**Migration and Invasion of Brain Tumors**

Richard A. Able, Jr.\*, Veronica Dudu\* and Maribel Vazquez

*Department of Biomedical Engineering The City College of The City University of New York (CCNY) U.S.A.* 

#### **1. Introduction**

Recent advances in molecular biology have led to new insights in the development, growth and infiltrative behaviors of primary brain tumors (Demuth and Berens, 2004; Huse and Holland, 2010; Johnson et al., 2009; Kanu et al., 2009). These tumors are derived from various brain cell lineages and have been historically classified on the basis of morphological and, more recently, immunohistochemical features with less emphasis on their underlying molecular pathogenesis (Huse and Holland, 2010). The detailed molecular characterization of brain tumors has laid the groundwork for augmentation of standard treatment with patient-specific designed targeted therapies (Johnson et al., 2009; Kanu et al., 2009). Nevertheless, these tumors are extremely aggressive in their infiltration of brain tissue (Altman et al., 2007; Hensel et al., 1998; Yamahara et al., 2010), as well as in their metastasis outside of brain (Algra et al., 1992). Further, it now appears that the physiological conditions of the normal brain itself constitute a biological environment conducive to the uncontrolled dissemination of primary tumors (Bellail et al., 2004; Sontheimer, 2004). This review surveys the latest research on the invasive behavior of two major types of primary brain tumors: gliomas and medulloblastomas - the most common tumors diagnosed within adult and pediatric brain, respectively (Rickert and Paulus, 2001). The material has been divided into five sections: i) Characteristics of malignant brain tumors; ii) Mechanisms of tumor cell migration; iii) Models for the study of brain tumor invasion *in vivo* and *ex vivo*; iv) Models for the study of brain tumor invasion *in vitro*; and v) Future prospects of anti-invasive brain tumor therapy.

#### **2. Characteristics of malignant brain tumors**

Gliomas, commonly found in the anterior cerebral hemisphere, are a group of tumors derived from glial cells - the most abundant cells in the brain (Larjavaara et al., 2007; Lim et al., 2007; Louis et al., 2007). They are classified based on well-characterized histological features (Louis, 2006; Scheithauer, 2009; Trembath et al., 2008). The World Health Organization (WHO) defines gliomas by cell type, location and grade, and categorizes them into four classes (Lassman, 2004): i) Grade I tumors, or pilocytic astrocytomas; ii) Grade II tumors, also called low-grade astrocytomas; iii) Grade III tumors, or anaplastic astrocytomas; and iv) Grade IV tumors, also known as glioblastoma multiforme (GBM).

<sup>\*</sup> Contributed equally

Migration and Invasion of Brain Tumors 227

grade-specific predictability (Zhou et al., 2010). Recent findings suggest the possibility that the recurrent growth of glioma is derived from chemo- and radio-resistant cancer stem cell renewal and/or growth of diffusively invasive cells (Hadjipanayis and Van Meir, 2009). Evidence emerging over the past decade has suggested the existence of stem-like cells within brain tumors, which are currently examined as potential sources of tumor resistance and recurrence (Galli et al., 2004; Lenkiewicz et al., 2009; Singh and Dirks, 2007). The inability to remove high-grade gliomas in their entirety, or to prohibit their migration to other parts of the brain has led to low survival rates among brain cancer patients (Demuth and Berens, 2004). Patients with GBM have a median survival of about 1 year, while patients with anaplastic gliomas can survive 2-3 years, and those with grade II gliomas often

Medulloblastomas (MBs) encompass a collection of clinically and molecularly diverse tumor subtypes, and are characterized by high tumor invasiveness to extraneural tissues and reoccurrence in the cerebellum after total resection (Dhall, 2009). Four different MB subtypes have been included in the current WHO classification (Louis et al., 2007): i) Classic MB; ii) Demoplastic/nodular MB; iii) MB with extensive nodularity; and iv) Anaplastic or large cell MB. Two other variants, medullomyoblastoma and melanotic MB, are much more rare. MBs are overwhelmingly found in pediatric patients, but can rarely occur within adult brain, where the tumor characteristics become very atypical. Adult MB is arguably a biologically distinct challenge in that it exhibits a higher proportion of desmoplastic histological characteristics, shows more proclivity toward cerebellar hemispheric origin, possesses different proliferative and apoptotic indices, and demonstrates a notorious tendency for late

relapse with respect to the pediatric variants (Chan et al., 2000; Sarkar et al., 2002).

cancer, because to date, few data link such genetic alterations to metastasis in MB.

reoccurrence and aggressive brain infiltration (Farin et al., 2006).

The treatment of patients with standard risk tumors, i.e. those who had tumors completely resected and with no evidence of dissemination to any other part of the body (Nishikawa, 2010), has been partially successful with survival rates of up to 40% for gliomas over the last five years (Van Meir et al., 2010) and 78% for MBs (Gajjar et al., 2006). In contrast, the cure of metastatic disease has been limited until recently to single cases (Fruhwald and Plass, 2002). Even though promising, the current treatment options for high-risk brain tumors are associated with neural and neuroendocrine side effects, with a tremendous decline in quality of life among survivors (Edelstein et al., 2011; Palmer et al., 2001), as well as growth

In the cases of both gliomas and MBs, the migration of cells from primary tumors to other locations, within brain or otherwise, has been one of the most clinically challenging and poorly understood processes that contributes to the poor life prognosis of patients. The

MBs are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) (Gibson et al., 2010) after aberrant activation of the Sonic Hedgehog (Shh) pathway (Figure 1B). A number of genetic alterations have been associated with MB (Biegel et al., 1997; Bigner et al., 1997; Herms et al., 2000; Yin et al., 2002). Studies of the receptors and intracellular signaling pathways that support proliferation and survival of GNPCs have shown a dysregulation of the Shh pathway, the canonical Wnt pathway, or the ERB-B pathway in both familiar and sporadic MBs (Gilbertson, 2004). A recent study showed that Wnt-subtype tumors infiltrate the dorsal brainstem, whereas Shhsubtype tumors are located within the cerebellar hemispheres (Gibson et al., 2010). These results have profound implications for future research and treatment of this childhood

survive 10-15 years (Louis et al., 2007).

Grade I tumors typically do not invade surrounding brain and are often curable with surgery, while tumors of grades II to IV are diffuse and invade normal brain, with grade III and IV tumors being most aggressive. Grade III and IV tumors are called "high-grade" or "malignant" tumors although they almost never metastasize to other tissues of the body (Lassman, 2004).

The etiological events causing glioma formation have not been clearly defined, but are thought to involve genetic alterations (Figure 1A). Such alterations disrupt cell cycle arrest pathways (Zhou et al., 2005; Zhou et al., 2010), and cause aberrant receptor tyrosine kinase activity in the brain cells (Dai et al., 2001). For instance, activation of receptors such as Hepatocyte Growth Factor Receptor (HGF) c-Met (Gentile et al., 2008), Platelet-Derived Growth Factor Receptor (PDGFR) (Cattaneo et al., 2006; Natarajan et al., 2006), and Epidermal Growth Factor Receptor (EGFR) (Chicoine and Silbergeld, 1997) is now wellknown to stimulate glioma motility. Additionally, marker specific glial progenitor populations, neural stem cells and cancer stem cells are being investigated for their roles as possible initiators of gliomagenesis (Briancon-Marjollet et al., 2010).

Fig. 1. Origin of brain tumors: development gone wrong. (A) During normal brain development, neural stem cells give rise to three main adult cell types: neurons, oligodendrocytes and astrocytes. Genetic alterations occur within these differentiated cells that can lead to the rise of malignant tumors. Alternatively, immature stem cells may serve as cancer stem cells that confer both radio- and chemoresistance phenotypes to gliomas. (B) Medulloblastomas originate in the cerebellum, from granule neuron precursor cells (GNPCs), upon un-controlled activation of Sonic Hedgehog (Shh) signaling pathway.

The current standard of care for gliomas is surgical removal of the tumor followed by postoperative radio- and chemotherapy (Stupp and Weber, 2005). However, due to their diffusely invasive properties, gliomas are one of the most difficult tumors to isolate or treat (Burger et al., 1985). Furthermore, while cell migration is fundamental to normal brain development and homeostasis, unconstrained migration of pathological and diseased cells makes the complete resection of tumor lesions, often performed for other types of tumors, an ineffective clinical treatment in brain. Prior to the advance of high-throughput genetic screening techniques clinicians depended primarily on glioma recurrence for prognosis of patient survival. Later, the generation of models that combined gene expression and molecular markers made it possible to subcategorize gliomas, enabling the increase in

Grade I tumors typically do not invade surrounding brain and are often curable with surgery, while tumors of grades II to IV are diffuse and invade normal brain, with grade III and IV tumors being most aggressive. Grade III and IV tumors are called "high-grade" or "malignant" tumors although they almost never metastasize to other tissues of the body

The etiological events causing glioma formation have not been clearly defined, but are thought to involve genetic alterations (Figure 1A). Such alterations disrupt cell cycle arrest pathways (Zhou et al., 2005; Zhou et al., 2010), and cause aberrant receptor tyrosine kinase activity in the brain cells (Dai et al., 2001). For instance, activation of receptors such as Hepatocyte Growth Factor Receptor (HGF) c-Met (Gentile et al., 2008), Platelet-Derived Growth Factor Receptor (PDGFR) (Cattaneo et al., 2006; Natarajan et al., 2006), and Epidermal Growth Factor Receptor (EGFR) (Chicoine and Silbergeld, 1997) is now wellknown to stimulate glioma motility. Additionally, marker specific glial progenitor populations, neural stem cells and cancer stem cells are being investigated for their roles as

possible initiators of gliomagenesis (Briancon-Marjollet et al., 2010).

Fig. 1. Origin of brain tumors: development gone wrong. (A) During normal brain development, neural stem cells give rise to three main adult cell types: neurons,

Medulloblastomas originate in the cerebellum, from granule neuron precursor cells (GNPCs), upon un-controlled activation of Sonic Hedgehog (Shh) signaling pathway.

oligodendrocytes and astrocytes. Genetic alterations occur within these differentiated cells that can lead to the rise of malignant tumors. Alternatively, immature stem cells may serve as cancer stem cells that confer both radio- and chemoresistance phenotypes to gliomas. (B)

The current standard of care for gliomas is surgical removal of the tumor followed by postoperative radio- and chemotherapy (Stupp and Weber, 2005). However, due to their diffusely invasive properties, gliomas are one of the most difficult tumors to isolate or treat (Burger et al., 1985). Furthermore, while cell migration is fundamental to normal brain development and homeostasis, unconstrained migration of pathological and diseased cells makes the complete resection of tumor lesions, often performed for other types of tumors, an ineffective clinical treatment in brain. Prior to the advance of high-throughput genetic screening techniques clinicians depended primarily on glioma recurrence for prognosis of patient survival. Later, the generation of models that combined gene expression and molecular markers made it possible to subcategorize gliomas, enabling the increase in

(Lassman, 2004).

grade-specific predictability (Zhou et al., 2010). Recent findings suggest the possibility that the recurrent growth of glioma is derived from chemo- and radio-resistant cancer stem cell renewal and/or growth of diffusively invasive cells (Hadjipanayis and Van Meir, 2009). Evidence emerging over the past decade has suggested the existence of stem-like cells within brain tumors, which are currently examined as potential sources of tumor resistance and recurrence (Galli et al., 2004; Lenkiewicz et al., 2009; Singh and Dirks, 2007). The inability to remove high-grade gliomas in their entirety, or to prohibit their migration to other parts of the brain has led to low survival rates among brain cancer patients (Demuth and Berens, 2004). Patients with GBM have a median survival of about 1 year, while patients with anaplastic gliomas can survive 2-3 years, and those with grade II gliomas often survive 10-15 years (Louis et al., 2007).

