cell metabolism was first described in the 1920s by Nobel laureate Otto Warburg and is to this day termed the Warburg effect, or aerobic glycolysis (Warburg et al., 1927). Most cancer cells, limited with regards to energy production by their shift away from the efficiency of the TCA cycle, rely upon an increased rate of glucose uptake for glycolytic ATP production. This allows the cell a number of advantages including the use of glycolytic intermediates for anabolic reactions, without which, rapidly proliferating cells in conditions of fluctuating oxygen availability could not survive (See review by Kroemer & Pouyssegur, 2008). All aerobic respiration generates ROS which induces oxidative damage within the cell (Balendiran et al., 2004). The enhanced metabolic activity of cancer cells raises ROS production to a level that demands adaptation by the cell to survive and proliferate despite the resulting high level of oxidative stress (Halliwell, 2007). The Warburg effect has been identified as a key factor in the increased oxidative stress that cancer cells face, and has also been directly implicated in the activation of oncogenes and the loss of tumour suppressor genes (Le et al., 2010).

#### **2.2 Glutathione synthesis response**

Glutathione (GSH) is a tripeptide thiol synthesized intracellularly from the amino acids glutamate, cysteine and glycine. In the cell it performs a number of functions, one of which is as the predominant cellular antioxidant in the body (Meister, 1995). GSH fulfils this role by acting as a substrate for several antioxidant enzymes as well as by acting directly upon free radicals in its reduced form, GSH, or in its oxidized form, glutathione disulfide (Meister, 1995). The rate-limiting step in GSH biosynthesis is the availability of cysteine, which in glioma cannot be synthesized intracellularly (Ishii et al., 1992). In glioma, increased oxidation of intracellular GSH and elevated oxidative stress induce the upregulation of cystine transport into the cell, allowing the dual processes of increased GSH biosynthesis, and increased cycling of the cystine/cysteine redox cycle, both of which counter the effects of ROS mediated damage (Banjac et al., 2008; Chung et al., 2005).

#### **3. The System xc - antiporter and glioma**

In several cancers including glioma, cysteine must be obtained through the import of cystine from the extracellular environment. Cystine is imported into the cell *via* the system xc cystine/glutamate antiporter; a transporter that is a feature of many cancer cell lines and endogenous to many tissues in the body. Cystine is the oxidized form of the amino acid, comprised of two cysteine molecules joined by a covalent double bond, and more prevalent in the oxidizing extracellular space. In the reducing environment of the cell, imported cystine is rapidly reduced to cysteine which is then incorporated as a substrate in GSH biosynthesis or serves to propel the cystine/cysteine redox cycle that plays a large role in maintaining extracellular redox balance (Ishii et al., 1992). The exported glutamate can have a number of deleterious effects upon the surrounding host tissue, many of which favour cancer cell survival and progression (Ishii et al., 1992).

#### **3.1 System xc -**

452 Glioma – Exploring Its Biology and Practical Relevance

Cancer cells exist under self-induced conditions of abnormally elevated oxidative stress resulting from a characteristic shift in glucose metabolism away from the TCA cycle and towards a high rate of aerobic glycolysis (Kroemer & Pouyssegur, 2008). This metabolic shift results in less efficient ATP production from glucose by the cell, but serves to confer unique benefits upon the cancer cell allowing survival in conditions of high proliferation, high oxidative stress, and varying access to blood vasculature. One adaptive characteristic of cancer cells is the upregulation of antioxidant defence mechanisms, necessary for protection

In normal cells, the metabolism of glucose generates ATP through glycolysis followed by a high rate of pyruvate metabolism through the TCA cycle. The final electron acceptor in the TCA cycle is oxygen, without which, the cycle ceases to function, and pyruvate is converted to lactate *via* anaerobic glycolysis (Kim & Dang, 2006). Anaerobic glycolysis is prevalent in hypoxic environments when the TCA cycle has no access to oxygen, however, glycolysis is also predominant in cancer cells even during aerobic conditions (Kim & Dang, 2006). This phenomenon of cancer cell metabolism was first described in the 1920s by Nobel laureate Otto Warburg and is to this day termed the Warburg effect, or aerobic glycolysis (Warburg et al., 1927). Most cancer cells, limited with regards to energy production by their shift away from the efficiency of the TCA cycle, rely upon an increased rate of glucose uptake for glycolytic ATP production. This allows the cell a number of advantages including the use of glycolytic intermediates for anabolic reactions, without which, rapidly proliferating cells in conditions of fluctuating oxygen availability could not survive (See review by Kroemer & Pouyssegur, 2008). All aerobic respiration generates ROS which induces oxidative damage within the cell (Balendiran et al., 2004). The enhanced metabolic activity of cancer cells raises ROS production to a level that demands adaptation by the cell to survive and proliferate despite the resulting high level of oxidative stress (Halliwell, 2007). The Warburg effect has been identified as a key factor in the increased oxidative stress that cancer cells face, and has also been directly implicated in the activation of oncogenes and the loss of tumour

Glutathione (GSH) is a tripeptide thiol synthesized intracellularly from the amino acids glutamate, cysteine and glycine. In the cell it performs a number of functions, one of which is as the predominant cellular antioxidant in the body (Meister, 1995). GSH fulfils this role by acting as a substrate for several antioxidant enzymes as well as by acting directly upon free radicals in its reduced form, GSH, or in its oxidized form, glutathione disulfide (Meister, 1995). The rate-limiting step in GSH biosynthesis is the availability of cysteine, which in glioma cannot be synthesized intracellularly (Ishii et al., 1992). In glioma, increased oxidation of intracellular GSH and elevated oxidative stress induce the upregulation of cystine transport into the cell, allowing the dual processes of increased GSH biosynthesis, and increased cycling of the cystine/cysteine redox cycle, both of which counter the effects

**2. Glioma metabolism and oxidative stress** 

from the high level of ROS generated by escalated glycolysis.

**2.1 Cancer cell metabolism** 

suppressor genes (Le et al., 2010).

**2.2 Glutathione synthesis response** 

of ROS mediated damage (Banjac et al., 2008; Chung et al., 2005).

System xc - is the name given to the Na+ independent electroneutral exchanger of cystine and glutamate first described in human fibroblasts by Bannai & Kitamura (1980), and later named by Makowske & Christensen (1982). It is classified within the family of heteromeric amino acid transporters, all of which are comprised of a single heavy polypeptide subunit (SLC3 family) and a single light subunit (SLC7 family) coupled *via* a disulfide bridge (Chillarón et al., 2001). These transporters are essential for the import of amino acids to the cell that cannot be intracellularly synthesized. In system xc - the heavy subunit is 4F2hc (SLC3A2), a type II membrane glycoprotein common to many amino acid transporters (Verrey et al., 2004). It plays a regulatory role, functioning to traffic and adhere the transporter complex to the cell membrane. It features one transmembrane domain, and has a molecular weight of ~85 kDa (Lim & Donaldson, 2010). 4F2hc is not essential to the transport action of system xc - , and can be supplanted with another heavy chain polypeptide with similar transport and adherence capabilities, (ex. rBAT) without losing antiporter function (Wang et al., 2003). The light subunit of system xc - is xCT (SLC7A11), which is entirely responsible for the amino acid exchange function of the transporter and unique to system xc -. It features 12 transmembrane domains, and has a molecular weight of ~55 kDa (Lim & Donaldson, 2010). Cystine and glutamate are exchanged with a 1:1 stoichiometry that does not require an ionic gradient, rather it is thought that glutamate, which must be eliminated from the cytosol to prevent toxicity, provides the concentration gradient necessary for transporter function (Bannai & Ishii, 1988).

#### **3.1.1 System xc in glia**

System xc - is expressed endogenously in a number of tissues in the body. In the human brain, it is a feature of both neurons and glial cells. Specifically, xCT was found to be expressed in neurons of the cerebral cortex, GFAP positive glial cells, vascular endothelial cells and the leptomeninges (Burdo et al., 2006). The prominence of system xc - in the brain is thought to be related to the organ's relatively high rate of glucose metabolism and the need for antioxidants to protect highly sensitive neurons from the resulting ROS production (Conrad & Sato, 2011). The expression of xCT fluctuates greatly, and can be readily induced under a number of stimuli including low levels of extracellular cystine (Bannai & Kitamura, 1982), and oxidative stress (Bannai et al., 1991). In astrocytes, the induced upregulation of xCT increased GSH synthesis and release and conferred antioxidant protection on immature

Oxidative Stress and Glutamate Release in Glioma 455

Fig. 1. Summary concept model demonstrating the impact of altered cancer cell metabolism

import of cystine and export of glutamate. Cystine import allows the synthesis of GSH and the cycling of the cystine/cysteine redox cycle. The export of glutamate has cytotoxic effects on brain cells within the tumour microenvironment, and autocrine and paracrine effects on


and cytotoxic treatments on cellular ROS, the upregulation of system xc

the glioma initiating growth and increased migration.

neurons in an *in vitro* co-culture model (Shih et al., 2006). Due to its critical role in maintaining antioxidant and glutamate balance, the misregulation of system xc - has the potential for great damage, and the transporter has been implicated in a number of CNS pathologies, including some characteristic features of morbidity in glioma.

#### **3.1.2 System xc - in glioma**

The ability of xCT to be readily induced upon exposure to oxidative stress or cysteine deficit is thought to be responsible for the presence of system xc - as a cell-culture induced artifact in some cell lines. In glioma cell lines this is not the case; system xc - has not only been demonstrated in established cell lines (Chung et al., 2005), but also in normal glia (Burdo et al., 2006), and in glioma tumour samples from patients (Lyons et al., 2007).

In glioma, glucose metabolism is significantly escalated, as is characteristic of cancer cell metabolism. The resulting increase in the production of ROS from aerobic glycolysis induces the upregulation of xCT expression (Kim et al., 2001). With 4F2hc present in abundance, this xCT increase is sufficient to initiate an upregulation of system xc - characteristic of glioma (Sontheimer, 2008). The consequences of this system xc upregulation are immensely detrimental to the patient as a result of both of the substrate actions of the antiporter. The greater import of cystine by system xc - allows the cell to survive and proliferate in conditions of oxidative stress that would be lethal to other cells. This increased resistance has a destructive outcome for the patient, allowing the glioma to survive and progress to a greater extent, and endowing the cell with resistance to cancer treatments, many of which attack cancer cells through increased oxidative stress. The simultaneous export of high levels of glutamate into the microenvironment of glutamate-sensitive brain tissues induces neuron and glial cell death and promotes the growth and migration of the tumour. These uniquely destructive outcomes from the action of system xc - ultimately aid the progression of the cancer. (See Fig. 1 concept model).

#### **4. System xc and oxidative stress in glioma**

The upregulation of xCT readily occurs in response to oxidative stress. To mediate oxidative damage, the system xc - antiporter acts in two cystine-dependent manners to provide antioxidant capabilities to the cell and surrounding microenvironment. By increasing the availability of intracellular cysteine, this rate-limiting substrate is provided for both the synthesis of GSH, and for the completion of one half of the cystine/cysteine redox cycle.

#### **4.1 System xc drives glutathione synthesis**

Within the cell, the tripeptide GSH is synthesized from its constituent amino acids, glycine, glutamate and cysteine *via* the enzymes -glutamylcysteine synthetase, adding glutamate; and glutathione synthetase, adding glycine in two steps (See Fig. 2). Oxidized GSH can be reduced back to its active form *via* glutathione reductase (Conrad & Sato, 2011). The ratelimiting factor in this pathway is the availability of intracellular cysteine (Ishii et al., 1987). Most mammalian cells have the ability to directly import cysteine with a number of transporters (Lo et al., 2008), however cysteine is not prevalent in the extracellular space to the degree of cystine. Upon export from the cell, cysteine, the reduced and more prominent intracellular form of the amino acid, is rapidly oxidized to cystine, which is vastly more

neurons in an *in vitro* co-culture model (Shih et al., 2006). Due to its critical role in

potential for great damage, and the transporter has been implicated in a number of CNS

The ability of xCT to be readily induced upon exposure to oxidative stress or cysteine deficit

demonstrated in established cell lines (Chung et al., 2005), but also in normal glia (Burdo et

In glioma, glucose metabolism is significantly escalated, as is characteristic of cancer cell metabolism. The resulting increase in the production of ROS from aerobic glycolysis induces the upregulation of xCT expression (Kim et al., 2001). With 4F2hc present in abundance, this

detrimental to the patient as a result of both of the substrate actions of the antiporter. The

of oxidative stress that would be lethal to other cells. This increased resistance has a destructive outcome for the patient, allowing the glioma to survive and progress to a greater extent, and endowing the cell with resistance to cancer treatments, many of which attack cancer cells through increased oxidative stress. The simultaneous export of high levels of glutamate into the microenvironment of glutamate-sensitive brain tissues induces neuron and glial cell death and promotes the growth and migration of the tumour. These uniquely

The upregulation of xCT readily occurs in response to oxidative stress. To mediate

provide antioxidant capabilities to the cell and surrounding microenvironment. By increasing the availability of intracellular cysteine, this rate-limiting substrate is provided for both the synthesis of GSH, and for the completion of one half of the cystine/cysteine

Within the cell, the tripeptide GSH is synthesized from its constituent amino acids, glycine, glutamate and cysteine *via* the enzymes -glutamylcysteine synthetase, adding glutamate; and glutathione synthetase, adding glycine in two steps (See Fig. 2). Oxidized GSH can be reduced back to its active form *via* glutathione reductase (Conrad & Sato, 2011). The ratelimiting factor in this pathway is the availability of intracellular cysteine (Ishii et al., 1987). Most mammalian cells have the ability to directly import cysteine with a number of transporters (Lo et al., 2008), however cysteine is not prevalent in the extracellular space to the degree of cystine. Upon export from the cell, cysteine, the reduced and more prominent intracellular form of the amino acid, is rapidly oxidized to cystine, which is vastly more









maintaining antioxidant and glutamate balance, the misregulation of system xc

pathologies, including some characteristic features of morbidity in glioma.

some cell lines. In glioma cell lines this is not the case; system xc

xCT increase is sufficient to initiate an upregulation of system xc

 **and oxidative stress in glioma** 

 **drives glutathione synthesis** 

(Sontheimer, 2008). The consequences of this system xc

destructive outcomes from the action of system xc

al., 2006), and in glioma tumour samples from patients (Lyons et al., 2007).

**3.1.2 System xc**

**-** 

greater import of cystine by system xc

cancer. (See Fig. 1 concept model).

oxidative damage, the system xc

**-**

**-**

**4. System xc**

redox cycle.

**4.1 System xc**

**in glioma** 

is thought to be responsible for the presence of system xc

Fig. 1. Summary concept model demonstrating the impact of altered cancer cell metabolism and cytotoxic treatments on cellular ROS, the upregulation of system xc -, and the consequent import of cystine and export of glutamate. Cystine import allows the synthesis of GSH and the cycling of the cystine/cysteine redox cycle. The export of glutamate has cytotoxic effects on brain cells within the tumour microenvironment, and autocrine and paracrine effects on the glioma initiating growth and increased migration.