Medulloblastomas (MBs) encompass a collection of clinically and molecularly diverse tumor subtypes, and are characterized by high tumor invasiveness to extraneural tissues and reoccurrence in the cerebellum after total resection (Dhall, 2009). Four different MB subtypes have been included in the current WHO classification (Louis et al., 2007): i) Classic MB; ii) Demoplastic/nodular MB; iii) MB with extensive nodularity; and iv) Anaplastic or large cell MB. Two other variants, medullomyoblastoma and melanotic MB, are much more rare. MBs are overwhelmingly found in pediatric patients, but can rarely occur within adult brain, where the tumor characteristics become very atypical. Adult MB is arguably a biologically distinct challenge in that it exhibits a higher proportion of desmoplastic histological characteristics, shows more proclivity toward cerebellar hemispheric origin, possesses different proliferative and apoptotic indices, and demonstrates a notorious tendency for late relapse with respect to the pediatric variants (Chan et al., 2000; Sarkar et al., 2002).

MBs are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) (Gibson et al., 2010) after aberrant activation of the Sonic Hedgehog (Shh) pathway (Figure 1B). A number of genetic alterations have been associated with MB (Biegel et al., 1997; Bigner et al., 1997; Herms et al., 2000; Yin et al., 2002). Studies of the receptors and intracellular signaling pathways that support proliferation and survival of GNPCs have shown a dysregulation of the Shh pathway, the canonical Wnt pathway, or the ERB-B pathway in both familiar and sporadic MBs (Gilbertson, 2004). A recent study showed that Wnt-subtype tumors infiltrate the dorsal brainstem, whereas Shhsubtype tumors are located within the cerebellar hemispheres (Gibson et al., 2010). These results have profound implications for future research and treatment of this childhood cancer, because to date, few data link such genetic alterations to metastasis in MB.

The treatment of patients with standard risk tumors, i.e. those who had tumors completely resected and with no evidence of dissemination to any other part of the body (Nishikawa, 2010), has been partially successful with survival rates of up to 40% for gliomas over the last five years (Van Meir et al., 2010) and 78% for MBs (Gajjar et al., 2006). In contrast, the cure of metastatic disease has been limited until recently to single cases (Fruhwald and Plass, 2002). Even though promising, the current treatment options for high-risk brain tumors are associated with neural and neuroendocrine side effects, with a tremendous decline in quality of life among survivors (Edelstein et al., 2011; Palmer et al., 2001), as well as growth reoccurrence and aggressive brain infiltration (Farin et al., 2006).

In the cases of both gliomas and MBs, the migration of cells from primary tumors to other locations, within brain or otherwise, has been one of the most clinically challenging and poorly understood processes that contributes to the poor life prognosis of patients. The

Migration and Invasion of Brain Tumors 229

During the mesenchymal-type migration often observed in gliomas, cells exhibit a highly polarized and fibroblastic morphology. Cells undergo the classical, overlapping processes generally exhibited during mammalian cell migration: cell polarization, protrusion of leading edge, traction at the trailing edge, and detachment (Lauffenburger and Horwitz, 1996). First, cells become highly fibroblast-like, with bipolar opposites. Second, a growing number of actin filaments begin to push the cell membrane outwards on the leading edge via the formation of lamellipodia or filopodia. Actin polymerization then initiates signal transduction pathways along the leading edge. Next, cell integrins come into contact with ECM ligands and cluster to recruit intracellular signaling proteins that induce phosphorylation signaling, or so-called "outside in" signaling (Hynes, 2002; Miyamoto et al., 1995) via focal adhesion kinases. Afterwards, surface proteases act to cleave ECM molecules via production of soluble matrix metalloproteases (MMPs) in order to degrade surrounding ECM. Finally, cell contraction occurs via myosin that leads to focal contact disassembly at

the trailing edge and actin cleavage and filament turnover (Wear et al., 2000).

cytoskeleton organization for increased dissemination (Wolf et al., 2003).

increase dissemination and metastatic spread (Friedl and Wolf, 2003).

**4. Models for the study of brain tumor invasion** *in vivo* **and** *ex vivo*

Glioma and MB models have been largely developed by studying altered oncogene expression through retroviral transfection of murine neural tissue of genetically engineered mouse models (GEMMs) (Fisher et al., 1999; Hatton et al., 2008; Heyer et al., 2010; Huse and

Contrarily, during amoeboid movement, cells utilize a "fast gliding" mechanism driven by weak interactions with the substrate. As such, cells like neutrophils and lymphocytes exhibit a shape-driven migration with appreciable lack of focal adhesions that allows them to circumnavigate rather than degrade surrounding ECM during migration (Friedl et al., 2001). The result is an increased cell motility, as well as cell ability to undergo early detachment and metastatic spread from primary tumors. Cancer cells may undergo conversion from mesenchymal to amoeboid type migration in order to alter integrin distribution and actin

Collective migration is a well-studied phenomenon that is characteristic of embryological development, such as during the migration of cell clusters or sheets in the ectoderm following closure of the neural tube (Davidson and Keller, 1999). *In vitro* studies (Friedl et al., 1995; Nabeshima et al., 1995) showed that cells can migrate as a functional unit, and that in contrast to single motile cells, cell-cell adhesion can lead to a particular form of cortical actin filament present along cell junctions. This enables formation of a larger, multicellular contractile body. Here, a select group of highly motile cells are designated as so-called "path-generating" cells that create migratory traction via pseudopod activity (Friedl et al., 1995; Hegerfeldt et al., 2002; Nabeshima et al., 1995). It is then believed that cells located in the inner and trailing regions are passively dragged behind during dissemination. In tumors, collective migration has been observed as protruding sheets that maintain contact with the primary site, or as cell clusters that detach from their origin and extend along paths of least resistance (Byers et al., 1995; Hashizume et al., 1996; Madhavan et al., 2001). Collective migration offers the advantage of protection from immunological response. Further, heterogeneous sets of cells that move as one functional unit can work together to promote the invasion of less motile, but potentially apoptosis-resistant, sub-populations that increase tumor survival. To complicate matters, cells may transition between collective and individual cell migration with dedifferentiated cells to

following sections will discuss the fundamental mechanisms of cellular migration, the common *in vivo* models used to examine tumor cell migration within brain, and current *in vitro* technologies used to characterize and better understand the migration of cells derived from gliomas and MBs.

#### **3. Mechanisms of tumor cell migration**

The migration of brain cancer cells is highly complex, involving interactions with extracellular matrix (ECM), and chemoattractants that either diffuse from blood vessels and/or are produced by neighboring cells (Condeelis and Segall, 2003; Sahai, 2005). As a consequence of such complexity, the molecular mechanisms of primary brain tumor migration and metastasis are poorly understood. Over the last several years, a group of critical growth factors has been the topic of research for their role as regulators of tumor biology and chemotaxis (Hamel and Westphal, 2000). It is believed that over time secreted cytokines diffuse and generate concentration gradients that are sensed by glioma and MBderived cells, leading to the detachment and migration of these cells away from the primary tumor (Chicoine and Silbergeld, 1997; Piperi et al., 2005). Therefore, brain tumor invasion is believed to be induced by soluble cytokines that stimulate directional and/or random tumor cell motility (Brockmann et al., 2003). Alternatively, cancer cells may communicate with specific distant targets through secreted microvesicles that contain growth factors and receptors, functional mRNAs, and miRNAs (Cocucci et al., 2009; Skog et al., 2008; Valadi et al., 2007). Such microvesicles are shed by most cell types, including cancer cells, and have been found in sera from numerous cancer patients (Cocucci et al., 2009; Skog et al., 2008).

While the effects of mitogens on the *in vitro* motility and invasion of glioma cells have been well documented using conventional assays, such as transwell chambers and spheroid models (discussed later in this chapter), the ability of soluble cytokines to drive various cellular functions (i.e. migration and/or proliferative growth) has been shown to depend upon several determinant factors. Some of these factors have been addressed in the literature, such as dosage-dependence (Gonzalez-Perez and Quinones-Hinojosa, 2010; Shih et al., 2004), contact inhibition (Weidner et al., 1990), as well as autocrine and paracrine signaling-driven tumor growth via extensive proliferation and aggressive recruitment of surrounding cells to the tumor (Betsholtz et al., 1984; Fomchenko and Holland, 2005; Hermansson et al., 1988; Rood et al., 2004). The diligent study of Central Nervous System tumor cell (CNSTC) invasion has identified four commonly overexpressed receptor tyrosine kinases as targets for anti-invasive therapies (EGFR, c-MET, PDGFR, and Vascular Epidermal Growth Factor Receptor (VEGFR)) (Abounader, 2009; Arora and Scholar, 2005; Huang et al., 2009; Zwick et al., 2001).

Cancer cell locomotion is highly sensitive to stimuli from the ECM as well as from the surrounding media. Receptors on the plasma membrane can activate cellular signaling pathways that alter the mechanotransduction of a cell via reorganization of motility-related organelles and cellular compartments. As an example, tumor-derived cells are known to increase cell motility in response to protease inhibitors and adhesion inhibitors (Sahai, 2005). The modes of cancer cell migration vary according to whether the cells undergo single cell chain, or collective migration. Tumor-derived cells disseminate from the bulk tumor mass individually via mesenchymal or amoeboid movement. However, in many tumors both single cell and collective cell migration may be present depending on the molecular cues dictating migration (Friedl and Wolf, 2003).

following sections will discuss the fundamental mechanisms of cellular migration, the common *in vivo* models used to examine tumor cell migration within brain, and current *in vitro* technologies used to characterize and better understand the migration of cells derived

The migration of brain cancer cells is highly complex, involving interactions with extracellular matrix (ECM), and chemoattractants that either diffuse from blood vessels and/or are produced by neighboring cells (Condeelis and Segall, 2003; Sahai, 2005). As a consequence of such complexity, the molecular mechanisms of primary brain tumor migration and metastasis are poorly understood. Over the last several years, a group of critical growth factors has been the topic of research for their role as regulators of tumor biology and chemotaxis (Hamel and Westphal, 2000). It is believed that over time secreted cytokines diffuse and generate concentration gradients that are sensed by glioma and MBderived cells, leading to the detachment and migration of these cells away from the primary tumor (Chicoine and Silbergeld, 1997; Piperi et al., 2005). Therefore, brain tumor invasion is believed to be induced by soluble cytokines that stimulate directional and/or random tumor cell motility (Brockmann et al., 2003). Alternatively, cancer cells may communicate with specific distant targets through secreted microvesicles that contain growth factors and receptors, functional mRNAs, and miRNAs (Cocucci et al., 2009; Skog et al., 2008; Valadi et al., 2007). Such microvesicles are shed by most cell types, including cancer cells, and have been found in sera from numerous cancer patients (Cocucci et al., 2009; Skog et al., 2008). While the effects of mitogens on the *in vitro* motility and invasion of glioma cells have been well documented using conventional assays, such as transwell chambers and spheroid models (discussed later in this chapter), the ability of soluble cytokines to drive various cellular functions (i.e. migration and/or proliferative growth) has been shown to depend upon several determinant factors. Some of these factors have been addressed in the literature, such as dosage-dependence (Gonzalez-Perez and Quinones-Hinojosa, 2010; Shih et al., 2004), contact inhibition (Weidner et al., 1990), as well as autocrine and paracrine signaling-driven tumor growth via extensive proliferation and aggressive recruitment of surrounding cells to the tumor (Betsholtz et al., 1984; Fomchenko and Holland, 2005; Hermansson et al., 1988; Rood et al., 2004). The diligent study of Central Nervous System tumor cell (CNSTC) invasion has identified four commonly overexpressed receptor tyrosine kinases as targets for anti-invasive therapies (EGFR, c-MET, PDGFR, and Vascular Epidermal Growth Factor Receptor (VEGFR)) (Abounader, 2009; Arora and Scholar, 2005;

Cancer cell locomotion is highly sensitive to stimuli from the ECM as well as from the surrounding media. Receptors on the plasma membrane can activate cellular signaling pathways that alter the mechanotransduction of a cell via reorganization of motility-related organelles and cellular compartments. As an example, tumor-derived cells are known to increase cell motility in response to protease inhibitors and adhesion inhibitors (Sahai, 2005). The modes of cancer cell migration vary according to whether the cells undergo single cell chain, or collective migration. Tumor-derived cells disseminate from the bulk tumor mass individually via mesenchymal or amoeboid movement. However, in many tumors both single cell and collective cell migration may be present depending on the molecular cues

from gliomas and MBs.

**3. Mechanisms of tumor cell migration** 

Huang et al., 2009; Zwick et al., 2001).

dictating migration (Friedl and Wolf, 2003).

During the mesenchymal-type migration often observed in gliomas, cells exhibit a highly polarized and fibroblastic morphology. Cells undergo the classical, overlapping processes generally exhibited during mammalian cell migration: cell polarization, protrusion of leading edge, traction at the trailing edge, and detachment (Lauffenburger and Horwitz, 1996). First, cells become highly fibroblast-like, with bipolar opposites. Second, a growing number of actin filaments begin to push the cell membrane outwards on the leading edge via the formation of lamellipodia or filopodia. Actin polymerization then initiates signal transduction pathways along the leading edge. Next, cell integrins come into contact with ECM ligands and cluster to recruit intracellular signaling proteins that induce phosphorylation signaling, or so-called "outside in" signaling (Hynes, 2002; Miyamoto et al., 1995) via focal adhesion kinases. Afterwards, surface proteases act to cleave ECM molecules via production of soluble matrix metalloproteases (MMPs) in order to degrade surrounding ECM. Finally, cell contraction occurs via myosin that leads to focal contact disassembly at the trailing edge and actin cleavage and filament turnover (Wear et al., 2000).

Contrarily, during amoeboid movement, cells utilize a "fast gliding" mechanism driven by weak interactions with the substrate. As such, cells like neutrophils and lymphocytes exhibit a shape-driven migration with appreciable lack of focal adhesions that allows them to circumnavigate rather than degrade surrounding ECM during migration (Friedl et al., 2001). The result is an increased cell motility, as well as cell ability to undergo early detachment and metastatic spread from primary tumors. Cancer cells may undergo conversion from mesenchymal to amoeboid type migration in order to alter integrin distribution and actin cytoskeleton organization for increased dissemination (Wolf et al., 2003).

Collective migration is a well-studied phenomenon that is characteristic of embryological development, such as during the migration of cell clusters or sheets in the ectoderm following closure of the neural tube (Davidson and Keller, 1999). *In vitro* studies (Friedl et al., 1995; Nabeshima et al., 1995) showed that cells can migrate as a functional unit, and that in contrast to single motile cells, cell-cell adhesion can lead to a particular form of cortical actin filament present along cell junctions. This enables formation of a larger, multicellular contractile body. Here, a select group of highly motile cells are designated as so-called "path-generating" cells that create migratory traction via pseudopod activity (Friedl et al., 1995; Hegerfeldt et al., 2002; Nabeshima et al., 1995). It is then believed that cells located in the inner and trailing regions are passively dragged behind during dissemination. In tumors, collective migration has been observed as protruding sheets that maintain contact with the primary site, or as cell clusters that detach from their origin and extend along paths of least resistance (Byers et al., 1995; Hashizume et al., 1996; Madhavan et al., 2001). Collective migration offers the advantage of protection from immunological response. Further, heterogeneous sets of cells that move as one functional unit can work together to promote the invasion of less motile, but potentially apoptosis-resistant, sub-populations that increase tumor survival. To complicate matters, cells may transition between collective and individual cell migration with dedifferentiated cells to increase dissemination and metastatic spread (Friedl and Wolf, 2003).

#### **4. Models for the study of brain tumor invasion** *in vivo* **and** *ex vivo*

Glioma and MB models have been largely developed by studying altered oncogene expression through retroviral transfection of murine neural tissue of genetically engineered mouse models (GEMMs) (Fisher et al., 1999; Hatton et al., 2008; Heyer et al., 2010; Huse and

Migration and Invasion of Brain Tumors 231

Fig. 2. Neoplastic cellular infiltration into surrounding non-neoplastic brain tissue in syngeneic rat (CNS-1) and mouse (GL26) GBM models and human glioma xenografts in nude mice (U251 and U87). Paraffin sections (5 μm) from GBM were stained with hematoxylin and eosin for evaluating neoplastic invasion. The numbers in low-

magnification microphotographs depict areas magnified in the microphotographs on the right. *Arrows* indicate malignant cells, clusters of GBM cells, and tumoral blood vessels infiltrating surrounding brain parenchyma. The indistinct tumor borders and the malignant

cells clearly entering the non-neoplastic brain tissue suggest an invasive phenotype.

(Courtesy of Candolfi et al., 2007)

Holland, 2010; Pazzaglia et al., 2002; Pazzaglia et al., 2006; Romer and Curran, 2004). Via this powerful methodology, diverse tumor types with distinct histological features have been generated dependent upon the specific genetic background of the cell of tumor origin and the disease location of interest (Furnari et al., 2007). In particular, the histological features of GEMM and implanted xenograph derived tumors have been shown to be similar to human brain tumors presented in identical CNS locations, and have shed light on the diverse nature of human gliomas found clinically (Candolfi et al., 2007).

Historically, it has been suggested that glioma cell infiltration throughout the brain primarily utilizes mechanisms of migration innately patterned by neural progenitors during normal brain development (Cayre et al., 2009; Kakita and Goldman, 1999; Scherer, 1940). Confirmation of this similarity has been accomplished *in vivo* via implanted xenographs that result in spontaneous intracranial GBMs in six different animal model variations that show reproducible invasion of tumor cells into non-neoplastic brain regions (Figure 2) (Candolfi et al., 2007). More recently, several labs began utilizing GEMMs to specifically examine glial progenitor recruitment *in vivo* (Assanah et al., 2006; Masui et al., 2010). For instance, Assanah and colleagues have demonstrated via histological analysis of cortical sections from GEMMs that overexpression of tumor inducing proteins like PDGF can induce malignant glioma cells to invade across the corpus callosum into the contralateral hemisphere and overlying cortex (Assanah et al., 2006; Assanah et al., 2009).

The diffusive invasion and increased recurrence of gliomas post-operatively have been attributed to the same therapies used to treat the disease. Narayana and colleagues reported clinical results of 61 high-grade GBM patients treated with an anti-angiogenesis drug, bevacizumab. Their results showed that 82% of the patients treated with bevacizumab suffer from tumor regrowth and 70% died from the disease within 19 months (Narayana et al., 2009). Pàez-Ribes and colleagues reported similar results showing that although the use of angiogenesis inhibitors, such as Sunitinib and SU10944, extend that survival time of treated mice to an additional 7 weeks versus non-treated mice, the kinase inhibitors tend to also evoke an increase in glioma cell invasion as well as to promote tumor progression (Pàez-Ribes et al., 2009). A closer examination using xenographs of human tumor spheroid implanted into rat brains, and further treated with the bevacizumab, led to a reduction in contrast enhancement in magnetic resonance imaging (MRI) analysis while enhancing glioma cell diffusion by 68% versus non-treated rats (Figure 3) (Keunen et al., 2011).

Characterization of MB migration *in vivo* has yet to be analyzed at large, as most of the reports to-date focused on tumor growth and not its dissemination. Nevertheless, a select number of *in vivo* studies examined tumor cell migration and invasion. Hatton and colleagues illustrated in a GEMM for MB that 94% of the mice developed MB by 2 months of age, and that these tumors frequently exhibited leptomeningeal spread, a common feature of the human disease (Hatton et al., 2008). MacDonald and colleagues implanted human MB cells in the brain of nude mice, and thereafter followed them *in vivo* at single-cell level via fluorescence microscopy (MacDonald et al., 1998). These MB cells were shown to invade the brain and to form distant micro-metastases. In another study, MB cells were engineered to overexpress HGF and were implanted subcutaneously and intra-cranially (Li et al., 2005). The study reported activated c-Met that strongly increased MB xenograft growth and invasive characteristics with finger-like protrusions, metastatic growth, and leptomeningeal spread. Such findings illustrate that the HGF/c-Met pathway is one of the mediators of MB malignancy.

Holland, 2010; Pazzaglia et al., 2002; Pazzaglia et al., 2006; Romer and Curran, 2004). Via this powerful methodology, diverse tumor types with distinct histological features have been generated dependent upon the specific genetic background of the cell of tumor origin and the disease location of interest (Furnari et al., 2007). In particular, the histological features of GEMM and implanted xenograph derived tumors have been shown to be similar to human brain tumors presented in identical CNS locations, and have shed light on the diverse nature

Historically, it has been suggested that glioma cell infiltration throughout the brain primarily utilizes mechanisms of migration innately patterned by neural progenitors during normal brain development (Cayre et al., 2009; Kakita and Goldman, 1999; Scherer, 1940). Confirmation of this similarity has been accomplished *in vivo* via implanted xenographs that result in spontaneous intracranial GBMs in six different animal model variations that show reproducible invasion of tumor cells into non-neoplastic brain regions (Figure 2) (Candolfi et al., 2007). More recently, several labs began utilizing GEMMs to specifically examine glial progenitor recruitment *in vivo* (Assanah et al., 2006; Masui et al., 2010). For instance, Assanah and colleagues have demonstrated via histological analysis of cortical sections from GEMMs that overexpression of tumor inducing proteins like PDGF can induce malignant glioma cells to invade across the corpus callosum into the contralateral hemisphere and

The diffusive invasion and increased recurrence of gliomas post-operatively have been attributed to the same therapies used to treat the disease. Narayana and colleagues reported clinical results of 61 high-grade GBM patients treated with an anti-angiogenesis drug, bevacizumab. Their results showed that 82% of the patients treated with bevacizumab suffer from tumor regrowth and 70% died from the disease within 19 months (Narayana et al., 2009). Pàez-Ribes and colleagues reported similar results showing that although the use of angiogenesis inhibitors, such as Sunitinib and SU10944, extend that survival time of treated mice to an additional 7 weeks versus non-treated mice, the kinase inhibitors tend to also evoke an increase in glioma cell invasion as well as to promote tumor progression (Pàez-Ribes et al., 2009). A closer examination using xenographs of human tumor spheroid implanted into rat brains, and further treated with the bevacizumab, led to a reduction in contrast enhancement in magnetic resonance imaging (MRI) analysis while enhancing

glioma cell diffusion by 68% versus non-treated rats (Figure 3) (Keunen et al., 2011).

Characterization of MB migration *in vivo* has yet to be analyzed at large, as most of the reports to-date focused on tumor growth and not its dissemination. Nevertheless, a select number of *in vivo* studies examined tumor cell migration and invasion. Hatton and colleagues illustrated in a GEMM for MB that 94% of the mice developed MB by 2 months of age, and that these tumors frequently exhibited leptomeningeal spread, a common feature of the human disease (Hatton et al., 2008). MacDonald and colleagues implanted human MB cells in the brain of nude mice, and thereafter followed them *in vivo* at single-cell level via fluorescence microscopy (MacDonald et al., 1998). These MB cells were shown to invade the brain and to form distant micro-metastases. In another study, MB cells were engineered to overexpress HGF and were implanted subcutaneously and intra-cranially (Li et al., 2005). The study reported activated c-Met that strongly increased MB xenograft growth and invasive characteristics with finger-like protrusions, metastatic growth, and leptomeningeal spread. Such findings illustrate that the HGF/c-Met pathway is one of the mediators of MB

of human gliomas found clinically (Candolfi et al., 2007).

overlying cortex (Assanah et al., 2006; Assanah et al., 2009).

malignancy.