Oxidative Stress and Glutamate Release in Glioma 457

As cysteine is rate-limiting in GSH synthesis, it is well expected that increased availability of

GSH levels. Increased cystine import in glioma has also been demonstrated to drive the cystine/cysteine redox cycle across the cell membrane, which acts independently of GSH to

promptly reduced in the cytoplasm, likely by GSH, and conversely cysteine is exported by the amino acid transporters system-L or system ASC to the extracellular environment where it is promptly oxidized (Conrad & Sato, 2011). It was discovered in xCT induced lymphoma cells that the cystine/cysteine cycle raised concentrations of extracellular cysteine and acted as an effective antioxidant even in cases of GSH depletion (Banjac et al., 2008). A subsequent study found that in cells negative for γ-glutamylcysteine synthetase and therefore unable to produce GSH, the cystine/cysteine cycle was sufficient to maintain oxidative stress protection (Mandal et al., 2010). This suggests that alternative redox systems can compensate for each other to the point of redundancy, and in this case, both cycles are driven by the import of cystine (Mandal et al., 2010). Both this redox cycle and GSH

The upregulation of antioxidant defences in glioma cells confers proliferation and survival benefits to glioma above those of normal cells without which, glioma could not thrive in their self-induced oxidative environment. The ability of glioma to upregulate antioxidant production in the face of ROS has long been suspected to contribute to the chemotherapy and radiation-resistance that is devastatingly common in the treatment of glioma, a condition already characterized by poor prognoses (Sontheimer, 2008). A large scale microarray to coordinate transporter gene expression in 60 cancer cell lines with the activity of 1400 anticancer drugs revealed 39 drugs that positively correlate with SLC7a11 (xCT) expression and 296 that negatively correlate (Huang & Sadée, 2006). An example of a positively correlating drug is L-alanosine, an amino acid analogue whose uptake is mediated by system






inhibition

mediated uptake. A negatively correlating drug is



 **drives the cystine/cysteine redox cycle** 

counter ROS. To cycle the amino acid , cystine is imported by system xc

**4.2 System xc**

**-**

the amino acid from system xc

oxidative stress to the cell.

xc -

system xc


**-**

The corollary effect of system xc

 **and glutamate** 

glutamate into the extracellular space, without which system xc

**5. System xc**

synthesis are enabled by the actions of system xc

. The authors demonstrated that pharmacologic system xc


is at least in part mediated through the availability of GSH (Pham et al., 2010).

geldanamycin, an antibiotic that targets heat shock protein 90 (Hsp90). System xc

increased the efficacy of geldanamycin through a reduction of intracellular GSH which reduced cellular resistance to the drug's cytotoxicity (Huang et al., 2005). Celastrol is another Hsp90 targeting drug that has demonstrated antitumoral properties specifically in glioma, and is also very negatively correlated with SLC7a11 expression (Huang et al., 2008). Inhibition of

did other negative modulators of GSH synthesis, indicating that celastrol resistance in glioma

demonstrated that glioma cells secrete amounts of glutamate *via* this mechanism that are significant enough to mediate excitotoxic cell death in the brain (Sontheimer, 2003; Takano

in celastrol-resistant glioma cells reduced chemoresistance to celastrol treatment, as

**4.3 Consequences of ROS resistance** 

L-alanosine by impeding its system xc

Fig. 2. Glutathione biosynthesis and the cystine/cysteine redox cycle as driven by amino acid transporters in glioma. Glutamate is secreted by system xc which requires both substrates to function.

common in circulation (Bannai & Ishii, 1988). Not all cells possess the molecular machinery to import cystine, however, many brain cells and consequently, glioma with their high expression of system xc - and abundance of intracellular glutamate have both the mechanisms and the gradient to drive cystine transport. Once inside the cell, cystine is reduced to cysteine where it can be incorporated into polypeptide synthesis including the synthesis of GSH (Savaskan & Eyüpoglu, 2010). System xc - is one of many cystine transporters in the CNS, however it has been identified as the only cystine transporter expressed in glioma (Chung et al., 2005). Many cancers including glioma have demonstrated increased basal levels of intracellular GSH (Louw et al., 1997). Pharmacological inhibition of system xc -, and therefore limitation or elimination of available intracellular cysteine is able to deplete intracellular GSH almost entirely in a dose and time-dependent manner in glioma cell lines (Chung et al., 2005; Chung & Sontheimer, 2009; Pham et al., 2010). The negative effects of this GSH limitation on cell growth can be rescued entirely by the introduction of membrane permeable exogenous GSH, suggesting that cysteine availability for GSH production is critical for glioma cell growth (Chung & Sontheimer, 2009).

Fig. 2. Glutathione biosynthesis and the cystine/cysteine redox cycle as driven by amino

common in circulation (Bannai & Ishii, 1988). Not all cells possess the molecular machinery to import cystine, however, many brain cells and consequently, glioma with their high

and the gradient to drive cystine transport. Once inside the cell, cystine is reduced to cysteine where it can be incorporated into polypeptide synthesis including the synthesis of

CNS, however it has been identified as the only cystine transporter expressed in glioma (Chung et al., 2005). Many cancers including glioma have demonstrated increased basal levels of intracellular GSH (Louw et al., 1997). Pharmacological inhibition of system xc

therefore limitation or elimination of available intracellular cysteine is able to deplete intracellular GSH almost entirely in a dose and time-dependent manner in glioma cell lines (Chung et al., 2005; Chung & Sontheimer, 2009; Pham et al., 2010). The negative effects of this GSH limitation on cell growth can be rescued entirely by the introduction of membrane permeable exogenous GSH, suggesting that cysteine availability for GSH production is





acid transporters in glioma. Glutamate is secreted by system xc

critical for glioma cell growth (Chung & Sontheimer, 2009).

GSH (Savaskan & Eyüpoglu, 2010). System xc

substrates to function.

expression of system xc

#### **4.2 System xc drives the cystine/cysteine redox cycle**

As cysteine is rate-limiting in GSH synthesis, it is well expected that increased availability of the amino acid from system xc - upregulation would have the observed positive impact on GSH levels. Increased cystine import in glioma has also been demonstrated to drive the cystine/cysteine redox cycle across the cell membrane, which acts independently of GSH to counter ROS. To cycle the amino acid , cystine is imported by system xc -, where it is promptly reduced in the cytoplasm, likely by GSH, and conversely cysteine is exported by the amino acid transporters system-L or system ASC to the extracellular environment where it is promptly oxidized (Conrad & Sato, 2011). It was discovered in xCT induced lymphoma cells that the cystine/cysteine cycle raised concentrations of extracellular cysteine and acted as an effective antioxidant even in cases of GSH depletion (Banjac et al., 2008). A subsequent study found that in cells negative for γ-glutamylcysteine synthetase and therefore unable to produce GSH, the cystine/cysteine cycle was sufficient to maintain oxidative stress protection (Mandal et al., 2010). This suggests that alternative redox systems can compensate for each other to the point of redundancy, and in this case, both cycles are driven by the import of cystine (Mandal et al., 2010). Both this redox cycle and GSH synthesis are enabled by the actions of system xc - and in glioma, both confer protection from oxidative stress to the cell.

#### **4.3 Consequences of ROS resistance**

The upregulation of antioxidant defences in glioma cells confers proliferation and survival benefits to glioma above those of normal cells without which, glioma could not thrive in their self-induced oxidative environment. The ability of glioma to upregulate antioxidant production in the face of ROS has long been suspected to contribute to the chemotherapy and radiation-resistance that is devastatingly common in the treatment of glioma, a condition already characterized by poor prognoses (Sontheimer, 2008). A large scale microarray to coordinate transporter gene expression in 60 cancer cell lines with the activity of 1400 anticancer drugs revealed 39 drugs that positively correlate with SLC7a11 (xCT) expression and 296 that negatively correlate (Huang & Sadée, 2006). An example of a positively correlating drug is L-alanosine, an amino acid analogue whose uptake is mediated by system xc - . The authors demonstrated that pharmacologic system xc - inhibition reduced the efficacy of L-alanosine by impeding its system xc mediated uptake. A negatively correlating drug is geldanamycin, an antibiotic that targets heat shock protein 90 (Hsp90). System xc inhibition increased the efficacy of geldanamycin through a reduction of intracellular GSH which reduced cellular resistance to the drug's cytotoxicity (Huang et al., 2005). Celastrol is another Hsp90 targeting drug that has demonstrated antitumoral properties specifically in glioma, and is also very negatively correlated with SLC7a11 expression (Huang et al., 2008). Inhibition of system xc in celastrol-resistant glioma cells reduced chemoresistance to celastrol treatment, as did other negative modulators of GSH synthesis, indicating that celastrol resistance in glioma is at least in part mediated through the availability of GSH (Pham et al., 2010).

#### **5. System xc and glutamate**

The corollary effect of system xc - mediated cystine uptake is the necessary secretion of glutamate into the extracellular space, without which system xc - cannot function. It has been demonstrated that glioma cells secrete amounts of glutamate *via* this mechanism that are significant enough to mediate excitotoxic cell death in the brain (Sontheimer, 2003; Takano

Oxidative Stress and Glutamate Release in Glioma 459

et al., 2008). Excitotoxicity is thought to be initiated as a result of excessive activation of glutamate receptors resulting in the uncontrolled increase of intracellular Ca2+ which stimulates the activation of cytotoxic enzymes (Choi, 1988). Neurons possess both the ionotropic α-amino-3-hydroxy-5-methyl-4-isoaxazolepropionate acid (AMPA) glutamate receptors, and N-methyl-D-aspartate (NMDA) glutamate receptors for excitatory glutamate signal transmission. Neurons in coculture and *in vivo* were shown to be highly sensitive to excitotoxic cell death when exposed to glutamate release from glioma (Takano et al., 2001). Treatment with the NMDA receptor antagonist MK801 reduced but did not entirely

Normal glial cells are also highly receptive to glutamate. Oligodendrocytes demonstrate a similar low tolerance to glutamate exposure as neurons, while astrocytes can tolerate much higher concentrations (Oka et al., 1993). Astrocytes normally function to remove glutamate from the extracellular space, so their tolerance to high glutamate concentrations is not surprising; however they too are eventually killed by an expanding glioma. Whether this cell death is also mediated by exposure to glutamate is not yet understood (de Groot &

It has also been reported that glutamate may have an autocrine or paracrine signalling effect on glioma cells. AMPA, NMDA, Kainate, and the metabotropic glutamate receptors mGluR3 and mGLuR5 have all been identified in glioma, and growth-effects have been demonstrated through manipulation of both AMPA and NMDA receptors. Most glioma express AMPA receptors that are permeable to Ca2+ upon activation by glutamate (Ishiuchi et al., 2007). Induced expression of the GluR2 receptor subunit which renders AMPA receptors Ca2+ impermeable sensitized glioma cells to apoptosis and reduced tumour growth *in vivo*, suggesting the ability of glioma-derived glutamate signalling through AMPA receptors to act in an autocrine/paracrine manner to stimulate cell growth (Ishiuchi

Exogenous glutamate has a stimulatory effect on growth when applied to glioma cells, and conversely, antiproliferative effects on glioma have been demonstrated individually with several AMPA receptor antagonists and several NMDA receptor antagonists (Rzeski et al., 2001). Inhibition of mGlu2/3 receptors with the antagonist LY341495 in glioma cells positive for both receptors also was able to reduce glioma cell growth both *in vitro* and *in vivo*  (Arcella et al., 2005). Taken together, these results obtained through the blockade of nearly all glutamate receptors expressed in glioma suggest a significant autocrine/paracrine effect

The myriad consequences originating from the upregulation of xCT in glioma have uncovered several novel possibilities for treatment of glioma. Any therapeutic targeting of the mechanisms of antioxidant production and glutamate release could prove to be critical in the treatment of glioma, as current therapies are limited in efficacy and often become redundant through acquired cell-resistance (Sontheimer, 2008). Symptom management may also arise from treating glutamate release, as it is hypothesized that frequent seizures, a morbidity that affects over 80% of glioma sufferers could be related to glutamate-induced hyperexcitability in the CNS, possibly in advance of neuron excitotoxic death and possibly

in the induction of these morbidities (Savaskan


and *in vivo,* indicating the role of system xc

eliminate this excitotoxicity (Takano et al., 2001).

on growth of glioma-derived glutamate.

an early indication of the cancer (de Groot & Sontheimer, 2010).

**6. Experimental therapeutics** 

Sontheimer, 2010).

et al., 2007).

et al., 2001; Ye & Sontheimer, 1999). The amino acid glutamate is most well known as the primary excitatory neurotransmitter in the CNS, however it also functions as a growth factor and motogen to different cell types in the brain (de Groot & Sontheimer, 2010), and mediates critical cell signalling in many non-neuronal tissues (Hinoi et al., 2004).

#### **5.1 Glutamate release**

The normal brain usually does not harbour extracellular glutamate in excess of 1-3µM, likely due to the glutamate reuptake mechanisms of glia (de Groot & Sontheimer, 2010). *In vitro*, astrocyte cultures demonstrate the ability to reduce extracellular glutamate concentrations to near 1µM from 92µM within 3 hours, while conversely several glioma cell lines raised extracellular glutamate to 400-500µM in a 12-hour period (Ye & Sontheimer, 1999). When neurons were grown in co-culture or treated with media from independent glioma cultures, neurons died from glutamate-mediated excitotoxicity (Ye & Sontheimer, 1999). In normal brain, glutamate released into the extracellular space is rapidly removed, either back into the presynaptic nerve terminal, or, more commonly, by glial cells *via* one of the excitatory amino acid transporters (EAAT1 or EAAT2) (Danbolt, 2001). Glutamate reuptake is a key feature of normal glial cells that surround the synaptic cleft, a mechanism that contributes to neuron protection and signal consistency (de Groot & Sontheimer, 2010). It has been demonstrated by microarray that EAAT2 expression in glioma is negatively correlated with tumour progression, and that induction of glioma with EAAT2 expression dose-dependently limits cell growth, suggesting that the loss of EAAT function in glioma cells may play a role in the accumulation of extracellular glutamate (de Groot et al., 2005). Glutamate release from glioma was confirmed *in vivo* through glioma cells implanted into rat brain. Glutamate was measured to be highest in peritumoral regions, significantly higher than in the normal brain and the tumour itself (Behrens et al., 2000; Takano et al., 2001). Cells of the same type cloned as to not release glutamate grew significantly smaller tumours than their glutamatereleasing counterparts (Takano et al., 2001). In glioma patients, despite conflicting reports, it appears that glutamate concentrations are significantly elevated in glioma in both the tumour (Behrens et al., 2000) and the peritumoural region (Roslin et al., 2003).

#### **5.2 Consequences of glutamate release**

Glutamate release into the peritumoral environment has a number of cytotoxic and cell signalling effects whose results are advantageous to glioma and seriously deleterious to the host. It has been suggested that glutamate release confers an adaptive advantage upon glioma (Sontheimer, 2003), but it is also possible that the release in great quantities of such a ubiquitous signalling molecule into a tissue that is highly sensitive to such molecules exerts a disruptive influence simply as a side-effect. This has also been demonstrated as a feature of glutamate-releasing cancers metastasized to bone, a tissue where glutamate is an important intercellular communication molecule (Seidlitz et al., 2010).

Glioma exist in an environment physically constrained to the cavity of the cranium, a space consumed by 85% tissue and 15% cerebrospinal fluid (CSF). To grow, glioma must create space to occupy, as compression cannot occur in a vessel filled with fluid. Glutamateinduced excitotoxic cell death is thought to be principally responsible for the clearance of brain cells along the tumour borders that allows glioma progression. Indicating the succeptability of brain tissues to glutamatergic disruption is that no cell type in the brain is without receptors for glutamate. The inhibition of system xc - in glioma significantly reduces extracellular glutamate levels, as well as neurodegeneration and cellular edema both *in vitro*

et al., 2001; Ye & Sontheimer, 1999). The amino acid glutamate is most well known as the primary excitatory neurotransmitter in the CNS, however it also functions as a growth factor and motogen to different cell types in the brain (de Groot & Sontheimer, 2010), and

The normal brain usually does not harbour extracellular glutamate in excess of 1-3µM, likely due to the glutamate reuptake mechanisms of glia (de Groot & Sontheimer, 2010). *In vitro*, astrocyte cultures demonstrate the ability to reduce extracellular glutamate concentrations to near 1µM from 92µM within 3 hours, while conversely several glioma cell lines raised extracellular glutamate to 400-500µM in a 12-hour period (Ye & Sontheimer, 1999). When neurons were grown in co-culture or treated with media from independent glioma cultures, neurons died from glutamate-mediated excitotoxicity (Ye & Sontheimer, 1999). In normal brain, glutamate released into the extracellular space is rapidly removed, either back into the presynaptic nerve terminal, or, more commonly, by glial cells *via* one of the excitatory amino acid transporters (EAAT1 or EAAT2) (Danbolt, 2001). Glutamate reuptake is a key feature of normal glial cells that surround the synaptic cleft, a mechanism that contributes to neuron protection and signal consistency (de Groot & Sontheimer, 2010). It has been demonstrated by microarray that EAAT2 expression in glioma is negatively correlated with tumour progression, and that induction of glioma with EAAT2 expression dose-dependently limits cell growth, suggesting that the loss of EAAT function in glioma cells may play a role in the accumulation of extracellular glutamate (de Groot et al., 2005). Glutamate release from glioma was confirmed *in vivo* through glioma cells implanted into rat brain. Glutamate was measured to be highest in peritumoral regions, significantly higher than in the normal brain and the tumour itself (Behrens et al., 2000; Takano et al., 2001). Cells of the same type cloned as to not release glutamate grew significantly smaller tumours than their glutamatereleasing counterparts (Takano et al., 2001). In glioma patients, despite conflicting reports, it appears that glutamate concentrations are significantly elevated in glioma in both the

mediates critical cell signalling in many non-neuronal tissues (Hinoi et al., 2004).

tumour (Behrens et al., 2000) and the peritumoural region (Roslin et al., 2003).

important intercellular communication molecule (Seidlitz et al., 2010).

without receptors for glutamate. The inhibition of system xc

Glutamate release into the peritumoral environment has a number of cytotoxic and cell signalling effects whose results are advantageous to glioma and seriously deleterious to the host. It has been suggested that glutamate release confers an adaptive advantage upon glioma (Sontheimer, 2003), but it is also possible that the release in great quantities of such a ubiquitous signalling molecule into a tissue that is highly sensitive to such molecules exerts a disruptive influence simply as a side-effect. This has also been demonstrated as a feature of glutamate-releasing cancers metastasized to bone, a tissue where glutamate is an

Glioma exist in an environment physically constrained to the cavity of the cranium, a space consumed by 85% tissue and 15% cerebrospinal fluid (CSF). To grow, glioma must create space to occupy, as compression cannot occur in a vessel filled with fluid. Glutamateinduced excitotoxic cell death is thought to be principally responsible for the clearance of brain cells along the tumour borders that allows glioma progression. Indicating the succeptability of brain tissues to glutamatergic disruption is that no cell type in the brain is

extracellular glutamate levels, as well as neurodegeneration and cellular edema both *in vitro*


**5.2 Consequences of glutamate release** 

**5.1 Glutamate release** 

and *in vivo,* indicating the role of system xc in the induction of these morbidities (Savaskan et al., 2008). Excitotoxicity is thought to be initiated as a result of excessive activation of glutamate receptors resulting in the uncontrolled increase of intracellular Ca2+ which stimulates the activation of cytotoxic enzymes (Choi, 1988). Neurons possess both the ionotropic α-amino-3-hydroxy-5-methyl-4-isoaxazolepropionate acid (AMPA) glutamate receptors, and N-methyl-D-aspartate (NMDA) glutamate receptors for excitatory glutamate signal transmission. Neurons in coculture and *in vivo* were shown to be highly sensitive to excitotoxic cell death when exposed to glutamate release from glioma (Takano et al., 2001). Treatment with the NMDA receptor antagonist MK801 reduced but did not entirely eliminate this excitotoxicity (Takano et al., 2001).