Fig. 2. Neoplastic cellular infiltration into surrounding non-neoplastic brain tissue in syngeneic rat (CNS-1) and mouse (GL26) GBM models and human glioma xenografts in nude mice (U251 and U87). Paraffin sections (5 μm) from GBM were stained with hematoxylin and eosin for evaluating neoplastic invasion. The numbers in lowmagnification microphotographs depict areas magnified in the microphotographs on the right. *Arrows* indicate malignant cells, clusters of GBM cells, and tumoral blood vessels infiltrating surrounding brain parenchyma. The indistinct tumor borders and the malignant cells clearly entering the non-neoplastic brain tissue suggest an invasive phenotype. (Courtesy of Candolfi et al., 2007)

Migration and Invasion of Brain Tumors 233

to characterize the expression molecular markers (Riffkin et al., 2001), and to evaluate the therapeutic potential of co-cultured T-cells for anti-tumor activity (Ahmed et al., 2007). By reducing the incidence of recurrent growth, clinicians envision the possibility of detecting and directly tracking migratory tumor cells *in vivo*, and therefore enabling operative procedures limited to a single total resection surgery. In order to accomplish this goal, there is a stringent need for development of enhanced imaging tools to allow visualization of migrating tumor cells. Meanwhile, the most successful quantitative assessment of CNSTCs migration has been accomplished outside of the brain itself, in engineered systems redesigned to mimic specific *in vivo* conditions. We will discuss these *in vitro* assays further, which have been utilized to evaluate a variety of cellular functions, from growth patterns and rates, to invasive motility of cells derived from highly malignant brain

Tumor cells of the brain have been characterized as having a highly infiltrative phenotype for spreading into the healthy surrounding parenchyma. This malignant property is arguably the principle reason for tumor recurrence and high mortality rates (Lim et al., 2007). The interaction of integrins, membrane anchored heterodimeric proteins, with various ECM proteins has been explored extensively, as it is one of the key events that occurs during the invasion of tumor cells within their local microenvironments (Teodorczyk and Martin-Villalba, 2010). Another key process of tumor invasion is the cellular secretion/production of proteases that degrade ECM proteins in order to create pores through which the cells may migrate; such proteases include serine proteases, various MMPs, and cathepsins (Rao, 2003). In addition to stimulating tumor invasion via degradation of ECM protein components, it is assumed that MMPs are capable of enhancing tumor growth by indirectly triggering the release of growth factors trapped within the basement membrane itself (Mott and Werb, 2004). Lastly, another key aspect of tumor dissemination is played by the activation of RTK signaling pathways. During the destruction of the basement membrane by MMPs, soluble growth factors are sequestered from the ECM and bind to their cognate cellular receptors to

trigger a cascade of events that enhance cellular migration (Zucker et al., 2000).

*In vitro* invasion assays are important tools for investigating the tumor-matrix interactions and the effects of extracellular macromolecules on these interactions. While not entirely identical to *in vivo* behavior, the study of tumor cell migration *in vitro* is advantageous due to the tightly-controlled experimental conditions, higher experimental throughput, and lower costs. The following section discusses the most commonly used *in vitro* assays, in the

Culture dish assays have the advantage of design simplicity and execution, while providing insightful information pertaining to cell-to-cell and cell-to-environment interactions. Coated culture dishes have been widely used to examine the roles played by specific ECM proteins, integrins, MMPs, and RTKs in stimulating the migration of brain tumor-derived cells, as

Integrins are membrane heterodimeric proteins that mediate cell-environment attachment (Hynes, 1987; Tucker, 2006). In addition to anchoring cells to their environment, integrins

**5. Models for the study of brain tumor invasion** *in vitro*

tumors.

order of increased complexity.

**5.1 Culture dish assays** 

detailed here.

Fig. 3. Changes in blood vessel morphology and tumor cell invasion after bev treatment. Immunostaining for von Willebrand factor (vWF) (A and B) and quantification thereof (C), indicating a significant reduction in the density of medium and large blood vessels and in total vessel number after bev treatment. (Scale bar: 200 μm.) Nestin-stained composite images (D and E) reveal a more homogeneous appearance of the treated compared with untreated tumors, also reflected in corresponding T2-weighted MRI images (F). Large vessels ("V") appear as dark tortuous lines in nestin and T2- weighted images and necrotic areas ("N") as brighter spots. Quantification of the nestin-positive cells outside the tumor core (G and H) shows a 68% increase in cell invasion after treatment (I). mi.v: microvessels; in.v: intermediate-sized vessels; ma.v: macrovessels; Ctrl: controls; Tr: treated. (Scale bars: ± SE.) \*\*\*P < 0.001. (Courtesy of Keunen et al., 2011)

The ability to visualize brain tumor invasion in direct response to specific genetic aberrations and alterations made to the immediate environment has been critical in understanding the characteristics of this process. An advance made in this direction was accomplished via the detection of specific biomarkers involved in the progression or migration of CNSTCs, such as Receptor Tyrosine Kinases (RTKs), using conjugated antibodies that enabled *in vivo* monitoring via MRI (Towner et al., 2008). Alternatively, to *in vivo* imaging procedures, *ex vivo* brain tumor invasion assays that enable the study of tissue outside of the living system have had a tremendous impact in the field. Brain slices from mice and rats have been used to quantify the invasion of human gliomas (Nakada et al., 2004), and have demonstrated suppressed invasion on 2D surfaces, suggesting that the brain environment alone is capable of regulating protein function and, consequentially, the pattern and directionality of glioma migration (Beadle et al., 2008). Additionally, not only have *ex vivo* cell cultures been used to study the invasive properties of CNS tumors, but also

Fig. 3. Changes in blood vessel morphology and tumor cell invasion after bev treatment. Immunostaining for von Willebrand factor (vWF) (A and B) and quantification thereof (C), indicating a significant reduction in the density of medium and large blood vessels and in total vessel number after bev treatment. (Scale bar: 200 μm.) Nestin-stained composite images (D and E) reveal a more homogeneous appearance of the treated compared with untreated tumors, also reflected in corresponding T2-weighted MRI images (F). Large vessels ("V") appear as dark tortuous lines in nestin and T2- weighted images and necrotic areas ("N") as brighter spots. Quantification of the nestin-positive cells outside the tumor core (G and H) shows a 68% increase in cell invasion after treatment (I). mi.v: microvessels; in.v: intermediate-sized vessels; ma.v: macrovessels; Ctrl: controls; Tr: treated. (Scale bars: ±

The ability to visualize brain tumor invasion in direct response to specific genetic aberrations and alterations made to the immediate environment has been critical in understanding the characteristics of this process. An advance made in this direction was accomplished via the detection of specific biomarkers involved in the progression or migration of CNSTCs, such as Receptor Tyrosine Kinases (RTKs), using conjugated antibodies that enabled *in vivo* monitoring via MRI (Towner et al., 2008). Alternatively, to *in vivo* imaging procedures, *ex vivo* brain tumor invasion assays that enable the study of tissue outside of the living system have had a tremendous impact in the field. Brain slices from mice and rats have been used to quantify the invasion of human gliomas (Nakada et al., 2004), and have demonstrated suppressed invasion on 2D surfaces, suggesting that the brain environment alone is capable of regulating protein function and, consequentially, the pattern and directionality of glioma migration (Beadle et al., 2008). Additionally, not only have *ex vivo* cell cultures been used to study the invasive properties of CNS tumors, but also

SE.) \*\*\*P < 0.001. (Courtesy of Keunen et al., 2011)

to characterize the expression molecular markers (Riffkin et al., 2001), and to evaluate the therapeutic potential of co-cultured T-cells for anti-tumor activity (Ahmed et al., 2007).

By reducing the incidence of recurrent growth, clinicians envision the possibility of detecting and directly tracking migratory tumor cells *in vivo*, and therefore enabling operative procedures limited to a single total resection surgery. In order to accomplish this goal, there is a stringent need for development of enhanced imaging tools to allow visualization of migrating tumor cells. Meanwhile, the most successful quantitative assessment of CNSTCs migration has been accomplished outside of the brain itself, in engineered systems redesigned to mimic specific *in vivo* conditions. We will discuss these *in vitro* assays further, which have been utilized to evaluate a variety of cellular functions, from growth patterns and rates, to invasive motility of cells derived from highly malignant brain tumors.

#### **5. Models for the study of brain tumor invasion** *in vitro*

Tumor cells of the brain have been characterized as having a highly infiltrative phenotype for spreading into the healthy surrounding parenchyma. This malignant property is arguably the principle reason for tumor recurrence and high mortality rates (Lim et al., 2007). The interaction of integrins, membrane anchored heterodimeric proteins, with various ECM proteins has been explored extensively, as it is one of the key events that occurs during the invasion of tumor cells within their local microenvironments (Teodorczyk and Martin-Villalba, 2010). Another key process of tumor invasion is the cellular secretion/production of proteases that degrade ECM proteins in order to create pores through which the cells may migrate; such proteases include serine proteases, various MMPs, and cathepsins (Rao, 2003). In addition to stimulating tumor invasion via degradation of ECM protein components, it is assumed that MMPs are capable of enhancing tumor growth by indirectly triggering the release of growth factors trapped within the basement membrane itself (Mott and Werb, 2004). Lastly, another key aspect of tumor dissemination is played by the activation of RTK signaling pathways. During the destruction of the basement membrane by MMPs, soluble growth factors are sequestered from the ECM and bind to their cognate cellular receptors to trigger a cascade of events that enhance cellular migration (Zucker et al., 2000).

*In vitro* invasion assays are important tools for investigating the tumor-matrix interactions and the effects of extracellular macromolecules on these interactions. While not entirely identical to *in vivo* behavior, the study of tumor cell migration *in vitro* is advantageous due to the tightly-controlled experimental conditions, higher experimental throughput, and lower costs. The following section discusses the most commonly used *in vitro* assays, in the order of increased complexity.

#### **5.1 Culture dish assays**

Culture dish assays have the advantage of design simplicity and execution, while providing insightful information pertaining to cell-to-cell and cell-to-environment interactions. Coated culture dishes have been widely used to examine the roles played by specific ECM proteins, integrins, MMPs, and RTKs in stimulating the migration of brain tumor-derived cells, as detailed here.

Integrins are membrane heterodimeric proteins that mediate cell-environment attachment (Hynes, 1987; Tucker, 2006). In addition to anchoring cells to their environment, integrins

Migration and Invasion of Brain Tumors 235

identify intracellular signaling proteins that negatively regulate MB migration/invasion and

Matrix-degrading proteases are involved in the hydrolytic breakdown of ECM proteins and have been shown to regulate tumor cell progression and invasion (Levicar et al., 2003; Rao, 2003; Rooprai and McCormick, 1997). Additionally, proteases have been well-studied and shown to display differential expression and activation patterns, correlated to their invasion-associated effects, i.e. angiogenesis (Forsyth et al., 1999; Thorns et al., 2003). These proteases are either located in the membrane of the cell or secreted into its surroundings, respectively denoted as MT-MMP and MMP. Diffusely invasive glioma cells express MMPs that enable them to catabolize ECM proteins that have been shown to prohibit the migration of other cells that lack these MMPs. For instance, specific membrane proteins expressed by CNS myelin have been shown to have anti-spreading functionality on neurite outgrowth,

The migration of glioma-, MB- and meningioma- cell lines on CNS myelin was found to be tumor grade-dependent and to involve active unspecified MMPs (Amberger et al., 1998). Culture dishes were coated with 15 g/dish rat spinal cord myelin, a concentration shown to reduce by 80% the fibroblast migration, followed by the seeding with various cell lines and the recording of cell ability to adhere and spread (Amberger et al., 1998). The results conclude that high grade GBMs, like U-251 MG, were able to strongly attach and spread, while low grade gliomas and MBs exhibited poor attachment and inhibited spreading (Amberger et al., 1998). Additionally, spreading of GBM and anaplastic astrocytomas cells on CNS myelin was strongly blocked when cells were treated with the MMP blocker Ophenanthroline (Felber et al., 1962), and temporarily inhibited with carbobenzoxy-Phe-Ala-Phe-Tyr-amide (Amberger et al., 1997) confirming the role played by MMPs in ECM

Belien and colleagues studied the role of MT1-MMP in enhanced spreading and migration of gliomas (Belien et al., 1999). As a substrate, they utilized myelin-coated culture dishes, since it was previously shown that invasion of gliomas predominantly occurs along the white matter of the CNS (Giese et al., 1996; Pedersen et al., 1995), which is heavily composed of myelin (Baumann and Pham-Dinh, 2001; McLaurin and Yong, 1995), to seed both gliomas and MT1-MMP-transfected fibroblasts. In this case, MT1-MMP was shown to be responsible for altering the cellular environment to enable migration of both gliomas and the transfected

When the invasiveness of five MB cell lines within a 3D *in vitro* collagen I or IV-based model was studied, the data showed that within hours of implantation, individual cells readily detached from the surface of the cell aggregates and invaded the collagen matrix, to distances of up to 1,200 µm and at rates of up to 300 µm per day (Ranger et al., 2010). Furthermore, MB invasiveness within this 3D model appears to depend upon a combination of metalloproteinase (MMP-1 and -2, TIMP-1 and -2) and cysteine protease activity (Ranger

The RTKs, like integrins, function as signal mediators of extracellular proteins yet in a different way. Integrins, as mentioned above, interact primarily with static, structural ECM proteins that are the composite materials of the cellular environment (Tucker, 2006). Meanwhile, RTKs interact with soluble macromolecules present in the environment, e.g. growth factors, that trigger a cascade of events in the cells, spanning from the extracellular surface of the plasma membrane to the nucleus, to elicit various cellular responses (Konopka and Bonni, 2003; Mueller et al., 2003; Teodorczyk and Martin-Villalba, 2010). Additionally,

astrocytes and fibroblasts (Schwab and Caroni, 1988; Spillmann et al., 1998).

modification as a precursory for migration.

fibroblasts (Belien et al., 1999).

et al., 2010).

proliferation.

have been shown to serve as signal mediators for ECM proteins that were found to stimulate tumor migration *in vitro* (Ohnishi et al., 1997; Tysnes et al., 1996). The most abundant ECM proteins found to interact with integrins in the brain are fibronectin, laminin, fibrinogen, tenascin-C, thrombodpondin, neuron-glia cell adhesion molecules (Ng-CAMs), and collagens IV and V (Rutka et al., 1988). Giese and colleagues evaluated astrocytoma migration as a function of integrin adhesiveness on various ECM proteins (collagen IV, fibronectin, laminin and vitronectin) (Giese et al., 1994). Based on the examination of eight different astrocytoma cell lines, the group concluded that the migration of glioma cells was subject to alteration depending on tumor expressed integrins and the availability of complementary matrix proteins. Furthermore, even though laminin frequently enabled tumor cells to adhere and migrate with increased adhesion, overall it was stated that there was no specific ECM protein that would always result in increased astrocytoma binding (Giese et al., 1994).

Friedlander and colleagues examined the migration trends of twenty-four excised human astrocytomas, ten GBM cell lines, and three MB cell lines on nine different ECM protein coated culture dishes (Friedlander et al., 1996). The comparative migration of astrocytomas (grades I, II and III), GBMs and MBs demonstrated that most tumor cells, regardless of their grade, were capable of migrating on fibronectin and laminin at rates exceeding 30 μm over a 16 hour period. A closer comparison between low-grade and high-grade tumor migration on all tested substrates revealed that, on average, high-grade tumors migrated approximately 14 μm more than low-grade tumor cells under similar conditions. Specifically, type IV collagen substrates induced a 4-fold increase in distances traveled by high-grade tumor cells over low-grade cells. Collagen IV coated substrates also stimulated approximately 100 μm migration over 16 hours of thirteen excised GBMs and eight well studied GBMs cell lines, with cell lines being more motile than the excised tumors (Friedlander et al., 1996). Finally, monoclonal antibodies specific for the v and 1 integrins were used to reduce the migration of four GBMs cell lines (U-373 MG, U-118 MG, U-251 MG and U-87 MG) on several migration enhancing ECM substrates, including collagen IV (Friedlander et al., 1996). These results illustrate that brain tumor-derived cells can migrate remarkably large distances within the brain, often to varied regions of the brain. However, tumor cell populations are very diverse, and such studies have not identified the lineage of motile cells, or whether certain sub-populations of cells could migrate farther than others within brain.

MB samples revealed type I collagen present in the leptomeninges, and in the ECM surrounding blood vessels and tumor cells (Liang et al., 2008). Expression of both type I collagen and 1 integrin, a subunit of a known type I collagen receptor, localized to the same area of MB. The same study showed that the adherence of MB cells to type I collagen matrix *in vitro* depends on the presence of 1 integrin (Liang et al., 2008).

A study by Corcoran and Del Maestro revealed that MB cell lines do not defer cell proliferation for migration across an uncoated surface or invasion of a type I collagen matrix, contrary to the "Go or Grow" hypothesis (Corcoran and Del Maestro, 2003). The "Go or Grow" hypothesis proposes that cell division and cell migration are temporally exclusive events, and that tumor cells defer cell division to migrate (Giese et al., 1996). Migrating and invading MBs continued to proliferate and migrate/invade, irrespective of the number of divisions that took place (Corcoran and Del Maestro, 2003). These findings emphasize the need to evaluate the effect of future therapies on both biological events and, if possible, to

have been shown to serve as signal mediators for ECM proteins that were found to stimulate tumor migration *in vitro* (Ohnishi et al., 1997; Tysnes et al., 1996). The most abundant ECM proteins found to interact with integrins in the brain are fibronectin, laminin, fibrinogen, tenascin-C, thrombodpondin, neuron-glia cell adhesion molecules (Ng-CAMs), and collagens IV and V (Rutka et al., 1988). Giese and colleagues evaluated astrocytoma migration as a function of integrin adhesiveness on various ECM proteins (collagen IV, fibronectin, laminin and vitronectin) (Giese et al., 1994). Based on the examination of eight different astrocytoma cell lines, the group concluded that the migration of glioma cells was subject to alteration depending on tumor expressed integrins and the availability of complementary matrix proteins. Furthermore, even though laminin frequently enabled tumor cells to adhere and migrate with increased adhesion, overall it was stated that there was no specific ECM protein that would always result in increased astrocytoma binding

Friedlander and colleagues examined the migration trends of twenty-four excised human astrocytomas, ten GBM cell lines, and three MB cell lines on nine different ECM protein coated culture dishes (Friedlander et al., 1996). The comparative migration of astrocytomas (grades I, II and III), GBMs and MBs demonstrated that most tumor cells, regardless of their grade, were capable of migrating on fibronectin and laminin at rates exceeding 30 μm over a 16 hour period. A closer comparison between low-grade and high-grade tumor migration on all tested substrates revealed that, on average, high-grade tumors migrated approximately 14 μm more than low-grade tumor cells under similar conditions. Specifically, type IV collagen substrates induced a 4-fold increase in distances traveled by high-grade tumor cells over low-grade cells. Collagen IV coated substrates also stimulated approximately 100 μm migration over 16 hours of thirteen excised GBMs and eight well studied GBMs cell lines, with cell lines being more motile than the excised tumors (Friedlander et al., 1996). Finally, monoclonal antibodies specific for the v and 1 integrins were used to reduce the migration of four GBMs cell lines (U-373 MG, U-118 MG, U-251 MG and U-87 MG) on several migration enhancing ECM substrates, including collagen IV (Friedlander et al., 1996). These results illustrate that brain tumor-derived cells can migrate remarkably large distances within the brain, often to varied regions of the brain. However, tumor cell populations are very diverse, and such studies have not identified the lineage of motile cells, or whether certain sub-populations of cells could migrate farther than others

MB samples revealed type I collagen present in the leptomeninges, and in the ECM surrounding blood vessels and tumor cells (Liang et al., 2008). Expression of both type I collagen and 1 integrin, a subunit of a known type I collagen receptor, localized to the same area of MB. The same study showed that the adherence of MB cells to type I collagen matrix

A study by Corcoran and Del Maestro revealed that MB cell lines do not defer cell proliferation for migration across an uncoated surface or invasion of a type I collagen matrix, contrary to the "Go or Grow" hypothesis (Corcoran and Del Maestro, 2003). The "Go or Grow" hypothesis proposes that cell division and cell migration are temporally exclusive events, and that tumor cells defer cell division to migrate (Giese et al., 1996). Migrating and invading MBs continued to proliferate and migrate/invade, irrespective of the number of divisions that took place (Corcoran and Del Maestro, 2003). These findings emphasize the need to evaluate the effect of future therapies on both biological events and, if possible, to

*in vitro* depends on the presence of 1 integrin (Liang et al., 2008).

(Giese et al., 1994).

within brain.

identify intracellular signaling proteins that negatively regulate MB migration/invasion and proliferation.

Matrix-degrading proteases are involved in the hydrolytic breakdown of ECM proteins and have been shown to regulate tumor cell progression and invasion (Levicar et al., 2003; Rao, 2003; Rooprai and McCormick, 1997). Additionally, proteases have been well-studied and shown to display differential expression and activation patterns, correlated to their invasion-associated effects, i.e. angiogenesis (Forsyth et al., 1999; Thorns et al., 2003). These proteases are either located in the membrane of the cell or secreted into its surroundings, respectively denoted as MT-MMP and MMP. Diffusely invasive glioma cells express MMPs that enable them to catabolize ECM proteins that have been shown to prohibit the migration of other cells that lack these MMPs. For instance, specific membrane proteins expressed by CNS myelin have been shown to have anti-spreading functionality on neurite outgrowth, astrocytes and fibroblasts (Schwab and Caroni, 1988; Spillmann et al., 1998).

The migration of glioma-, MB- and meningioma- cell lines on CNS myelin was found to be tumor grade-dependent and to involve active unspecified MMPs (Amberger et al., 1998). Culture dishes were coated with 15 g/dish rat spinal cord myelin, a concentration shown to reduce by 80% the fibroblast migration, followed by the seeding with various cell lines and the recording of cell ability to adhere and spread (Amberger et al., 1998). The results conclude that high grade GBMs, like U-251 MG, were able to strongly attach and spread, while low grade gliomas and MBs exhibited poor attachment and inhibited spreading (Amberger et al., 1998). Additionally, spreading of GBM and anaplastic astrocytomas cells on CNS myelin was strongly blocked when cells were treated with the MMP blocker Ophenanthroline (Felber et al., 1962), and temporarily inhibited with carbobenzoxy-Phe-Ala-Phe-Tyr-amide (Amberger et al., 1997) confirming the role played by MMPs in ECM modification as a precursory for migration.

Belien and colleagues studied the role of MT1-MMP in enhanced spreading and migration of gliomas (Belien et al., 1999). As a substrate, they utilized myelin-coated culture dishes, since it was previously shown that invasion of gliomas predominantly occurs along the white matter of the CNS (Giese et al., 1996; Pedersen et al., 1995), which is heavily composed of myelin (Baumann and Pham-Dinh, 2001; McLaurin and Yong, 1995), to seed both gliomas and MT1-MMP-transfected fibroblasts. In this case, MT1-MMP was shown to be responsible for altering the cellular environment to enable migration of both gliomas and the transfected fibroblasts (Belien et al., 1999).