Normal glial cells are also highly receptive to glutamate. Oligodendrocytes demonstrate a similar low tolerance to glutamate exposure as neurons, while astrocytes can tolerate much higher concentrations (Oka et al., 1993). Astrocytes normally function to remove glutamate from the extracellular space, so their tolerance to high glutamate concentrations is not surprising; however they too are eventually killed by an expanding glioma. Whether this cell death is also mediated by exposure to glutamate is not yet understood (de Groot & Sontheimer, 2010).

It has also been reported that glutamate may have an autocrine or paracrine signalling effect on glioma cells. AMPA, NMDA, Kainate, and the metabotropic glutamate receptors mGluR3 and mGLuR5 have all been identified in glioma, and growth-effects have been demonstrated through manipulation of both AMPA and NMDA receptors. Most glioma express AMPA receptors that are permeable to Ca2+ upon activation by glutamate (Ishiuchi et al., 2007). Induced expression of the GluR2 receptor subunit which renders AMPA receptors Ca2+ impermeable sensitized glioma cells to apoptosis and reduced tumour growth *in vivo*, suggesting the ability of glioma-derived glutamate signalling through AMPA receptors to act in an autocrine/paracrine manner to stimulate cell growth (Ishiuchi et al., 2007).

Exogenous glutamate has a stimulatory effect on growth when applied to glioma cells, and conversely, antiproliferative effects on glioma have been demonstrated individually with several AMPA receptor antagonists and several NMDA receptor antagonists (Rzeski et al., 2001). Inhibition of mGlu2/3 receptors with the antagonist LY341495 in glioma cells positive for both receptors also was able to reduce glioma cell growth both *in vitro* and *in vivo*  (Arcella et al., 2005). Taken together, these results obtained through the blockade of nearly all glutamate receptors expressed in glioma suggest a significant autocrine/paracrine effect on growth of glioma-derived glutamate.

#### **6. Experimental therapeutics**

The myriad consequences originating from the upregulation of xCT in glioma have uncovered several novel possibilities for treatment of glioma. Any therapeutic targeting of the mechanisms of antioxidant production and glutamate release could prove to be critical in the treatment of glioma, as current therapies are limited in efficacy and often become redundant through acquired cell-resistance (Sontheimer, 2008). Symptom management may also arise from treating glutamate release, as it is hypothesized that frequent seizures, a morbidity that affects over 80% of glioma sufferers could be related to glutamate-induced hyperexcitability in the CNS, possibly in advance of neuron excitotoxic death and possibly an early indication of the cancer (de Groot & Sontheimer, 2010).

Oxidative Stress and Glutamate Release in Glioma 461

diagnosed glioma patients (de Groot & Sontheimer, 2010). The upcoming results of this trial

contribute greatly to the establishment of the role of this critical transporter in glioma

Glioma exist in conditions of high oxidative stress as a result of the metabolic shift away from the TCA cycle and towards increased rates of glycolysis. This metabolic shift, the Warburg effect, is characteristic of cancer cells and confers unique benefits to the cell which allow survival and proliferation in conditions of rapid growth, division and variable access to blood vasculature. However, a result of this reliance on glycolysis is the increased prevalence of ROS in the cancer cell. To survive these conditions, cancer cells must possess upregulated mechanisms of antioxidation. Glioma exhibit an oxidative stress-mediated upregulation of the xCT coding gene SLC7a11, which, along with the membrane-anchoring protein 4F2hc comprise the two subunits of the Na+ independent electroneutral

that is ultimately responsible for several of the features of morbidity in glioma, as well as its characteristic chemo- and radiation-resistance. The import of cystine into the cell increases the availability of cysteine for the GSH synthesis pathway and for the cystine/cysteine redox cycle. These two antioxidant pathways function to relieve the glioma cell of significant oxidative stress, allowing increased proliferation and survival of the cancer cells. This also allows resistance of glioma to oxidative stress-inducing radiation and chemotherapies. Conversely, the export of glutamate results in the neurotoxic death of neurons and glial cells in the vicinity of the tumour, and acts in an autocrine/paracrine manner to stimulate glioma proliferation and migration. Therapies are currently under development with these mechanisms in mind. Glutamate receptor antagonists have been demonstrated to limit brain cell death and to inhibit tumour growth *in vitro* and in xenograft

antioxidant purposes and also prevent the release of glutamate into the extracellular environment have also demonstrated success *in vitro* and *in vivo*, and a clinical trial is currently underway with the inhibitor sulfasalazine. This work opens new pathways for investigation in a condition well known for poor prognoses and limited treatment options.

Arcella, A., Carpinelli, G., Battaglia, G., D'Onofrio, M., Santoro, F., Ngomba, R. T., et al.

Banjac, A., Perisic, T, Sato, H, Seiler, A, Bannai, S, Weiss, N, et al. (2008). The

reduces the growth of glioma cells in vivo. *Neuro-oncology*, *7*(3), 236-45. Balendiran, G. K., Dabur, R., & Fraser, D. (2004). The role of glutathione in cancer. *Cell* 

(2005). Pharmacological blockade of group II metabotropic glutamate receptors

cystine/cysteine cycle: a redox cycle regulating susceptibility versus resistance to

mechanism may soon become of great importance to cancer treatment.

*biochemistry and function*, *22*(6), 343-52.

cell death. *Oncogene*, *27*, 1618-1628.





will be the first clinical evidence of treatments directed at system xc

morbidity and treatment.

cystine/glutamate antiporter called system xc

animal models of glioma. System xc

The evidence of system xc

**8. References** 

**7. Conclusion** 

#### **6.1 Targeting glutamate receptors**

AMPA receptor targeting has emerged as the most prolific avenue of interest for treatment from glioma glutamate-release work. An AMPA antagonist called talampanel is currently the most likely candidate for glioma treatment in this manner in large part because it does not exhibit the side-effects of most glutamate receptor antagonists in the CNS, and it has been shown to increase the lifespan of mice xenografted with human glioma (Goudar et al., 2004). Two clinical trials have developed from these findings. The first, begun in 2009, was a phase II trial designed to examine the efficacy of talampanel in conjunction with standard radiation and temozolomide treatments in improving survival in adults with newly diagnosed glioblastoma (Grossman et al., 2009). This trial concluded that patients treated with talampanel demonstrated significantly longer survival than those who received standard care alone (Grossman et al., 2010). While this is promising and certainly demands further investigation, this study alone cannot be deemed conclusive. The second trial, a smaller phase II trial, examined the effects of talampanel alone on 6-month survival of patients with recurrent malignant glioma (Iwamoto et al., 2010). This trial determined that talampanel alone conferred no obvious advantage on patient survival, but the drug was tolerated well with no severe side-effects (Iwamoto et al., 2010).

Inhibitors of other glutamate receptors have not yet been clinically evaluated, however the preliminary success of animal models of glioma treatment as mentioned above will certainly lead to trials of other glutamate receptor antagonists in the near future. Significant promise is held by these inhibitors as both candidate adjuvant therapies capable of supplementing treatment cytotoxicity or of mediating the effects of glutamate on the brain.

#### **6.2 System xc - Inhibition**

While the above-mentioned therapies for mediating the excess glutamate released by glioma are promising, certainly the most attractive potential therapies to arise from these studies are those that involve the inhibition of system xc -. Rather than mediate the consequences of destructive glutamate release and treatment-resistance, system xc - inhibition could eliminate the function of the transporter responsible for the excess glutamate, and consequently limit the multiple morbidities of glutamate release rather than manage its downstream effects. In addition, system xc - inhibition would limit cysteine availability to the glioma cell and therefore inhibit its antioxidative capabilities by way of both limiting glutathione synthesis and halting the drive of the cystine/cysteine redox cycle. There are many chemical inhibitors of system xc - ; of these, the cyclic glutamate analogue S-(4)-carboxyphenylglycine has emerged as the most potent inhibitor (Patel et al., 2004), and the FDA approved antiinflammatory drug sulfasalazine has garnered the most clinical interest. Sulfasalazine has been demonstrated in animal models to effectively slow the growth of glioma and reduce levels of both intracellular GSH and extracellular glutamate (Chung et al., 2005; Chung & Sontheimer, 2009). A phase I clinical trial of sulfasalazine to evaluate drug safety and effects on tumour growth in the treatment of grade 3 glioma in a small number of patients was prematurely terminated due to several adverse effects during treatment (Robe et al., 2009; 2006). This study was initiated on the basis on sulfasalazine acting as an inhibitor of NFkB, however treatment did not differ from that required for system xc - inhibition. Although the poor outcomes from this trial are unfortunate, they do little to dampen the potential of sulfasalazine for glioma treatment or system xc - as a therapeutic target. Another phase I clinical trial has just recently been initiated with the intent to examine the effects of sulfasalazine on glutamate release in the brain, and on seizures in low-grade, newlydiagnosed glioma patients (de Groot & Sontheimer, 2010). The upcoming results of this trial will be the first clinical evidence of treatments directed at system xc - inhibition, and will contribute greatly to the establishment of the role of this critical transporter in glioma morbidity and treatment.

#### **7. Conclusion**

460 Glioma – Exploring Its Biology and Practical Relevance

AMPA receptor targeting has emerged as the most prolific avenue of interest for treatment from glioma glutamate-release work. An AMPA antagonist called talampanel is currently the most likely candidate for glioma treatment in this manner in large part because it does not exhibit the side-effects of most glutamate receptor antagonists in the CNS, and it has been shown to increase the lifespan of mice xenografted with human glioma (Goudar et al., 2004). Two clinical trials have developed from these findings. The first, begun in 2009, was a phase II trial designed to examine the efficacy of talampanel in conjunction with standard radiation and temozolomide treatments in improving survival in adults with newly diagnosed glioblastoma (Grossman et al., 2009). This trial concluded that patients treated with talampanel demonstrated significantly longer survival than those who received standard care alone (Grossman et al., 2010). While this is promising and certainly demands further investigation, this study alone cannot be deemed conclusive. The second trial, a smaller phase II trial, examined the effects of talampanel alone on 6-month survival of patients with recurrent malignant glioma (Iwamoto et al., 2010). This trial determined that talampanel alone conferred no obvious advantage on patient survival, but the drug was

Inhibitors of other glutamate receptors have not yet been clinically evaluated, however the preliminary success of animal models of glioma treatment as mentioned above will certainly lead to trials of other glutamate receptor antagonists in the near future. Significant promise is held by these inhibitors as both candidate adjuvant therapies capable of supplementing

While the above-mentioned therapies for mediating the excess glutamate released by glioma are promising, certainly the most attractive potential therapies to arise from these studies

the function of the transporter responsible for the excess glutamate, and consequently limit the multiple morbidities of glutamate release rather than manage its downstream effects. In

therefore inhibit its antioxidative capabilities by way of both limiting glutathione synthesis and halting the drive of the cystine/cysteine redox cycle. There are many chemical

has emerged as the most potent inhibitor (Patel et al., 2004), and the FDA approved antiinflammatory drug sulfasalazine has garnered the most clinical interest. Sulfasalazine has been demonstrated in animal models to effectively slow the growth of glioma and reduce levels of both intracellular GSH and extracellular glutamate (Chung et al., 2005; Chung & Sontheimer, 2009). A phase I clinical trial of sulfasalazine to evaluate drug safety and effects on tumour growth in the treatment of grade 3 glioma in a small number of patients was prematurely terminated due to several adverse effects during treatment (Robe et al., 2009; 2006). This study was initiated on the basis on sulfasalazine acting as an inhibitor of NFkB,

poor outcomes from this trial are unfortunate, they do little to dampen the potential of

clinical trial has just recently been initiated with the intent to examine the effects of sulfasalazine on glutamate release in the brain, and on seizures in low-grade, newly-


; of these, the cyclic glutamate analogue S-(4)-carboxyphenylglycine





**6.1 Targeting glutamate receptors** 

**6.2 System xc**

addition, system xc

inhibitors of system xc

**-**

 **Inhibition** 

are those that involve the inhibition of system xc


sulfasalazine for glioma treatment or system xc

tolerated well with no severe side-effects (Iwamoto et al., 2010).

destructive glutamate release and treatment-resistance, system xc

however treatment did not differ from that required for system xc

treatment cytotoxicity or of mediating the effects of glutamate on the brain.

Glioma exist in conditions of high oxidative stress as a result of the metabolic shift away from the TCA cycle and towards increased rates of glycolysis. This metabolic shift, the Warburg effect, is characteristic of cancer cells and confers unique benefits to the cell which allow survival and proliferation in conditions of rapid growth, division and variable access to blood vasculature. However, a result of this reliance on glycolysis is the increased prevalence of ROS in the cancer cell. To survive these conditions, cancer cells must possess upregulated mechanisms of antioxidation. Glioma exhibit an oxidative stress-mediated upregulation of the xCT coding gene SLC7a11, which, along with the membrane-anchoring protein 4F2hc comprise the two subunits of the Na+ independent electroneutral cystine/glutamate antiporter called system xc -. This antiporter drives the molecular turnover that is ultimately responsible for several of the features of morbidity in glioma, as well as its characteristic chemo- and radiation-resistance. The import of cystine into the cell increases the availability of cysteine for the GSH synthesis pathway and for the cystine/cysteine redox cycle. These two antioxidant pathways function to relieve the glioma cell of significant oxidative stress, allowing increased proliferation and survival of the cancer cells. This also allows resistance of glioma to oxidative stress-inducing radiation and chemotherapies. Conversely, the export of glutamate results in the neurotoxic death of neurons and glial cells in the vicinity of the tumour, and acts in an autocrine/paracrine manner to stimulate glioma proliferation and migration. Therapies are currently under development with these mechanisms in mind. Glutamate receptor antagonists have been demonstrated to limit brain cell death and to inhibit tumour growth *in vitro* and in xenograft animal models of glioma. System xc - inhibitors that prevent the import of cystine for antioxidant purposes and also prevent the release of glutamate into the extracellular environment have also demonstrated success *in vitro* and *in vivo*, and a clinical trial is currently underway with the inhibitor sulfasalazine. This work opens new pathways for investigation in a condition well known for poor prognoses and limited treatment options. The evidence of system xc - in other cancers in addition to glioma suggests that this mechanism may soon become of great importance to cancer treatment.

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**21** 

*Belarus* 

Vladimir A. Kulchitsky et al.\*

**Improving the Efficiency of Chemotherapeutic** 

*Institute of Physiology, National Academy of Sciences of Belarus, Minsk* 

**Drugs by the Action on Neuroepithelial Tumors** 

The problem of cancer embraces a lot of unresolved issues, among which there dominates the problem of ascertainment of the mechanisms of uncontrolled growth and cellular spill of tumor neoplasm, composed of dividing cancer cells and cancer stem cells (Schatton & Frank MH, 2009; Schatton et al., 2009; Frank NY et al., 2010). It is impossible to answer the question about a complete removal of the tumor tissue and simultaneous minimizing the adverse effects of surgical and other manipulations while removing the tumor without solving this problem. This is a particularly relevant goal for physicians who are engaged in treatment of brain tumors. The destruction of nerve tissue nonaffected by tumor growth has a negative impact on the integrative brain activity and at least on the central control of all bodily functions and homeostasis maintenance. How is it possible to reduce by-effects of major therapeutic technologies in neurooncology (surgical, radiological, chemotherapeutic),

Since you choose chemotherapy as one of the ways to impact on tumor tissue, it is impossible not to mention the commonly known toxic effect of chemotherapeutic agents on all body tissues. Destroying the tumor cells, cytotoxic agents kill healthy cells and tissues. Thus, local or systemic applying the chemotherapy leads inevitably to the destruction of healthy brain cells in case of the tumor localization within the cranial cavity and the spinal canal. Thus, one of the objectives of this work was to develop methodic of leveling the general toxic effects of chemotherapy while strengthening their local destructive

Tumors of the brain and spinal cord have extremely variety (set) of histological forms which are accounted for their origin from the elements of various tissues and for peculiarity of

\*Michael V. Talabaev2, Alexander N. Chernov1, Dmitry G. Grigoriev3, Yuri E. Demidchik4, Dmitry G. Shcharbin5, Nicholas M. Chekan6, Vladimir V.Kazbanov1, Tatiana A. Gurinovich1, Anatoly

*5Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Minsk; Belarus 6Physical-Technical Institute, National Academy of Sciences of Belarus, Minsk; Belarus 7Institute of Physical Organic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus*

having preserved or enhanced their selective tumor damaging action?

effect on tumor tissue. What ways have been chosen to solve this problem?