When the invasiveness of five MB cell lines within a 3D *in vitro* collagen I or IV-based model was studied, the data showed that within hours of implantation, individual cells readily detached from the surface of the cell aggregates and invaded the collagen matrix, to distances of up to 1,200 µm and at rates of up to 300 µm per day (Ranger et al., 2010). Furthermore, MB invasiveness within this 3D model appears to depend upon a combination of metalloproteinase (MMP-1 and -2, TIMP-1 and -2) and cysteine protease activity (Ranger et al., 2010).

The RTKs, like integrins, function as signal mediators of extracellular proteins yet in a different way. Integrins, as mentioned above, interact primarily with static, structural ECM proteins that are the composite materials of the cellular environment (Tucker, 2006). Meanwhile, RTKs interact with soluble macromolecules present in the environment, e.g. growth factors, that trigger a cascade of events in the cells, spanning from the extracellular surface of the plasma membrane to the nucleus, to elicit various cellular responses (Konopka and Bonni, 2003; Mueller et al., 2003; Teodorczyk and Martin-Villalba, 2010). Additionally,

Migration and Invasion of Brain Tumors 237

Spheroids grown from several different GBM cell lines were placed on uncoated 24-well dishes and treated with EGF, which triggered a strong stimulation of cellular invasion and increased growth (Lund-Johansen et al., 1990). Similarly, spheroids grown from several human glioma cell lines exhibited enhanced growth and directional migration when cultured in 10 ng/ml EGF or 10 ng/ml bFGF concentrations, compared to control and other growth factors, such as PDGFBB (Engebraaten et al., 1993). When MB cultures were induced to generate spheroids, gene expression of CD133 (a hallmark of the brain cancer stem cells and radioresistant tumors), MT1-MMP, and MMP-9 were induced and correlated with increased invasiveness of the spheroid cells (Annabi et al., 2008). Additionally, Corcoran and Del Maestro revealed that MB cells from an established cell line, UW228-13, could exhibit elevated levels of invasion into a 3D matrix of type 1 collagen compared to biopsied

Fig. 4. First and last images extracted from time-lapse videos of DAOY (A and B) and UW228-3 (C and D) spheroids invading Type I collagen matrices. Spheroids were recorded 48 hours after implantation. The numbers identify cells that divided zero, one, or two times in 40 hours; parent cells are labeled in A and C and daughter cells in B and D. The number of hours elapsed from the start time of the videos (t) are indicated at the top right corner of

Wild-Bode and colleagues grew glioma spheroids on agar base-coated culture flasks until they were ~200 m in diameter, followed by their transfer to 96-well plates. In order to examine the cause of glioma relapse in close proximity to the excised lesion, they measured the radial distance of migration as function of irradiation at 3 Gy. Irradiation led to increased migration of all cell lines, compared to non-irradiated cells, a phenomenon which was linked to increased expression and activation of MMP-2, MMP-9 and MT1-MMP (Wild-

images. Scale bars, 250 μm. (Courtesy of Corcoran and DelMaestro, 2003)

Bode et al., 2001).

DAOY cells (Figure 4) (Corcoran and Del Maestro, 2003).

genetically modified and overexpressed RTKs are capable of eliciting cellular signals in the absence of ligand binding, thus bypassing the need for an extracellular trigger (Akbasak and Sunar-Akbasak, 1992; Dong et al., 2011; Strommer et al., 1990; Torp et al., 2007). Ding and colleagues employed U-87 MG cells grown on coated cultured dishes to demonstrate a strong adhesion to vitronectin, as opposed to collagen or laminin, which was mediated through the v3 and v5 integrins (Ding et al., 2003). Additionally, the group was able a to link the specific cooperative interaction between the RTK PDGFR and the v3 integrin to induce migration of U-87 MG cells into the wounded area in a scratch-wound assay after stimulation of the culture with PDGF (Ding et al., 2003). Therefore, these results suggest the direct correlation between the ability of RTKs to transmit extracellular signals into the cell and to convert these signals into direct and measurable cellular response.

As an improvement upon the simple use of cell culture dishes for study of tumor cell migration, micropipette turning assays have been used to create gradients within culture dishes that enable changes in cell migration with micropipette position. Gradients, defined as fields where biochemical concentrations are varied along a specific distance, are generated via simple diffusion of biological molecules from the micropipette into the culture medium. Gradient formation and stability are functions of the molecular properties of the stimulant being used (i.e. diffusivity constant and molecular weight), as well as pipette mechanics (i.e. dimensions and flow rates) (Lohof et al., 1992), and for these reasons make gradient measurement difficult. Wyckoff and colleagues used the micropipette method to collect subpopulations of motile mammary carcinoma and macrophage cells into microneedles filled with Matrigel™ and a range of EGF concentrations from confluent culture dishes (Wyckoff et al., 2000). The Matrigel™ matrix is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma (Ohashi et al., 2006; Reed et al., 2009), commonly used in cell-matrix interaction studies. The contents of the microneedles were then emptied into new culture dishes and allowed to grow for at least 6 days before quantifying cell populations. A bell-shaped curve of normalized cell numbers was reported and had the maximal number of collected cells for 25 nM EGF, 8-fold greater than controls (Wyckoff et al., 2000). Such results illustrate that growth factor concentration gradients, or differences in concentrations along given distances, stimulate brain tumor cell migration.

#### **5.2 Spheroids**

A key question regarding cancer cell migration and invasion is based on determining the reason why tumor cells detach from the bulk tumor mass. Some studies have suggested that lack of contact-inhibition may be responsible for cell migration away from the bulk (Pedersen et al., 1995). While normal cells go into a quiescent state that allows apoptosis during nutrient depleted states, cancer cells do not rely on contact-dependent growth and therefore can detach and venture out to diffusely invade the parenchyma. The Spheroid Model utilizes the natural tendency of cancer cells to form colonies and to grow into localized spheres (Santini and Rainaldi, 1999; Zhang et al., 2005). This model mimics the 3D characteristics of cell migration, while culture dish experiments described in the previous subsection provide important data on 2D cell migration. As a result, the spheroid model has been used to study the directional migration of tumor cells from the bulk spheroid mass in response to specific motogens and chemotherapeutic agents, as well as to measure the penetration of various molecules into the tumor (Carlsson and Nederman, 1983; Nederman et al., 1983).

genetically modified and overexpressed RTKs are capable of eliciting cellular signals in the absence of ligand binding, thus bypassing the need for an extracellular trigger (Akbasak and Sunar-Akbasak, 1992; Dong et al., 2011; Strommer et al., 1990; Torp et al., 2007). Ding and colleagues employed U-87 MG cells grown on coated cultured dishes to demonstrate a strong adhesion to vitronectin, as opposed to collagen or laminin, which was mediated through the v3 and v5 integrins (Ding et al., 2003). Additionally, the group was able a to link the specific cooperative interaction between the RTK PDGFR and the v3 integrin to induce migration of U-87 MG cells into the wounded area in a scratch-wound assay after stimulation of the culture with PDGF (Ding et al., 2003). Therefore, these results suggest the direct correlation between the ability of RTKs to transmit extracellular signals into the cell

As an improvement upon the simple use of cell culture dishes for study of tumor cell migration, micropipette turning assays have been used to create gradients within culture dishes that enable changes in cell migration with micropipette position. Gradients, defined as fields where biochemical concentrations are varied along a specific distance, are generated via simple diffusion of biological molecules from the micropipette into the culture medium. Gradient formation and stability are functions of the molecular properties of the stimulant being used (i.e. diffusivity constant and molecular weight), as well as pipette mechanics (i.e. dimensions and flow rates) (Lohof et al., 1992), and for these reasons make gradient measurement difficult. Wyckoff and colleagues used the micropipette method to collect subpopulations of motile mammary carcinoma and macrophage cells into microneedles filled with Matrigel™ and a range of EGF concentrations from confluent culture dishes (Wyckoff et al., 2000). The Matrigel™ matrix is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma (Ohashi et al., 2006; Reed et al., 2009), commonly used in cell-matrix interaction studies. The contents of the microneedles were then emptied into new culture dishes and allowed to grow for at least 6 days before quantifying cell populations. A bell-shaped curve of normalized cell numbers was reported and had the maximal number of collected cells for 25 nM EGF, 8-fold greater than controls (Wyckoff et al., 2000). Such results illustrate that growth factor concentration gradients, or differences in concentrations along given distances, stimulate

A key question regarding cancer cell migration and invasion is based on determining the reason why tumor cells detach from the bulk tumor mass. Some studies have suggested that lack of contact-inhibition may be responsible for cell migration away from the bulk (Pedersen et al., 1995). While normal cells go into a quiescent state that allows apoptosis during nutrient depleted states, cancer cells do not rely on contact-dependent growth and therefore can detach and venture out to diffusely invade the parenchyma. The Spheroid Model utilizes the natural tendency of cancer cells to form colonies and to grow into localized spheres (Santini and Rainaldi, 1999; Zhang et al., 2005). This model mimics the 3D characteristics of cell migration, while culture dish experiments described in the previous subsection provide important data on 2D cell migration. As a result, the spheroid model has been used to study the directional migration of tumor cells from the bulk spheroid mass in response to specific motogens and chemotherapeutic agents, as well as to measure the penetration of various molecules into the tumor (Carlsson and Nederman, 1983; Nederman

and to convert these signals into direct and measurable cellular response.

brain tumor cell migration.

**5.2 Spheroids** 

et al., 1983).

Spheroids grown from several different GBM cell lines were placed on uncoated 24-well dishes and treated with EGF, which triggered a strong stimulation of cellular invasion and increased growth (Lund-Johansen et al., 1990). Similarly, spheroids grown from several human glioma cell lines exhibited enhanced growth and directional migration when cultured in 10 ng/ml EGF or 10 ng/ml bFGF concentrations, compared to control and other growth factors, such as PDGFBB (Engebraaten et al., 1993). When MB cultures were induced to generate spheroids, gene expression of CD133 (a hallmark of the brain cancer stem cells and radioresistant tumors), MT1-MMP, and MMP-9 were induced and correlated with increased invasiveness of the spheroid cells (Annabi et al., 2008). Additionally, Corcoran and Del Maestro revealed that MB cells from an established cell line, UW228-13, could exhibit elevated levels of invasion into a 3D matrix of type 1 collagen compared to biopsied DAOY cells (Figure 4) (Corcoran and Del Maestro, 2003).

Fig. 4. First and last images extracted from time-lapse videos of DAOY (A and B) and UW228-3 (C and D) spheroids invading Type I collagen matrices. Spheroids were recorded 48 hours after implantation. The numbers identify cells that divided zero, one, or two times in 40 hours; parent cells are labeled in A and C and daughter cells in B and D. The number of hours elapsed from the start time of the videos (t) are indicated at the top right corner of images. Scale bars, 250 μm. (Courtesy of Corcoran and DelMaestro, 2003)

Wild-Bode and colleagues grew glioma spheroids on agar base-coated culture flasks until they were ~200 m in diameter, followed by their transfer to 96-well plates. In order to examine the cause of glioma relapse in close proximity to the excised lesion, they measured the radial distance of migration as function of irradiation at 3 Gy. Irradiation led to increased migration of all cell lines, compared to non-irradiated cells, a phenomenon which was linked to increased expression and activation of MMP-2, MMP-9 and MT1-MMP (Wild-Bode et al., 2001).

Migration and Invasion of Brain Tumors 239

dependent migration and invasion of five different human glioma cell lines toward various concentrations of HGF, in addition to reducing basal migration of these cells using an anti-HGF neutralizing antibody (Koochekpour et al., 1997). Brockmann and colleagues reported increases in U-87 MG migration, as high as 33-fold greater than controls, in the presence of 100 pM HGF concentrations. In the same study, 1 nM TGF and 50 nM FGF1 stimulated U-87 MG migration 17- and 4-fold, respectively (Brockmann et al., 2003). Transwell assays were used to demonstrate the chemotaxis of metastatic breast adenocarcinomas toward bone and brain extracts, rather than extracts from liver or lung (Hujanen and Terranova, 1985). Interestingly, it was found that C6-GFP rat glioma cells could extend their leading cytoplasmic processes through membrane pores, as a function of actin dynamics alone, but they required myosin IIA/B to generate additional cytoplasmic contractile forces to push the

Fig. 5. Boyden chamber assay. (A) Transwell migration assays are composed of a well insert

A study that looked at the inhibitory effect of dietary-derived flavonols on the HGF receptor c-Met activity suggests that such an effect may contribute to the chemopreventive properties of these molecules (Labbe et al., 2009). The authors showed that the flavonols quercetin, kaempferol, and myricetin inhibited HGF/c-Met signaling in MB, preventing the formation of actin-rich membrane ruffles and resulting in the inhibition of c-Met-induced cell migration in Boyden chambers (Labbe et al., 2009). Furthermore, quercetin and kaempferol

While investigating the effect of ionizing irradiation on the invasiveness of glioma cells via transwell assays, Park and colleagues reported increased Matrigel™ invasion of PTEN null gliomas, U-251 MG and U-373 MG, as a result of elevated levels irradiation treatment, which the group suggested correlates with increases in MMP-2 secretion (Park et al., 2006). Similarly, Wild-Bode and colleagues found that the sublethal irradiation doses of 1, 3, and 6 Gy increased the chemotactic migration and invasion of three different human glioma cell lines with increasing dosage (Wild-Bode et al., 2001). Similarly to glioma, radiation enhanced invasion and migration of 7 Gy irradiated MB compared to non-irradiated MB cells, as assessed via Boyden chamber assays (Nalla et al., 2010). Increased expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), focal adhesion kinase (FAK), N-cadherin and integrin subunits (e.g., α3, α5 and β1) was detected in irradiated cells.

with a porous filter bottom that temporarily separates the cell solution from the test solution. (B) The filter has randomly distributed microscale diameter pores as shown by arrows. (C) Invasive Daoy cells on the underside of the filter stained and imaged for

also strongly diminished HGF-mediated Akt activation (Labbe et al., 2009).

migration analysis. Scale bar = 50 μm.

nucleus through pores having a smaller diameter (Beadle et al., 2008).

Just as the spheroid model employs a 3D environment in order to better mimic *in vivo*  conditions, other *in vitro* technologies were developed to imitate the biochemical environment of the brain. In particular, transwell assays were developed in order to expose tumor-derived cells to different concentration gradients of cytokines present within brain.

#### **5.3 Transwell migration assays**

The transwell migration assay is a commonly-used test to study the migratory response of cells to inducers or inhibitors. This assay is also known as the Boyden or modified Boyden chamber assay, and was originally used to evaluate leukocyte chemotaxis (Boyden, 1962). In this assay, a chamber that is separated into two compartments by a polyethylene terephthalate filter (Figure 5A) and the cells placed into the upper compartment are allowed to settle, while the solution being tested for chemotactic activity is placed in the lower compartment. The membrane contains randomly distributed pores through which the cells migrate (Figure 5B), in response to the chemoattractant from the bottom compartment. Invasive cells migrated to the underside of the filter can be stained and quantified (Figure 5C).

Different ECM components can be used to coat the filter in order to mimic the basement membrane that cells must penetrate while invading *in vivo*, while exposing the cells to various chemicals for different time lengths. The main advantage of this assay is its detection sensitivity. Migration through the permeable membrane can be caused by very low levels of chemoattractants (Kreutzer et al., 1978). Prolonged studies are difficult, due to the fact that the chemoattractant concentration will quickly equalize between the compartment below the membrane and the compartment above the membrane. Another disadvantage is the relative difficulty in setting up the transwells. Despite these disadvantages, transwell assays are commonly the test of choice for migration and invasion studies *in vitro*.

Over the last 50-years, several modifications have been implemented to this technology by various research groups in order to circumvent difficulties encountered with its use. For instance, upon crossing the membrane and reaching the lower surface of the chamber, cells may detach from the filter, thus resulting in an underestimate of transmigrated cells (Li and Zhu, 1999). Albini and colleagues were among the first researchers to use filters coated with ECM. They used radiolabeled proteins to demonstrate an 8-10 hour gradient stabilization period within the Boyden chamber, and showed that cell invasion time was very much dependent on the volume of the coated matrix barrier (Albini et al., 1987). Li and Zhu pioneered the use of different cell populations to attract other cells, by growing a monolayer of bovine aortic endothelial cells on filters, and investigating the transendothelial migration of six cell lines of different human tumors (Li and Zhu, 1999).

Chemotactic migration of GMB cells in response to several growth factors, predominately PDGF, EGF, and HGF, has been extensively studied (Hoelzinger et al., 2007). These studies have demonstrated dosage-dependent motogenic responses to various concentrations and combinations of these cytokines (Brockmann et al., 2003). For example, Moriyama and colleagues demonstrated, through a checkerboard analysis of various HGF concentrations, that a dose-dependent response to HGF induces both chemotaxis and chemokinesis of U-251 MG cells (Moriyama et al., 1996). In this study the concept of an "optimal concentration" was introduced, and the authors reported a decline in the chemotactic activity of U-251 MG cells at concentrations exceeding the reported optimal concentration of 50 ng/ml (Moriyama et al., 1996). Similarly, Koochekpour and colleagues used transwell assays to show dose-

Just as the spheroid model employs a 3D environment in order to better mimic *in vivo*  conditions, other *in vitro* technologies were developed to imitate the biochemical environment of the brain. In particular, transwell assays were developed in order to expose tumor-derived cells to different concentration gradients of cytokines present within brain.

The transwell migration assay is a commonly-used test to study the migratory response of cells to inducers or inhibitors. This assay is also known as the Boyden or modified Boyden chamber assay, and was originally used to evaluate leukocyte chemotaxis (Boyden, 1962). In this assay, a chamber that is separated into two compartments by a polyethylene terephthalate filter (Figure 5A) and the cells placed into the upper compartment are allowed to settle, while the solution being tested for chemotactic activity is placed in the lower compartment. The membrane contains randomly distributed pores through which the cells migrate (Figure 5B), in response to the chemoattractant from the bottom compartment. Invasive cells migrated to the underside of the filter can be stained and quantified (Figure

Different ECM components can be used to coat the filter in order to mimic the basement membrane that cells must penetrate while invading *in vivo*, while exposing the cells to various chemicals for different time lengths. The main advantage of this assay is its detection sensitivity. Migration through the permeable membrane can be caused by very low levels of chemoattractants (Kreutzer et al., 1978). Prolonged studies are difficult, due to the fact that the chemoattractant concentration will quickly equalize between the compartment below the membrane and the compartment above the membrane. Another disadvantage is the relative difficulty in setting up the transwells. Despite these disadvantages, transwell assays are commonly the test of choice for migration and invasion

Over the last 50-years, several modifications have been implemented to this technology by various research groups in order to circumvent difficulties encountered with its use. For instance, upon crossing the membrane and reaching the lower surface of the chamber, cells may detach from the filter, thus resulting in an underestimate of transmigrated cells (Li and Zhu, 1999). Albini and colleagues were among the first researchers to use filters coated with ECM. They used radiolabeled proteins to demonstrate an 8-10 hour gradient stabilization period within the Boyden chamber, and showed that cell invasion time was very much dependent on the volume of the coated matrix barrier (Albini et al., 1987). Li and Zhu pioneered the use of different cell populations to attract other cells, by growing a monolayer of bovine aortic endothelial cells on filters, and investigating the transendothelial migration

Chemotactic migration of GMB cells in response to several growth factors, predominately PDGF, EGF, and HGF, has been extensively studied (Hoelzinger et al., 2007). These studies have demonstrated dosage-dependent motogenic responses to various concentrations and combinations of these cytokines (Brockmann et al., 2003). For example, Moriyama and colleagues demonstrated, through a checkerboard analysis of various HGF concentrations, that a dose-dependent response to HGF induces both chemotaxis and chemokinesis of U-251 MG cells (Moriyama et al., 1996). In this study the concept of an "optimal concentration" was introduced, and the authors reported a decline in the chemotactic activity of U-251 MG cells at concentrations exceeding the reported optimal concentration of 50 ng/ml (Moriyama et al., 1996). Similarly, Koochekpour and colleagues used transwell assays to show dose-

of six cell lines of different human tumors (Li and Zhu, 1999).

**5.3 Transwell migration assays** 

5C).

studies *in vitro*.

dependent migration and invasion of five different human glioma cell lines toward various concentrations of HGF, in addition to reducing basal migration of these cells using an anti-HGF neutralizing antibody (Koochekpour et al., 1997). Brockmann and colleagues reported increases in U-87 MG migration, as high as 33-fold greater than controls, in the presence of 100 pM HGF concentrations. In the same study, 1 nM TGF and 50 nM FGF1 stimulated U-87 MG migration 17- and 4-fold, respectively (Brockmann et al., 2003). Transwell assays were used to demonstrate the chemotaxis of metastatic breast adenocarcinomas toward bone and brain extracts, rather than extracts from liver or lung (Hujanen and Terranova, 1985). Interestingly, it was found that C6-GFP rat glioma cells could extend their leading cytoplasmic processes through membrane pores, as a function of actin dynamics alone, but they required myosin IIA/B to generate additional cytoplasmic contractile forces to push the nucleus through pores having a smaller diameter (Beadle et al., 2008).

Fig. 5. Boyden chamber assay. (A) Transwell migration assays are composed of a well insert with a porous filter bottom that temporarily separates the cell solution from the test solution. (B) The filter has randomly distributed microscale diameter pores as shown by arrows. (C) Invasive Daoy cells on the underside of the filter stained and imaged for migration analysis. Scale bar = 50 μm.

A study that looked at the inhibitory effect of dietary-derived flavonols on the HGF receptor c-Met activity suggests that such an effect may contribute to the chemopreventive properties of these molecules (Labbe et al., 2009). The authors showed that the flavonols quercetin, kaempferol, and myricetin inhibited HGF/c-Met signaling in MB, preventing the formation of actin-rich membrane ruffles and resulting in the inhibition of c-Met-induced cell migration in Boyden chambers (Labbe et al., 2009). Furthermore, quercetin and kaempferol also strongly diminished HGF-mediated Akt activation (Labbe et al., 2009).

While investigating the effect of ionizing irradiation on the invasiveness of glioma cells via transwell assays, Park and colleagues reported increased Matrigel™ invasion of PTEN null gliomas, U-251 MG and U-373 MG, as a result of elevated levels irradiation treatment, which the group suggested correlates with increases in MMP-2 secretion (Park et al., 2006). Similarly, Wild-Bode and colleagues found that the sublethal irradiation doses of 1, 3, and 6 Gy increased the chemotactic migration and invasion of three different human glioma cell lines with increasing dosage (Wild-Bode et al., 2001). Similarly to glioma, radiation enhanced invasion and migration of 7 Gy irradiated MB compared to non-irradiated MB cells, as assessed via Boyden chamber assays (Nalla et al., 2010). Increased expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), focal adhesion kinase (FAK), N-cadherin and integrin subunits (e.g., α3, α5 and β1) was detected in irradiated cells.