I. Gordienko6, Elena K. Sergeeva6, Vladimir I. Potkin7 and Vladimir N. Kalunov1 *1Institute of Physiology, National Academy of Sciences of Belarus, Minsk;Belarus 2 Clinical Hospital of Emergency, Minsk;Belarus* 

*4Belarusian Medical Academy of Post-Graduate Education, Minsk;Belarus* 

*3Belarusian State Medical University, Minsk;Belarus* 

**1. Introduction**


## **Improving the Efficiency of Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors**

Vladimir A. Kulchitsky et al.\*

*Institute of Physiology, National Academy of Sciences of Belarus, Minsk Belarus* 

#### **1. Introduction**

464 Glioma – Exploring Its Biology and Practical Relevance

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6372-7.

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metabolism and tumor microenvironment normalization. *Annals of anatomy =* 

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Cystine/glutamate exchange modulates glutathione supply for neuroprotection from oxidative stress and cell proliferation. *The Journal of neuroscience : the official* 

(2004). CATs and HATs: the SLC7 family of amino acid transporters. *Pflügers Archiv* 

Expression of the activity of cystine/glutamate exchange transporter, system x(c)(-), by xCT and rBAT. *Biochemical and biophysical research communications*, *305*(3), 611-8. Warburg, O., Wind, F., & Negelein, E. (1927). The metabolism of tumours in the body. *The*  The problem of cancer embraces a lot of unresolved issues, among which there dominates the problem of ascertainment of the mechanisms of uncontrolled growth and cellular spill of tumor neoplasm, composed of dividing cancer cells and cancer stem cells (Schatton & Frank MH, 2009; Schatton et al., 2009; Frank NY et al., 2010). It is impossible to answer the question about a complete removal of the tumor tissue and simultaneous minimizing the adverse effects of surgical and other manipulations while removing the tumor without solving this problem. This is a particularly relevant goal for physicians who are engaged in treatment of brain tumors. The destruction of nerve tissue nonaffected by tumor growth has a negative impact on the integrative brain activity and at least on the central control of all bodily functions and homeostasis maintenance. How is it possible to reduce by-effects of major therapeutic technologies in neurooncology (surgical, radiological, chemotherapeutic), having preserved or enhanced their selective tumor damaging action?

Since you choose chemotherapy as one of the ways to impact on tumor tissue, it is impossible not to mention the commonly known toxic effect of chemotherapeutic agents on all body tissues. Destroying the tumor cells, cytotoxic agents kill healthy cells and tissues. Thus, local or systemic applying the chemotherapy leads inevitably to the destruction of healthy brain cells in case of the tumor localization within the cranial cavity and the spinal canal. Thus, one of the objectives of this work was to develop methodic of leveling the general toxic effects of chemotherapy while strengthening their local destructive effect on tumor tissue. What ways have been chosen to solve this problem?

Tumors of the brain and spinal cord have extremely variety (set) of histological forms which are accounted for their origin from the elements of various tissues and for peculiarity of

<sup>\*</sup>Michael V. Talabaev2, Alexander N. Chernov1, Dmitry G. Grigoriev3, Yuri E. Demidchik4, Dmitry

G. Shcharbin5, Nicholas M. Chekan6, Vladimir V.Kazbanov1, Tatiana A. Gurinovich1, Anatoly

I. Gordienko6, Elena K. Sergeeva6, Vladimir I. Potkin7 and Vladimir N. Kalunov1

*<sup>1</sup>Institute of Physiology, National Academy of Sciences of Belarus, Minsk;Belarus 2 Clinical Hospital of Emergency, Minsk;Belarus* 

*<sup>3</sup>Belarusian State Medical University, Minsk;Belarus* 

*<sup>4</sup>Belarusian Medical Academy of Post-Graduate Education, Minsk;Belarus* 

*<sup>5</sup>Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Minsk; Belarus 6Physical-Technical Institute, National Academy of Sciences of Belarus, Minsk; Belarus 7Institute of Physical Organic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus*

Improving the Efficiency of

Polo, 1983; Xie et al., 2005).

2006).

Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors 467

deviation from the motor areas, speech, vision, hearing, stop of growth, and cognitive deficits with reduced intelligence), what in totality leads to disability (Kemper et al., 2004;

The postulates set forward underline the actuality of further improving of existing strategies and creating new ones for earlier diagnosis, prognosis and a more successful fight against cancer. One of the ways to the aim is a broad involvement in oncology of a very representative class of multifunctional endogenous biological regulators of peptide nature, generally called as growth factors. In own structure they number a series of families, among which are neurotrophins (Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophins 3, 4, 5 (NT), Transforming Growth Factor (TGF), Fibroblast Growth Factor ( FGF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Insulin-Like Growth factor (IGF), and others, rating more than 50 different variants (Antonelli et al., 2007; Beebe et al., 2003). The key role belongs to growth factors in the capacity of epigenetic "directive" signals in the control of such fundamental morphogenetic processes in ontogenesis as growth, survival, proliferation, differentiation (i.e., the selection of the terminal tract of specialization by stem and progenitor cells), guided migration and elongation processes, synaptogenesis, regulation of cell homeostasis by apoptosis, regeneration, as well as maintaining a normal cyto-biochemical status of mature cells and their resistance to damaging factors (Alam et al., 2010; Charles et al., 2011; Vinores & Perez-

The interest to determine the significance of these compounds in tumor formation and in their reverse transforming potential has just relatively recently emerged, when it became vivid that virtually all types of neoplasias are synthesized not only by growth factors but actively expressize their receptors (Antonelli et al., 2007; Barnes et al., 2009; Blum & Konnerth, 2005; Brossard et al., 2009; Evangelopoulos et al, 2004; Krűttgen et al., 2006; Nakagawara, 2001). At the same time we point out a startling fact: NGF was purified for the first time from sarcoma secretions S180, and protooncogenes sites, which take it, were purified from high-affinity TrkA and low relational P75 from biopsies of colon carcinoma (*intestinum crassum*) and human melanoma. It is much more interesting that bio testing of growth factors on its activity is performed on rat pheochromocytoma PC12 (Krűttgen et al.,

The experiments proved that *in vitro* the cell lines of neuroblastoma (IMR-32, SY-5Y, SK-N-SH, NB-GR) and medullary pheochromocytoma cease to divide in the presence of NGF and are transformed into neuron-like elements at braking formation of DNA, at the intensification of including labeled amino acids, at the appearance of sprouts, at the growth of size, substrate adhesion together with the formation of pseudoganglies and at the occurrence in membrane of electrical excitability (Krűttgen et al., 2006; Poluha et al., 1995). On the other hand, the ability of NGF *in vivo* to reduce the number of induced nitrosourea by neurinomas was revealed, as well as the speed of their development, and the ability to reduce the volume and to prolong animal survival after subcutaneous injection or intracerebral implantation of anaplastic glioma cell F98, T9, and neurinomas (Yaeger et al., 1992). The predictive value of identification of perceiving Neurotrophins receptors is overviewed. So thus the over expression of TrkA and C (for NT-3) in neuro-, medulla and glioblastomas promises a favorable outcome, due to a spontaneous regression (through differentiation), the inclusion of suicide programs or autophagy (Blum & Konnerth, 2005; Collins, 2004; Krűttgen et al., 2006; Yamaguchi et al., 2007). Patients with low or no detectable levels of these receptors in medulloblastoma exhibit 5-fold risk of death than with

Vega et al., 2003); vi) a limited range of available medications.

genesis not solved up to the end in respect to especially neuroepithelial tumors (Hirose & Yoshida, 2006; Nicholas et al., 2011; Van den Eynde et al., 2011). The nervous system tumors account for almost 10% of all human neoplasms, and up to 20% for children (Alomar, 2010; Davis et al., 2010; Myung et al., 2010). Neoplasms of brain and spinal cord are the most common solid tumors in childhood. They take the second place in frequency after leukemia of all child malignancies. In childhood (under 14 years) the vast majority of cases occur in neuroepithelial tumors. Of these, about 80% are of six histological forms: medulloblastoma, juvenile pilocytic astrocytoma, diffuse astrocytoma, ependymoma, craniopharyngioma, and neuroblastoma.

Among the tumors of neuroepithelial tissues 9 sub-groups are selected out: astrocytic, oligodendroglial, ependymal, oligoastrocytic tumors (mixed gliomas), tumors of vascular plexus, and other neuroepithelial tumors (glial tumors of unspecified origin), neuronal and neuronal-glial, pineal, and embryonal tumors. In most cases glial tumors (gliomas) such as astrocytic, oligodendroglial, and ependymal glioma ones are detected. Histological diagnosis was based on identifying the predominant cell type.

Combined treatment of the brain cancer (children including), which supposes surgery interference together with chemo- and radiotherapy, does not so far reach the desired impact. According to statistics concerning only medulloblastoma, the average diseasefree and overall survival during 2-7 years for patients up to 4 years old is respectively 46% and 54%. So, tiny comforting is aggravated by data that uniquely ascertain the growth of intracerebral cancer. Thus, in the U.S. during 30 years from 1973 to 2003 it increased from 4.1 to 5.2 for women and from 5.9 to 7.0 for men per 100 000 population (Alomar, 2010). Similar dynamics were recorded by The Child Cancer Registry of the Belarusian Republic. The percentage of brain tumors rose from 2.5 to 3.3 per 100 000 children living in Belarus from 1989 to 2005.

A number of intractable causes is due to a low curability of brain tumors. The most productive radical method of removal of cancer neoplasm is often limited by the fact of their location near vital centers. Courses of medical and radiation treatment in accordance with approved protocols HIT`91, PO/02-PO/04 (Dunkel et al., 2010; Rosenfeld et al., 2010) along with known positive ones embrace recognizable negative aspects. To the negative ones there pertain as follows: i) a low permeability of the blood-brain barrier being created by tight contacts of micro vessel intracerebral network, which became completed with a set of proteins (Claudine 1,5, Occludin and others), which are impermeable to hydrophilic low soluble compounds exceeding a diameter of 18 Å and a molecular weight of 180 Da. The vast majority of cytotoxic drugs pertain to them (Erdlenbruch et al., 2000; Kemper et al., 2004; Xie et al., 2005); ii) a weak selectivity of the concentration of cytostatic in the place of neoplasm (Cragnolini & Friedman, 2008); iii) an insufficiently studied mechanism of the action of traditional and new pharmacological agents, as well as their clearance, which hinders a reasonable estimation of the amount of molecules that come in direct contact with tumor cells (Gerstner & Fine, 2007; Ta et al., 2009); iv) a lack of unified schemes of medical product application, taking into account the morphological structure of complex heterogeneous neoplasias, stages of their malignancy, and individual sensitivity of the tumor cells to them and patient age (Alomar, 2010; Myung, et al., 2010); v) a symptomatic toxic side effect, reduced to a violation of general tissue metabolism and endocrine function, to immunity suppression, involvement in a destructive reaction of abnormal elements along with intact (healthy) cell ones, as well as the development of complications (acute arachnoiditis, meningo encephalopathy, renal failure, passing

genesis not solved up to the end in respect to especially neuroepithelial tumors (Hirose & Yoshida, 2006; Nicholas et al., 2011; Van den Eynde et al., 2011). The nervous system tumors account for almost 10% of all human neoplasms, and up to 20% for children (Alomar, 2010; Davis et al., 2010; Myung et al., 2010). Neoplasms of brain and spinal cord are the most common solid tumors in childhood. They take the second place in frequency after leukemia of all child malignancies. In childhood (under 14 years) the vast majority of cases occur in neuroepithelial tumors. Of these, about 80% are of six histological forms: medulloblastoma, juvenile pilocytic astrocytoma, diffuse astrocytoma, ependymoma, craniopharyngioma, and

Among the tumors of neuroepithelial tissues 9 sub-groups are selected out: astrocytic, oligodendroglial, ependymal, oligoastrocytic tumors (mixed gliomas), tumors of vascular plexus, and other neuroepithelial tumors (glial tumors of unspecified origin), neuronal and neuronal-glial, pineal, and embryonal tumors. In most cases glial tumors (gliomas) such as astrocytic, oligodendroglial, and ependymal glioma ones are detected.

Combined treatment of the brain cancer (children including), which supposes surgery interference together with chemo- and radiotherapy, does not so far reach the desired impact. According to statistics concerning only medulloblastoma, the average diseasefree and overall survival during 2-7 years for patients up to 4 years old is respectively 46% and 54%. So, tiny comforting is aggravated by data that uniquely ascertain the growth of intracerebral cancer. Thus, in the U.S. during 30 years from 1973 to 2003 it increased from 4.1 to 5.2 for women and from 5.9 to 7.0 for men per 100 000 population (Alomar, 2010). Similar dynamics were recorded by The Child Cancer Registry of the Belarusian Republic. The percentage of brain tumors rose from 2.5 to 3.3 per 100 000 children living in Belarus

A number of intractable causes is due to a low curability of brain tumors. The most productive radical method of removal of cancer neoplasm is often limited by the fact of their location near vital centers. Courses of medical and radiation treatment in accordance with approved protocols HIT`91, PO/02-PO/04 (Dunkel et al., 2010; Rosenfeld et al., 2010) along with known positive ones embrace recognizable negative aspects. To the negative ones there pertain as follows: i) a low permeability of the blood-brain barrier being created by tight contacts of micro vessel intracerebral network, which became completed with a set of proteins (Claudine 1,5, Occludin and others), which are impermeable to hydrophilic low soluble compounds exceeding a diameter of 18 Å and a molecular weight of 180 Da. The vast majority of cytotoxic drugs pertain to them (Erdlenbruch et al., 2000; Kemper et al., 2004; Xie et al., 2005); ii) a weak selectivity of the concentration of cytostatic in the place of neoplasm (Cragnolini & Friedman, 2008); iii) an insufficiently studied mechanism of the action of traditional and new pharmacological agents, as well as their clearance, which hinders a reasonable estimation of the amount of molecules that come in direct contact with tumor cells (Gerstner & Fine, 2007; Ta et al., 2009); iv) a lack of unified schemes of medical product application, taking into account the morphological structure of complex heterogeneous neoplasias, stages of their malignancy, and individual sensitivity of the tumor cells to them and patient age (Alomar, 2010; Myung, et al., 2010); v) a symptomatic toxic side effect, reduced to a violation of general tissue metabolism and endocrine function, to immunity suppression, involvement in a destructive reaction of abnormal elements along with intact (healthy) cell ones, as well as the development of complications (acute arachnoiditis, meningo encephalopathy, renal failure, passing

Histological diagnosis was based on identifying the predominant cell type.

neuroblastoma.

from 1989 to 2005.

deviation from the motor areas, speech, vision, hearing, stop of growth, and cognitive deficits with reduced intelligence), what in totality leads to disability (Kemper et al., 2004; Vega et al., 2003); vi) a limited range of available medications.

The postulates set forward underline the actuality of further improving of existing strategies and creating new ones for earlier diagnosis, prognosis and a more successful fight against cancer. One of the ways to the aim is a broad involvement in oncology of a very representative class of multifunctional endogenous biological regulators of peptide nature, generally called as growth factors. In own structure they number a series of families, among which are neurotrophins (Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophins 3, 4, 5 (NT), Transforming Growth Factor (TGF), Fibroblast Growth Factor ( FGF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Insulin-Like Growth factor (IGF), and others, rating more than 50 different variants (Antonelli et al., 2007; Beebe et al., 2003). The key role belongs to growth factors in the capacity of epigenetic "directive" signals in the control of such fundamental morphogenetic processes in ontogenesis as growth, survival, proliferation, differentiation (i.e., the selection of the terminal tract of specialization by stem and progenitor cells), guided migration and elongation processes, synaptogenesis, regulation of cell homeostasis by apoptosis, regeneration, as well as maintaining a normal cyto-biochemical status of mature cells and their resistance to damaging factors (Alam et al., 2010; Charles et al., 2011; Vinores & Perez-Polo, 1983; Xie et al., 2005).

The interest to determine the significance of these compounds in tumor formation and in their reverse transforming potential has just relatively recently emerged, when it became vivid that virtually all types of neoplasias are synthesized not only by growth factors but actively expressize their receptors (Antonelli et al., 2007; Barnes et al., 2009; Blum & Konnerth, 2005; Brossard et al., 2009; Evangelopoulos et al, 2004; Krűttgen et al., 2006; Nakagawara, 2001). At the same time we point out a startling fact: NGF was purified for the first time from sarcoma secretions S180, and protooncogenes sites, which take it, were purified from high-affinity TrkA and low relational P75 from biopsies of colon carcinoma (*intestinum crassum*) and human melanoma. It is much more interesting that bio testing of growth factors on its activity is performed on rat pheochromocytoma PC12 (Krűttgen et al., 2006).

The experiments proved that *in vitro* the cell lines of neuroblastoma (IMR-32, SY-5Y, SK-N-SH, NB-GR) and medullary pheochromocytoma cease to divide in the presence of NGF and are transformed into neuron-like elements at braking formation of DNA, at the intensification of including labeled amino acids, at the appearance of sprouts, at the growth of size, substrate adhesion together with the formation of pseudoganglies and at the occurrence in membrane of electrical excitability (Krűttgen et al., 2006; Poluha et al., 1995). On the other hand, the ability of NGF *in vivo* to reduce the number of induced nitrosourea by neurinomas was revealed, as well as the speed of their development, and the ability to reduce the volume and to prolong animal survival after subcutaneous injection or intracerebral implantation of anaplastic glioma cell F98, T9, and neurinomas (Yaeger et al., 1992). The predictive value of identification of perceiving Neurotrophins receptors is overviewed. So thus the over expression of TrkA and C (for NT-3) in neuro-, medulla and glioblastomas promises a favorable outcome, due to a spontaneous regression (through differentiation), the inclusion of suicide programs or autophagy (Blum & Konnerth, 2005; Collins, 2004; Krűttgen et al., 2006; Yamaguchi et al., 2007). Patients with low or no detectable levels of these receptors in medulloblastoma exhibit 5-fold risk of death than with

Improving the Efficiency of

**2.2 Cytoscopic study** 

**2.3 Cultural studies** 

attachment to the substrate.

**2.4 Clinical, laboratory and instrumental methods** 

StatPlus 2005. Differences were estimated to be significant at *P* <0.05.

case of choosing the treatment tactics.

Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors 469

of Cell Monitoring to assess individual sensitivity of tumor cells to chemotherapeutic drugs *in vitro*. After the mentioned period 0.5 ml of chemotherapy was added in doses approved by instructions and converted either to a square cup (10.0 cm2) or to β-subunit of recombinant human NGF (Sigma-Aldrich, USA, 1.0 μg / ml) or to Dendrimers (PAMAM 0.1, 1.0, 10.0 μg / ml, Sigma-Aldrich, USA), or to heterocyclic compounds (0.1, 1.0, 10.0 μg / ml), or to these or others in various combinations. Each series of observations *in vitro*  consisted of 30 applications (n = 30). Assessment of the viability of tumor cells was estimated in 24 hours after putting test compounds into the environment for their ability to incorporate trypan blue. For this matter the cell suspension was mixed with 2% dye solution in saline buffer pH = 7.2 at a ratio of 1:10 and transferred into Goryaev's chamber, where the number of dead (paint over) and living (light) elements was counted at a percentage. The obtained data were reported to neurosurgeons who together with other specialists

Cytoscopic study of surgical specimens was carried out after using the methods of frozen sections, or the crushed drop. The final conclusion of the histological form of the tumor and its malignancy degree was made after alcohol treatment, filling material in paraffin, sectioning and staining by the following methodic: a method of staining with hematoxylin and eosin; histochemical methods for the detection of glial filaments, collagen and reticulin fibers; immunohistochemical studies for detection of acidic glial fibrillary protein, neurofilaments, synaptophysin, and neuron-specific enolase; a definition of PCNA, Ki-67, cyclins in order to clarify the nature of proliferative activity. The conducted cytoscopic research allowed to identify indications of malignancy and was a guide to neurosurgeons in

Pieces of biopsy material were washed from the blood and mechanically comminuted in Hank's solution (Sigma-Aldrich, USA) with Gentamicin sulfate added, and then for 30 min they were put in a mixture of 0.25% trypsin solution in Ethylenediaminetetraacetic acid (EDTA) (2 ml) at a ratio of 1:3. The effect of the enzyme was inhibited by adding 3 ml Fetal Calf Serum – FCS (Sigma-Aldrich, USA) for a period of 3-5 minutes. The material treated in such a way was crushed under a microscope with a sterile blade up to pasty consistency and then was taken to a sterile Petri dish with medium Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, USA), with adding ETS at a ratio of 1:10 and 4% sulfate solution Gentamicin (10-4 g / l). The cells obtained from the substrate were grown in a medium of this composition for 2-7 days at 37ºC, 95% humidity and 5% partial pressure of CO2 (Chekan et al., 2009). Stay duration of the cells *in vitro* was dictated by the speed of the

Clinical, laboratory and instrumental methods of the study included a list of routine clinical examination methods and laboratory diagnostics, as well as computed tomography and nuclear magnetic resonance. The credibility of differences between the average values was set by a Mann-Whitney test for nonparametric samples using the computer program

developed the tactics of post-operative treatment and determined prognosis.

a high level (Krűttgen et al., 2006). *Per contra*, the enhanced expression of TrkB (ligand BDNF), which takes place in aggressive tumors, where isoforms often truncated and lacked of intracellular domain, goes with the pessimistic ending.

In this light the aim of the research was to study individual and combined effects of some of cytostatic and growth factors (Liu et al., 2010), which are in circulation, on the survival of primary culture of cells. Alongside an attempt of the combined action of cytostatic, that is the factor of Nerve Growth and Dendrimers (or Heterocyclic Compounds) on primary culture cells of neuroepithelial tumors was undertaken. Dendrimers are the extended threedimensional molecules which contain a large number of active functional groups on the outer surface (Morgan et al., 2006; Waite & Roth, 2009). We focused on one of the most common types of Dendrimers – Polyamidoamine, (PAMAM), containing ethylenediamine core and branching of methyl acrylate and ethylenediamine (Kang et al., 2010). We aimed at verifying the hypothesis about the possibility of dose reducing of cytostatic at combined application of chemotherapy with growth factors and nanoparticles, in particular Dendrimers in these experiments. The fifth generation of Dendrimer (PAMAM G5) was used. The effect of combinations of cytostatic, Nerve Growth Factor and Dendrimers or the cytostatic agent, Nerve Growth Factor and Heterocyclic Compounds was studied in separate series of experiments. Heterocycles of isoxazole series (Isoxazole) and isothiazolyl (Isothiazole) (typical representatives of 1,2-azoles) are structural fragments of a wide range molecules of active physiologically substances, what causes a growing interest in the research of the synthesis and in the study of the biological properties of these compounds. Isoxazole heterocycle is a compound part of molecules of the cytotoxic, antitubercular agents, anticonvulsants, and pesticides.

The compounds to be perspective for the treatment of Alzheimer's disease and inflammatory, antithrombosis and anticonvulsive drugs were identified among the derivatives of isothiazolyl. It was recently found that some isothiazolyls were inhibitors of kinases and could be used in the treatment of tumors. For example, isothiazolyl with urea function in position 3 is an inhibitor of tyrosine kinases and now it is under studying as an anticancer drug CP-547, 632 (Beebe et al., 2003). The fellow-colleagues of Institute of Physical Organic Chemistry NAS of Belarus developed methods for the synthesis of new 5 substituted 1,2-thiazol-3-ilcarbamids and their heteroanalogs – 1,2-oxazole-3-ilcarbamids – isosteres known as inhibitors of tyrosine kinases, which are of some interest for testing in our planned experiments. The goal of these experiments was to find ways of reducing the dose of cytotoxic drugs, under condition of preserving or increasing the toxic effects of chemotherapy on tumor tissue.

#### **2. Methodic**

The biopsy material was taken from 67 children aged from 1 to 15 years old who were treated at the children's neurosurgical department of Municipal Clinical Emergency Hospital in Minsk from November 2008 to December 2010.

#### **2.1 The research protocol**

The biopsy material taken during the fine-needle stereotactic or neurosurgical operation was transported in an hour to the Pathology Laboratory to determine the histology forms of the tumor and degrees of its malignancy, and was simultaneously delivered to the Laboratory of Cell Monitoring to assess individual sensitivity of tumor cells to chemotherapeutic drugs *in vitro*. After the mentioned period 0.5 ml of chemotherapy was added in doses approved by instructions and converted either to a square cup (10.0 cm2) or to β-subunit of recombinant human NGF (Sigma-Aldrich, USA, 1.0 μg / ml) or to Dendrimers (PAMAM 0.1, 1.0, 10.0 μg / ml, Sigma-Aldrich, USA), or to heterocyclic compounds (0.1, 1.0, 10.0 μg / ml), or to these or others in various combinations. Each series of observations *in vitro*  consisted of 30 applications (n = 30). Assessment of the viability of tumor cells was estimated in 24 hours after putting test compounds into the environment for their ability to incorporate trypan blue. For this matter the cell suspension was mixed with 2% dye solution in saline buffer pH = 7.2 at a ratio of 1:10 and transferred into Goryaev's chamber, where the number of dead (paint over) and living (light) elements was counted at a percentage. The obtained data were reported to neurosurgeons who together with other specialists developed the tactics of post-operative treatment and determined prognosis.

#### **2.2 Cytoscopic study**

468 Glioma – Exploring Its Biology and Practical Relevance

a high level (Krűttgen et al., 2006). *Per contra*, the enhanced expression of TrkB (ligand BDNF), which takes place in aggressive tumors, where isoforms often truncated and lacked

In this light the aim of the research was to study individual and combined effects of some of cytostatic and growth factors (Liu et al., 2010), which are in circulation, on the survival of primary culture of cells. Alongside an attempt of the combined action of cytostatic, that is the factor of Nerve Growth and Dendrimers (or Heterocyclic Compounds) on primary culture cells of neuroepithelial tumors was undertaken. Dendrimers are the extended threedimensional molecules which contain a large number of active functional groups on the outer surface (Morgan et al., 2006; Waite & Roth, 2009). We focused on one of the most common types of Dendrimers – Polyamidoamine, (PAMAM), containing ethylenediamine core and branching of methyl acrylate and ethylenediamine (Kang et al., 2010). We aimed at verifying the hypothesis about the possibility of dose reducing of cytostatic at combined application of chemotherapy with growth factors and nanoparticles, in particular Dendrimers in these experiments. The fifth generation of Dendrimer (PAMAM G5) was used. The effect of combinations of cytostatic, Nerve Growth Factor and Dendrimers or the cytostatic agent, Nerve Growth Factor and Heterocyclic Compounds was studied in separate series of experiments. Heterocycles of isoxazole series (Isoxazole) and isothiazolyl (Isothiazole) (typical representatives of 1,2-azoles) are structural fragments of a wide range molecules of active physiologically substances, what causes a growing interest in the research of the synthesis and in the study of the biological properties of these compounds. Isoxazole heterocycle is a compound part of molecules of the cytotoxic, antitubercular

The compounds to be perspective for the treatment of Alzheimer's disease and inflammatory, antithrombosis and anticonvulsive drugs were identified among the derivatives of isothiazolyl. It was recently found that some isothiazolyls were inhibitors of kinases and could be used in the treatment of tumors. For example, isothiazolyl with urea function in position 3 is an inhibitor of tyrosine kinases and now it is under studying as an anticancer drug CP-547, 632 (Beebe et al., 2003). The fellow-colleagues of Institute of Physical Organic Chemistry NAS of Belarus developed methods for the synthesis of new 5 substituted 1,2-thiazol-3-ilcarbamids and their heteroanalogs – 1,2-oxazole-3-ilcarbamids – isosteres known as inhibitors of tyrosine kinases, which are of some interest for testing in our planned experiments. The goal of these experiments was to find ways of reducing the dose of cytotoxic drugs, under condition of preserving or increasing the toxic effects of

The biopsy material was taken from 67 children aged from 1 to 15 years old who were treated at the children's neurosurgical department of Municipal Clinical Emergency

The biopsy material taken during the fine-needle stereotactic or neurosurgical operation was transported in an hour to the Pathology Laboratory to determine the histology forms of the tumor and degrees of its malignancy, and was simultaneously delivered to the Laboratory

of intracellular domain, goes with the pessimistic ending.

agents, anticonvulsants, and pesticides.

chemotherapy on tumor tissue.

**2.1 The research protocol** 

Hospital in Minsk from November 2008 to December 2010.

**2. Methodic** 

Cytoscopic study of surgical specimens was carried out after using the methods of frozen sections, or the crushed drop. The final conclusion of the histological form of the tumor and its malignancy degree was made after alcohol treatment, filling material in paraffin, sectioning and staining by the following methodic: a method of staining with hematoxylin and eosin; histochemical methods for the detection of glial filaments, collagen and reticulin fibers; immunohistochemical studies for detection of acidic glial fibrillary protein, neurofilaments, synaptophysin, and neuron-specific enolase; a definition of PCNA, Ki-67, cyclins in order to clarify the nature of proliferative activity. The conducted cytoscopic research allowed to identify indications of malignancy and was a guide to neurosurgeons in case of choosing the treatment tactics.

#### **2.3 Cultural studies**

Pieces of biopsy material were washed from the blood and mechanically comminuted in Hank's solution (Sigma-Aldrich, USA) with Gentamicin sulfate added, and then for 30 min they were put in a mixture of 0.25% trypsin solution in Ethylenediaminetetraacetic acid (EDTA) (2 ml) at a ratio of 1:3. The effect of the enzyme was inhibited by adding 3 ml Fetal Calf Serum – FCS (Sigma-Aldrich, USA) for a period of 3-5 minutes. The material treated in such a way was crushed under a microscope with a sterile blade up to pasty consistency and then was taken to a sterile Petri dish with medium Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, USA), with adding ETS at a ratio of 1:10 and 4% sulfate solution Gentamicin (10-4 g / l). The cells obtained from the substrate were grown in a medium of this composition for 2-7 days at 37ºC, 95% humidity and 5% partial pressure of CO2 (Chekan et al., 2009). Stay duration of the cells *in vitro* was dictated by the speed of the attachment to the substrate.

#### **2.4 Clinical, laboratory and instrumental methods**

Clinical, laboratory and instrumental methods of the study included a list of routine clinical examination methods and laboratory diagnostics, as well as computed tomography and nuclear magnetic resonance. The credibility of differences between the average values was set by a Mann-Whitney test for nonparametric samples using the computer program StatPlus 2005. Differences were estimated to be significant at *P* <0.05.

Improving the Efficiency of

Fig. 2. Atypical teratoid / rhabdoid tumor

magnification is x 312.5.

and especially about hemangioblastoma (74.7 ± 3.9%).

Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors 471

The same can be said about Etoposide (Ebewe Artsnaym., Austria, 1.0 μg / ml) in relation to the protoplasmic astrocytoma cells (61.0 ± 4.7%), oligoastrocytoma (55.1 ± 8.5%) (Fig. 3 a, b)

a b c

Cytarabine (Belmedpreparaty, Belarus, 1.0 μg / ml) effectively suppressed the cell vitality in cultures of anaplastic astrocytoma (51.4 ± 5.9%) and immature teratoma (90.6 ± 5.0%). Other substances – Methotrexate (Ebewe Artsnaym., Austria, 50.0 μg / ml) and Gemcitabine (Wee-Em-Gee Pharmaceuticals Pvt. Ltd., India, 2.0 μg / ml) though were not included in the list of

Fig. 3. Oligoastrocytoma cell survival in a day after the application of Etoposide (b) and β-

NGF (c) in comparison with intact cells of primary culture of the tumor (a). The

### **3. Results**

The following types of cancer were included according to the histological conclusions based on classification of brain tumors (WHO, 2007) into the observations: astrocytic, embryonic, and compiled under the title "other types" of neoplasm. In the group of cancers of astrocytic origin were present: pilocytic, pilomixoidnic, protoplasmic, pleomorphic, anaplastic neoplasm. Medulloblastoma, atypical teratoidnic/rabdoidnic tumor of posterior fossa, and malignant neuroectodermal tumor of the left temporal lobe were in the category of embryonic tumors. The category of "the other options" of neoplasm included: anaplastic oligodendrogliomas, oligoastrocytomas of a cerebellar vermis, ependymoma, gangliogliomas of a mixed neuroglial type, immature teratoma of a pineal region, which was attributable to germinocell tumors, glioma of an optic chiasm, and a gemangioblastoma to be regarded as an intramedullary tumor of a cervical spinal cord.

It was revealed in the experiments *in vitro* that Cisplatin (Merck, USA, 1.0 μg / ml) took priority in the samples of pilocytic astrocytoma, medulloblastoma, and malignant neuroectodermal tumors of the temporal lobe, where the percentage of dead cells reached respectively 61.9 ± 12.9, 40.9 ± 11.4, 41.6 ± 8.5, 68.6 ± 7.8 reliably exceeding the one in those experiments where the cells of a primary culture of neuroepithelial tissue devolved out of contact with chemotherapy (14.3 ± 5.0%). A similar position was taken by Carboplatin (Merck, USA, 4.0 μg / ml) in biopsies from pleyomorfic xanthoastrocytoma, anaplastic oligodendroglioma (Fig. 1), optic chiasm glioma and atypical teratoid / rhabdoid tumors (Fig. 2) with lethality according to the order of enumeration 57.9 ± 5.9, 68.4 ± 10.6, 78.9 ± 4.1 and 79.5 ± 1.0%.

Fig. 1. Anaplastic oligodendroglioma

The following types of cancer were included according to the histological conclusions based on classification of brain tumors (WHO, 2007) into the observations: astrocytic, embryonic, and compiled under the title "other types" of neoplasm. In the group of cancers of astrocytic origin were present: pilocytic, pilomixoidnic, protoplasmic, pleomorphic, anaplastic neoplasm. Medulloblastoma, atypical teratoidnic/rabdoidnic tumor of posterior fossa, and malignant neuroectodermal tumor of the left temporal lobe were in the category of embryonic tumors. The category of "the other options" of neoplasm included: anaplastic oligodendrogliomas, oligoastrocytomas of a cerebellar vermis, ependymoma, gangliogliomas of a mixed neuroglial type, immature teratoma of a pineal region, which was attributable to germinocell tumors, glioma of an optic chiasm, and a gemangioblastoma

It was revealed in the experiments *in vitro* that Cisplatin (Merck, USA, 1.0 μg / ml) took priority in the samples of pilocytic astrocytoma, medulloblastoma, and malignant neuroectodermal tumors of the temporal lobe, where the percentage of dead cells reached respectively 61.9 ± 12.9, 40.9 ± 11.4, 41.6 ± 8.5, 68.6 ± 7.8 reliably exceeding the one in those experiments where the cells of a primary culture of neuroepithelial tissue devolved out of contact with chemotherapy (14.3 ± 5.0%). A similar position was taken by Carboplatin (Merck, USA, 4.0 μg / ml) in biopsies from pleyomorfic xanthoastrocytoma, anaplastic oligodendroglioma (Fig. 1), optic chiasm glioma and atypical teratoid / rhabdoid tumors (Fig. 2) with lethality according to the order of enumeration 57.9 ± 5.9, 68.4 ± 10.6, 78.9 ± 4.1

to be regarded as an intramedullary tumor of a cervical spinal cord.

**3. Results** 

and 79.5 ± 1.0%.

Fig. 1. Anaplastic oligodendroglioma

Fig. 2. Atypical teratoid / rhabdoid tumor

The same can be said about Etoposide (Ebewe Artsnaym., Austria, 1.0 μg / ml) in relation to the protoplasmic astrocytoma cells (61.0 ± 4.7%), oligoastrocytoma (55.1 ± 8.5%) (Fig. 3 a, b) and especially about hemangioblastoma (74.7 ± 3.9%).

Fig. 3. Oligoastrocytoma cell survival in a day after the application of Etoposide (b) and β-NGF (c) in comparison with intact cells of primary culture of the tumor (a). The magnification is x 312.5.

Cytarabine (Belmedpreparaty, Belarus, 1.0 μg / ml) effectively suppressed the cell vitality in cultures of anaplastic astrocytoma (51.4 ± 5.9%) and immature teratoma (90.6 ± 5.0%). Other substances – Methotrexate (Ebewe Artsnaym., Austria, 50.0 μg / ml) and Gemcitabine (Wee-Em-Gee Pharmaceuticals Pvt. Ltd., India, 2.0 μg / ml) though were not included in the list of

Improving the Efficiency of

Fig. 5. Undifferentiated medulloblastoma

Fig. 6. Anaplastic astrocytoma. Moderate cellularity. Nuclear polymorphism

cytostatics.

Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors 473

the contrast, the derivatives of pleomorphic xanthoastrocytoma responded to β-NGF by a significant increase in its resistance to the toxin (an increase of vitality). Their death was 13.6 ± 2.9% in relation to the fixed one in the tumor cells which were not subjected to any influence – 31.7 ± 0.5%, whereas the culture of ganglioglioma was generally indifferent to

leading substances, often successfully shared with them the second or third position. However, in a case of anaplastic oligodendroglioma the latter was not effective. Moreover, in its presence the cells of pleomorphic xanthoastrocytoma like Etoposide ones demonstrated a paradoxical reaction – a statistically significant increase of survival potential. The same is with carboplatin. Maximum susceptibility to it is shown by document in the samples of four neoplasm, and first of all of atypical teratoid / rabdoid tumor and optic chiasm glioma to have lost 79.5 ± 1.0 and 78.9 ± 4.1% viable units. These compounds are followed by Etoposide, which predominant efficacy was also recorded in three types of tumor cells, especially in glioblastoma.

Fig. 4. Glioblastoma with marked vascular proliferation and the formation of glomerular structures.

It is right to note the following fact: culture objects manifested almost the same (competitive) sensitivity to two or more agents in virtually all cases, if one takes into consideration the closeness of a number of infected cells, differences of which were not included in the validation category. As for β-NGF, its superiority (although not statistically significant) over the cytostatic regarding a number of perished *in vitro* units occurred in biopsies of pilomixoid astrocytoma (47.6 ± 1.3% vs. 40.9 ± 11.4% of Cisplatin), medulloblastoma (Fig. 5) (44.9 ± 2.9 vs. 41.6 ± 8.5% from the same preparation), oligoastrocytoma (78.3 ± 0.9 vs. 55.1 ± 8.5% of Etoposide) (Fig. 3 a, b, c) and ependymoma (62.4 ± 6.0 vs. 58.6 ± 4.4% of Carboplatin).

The efficiency of β-NGF (68.2 ± 0.5%) almost coincided with that of Cytarabine (68.9 ± 11.9%), and under the contact with the cells of anaplastic astrocytomas (Fig. 6) (38.2 ± 2.3%) reached the 2nd position after for Cytarabine (54.4 ± 5.9%), pushing on the third place Carboplatin (35.9 ± 1.8%) when glioblastoma was applied to the cells the primary culture. In

leading substances, often successfully shared with them the second or third position. However, in a case of anaplastic oligodendroglioma the latter was not effective. Moreover, in its presence the cells of pleomorphic xanthoastrocytoma like Etoposide ones demonstrated a paradoxical reaction – a statistically significant increase of survival potential. The same is with carboplatin. Maximum susceptibility to it is shown by document in the samples of four neoplasm, and first of all of atypical teratoid / rabdoid tumor and optic chiasm glioma to have lost 79.5 ± 1.0 and 78.9 ± 4.1% viable units. These compounds are followed by Etoposide, which predominant efficacy was also recorded in

three types of tumor cells, especially in glioblastoma.

Fig. 4. Glioblastoma with marked vascular proliferation and the formation

It is right to note the following fact: culture objects manifested almost the same (competitive) sensitivity to two or more agents in virtually all cases, if one takes into consideration the closeness of a number of infected cells, differences of which were not included in the validation category. As for β-NGF, its superiority (although not statistically significant) over the cytostatic regarding a number of perished *in vitro* units occurred in biopsies of pilomixoid astrocytoma (47.6 ± 1.3% vs. 40.9 ± 11.4% of Cisplatin), medulloblastoma (Fig. 5) (44.9 ± 2.9 vs. 41.6 ± 8.5% from the same preparation), oligoastrocytoma (78.3 ± 0.9 vs. 55.1 ± 8.5% of Etoposide) (Fig. 3 a, b, c) and ependymoma (62.4 ± 6.0 vs. 58.6 ± 4.4% of

The efficiency of β-NGF (68.2 ± 0.5%) almost coincided with that of Cytarabine (68.9 ± 11.9%), and under the contact with the cells of anaplastic astrocytomas (Fig. 6) (38.2 ± 2.3%) reached the 2nd position after for Cytarabine (54.4 ± 5.9%), pushing on the third place Carboplatin (35.9 ± 1.8%) when glioblastoma was applied to the cells the primary culture. In

of glomerular structures.

Carboplatin).

the contrast, the derivatives of pleomorphic xanthoastrocytoma responded to β-NGF by a significant increase in its resistance to the toxin (an increase of vitality). Their death was 13.6 ± 2.9% in relation to the fixed one in the tumor cells which were not subjected to any influence – 31.7 ± 0.5%, whereas the culture of ganglioglioma was generally indifferent to cytostatics.

Fig. 5. Undifferentiated medulloblastoma

Fig. 6. Anaplastic astrocytoma. Moderate cellularity. Nuclear polymorphism

Improving the Efficiency of

(13.3 ± 2.9%) compared with control (32.9 ± 3.0%).

example of such a design. The data are presented in Table 1.

Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors 475

The dependence of the destructive action of cytostatic and β-NGF also correlated with the age of the patients. Differences in digital indicators which characterize the percentage of cell loss of primary tumor culture *in vitro* gave evidence of the complex mechanisms of interaction between tumor cells with cytotoxic drugs coming in the triad of the most active compounds. So, at the child age under 3 years old the tumor generating effect was observed in Carboplatin (59.7 ± 4.1%), Cisplatin (51.4 ± 4.5%) and Cytarabine (48.3 ± 2.4%), and at the age from 4 to 6 years old β-NGF (63.2 ± 4.8%), Cisplatin (62.3 ± 7.6%) and Methotrexate (49.4 ± 5.8%) were in the lead. At the age from 7 to 10 years old the toxic substances rating looked like follows: Carboplatin (38.1 ± 8.8), β-NGF (27.1 ± 6.1%) and Methotrexate (26.2 ± 3.1%). At the age from 11 to 15 years old Methotrexate moved to the first position (56.0 ± 3.8%), slightly ahead of Carboplatin (54.4 ± 2.9%) and Cisplatin (53.6 ± 3.7%). β-NGF, taking the first place in terms of suppression of cell survival in the intermediate age groups, showed in the fourth (most mature) group an inverted effect, which increased cell survival significantly

Experiments conducted *in vitro* demonstrated once again the well-known position of the highest individual sensitivity of glioma to cytostatics. No one has ever managed to fix the same sequence in the effectiveness of tumor destroying the action by the protocol approved cytostatic at absolutely identical histological diagnoses. Registered dependence of differences of effects from sex and age did not permit to explain the inner workings of such a high individual sensitivity of glioma to chemotherapy. It is obvious that one of explanations for this phenomenon can be a different degree of presence of stem tumor cells in tumor tissue. The high stability of stem tumor cells to damaging agents is well known. We are therefore got interested in the effect of enhancing the anticancer effect of cytostatics and NGF presence. As a hypothesis we can suggest that a strengthening of an anticancer effect of a combination of chemotherapeutic drugs and NGF is determined by an influence of drugs not only on dividing tumor cells but also on stem tumor cells. If it is so, then it is advisable to try to test different combinations of chemotherapy with cytotoxic substances of a new generation. In particular, we talk about the heterocyclic compounds, many of which are capable of inhibiting the intracellular tyrosine kinas path and, thus, to initiate the mechanisms of apoptosis in tumor tissue. The use of nanoparticles, in particular, Fullerenes or Dendrimers seems making a promise for these tasks. More details will be discussed below. There are many more challenges in oncology on the way to more effective cancer therapy. The well known high toxicity of chemotherapeutic drugs for all organs and systems of a living organism stimulates scientists and oncologists to find ways of reducing the general toxic action of cytostatic, and maintaining their anti-tumor effect. The result in these experiments on primary culture oligoastrocytoma cerebellar vermis can be given as an

The following fact draws attention at analyzing the data. In comparison with the natural death of cells the addition of Carboplatin, Methotrexate or Cisplatin was accompanied by an increase in the percentage of dead cells in a Petri dish from 35% to 47 % in the control (when cells of primary culture of oligoastrocytoma cerebellar vermis were developing in the culture medium without any contact with the chemotherapy). The combination of one of these three chemotherapy drugs with Nerve Growth Factor under decreasing doses of the cytostatic factor led in 10 times to the preservation or even an increase of the cytostatic effect (Table 1). The effect of two cytostatics - Carboplatin and Methotrexate increased especially

The access to the analysis of a destructive influence of combined test compounds application, while keeping in mind the possibility of potentiation of their individual effects was the logical corollary from an analysis of the brought materials. It turned out that in most combinations (excluding β-NGF + Methotrexate in the case of pilocytic astrocytoma), a marked tendency of a destructive cellular reactions increase in comparison with those described in the isolated introduction of preparation) was observed. A combination of Cisplatin + Etoposide (89.5 ± 0.8%), Carboplatin + Cytarabine (81.4 ± 2.0%), Carboplatin + Etoposide (69.2 ± 3.0%) and Cisplatin + Carboplatin (68.8 ± 7.3%) for cells oligoastrocytoma, and Cisplatin + Carboplatin (78.1 ± 1.9%) and β-NGF + Cisplatin (55.5 ± 3.2%) shown with an example of cells of ganglioglioma was especially attractive *in vitro* conditions. In these cases, the combined effect of cytotoxic drugs significantly exceeded observed ones under individual applications of each component from every pair of drugs. It's worth noting that β-NGF, despite the decline of its performance in a variant of samples from oligoastrocytoma, tends as a rule to significantly increasing toxic effects on the tumor cells survival in a combination with cytotoxic drugs. This raises a question of reasonableness of the further research in terms of the simultaneous application of these agents. For the sake of fairness it should be emphasized that in any case of their combination one failed to reveal signs of synergy, where the reaction to the combination would have excelled the arithmetic sum of the personal effects, included as components in its structure. This can be conditioned by complex polymorphism of brain tumors, including dividing tumor cells and stem tumor cells at that. The survival of an even single cancer stem cell could lead to a recurrence of tumor metastasis and uncontrolled process in other parts of the central nervous system.

It is important to point out that varying selective affinity touched not only biopsy samples belonging to different types of tumors, but also tumors taken away from an absolutely analogous composition accordingly to histological compounds and a measure of tumor progression. For example, the maximum destruction of cultured cells was recorded against a background application Cytarabine (68.3 ± 6.9%, *P* <0.05), and to a lesser extent Cisplatin (49.4 ± 5.4%, P <0.05), and Methotrexate (39.7 ± 7.2%, *P* <0.05) in three patients with medulloblastoma of the IV stage. The other chemotherapy drugs effect on cells of tumor had actually no difference in growth of cell culture without the application of cytostatic. This circumstance makes relevant carrying out a preliminary rapid assessment of individual susceptibility of patients to chemotherapeutic drugs with involvement of cultural systems.

Moreover, it was possible to show by a document a certain correlation between the effectiveness of test compounds and the degree of malignancy at the same astrocytic nature of the tumors. Let us demonstrate this by the following examples. Thus Cisplatin (61.9 ± 12.9%), Carboplatin (53.8 ± 7.9%) and Methotrexate (47.0 ± 7.2%) came into the triad of superior according to induction of cell lethality at the Ist stage. At the IInd stage Carboplatin (57.9 ± 5.9%), Methotrexate (51.1 ± 4.0%) and Cytarabine (50.2 ± 9.4%) occupied the dominant position. On the IIId – Cytarabine (54.4 ± 5.9%), Carboplatin (35.9 ± 1.8%) and Etoposide (34.5 ± 4.4%), and at the IVth – Cytarabine (68.9 ± 11.9%), Etoposide (63.1 ± 0.4%) and Gemcitabine (61.5 ± 1.7%). As a matter of fact the mentioned graduation should be viewed with a certain degree of conditionality, since statistically significant differences between the members of each triad could not be established. It's noteworthy, however, that β-NGF, occupying a relatively modest position in the first phase of malignancy acquired a tough competition (68.2 ± 0.5%) leading to the IVth stage of Cytarabine (68.9 ± 11.9%) and bunched over (38.2 ± 2.3%) from the 2nd stage (35.9 ± 1.8%) of Carboplatin (please, bear in mind: at isolated applications of β-NGF).

The access to the analysis of a destructive influence of combined test compounds application, while keeping in mind the possibility of potentiation of their individual effects was the logical corollary from an analysis of the brought materials. It turned out that in most combinations (excluding β-NGF + Methotrexate in the case of pilocytic astrocytoma), a marked tendency of a destructive cellular reactions increase in comparison with those described in the isolated introduction of preparation) was observed. A combination of Cisplatin + Etoposide (89.5 ± 0.8%), Carboplatin + Cytarabine (81.4 ± 2.0%), Carboplatin + Etoposide (69.2 ± 3.0%) and Cisplatin + Carboplatin (68.8 ± 7.3%) for cells oligoastrocytoma, and Cisplatin + Carboplatin (78.1 ± 1.9%) and β-NGF + Cisplatin (55.5 ± 3.2%) shown with an example of cells of ganglioglioma was especially attractive *in vitro* conditions. In these cases, the combined effect of cytotoxic drugs significantly exceeded observed ones under individual applications of each component from every pair of drugs. It's worth noting that β-NGF, despite the decline of its performance in a variant of samples from oligoastrocytoma, tends as a rule to significantly increasing toxic effects on the tumor cells survival in a combination with cytotoxic drugs. This raises a question of reasonableness of the further research in terms of the simultaneous application of these agents. For the sake of fairness it should be emphasized that in any case of their combination one failed to reveal signs of synergy, where the reaction to the combination would have excelled the arithmetic sum of the personal effects, included as components in its structure. This can be conditioned by complex polymorphism of brain tumors, including dividing tumor cells and stem tumor cells at that. The survival of an even single cancer stem cell could lead to a recurrence of tumor metastasis and uncontrolled process in other parts of the central nervous system. It is important to point out that varying selective affinity touched not only biopsy samples belonging to different types of tumors, but also tumors taken away from an absolutely analogous composition accordingly to histological compounds and a measure of tumor progression. For example, the maximum destruction of cultured cells was recorded against a background application Cytarabine (68.3 ± 6.9%, *P* <0.05), and to a lesser extent Cisplatin (49.4 ± 5.4%, P <0.05), and Methotrexate (39.7 ± 7.2%, *P* <0.05) in three patients with medulloblastoma of the IV stage. The other chemotherapy drugs effect on cells of tumor had actually no difference in growth of cell culture without the application of cytostatic. This circumstance makes relevant carrying out a preliminary rapid assessment of individual susceptibility of patients to chemotherapeutic drugs with involvement of cultural systems. Moreover, it was possible to show by a document a certain correlation between the effectiveness of test compounds and the degree of malignancy at the same astrocytic nature of the tumors. Let us demonstrate this by the following examples. Thus Cisplatin (61.9 ± 12.9%), Carboplatin (53.8 ± 7.9%) and Methotrexate (47.0 ± 7.2%) came into the triad of superior according to induction of cell lethality at the Ist stage. At the IInd stage Carboplatin (57.9 ± 5.9%), Methotrexate (51.1 ± 4.0%) and Cytarabine (50.2 ± 9.4%) occupied the dominant position. On the IIId – Cytarabine (54.4 ± 5.9%), Carboplatin (35.9 ± 1.8%) and Etoposide (34.5 ± 4.4%), and at the IVth – Cytarabine (68.9 ± 11.9%), Etoposide (63.1 ± 0.4%) and Gemcitabine (61.5 ± 1.7%). As a matter of fact the mentioned graduation should be viewed with a certain degree of conditionality, since statistically significant differences between the members of each triad could not be established. It's noteworthy, however, that β-NGF, occupying a relatively modest position in the first phase of malignancy acquired a tough competition (68.2 ± 0.5%) leading to the IVth stage of Cytarabine (68.9 ± 11.9%) and bunched over (38.2 ± 2.3%) from the 2nd stage (35.9 ± 1.8%) of Carboplatin (please, bear in

mind: at isolated applications of β-NGF).

The dependence of the destructive action of cytostatic and β-NGF also correlated with the age of the patients. Differences in digital indicators which characterize the percentage of cell loss of primary tumor culture *in vitro* gave evidence of the complex mechanisms of interaction between tumor cells with cytotoxic drugs coming in the triad of the most active compounds. So, at the child age under 3 years old the tumor generating effect was observed in Carboplatin (59.7 ± 4.1%), Cisplatin (51.4 ± 4.5%) and Cytarabine (48.3 ± 2.4%), and at the age from 4 to 6 years old β-NGF (63.2 ± 4.8%), Cisplatin (62.3 ± 7.6%) and Methotrexate (49.4 ± 5.8%) were in the lead. At the age from 7 to 10 years old the toxic substances rating looked like follows: Carboplatin (38.1 ± 8.8), β-NGF (27.1 ± 6.1%) and Methotrexate (26.2 ± 3.1%). At the age from 11 to 15 years old Methotrexate moved to the first position (56.0 ± 3.8%), slightly ahead of Carboplatin (54.4 ± 2.9%) and Cisplatin (53.6 ± 3.7%). β-NGF, taking the first place in terms of suppression of cell survival in the intermediate age groups, showed in the fourth (most mature) group an inverted effect, which increased cell survival significantly (13.3 ± 2.9%) compared with control (32.9 ± 3.0%).

Experiments conducted *in vitro* demonstrated once again the well-known position of the highest individual sensitivity of glioma to cytostatics. No one has ever managed to fix the same sequence in the effectiveness of tumor destroying the action by the protocol approved cytostatic at absolutely identical histological diagnoses. Registered dependence of differences of effects from sex and age did not permit to explain the inner workings of such a high individual sensitivity of glioma to chemotherapy. It is obvious that one of explanations for this phenomenon can be a different degree of presence of stem tumor cells in tumor tissue. The high stability of stem tumor cells to damaging agents is well known.

We are therefore got interested in the effect of enhancing the anticancer effect of cytostatics and NGF presence. As a hypothesis we can suggest that a strengthening of an anticancer effect of a combination of chemotherapeutic drugs and NGF is determined by an influence of drugs not only on dividing tumor cells but also on stem tumor cells. If it is so, then it is advisable to try to test different combinations of chemotherapy with cytotoxic substances of a new generation. In particular, we talk about the heterocyclic compounds, many of which are capable of inhibiting the intracellular tyrosine kinas path and, thus, to initiate the mechanisms of apoptosis in tumor tissue. The use of nanoparticles, in particular, Fullerenes or Dendrimers seems making a promise for these tasks. More details will be discussed below. There are many more challenges in oncology on the way to more effective cancer therapy. The well known high toxicity of chemotherapeutic drugs for all organs and systems of a living organism stimulates scientists and oncologists to find ways of reducing the general toxic action of cytostatic, and maintaining their anti-tumor effect. The result in these experiments on primary culture oligoastrocytoma cerebellar vermis can be given as an example of such a design. The data are presented in Table 1.

The following fact draws attention at analyzing the data. In comparison with the natural death of cells the addition of Carboplatin, Methotrexate or Cisplatin was accompanied by an increase in the percentage of dead cells in a Petri dish from 35% to 47 % in the control (when cells of primary culture of oligoastrocytoma cerebellar vermis were developing in the culture medium without any contact with the chemotherapy). The combination of one of these three chemotherapy drugs with Nerve Growth Factor under decreasing doses of the cytostatic factor led in 10 times to the preservation or even an increase of the cytostatic effect (Table 1). The effect of two cytostatics - Carboplatin and Methotrexate increased especially

Improving the Efficiency of

from the edge of the sample (B)

(B)

those which coating consisted of silver nanoparticles.

A B

A B

Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors 477

Fig. 7. Oligodendrogliomas cell distribution in twenty-four hours at 40 mm from the

Fig. 8. Oligodendrogliomas cell distribution in twenty-four hours at 40 mm from the titanium sample with titanium dioxide (TiO2) (A), and at 1 mm from the edge of the sample

titanium sample with diamond-like coating containing silver nanoparticles (A), and at 1 mm

It was established that if the titanium samples were coated with titanium dioxide (TiO2), a cytotoxic effect of these samples (Fig. 8 A and B) would not differ from a cytotoxic effect of

demonstratively. If you are not going to speculate on possible mechanisms of this phenomenon, then one is competent to conclude that the concentration of cytostatic can be significantly reduced in situations of combined use of chemotherapy with NGF. This reduction of dosage will be accompanied by a decline in general toxic action of chemotherapy drugs (which is critical for every cancer patient) and a persistence of the cytostatic action in relation to tumor cells (what is critical for a patient and a physician). A similar cytotoxic effect was observed earlier while applying diamond-like structures (Chekan et al., 2009). Experiments *in vitro* decreased survival of rat C6 glioma cells in the presence of implants made of titanium alloy VT-16. Putting diamond-like carbon coatings on the alloy VT-16 was accompanied with an increase in the percentage of cell death on the fifth day of cultivation, compared with the control: 39.9 ± 2.1% and 5.4 ± 0.3%, respectively. A more significant decrease in mitotic activity and cell viability was observed when C6 glioma cells contacted with diamond-like carbon coating, comprising silver nanoparticles. The number of cell destruction of glioma C6 at contact with the diamond-like carbon covering, including up to 3.5 % Silver nanoparticles, made 53.7 ± 2.1%, and at doping up to 6.7 % Silver the cell destruction reached 66.7 ± 3.2% (P <0.05) in comparison with the control. Hence, the maximum toxic effect in regard to C6 glioma was detected in samples coated with diamond-like film, including silver nanoparticles. Similar results were obtained in the application of diamond coatings on the primary culture of human gliomas. If the surface of titanium samples with a diamond-like coatings included additional silver nanoparticles, the cytotoxic effect on the second day after exposure of cells of oligodendrogliomas with the surface of the samples would be disastrous for the viability of these cells. As it is shown in Fig. 7 A, processes of proliferation are continuing in tumor cells outside the titanium samples, while at the site location on a Petri dish of titanium sample almost all cells died (Fig. 7 B).


Table 1. Percentage of cell death oligoastrocytoma cerebellar vermis at a combination of different doses of cytostatics with Nerve Growth Factor (NGF). (The asterisk \* denotes the reliability of *P* <0.05)

demonstratively. If you are not going to speculate on possible mechanisms of this phenomenon, then one is competent to conclude that the concentration of cytostatic can be significantly reduced in situations of combined use of chemotherapy with NGF. This reduction of dosage will be accompanied by a decline in general toxic action of chemotherapy drugs (which is critical for every cancer patient) and a persistence of the cytostatic action in relation to tumor cells (what is critical for a patient and a physician). A similar cytotoxic effect was observed earlier while applying diamond-like structures (Chekan et al., 2009). Experiments *in vitro* decreased survival of rat C6 glioma cells in the presence of implants made of titanium alloy VT-16. Putting diamond-like carbon coatings on the alloy VT-16 was accompanied with an increase in the percentage of cell death on the fifth day of cultivation, compared with the control: 39.9 ± 2.1% and 5.4 ± 0.3%, respectively. A more significant decrease in mitotic activity and cell viability was observed when C6 glioma cells contacted with diamond-like carbon coating, comprising silver nanoparticles. The number of cell destruction of glioma C6 at contact with the diamond-like carbon covering, including up to 3.5 % Silver nanoparticles, made 53.7 ± 2.1%, and at doping up to 6.7 % Silver the cell destruction reached 66.7 ± 3.2% (P <0.05) in comparison with the control. Hence, the maximum toxic effect in regard to C6 glioma was detected in samples coated with diamond-like film, including silver nanoparticles. Similar results were obtained in the application of diamond coatings on the primary culture of human gliomas. If the surface of titanium samples with a diamond-like coatings included additional silver nanoparticles, the cytotoxic effect on the second day after exposure of cells of oligodendrogliomas with the surface of the samples would be disastrous for the viability of these cells. As it is shown in Fig. 7 A, processes of proliferation are continuing in tumor cells outside the titanium samples, while at the site location on a Petri dish of titanium sample

> **Title Series Cell death, %**  Control 12.5 ± 4.2

Carboplatin 4.0 μg / ml 35.3 ± 0.9\*

Methotrexate 50.0 μg / ml 43.7 ± 8.6\*

Cisplatin 1.0 μg / ml 47.4 ± 3.0\*

Table 1. Percentage of cell death oligoastrocytoma cerebellar vermis at a combination of different doses of cytostatics with Nerve Growth Factor (NGF). (The asterisk \* denotes the

Carboplatin 0.4 μg / ml + β NGF 0.1 μg / ml 57.1 ± 12.5\*

Methotrexate 5.0 μg / ml + β NGF 0.1 μg / ml 72.4 ± 2.5\*

Cisplatin 0.1 μg / ml + β NGF 0.1 μg / ml 50.0 ± 8.1\*

almost all cells died (Fig. 7 B).

reliability of *P* <0.05)

Fig. 7. Oligodendrogliomas cell distribution in twenty-four hours at 40 mm from the titanium sample with diamond-like coating containing silver nanoparticles (A), and at 1 mm from the edge of the sample (B)

It was established that if the titanium samples were coated with titanium dioxide (TiO2), a cytotoxic effect of these samples (Fig. 8 A and B) would not differ from a cytotoxic effect of those which coating consisted of silver nanoparticles.

Fig. 8. Oligodendrogliomas cell distribution in twenty-four hours at 40 mm from the titanium sample with titanium dioxide (TiO2) (A), and at 1 mm from the edge of the sample (B)

Improving the Efficiency of

demonstration.

Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors 479

Chimera. Evaluation of energy characteristics of the van der Waals and electrostatic interaction suggests the possibility of efficient binding of 1,2-azole ligand protein. These studies helped to choose the best version of heterocyclic compounds (Carbamide), which application in a combination with cytostatic agents and Nerve Growth Factor allowed reducing the dose of the cytostatic factor in 10 times at maintaining *in vitro* the tumor generating effect on primary cell cultures. There are the above listed drawings as a

Fig. 9. Atypical teratoid / rhabdoid tumor cells remaining in a day after the application of Cisplatin (1.0 μg / ml), β-NGF (0.1 μg / ml), Polyamidoamine Dendrimer (30 mM, 10.0 μl)

Fig. 10. Atypical teratoid / rhabdoid tumor cells remaining in a day after the application of Cisplatin (0.1 μg / ml), β-NGF (0.1 μg / ml), Polyamidoamine Dendrimer (30 mM, 10.0 μl)


The highest potentiating effect of the combination of chemotherapy and nanoparticles was obtained by the application of Dendrimers. Series of experiments of a Cisplatin combination with PAMAM on the primary culture of medulloblastoma are in Table 2.

Table 2. Percentage of cell death medulloblastoma IV at a combination of different doses of Cisplatin with Polyamidoamine (PAMAM) Dendrimer (the asterisk \* denotes the reliability of *P* <0.05)

As it is seen in Table 2, the percentage of cell death of malignant medulloblastoma increased significantly in primary culture under the action of Cisplatin and paradoxically reduced by a combination of Cisplatin with NGF. This fact does illustrate once again the high specificity of each particular tumor in each patient, what determines the choice of individual treatment strategy in each case. This choice should be guided by research data and the sensitivity of cells in primary culture *in vitro*.

Application of PAMAM 30 mM (10.0 μl) in a Petri dish was accompanied by increased cell death in comparison with control ones. In principle, the anticancer effect of Dendrimer is described in literature but has not been studied in detail (Bei et al., 2010). At this stage we have only stated such an action of PAMAM. Surprisingly a stable toxic effect of a combination Cisplatin with PAMAM was demonstrated at using two different concentrations of chemotherapeutic drugs (Table 2). The anticancer effect of this drug combination ranged from 92% to 98% (Fig. 9, and Fig. 10). Therefore it is very important to find such a combination chemotherapy with growth factors and nanoparticles, which would reduce the dose of cytostatics, when we tried to determine the sensitivity of individual tumor *in vitro* to cytostatic in the next phase of the research. At the same cytotoxic effect of substances used must be maintained at maximum levels. Dendrimers belong to a class of polymeric compounds whose molecules have a large number of branches. At their acquisition a number of branches of the molecule increase with every elementary act of growth. As a result, the shape and rigidity of the molecules change with increasing molecular weight of these compounds, what is usually accompanied with changes in physical and chemical properties of Dendrimers. Inside Dendrimers cavities are formed which can be filled in with a variety of substances, such as cytostatic. This ability of Dendrimers was one of the factors to determine the decision to use them for enhancement of their anticancer effect of chemotherapy.

The paper drew attention also to other compounds that might be effective against tumor growth. The input material for the synthesis of heterocyclic compounds was 1,2-azole-3 carboxylic acids, which were consistently converted into azides or carbamide. Implemented computer modeling of ligand-protein complexes of carbamide was carried out in the framework of the methods of molecular mechanics using the program Dock 6.4 and USF

The highest potentiating effect of the combination of chemotherapy and nanoparticles was obtained by the application of Dendrimers. Series of experiments of a Cisplatin combination

> **Title Series Cell death**, % Control 20.7 ± 3.3 Cisplatin 1.0 μg / ml 67.6 ± 7.4**\***

> > 92.7 ± 4.9**\***

98.4 ± 1.5**\***

with PAMAM on the primary culture of medulloblastoma are in Table 2.

Cisplatin 1.0 μg / ml + β NGF 0.1 μg / ml 49.2 ± 6.8**\***

Cisplatin 1.0 μg / ml + PAMAM 30 mM (10.0 μl )

Cisplatin 0.1 μg / ml + PAMAM 30 mM (10.0 μl )

cells in primary culture *in vitro*.

their anticancer effect of chemotherapy.

of *P* <0.05)

PAMAM 30 mM (10.0 μl ) 45.5 ± 6.7**\***

Table 2. Percentage of cell death medulloblastoma IV at a combination of different doses of Cisplatin with Polyamidoamine (PAMAM) Dendrimer (the asterisk \* denotes the reliability

As it is seen in Table 2, the percentage of cell death of malignant medulloblastoma increased significantly in primary culture under the action of Cisplatin and paradoxically reduced by a combination of Cisplatin with NGF. This fact does illustrate once again the high specificity of each particular tumor in each patient, what determines the choice of individual treatment strategy in each case. This choice should be guided by research data and the sensitivity of

Application of PAMAM 30 mM (10.0 μl) in a Petri dish was accompanied by increased cell death in comparison with control ones. In principle, the anticancer effect of Dendrimer is described in literature but has not been studied in detail (Bei et al., 2010). At this stage we have only stated such an action of PAMAM. Surprisingly a stable toxic effect of a combination Cisplatin with PAMAM was demonstrated at using two different concentrations of chemotherapeutic drugs (Table 2). The anticancer effect of this drug combination ranged from 92% to 98% (Fig. 9, and Fig. 10). Therefore it is very important to find such a combination chemotherapy with growth factors and nanoparticles, which would reduce the dose of cytostatics, when we tried to determine the sensitivity of individual tumor *in vitro* to cytostatic in the next phase of the research. At the same cytotoxic effect of substances used must be maintained at maximum levels. Dendrimers belong to a class of polymeric compounds whose molecules have a large number of branches. At their acquisition a number of branches of the molecule increase with every elementary act of growth. As a result, the shape and rigidity of the molecules change with increasing molecular weight of these compounds, what is usually accompanied with changes in physical and chemical properties of Dendrimers. Inside Dendrimers cavities are formed which can be filled in with a variety of substances, such as cytostatic. This ability of Dendrimers was one of the factors to determine the decision to use them for enhancement of

The paper drew attention also to other compounds that might be effective against tumor growth. The input material for the synthesis of heterocyclic compounds was 1,2-azole-3 carboxylic acids, which were consistently converted into azides or carbamide. Implemented computer modeling of ligand-protein complexes of carbamide was carried out in the framework of the methods of molecular mechanics using the program Dock 6.4 and USF Chimera. Evaluation of energy characteristics of the van der Waals and electrostatic interaction suggests the possibility of efficient binding of 1,2-azole ligand protein. These studies helped to choose the best version of heterocyclic compounds (Carbamide), which application in a combination with cytostatic agents and Nerve Growth Factor allowed reducing the dose of the cytostatic factor in 10 times at maintaining *in vitro* the tumor generating effect on primary cell cultures. There are the above listed drawings as a demonstration.

Fig. 9. Atypical teratoid / rhabdoid tumor cells remaining in a day after the application of Cisplatin (1.0 μg / ml), β-NGF (0.1 μg / ml), Polyamidoamine Dendrimer (30 mM, 10.0 μl)

Fig. 10. Atypical teratoid / rhabdoid tumor cells remaining in a day after the application of Cisplatin (0.1 μg / ml), β-NGF (0.1 μg / ml), Polyamidoamine Dendrimer (30 mM, 10.0 μl)

Improving the Efficiency of

of 1.0 mg / ml

**4. Conclusion** 

Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors 481

Fig. 13. Pilocytic astrocytoma cells in a day after application of Carbamide at a concentration

The data obtained are the basis to discuss several aspects of increasing the chemotherapy effectiveness problem. Being general toxic poisons in the action by their nature, chemotherapy drugs come out as an essential attribute of anticancer therapy in accordance with the majority of the approved treatment protocols. There are no alternatives to their use considering the presence of appropriate evidence. In such a case, how can one reduce the general antineoplastic action of cytostatics and keep their strong anticancer effect? The anticancer effect is under discussion in respect of not only dividing tumor cells, but also stem tumor cells. The advantage of a mutual use of cytostatics and NGF compared with their individual use was confirmed in the work. It puts on the agenda the top-priority intensively developing problem, that is of improving the permeability of the blood-brain barrier at systemic application of chemotherapy and the search for "circuitous" ways to the desired delivery of biologically active compounds to tumor tissues (Alam et al., 2010; Chen et al., 2011; Gerstner & Fine, 2007). In order to implement the first plan several directions are supposed. One of them is to attract hyperosmotic solutions containing histamine, bradykinin, mannitol and so on, which will contribute to achieving the cytostatic targets in the brain at their systematic putting into operation (Kemper et al., 2004; Xie et al., 2005). But the transient opening by them of the entrance gate provides at the same time an opportunity of entering via them for the neurotoxic substance what is especially dangerous in case of a more complex operative procedure of an intracarotid infusion of chemotherapeutic agents.

This may be accompanied by disorders of speech, movement, visual perception.

One more trend is based on creation of conjugates of an active commencement with proteins, which are able to recognize the integral components of cerebrovascular structures, such as antibodies to the receptor ferritin in relation to the NGF. This ensures its effectiveness in a rank of low concentrations. The simultaneous application of anticancer

Anticancer effects of heterocyclic compounds in one more series of experiments were approved (Figs. 11-13). The Fig. 11 shows the primary tumor cells of pilocytic astrocytoma in two days after passage. Figure 12 shows primary tumor cells pilocytic astrocytoma in two days after the addition of Azide at a concentration of 1.0 mg / ml. Figure 13 shows primary tumor cells pilocytic astrocytoma in two days after the addition of Carbamide at a concentration of 1.0 mg / ml. Toxic properties of Azides and Carbamide can be explained by their ability to inhibit the tyrosine kinase pathway. This mechanism can be implemented in the subsequent initiation of apoptosis in tumor cells.

Fig. 11. Pilocytic astrocytoma cells in a day after passage

Fig. 12. Pilocytic astrocytoma cells in a day after application of Azide at a concentration of 1.0 mg / ml

Fig. 13. Pilocytic astrocytoma cells in a day after application of Carbamide at a concentration of 1.0 mg / ml

#### **4. Conclusion**

480 Glioma – Exploring Its Biology and Practical Relevance

Anticancer effects of heterocyclic compounds in one more series of experiments were approved (Figs. 11-13). The Fig. 11 shows the primary tumor cells of pilocytic astrocytoma in two days after passage. Figure 12 shows primary tumor cells pilocytic astrocytoma in two days after the addition of Azide at a concentration of 1.0 mg / ml. Figure 13 shows primary tumor cells pilocytic astrocytoma in two days after the addition of Carbamide at a concentration of 1.0 mg / ml. Toxic properties of Azides and Carbamide can be explained by their ability to inhibit the tyrosine kinase pathway. This mechanism can be implemented in

Fig. 12. Pilocytic astrocytoma cells in a day after application of Azide at a concentration of

the subsequent initiation of apoptosis in tumor cells.

Fig. 11. Pilocytic astrocytoma cells in a day after passage

1.0 mg / ml

The data obtained are the basis to discuss several aspects of increasing the chemotherapy effectiveness problem. Being general toxic poisons in the action by their nature, chemotherapy drugs come out as an essential attribute of anticancer therapy in accordance with the majority of the approved treatment protocols. There are no alternatives to their use considering the presence of appropriate evidence. In such a case, how can one reduce the general antineoplastic action of cytostatics and keep their strong anticancer effect? The anticancer effect is under discussion in respect of not only dividing tumor cells, but also stem tumor cells. The advantage of a mutual use of cytostatics and NGF compared with their individual use was confirmed in the work. It puts on the agenda the top-priority intensively developing problem, that is of improving the permeability of the blood-brain barrier at systemic application of chemotherapy and the search for "circuitous" ways to the desired delivery of biologically active compounds to tumor tissues (Alam et al., 2010; Chen et al., 2011; Gerstner & Fine, 2007). In order to implement the first plan several directions are supposed. One of them is to attract hyperosmotic solutions containing histamine, bradykinin, mannitol and so on, which will contribute to achieving the cytostatic targets in the brain at their systematic putting into operation (Kemper et al., 2004; Xie et al., 2005). But the transient opening by them of the entrance gate provides at the same time an opportunity of entering via them for the neurotoxic substance what is especially dangerous in case of a more complex operative procedure of an intracarotid infusion of chemotherapeutic agents. This may be accompanied by disorders of speech, movement, visual perception.

One more trend is based on creation of conjugates of an active commencement with proteins, which are able to recognize the integral components of cerebrovascular structures, such as antibodies to the receptor ferritin in relation to the NGF. This ensures its effectiveness in a rank of low concentrations. The simultaneous application of anticancer

Improving the Efficiency of

than 10 times;

cytostatic effect;

**5. Acknowledgment** 

**6. References** 

characteristics of patients;

percentage of dying in a culture of cellular elements;

*Sciences*, Vol.40, No.5, pp. 385-403.

No.2, pp. 94-101.

*Diagnostic Pathology*, Vol.27, No.2, pp. 97-104.

Chemotherapeutic Drugs by the Action on Neuroepithelial Tumors 483

i. Sensitivity of glioma cells to chemotherapeutic agents, Nerve Growth Factor, and Dendrimers, Diamond like coated samples, heterocyclic compaunds *in vitro* depends on the origin, histological type tumors, the degree of malignancy, age and individual

ii. Combined application of Nerve Growth Factor and chemotherapy increases the

iii. A complementary effect of Nerve Growth Factor appears to enhance cytotoxic effects of chemotherapy. This addictiveness reduces the effective dose of cytotoxic drugs in more

iv. The presence of growth factor and/or Dendrimers, heterocyclic compounds do reduce the toxic dose of the chemotherapeutic drugs simultaneously maintaining a high

v. Detection *in vitro* of high individual sensitivity of brain tumors to cytostatics confirms

vi. It is appropriate to take into account the results of experiments in order to determine the sensitivity of tumor cells of primary culture to cytostatic drugs in the development of new specialized treatment protocols for brain tumors (mono- or polychemotherapy).

We express our deep appreciation to Professor Joseph Zalutsky for supporting this work, and Professor Eugene Cherstvoy for the attention and assistance in the histological studies.

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drugs with inhibitors of a large glycoprotein P, which prevents the movement of cytotoxic drugs through the blood-brain barrier (Gerstner & Fine, 2007; Kemper et al., 2004) is promising. Preclinical trials with Paclitaxel have confirmed the perspectives of such a method, making reasonable transition to clinical trials.

Attempts of a different kind are being made, namely the involvement of the bradykinin analog BMP-7, endowed with a more extended half-life period and with selectivity in respect to the receptor B2 in comparison with the being endogenously synthesized compound (Kemper et al., 2004; Ta et al., 2009; Xie et al., 2005). Being associated with them, BMP-7 leads to the opening of calcium channels in cells, an increase in their level of free cations. That leads to a reaction of endothelial cells and to weakening of intercellular contacts. The vascular permeability increases in addition. The recorded concentrations of growth factors were achieved in 30 minutes with the highest representation in the striatum, hippocampus, cerebral cortex and relatively low concentrations in the olfactory bulb, cerebellum and brainstem at joint custody of NGF and BMP-7 into stabilized liposomes, which are smaller than 100 nm. There was a good agreement between the permeability coefficient of the drug and its targeted action (Xie et al., 2005) at that. Under intracarotid administration of BMP-7 with Carboplatin in rat models of gliomas such a combination has significantly reduced the number of cytostatic normally used in humans. Phase II in clinical trials conducted on 87 patients with recurrence of malignant gliomas recorded a noticeable advantage of processing BMP-7 + Carboplatin before individually using only Carboplatin.

The advances in molecular biology and genetic engineering, which ensured the implementation of experimental and clinical use of low molecular weight peptides, proteins, oligonucleotides, monoclonal antibodies, etc. (Kruttgen et al., 2006; Xie et al., 2005) contributed to considerable progress in the development of a "roundabout" ways of delivery of drugs. In total they have created a fertile ground not only for in-depth understanding the basic mechanisms of carcinogenesis, but also simultaneously for widening the means of early diagnosis, the front of anticancer attack and optimization of medical schemes.

In a series of widely exploited methods there is a stereotactic implantation in the bed, formed after excision of tumor, indifferent biodegradable polymer substrates, impregnated in that or another way with an active principle. In this case, the cytostatic penetrates in measured doses into the surrounding tissue during a long period of time and destroy the remaining infected cells (Kemper et al., 2004; Ta et al., 2009). The concentration of drugs, developed in this case, can exceed observed ones with intravenous injection from 4 to 1200 times. In one of the clinical trial performed on 222 adult patients with recurrent gliomas mortality of persons who were within 6 months receiving such a method Cisplatin was 44% versus 67% among patients treated with placebo (*P* < 0.02).

The use of polymer capsules can also supply the brain with cells (transfected with viral vectors) which express a particular desired target gene which products act targeteous on the relevant parts of oncogenesis. The primary fibroblasts, astrocytes, ependimocytes, stem or progenitor cells serve as usual objects. For example, subclones Cyclin-Dependent Kinase 2 Interacting Protein (CINP) releasing *in vitro* NGF at 2 ng/ h/10-5 cells over 10 weeks was isolated from conventionally immortalized progenitor neuroblasts of the central nervous system of rat embryos with embedded DNA of growth factors. Being introduced into the brain such cells survived well, migrated at a distance of 15 mm from the implant site and integrated with the host tissue without any signs of growth or tumor formation.

Thus, we can conclude:


#### **5. Acknowledgment**

We express our deep appreciation to Professor Joseph Zalutsky for supporting this work, and Professor Eugene Cherstvoy for the attention and assistance in the histological studies.

#### **6. References**

482 Glioma – Exploring Its Biology and Practical Relevance

drugs with inhibitors of a large glycoprotein P, which prevents the movement of cytotoxic drugs through the blood-brain barrier (Gerstner & Fine, 2007; Kemper et al., 2004) is promising. Preclinical trials with Paclitaxel have confirmed the perspectives of such a

Attempts of a different kind are being made, namely the involvement of the bradykinin analog BMP-7, endowed with a more extended half-life period and with selectivity in respect to the receptor B2 in comparison with the being endogenously synthesized compound (Kemper et al., 2004; Ta et al., 2009; Xie et al., 2005). Being associated with them, BMP-7 leads to the opening of calcium channels in cells, an increase in their level of free cations. That leads to a reaction of endothelial cells and to weakening of intercellular contacts. The vascular permeability increases in addition. The recorded concentrations of growth factors were achieved in 30 minutes with the highest representation in the striatum, hippocampus, cerebral cortex and relatively low concentrations in the olfactory bulb, cerebellum and brainstem at joint custody of NGF and BMP-7 into stabilized liposomes, which are smaller than 100 nm. There was a good agreement between the permeability coefficient of the drug and its targeted action (Xie et al., 2005) at that. Under intracarotid administration of BMP-7 with Carboplatin in rat models of gliomas such a combination has significantly reduced the number of cytostatic normally used in humans. Phase II in clinical trials conducted on 87 patients with recurrence of malignant gliomas recorded a noticeable advantage of processing BMP-7 + Carboplatin before individually using only Carboplatin. The advances in molecular biology and genetic engineering, which ensured the implementation of experimental and clinical use of low molecular weight peptides, proteins, oligonucleotides, monoclonal antibodies, etc. (Kruttgen et al., 2006; Xie et al., 2005) contributed to considerable progress in the development of a "roundabout" ways of delivery of drugs. In total they have created a fertile ground not only for in-depth understanding the basic mechanisms of carcinogenesis, but also simultaneously for widening the means of

early diagnosis, the front of anticancer attack and optimization of medical schemes.

versus 67% among patients treated with placebo (*P* < 0.02).

Thus, we can conclude:

In a series of widely exploited methods there is a stereotactic implantation in the bed, formed after excision of tumor, indifferent biodegradable polymer substrates, impregnated in that or another way with an active principle. In this case, the cytostatic penetrates in measured doses into the surrounding tissue during a long period of time and destroy the remaining infected cells (Kemper et al., 2004; Ta et al., 2009). The concentration of drugs, developed in this case, can exceed observed ones with intravenous injection from 4 to 1200 times. In one of the clinical trial performed on 222 adult patients with recurrent gliomas mortality of persons who were within 6 months receiving such a method Cisplatin was 44%

The use of polymer capsules can also supply the brain with cells (transfected with viral vectors) which express a particular desired target gene which products act targeteous on the relevant parts of oncogenesis. The primary fibroblasts, astrocytes, ependimocytes, stem or progenitor cells serve as usual objects. For example, subclones Cyclin-Dependent Kinase 2 Interacting Protein (CINP) releasing *in vitro* NGF at 2 ng/ h/10-5 cells over 10 weeks was isolated from conventionally immortalized progenitor neuroblasts of the central nervous system of rat embryos with embedded DNA of growth factors. Being introduced into the brain such cells survived well, migrated at a distance of 15 mm from the implant site and

integrated with the host tissue without any signs of growth or tumor formation.

method, making reasonable transition to clinical trials.


Improving the Efficiency of

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### *Edited by Anirban Ghosh*

The tittle 'Glioma - Exploring Its Biology and Practical Relevance' is indicative of its content. This volume contains 21 chapters basically intended to explore glioma biology and discussing the experimental model systems for the purpose. It is hoped that the present volume will provide supportive and relevant awareness and understanding on the fundamental advances of the subject to the professionals from any sphere interested about glioma.

Glioma - Exploring Its Biology and Practical Relevance

Glioma

Exploring Its Biology

and Practical Relevance

*Edited by Anirban Ghosh*

Photo by CreVis2 / iStock