Migration and Invasion of Brain Tumors 241

cell locomotion (Guck et al., 2010). PDMS microsystems pre-define the cell migratory path within microsized channels that mimic *in vivo* conditions. As such, cell motility and directionality can be examined and measured via conventional time-lapse imaging. Pioneering applications of microdevices for cancer cell study utilized microchannels coated and filled with various extracellular matrixes in 2D and 3D (Schoen et al., 2010; Sung et al., 2009) to illustrate the selectivity of cancer cell migration on distinct ECM, as well as to measure traction forces and leading edge protrusions of a variety of cancer cell types (Li et al., 2009). More recently, biomedical engineers have begun to develop systems that generate linear and non-linear cytokine gradients in order to more accurately investigate the

Establishment of steady-state gradient profiles has been examined using flow-based gradient generators, diffusion-based gradient generators, as well as hybrid generators (mixture of convection and diffusion). One of the original microdesigns for migration study was developed by Li Jeon and colleagues, in a gradient mixer design initially used for neutrophil chemotaxis (Li Jeon et al., 2002). This device contains multiple inlets that enable the loading of different ligand solutions that are then mixed in channels perpendicular to the flow direction (Figure 6A). Subsequently, a variety of system designs have been developed to generate alterative gradient shapes for further chemotaxis study (Kim et al., 2010). In such flow-based designs, two concentrations of biomolecules flow separately into a network of microchannels, where mixers are patterned to combine adjacent streams via convection in order to generate a chemical gradient. While flow-based devices are able to finely control the spatiotemporal resolution of the gradient, they require constant flow rates of reagents that remove molecules secreted from cells that are critical to regulation of tumor

Fig. 6. Schematics of microdevices currently used to generate concentration gradients (not to scale). The flow mixer device was first proposed by Li Jeon and colleagues to create highly controllable concentrations along specific distances via continuous convective flow. (A) Schematic representation of a premixing gradient generating device pioneered by Li Jeon and colleagues. (B) A hybrid microlane system uses interconnected reservoirs to create concentration gradients via both convection and diffusion. The microchannel approximately measures 13 mm in length, 90 μm in depth and 100 μm in width (averaged with the upper side of 95 μm and the lower side of 105 μm), as its semi-hemispherical cross-section shown in inset.

chemotactic behavior of cells derived from primary tumors.

cell migration (Huang et al., 2011).

(Courtesy of Kong et al., 2010)

Conversely, down-regulation of uPAR reduced the radiation-induced adhesion, migration and invasion of the irradiated cells, primarily by inhibiting phosphorylation of FAK, Paxillin and Rac-1/Cdc42 (Nalla et al., 2010).

Transwell assays were also used to study the activated RTK-dependent MB migration. These studies have shown that MB migration is dependent on estrogen-receptor (Belcher et al., 2009), c-Met (Guessous et al., 2010), and PDGFR-β activation (Yuan et al., 2010). Via a combination of wound-healing assays and modified Boyden chamber assays, two groups showed that PDGF-induced overexpression of Rac1, a Rho GTPase, is involved in MB cell migration and invasion, whereas knockdown of Rac1 expression dramatically inhibited migration and invasion of MBs (Chen et al., 2011; Yuan et al., 2010). These findings may promote the evaluation of Rac1 as a novel therapeutic agent impairing medulloblastoma PDGF-induced migration/invasion. Additional work has demonstrated that PDGFR-β activity may guide the migration of MB by transactivating EGFR (Abouantoun and MacDonald, 2009). These results are of particular interest, since EGFR is known to be expressed in GNPCs of the human cerebellum, participating in its normal development and function (Seroogy et al., 1995). Recently, the multifunctional signaling protein neurotrophin receptor p75NTR was shown to be a central regulator for GBM (Johnston et al., 2007) and MB spinal invasion while γ-secretase inhibitor, which blocks p75NTR proteolytic processing, significantly abrogates p75NTR induced MB migration and invasion *in vitro* and *in vivo* (Wang et al., 2010).

The transwell assays, even though used commonly for cell migration experiments, often yield inconsistent results across research groups due to the experimental individual assay modifications made by each group. For example, the ECM component used for filter coating can serve as a chemoattractant via integrin signaling (triggered by the interaction with laminin contained in the matrix), or via the release of growth factors embedded in the matrix itself. Although a reduced-growth factor form of Matrigel™ is generally used (i.e. reduced amounts of the above mentioned molecules are present in the matrix), there are a plethora of ECM proteins and growth factors, reconstituted along with Matrigel™, whose concentrations may vary with each batch purchased, and cause variations in the results. Yet, perhaps the largest shortcoming of transwell assays with respect to quantifying migration is that the cytokine microenvironments they create are very complex to model mathematically. Diffusion gradients of molecules across the membrane pores are difficult to measure or predict analytically, with or without matrix coating. Among *in vitro* approaches, microfluidics has proven to be a powerful technology to study cell migration over the past few years, due to the ability to generate a precise cell microenvironment that can be both predicted by analytical models and validated experimentally (Kong et al., 2010), as summarized further on.

#### **5.4 Microdevices**

Advances in microfabrication have made microfluidics systems easier to design and manufacture. Currently, the majority of devices are constructed of polydimethylsiloxane (PDMS) via soft lithography pioneered by the Whitesides group (McDonald and Whitesides, 2002). The polymer allows the construction of systems with high transparency and low thickness that are highly-compatible with biological microscopy. As dissemination of glioma or MB cells can often follow the path of white matter tracks or other heterogeneous structures, mechanical properties of the microenvironment play significant roles in tumor

Conversely, down-regulation of uPAR reduced the radiation-induced adhesion, migration and invasion of the irradiated cells, primarily by inhibiting phosphorylation of FAK, Paxillin

Transwell assays were also used to study the activated RTK-dependent MB migration. These studies have shown that MB migration is dependent on estrogen-receptor (Belcher et al., 2009), c-Met (Guessous et al., 2010), and PDGFR-β activation (Yuan et al., 2010). Via a combination of wound-healing assays and modified Boyden chamber assays, two groups showed that PDGF-induced overexpression of Rac1, a Rho GTPase, is involved in MB cell migration and invasion, whereas knockdown of Rac1 expression dramatically inhibited migration and invasion of MBs (Chen et al., 2011; Yuan et al., 2010). These findings may promote the evaluation of Rac1 as a novel therapeutic agent impairing medulloblastoma PDGF-induced migration/invasion. Additional work has demonstrated that PDGFR-β activity may guide the migration of MB by transactivating EGFR (Abouantoun and MacDonald, 2009). These results are of particular interest, since EGFR is known to be expressed in GNPCs of the human cerebellum, participating in its normal development and function (Seroogy et al., 1995). Recently, the multifunctional signaling protein neurotrophin receptor p75NTR was shown to be a central regulator for GBM (Johnston et al., 2007) and MB spinal invasion while γ-secretase inhibitor, which blocks p75NTR proteolytic processing, significantly abrogates p75NTR induced MB migration and invasion *in vitro* and *in vivo*

The transwell assays, even though used commonly for cell migration experiments, often yield inconsistent results across research groups due to the experimental individual assay modifications made by each group. For example, the ECM component used for filter coating can serve as a chemoattractant via integrin signaling (triggered by the interaction with laminin contained in the matrix), or via the release of growth factors embedded in the matrix itself. Although a reduced-growth factor form of Matrigel™ is generally used (i.e. reduced amounts of the above mentioned molecules are present in the matrix), there are a plethora of ECM proteins and growth factors, reconstituted along with Matrigel™, whose concentrations may vary with each batch purchased, and cause variations in the results. Yet, perhaps the largest shortcoming of transwell assays with respect to quantifying migration is that the cytokine microenvironments they create are very complex to model mathematically. Diffusion gradients of molecules across the membrane pores are difficult to measure or predict analytically, with or without matrix coating. Among *in vitro* approaches, microfluidics has proven to be a powerful technology to study cell migration over the past few years, due to the ability to generate a precise cell microenvironment that can be both predicted by analytical models and validated experimentally (Kong et al., 2010), as

Advances in microfabrication have made microfluidics systems easier to design and manufacture. Currently, the majority of devices are constructed of polydimethylsiloxane (PDMS) via soft lithography pioneered by the Whitesides group (McDonald and Whitesides, 2002). The polymer allows the construction of systems with high transparency and low thickness that are highly-compatible with biological microscopy. As dissemination of glioma or MB cells can often follow the path of white matter tracks or other heterogeneous structures, mechanical properties of the microenvironment play significant roles in tumor

and Rac-1/Cdc42 (Nalla et al., 2010).

(Wang et al., 2010).

summarized further on.

**5.4 Microdevices** 

cell locomotion (Guck et al., 2010). PDMS microsystems pre-define the cell migratory path within microsized channels that mimic *in vivo* conditions. As such, cell motility and directionality can be examined and measured via conventional time-lapse imaging. Pioneering applications of microdevices for cancer cell study utilized microchannels coated and filled with various extracellular matrixes in 2D and 3D (Schoen et al., 2010; Sung et al., 2009) to illustrate the selectivity of cancer cell migration on distinct ECM, as well as to measure traction forces and leading edge protrusions of a variety of cancer cell types (Li et al., 2009). More recently, biomedical engineers have begun to develop systems that generate linear and non-linear cytokine gradients in order to more accurately investigate the chemotactic behavior of cells derived from primary tumors.

Establishment of steady-state gradient profiles has been examined using flow-based gradient generators, diffusion-based gradient generators, as well as hybrid generators (mixture of convection and diffusion). One of the original microdesigns for migration study was developed by Li Jeon and colleagues, in a gradient mixer design initially used for neutrophil chemotaxis (Li Jeon et al., 2002). This device contains multiple inlets that enable the loading of different ligand solutions that are then mixed in channels perpendicular to the flow direction (Figure 6A). Subsequently, a variety of system designs have been developed to generate alterative gradient shapes for further chemotaxis study (Kim et al., 2010). In such flow-based designs, two concentrations of biomolecules flow separately into a network of microchannels, where mixers are patterned to combine adjacent streams via convection in order to generate a chemical gradient. While flow-based devices are able to finely control the spatiotemporal resolution of the gradient, they require constant flow rates of reagents that remove molecules secreted from cells that are critical to regulation of tumor cell migration (Huang et al., 2011).

Fig. 6. Schematics of microdevices currently used to generate concentration gradients (not to scale). The flow mixer device was first proposed by Li Jeon and colleagues to create highly controllable concentrations along specific distances via continuous convective flow. (A) Schematic representation of a premixing gradient generating device pioneered by Li Jeon and colleagues. (B) A hybrid microlane system uses interconnected reservoirs to create concentration gradients via both convection and diffusion. The microchannel approximately measures 13 mm in length, 90 μm in depth and 100 μm in width (averaged with the upper side of 95 μm and the lower side of 105 μm), as its semi-hemispherical cross-section shown in inset. (Courtesy of Kong et al., 2010)

Migration and Invasion of Brain Tumors 243

umbilical cord blood-derived stem cells can integrate into human MB after local delivery, and that MMP-2 expression by the tumor cells mediates this response through the SDF1/CXCR4 pathway (Bhoopathi et al., 2011). These results offer a new promising therapeutic modality that uses human stem cells for targeting intra-cranial as well as

Although significant results are being generated from the stem cell community, brain tumor researchers have only recently begun to reflect on the specifics of glial progenitor recruitment as a form of treatment for the disease. Glial progenitor cells have shown increased healing potential after supplementing the cultures with exogenous concentrations of VEGF D when compared to controls (Kranich et al., 2009). Using transwell assays, a dose dependent invasive response of murine neural stem cells towards human glioma conditioned media (Heese et al., 2005) and bi-potential O-2A progenitors toward PDGF

Ideally, as has been the case in previous years, the focus should be to target cytokines and their cognate receptors involved in glioma and MB chemotaxis signaling events. Moving forward, the community is in great need of technologies and strategies that can both approximate the chemical microenvironment present in the *in vivo* brain, and replicate these environments *in vitro*. In so doing, migration strategies can be developed that examine how combinations of cytokine and/or pharmacological cocktails can be used to limit the

The migration of glioma and medulloblastoma tumor cells into healthy brain tissue is a critical, yet poorly-understood, component of the tumor invasion and metastasis that contributes to poor patient prognosis. Extensive *in vivo* studies of brain tumors have generated invaluable data to elucidate the molecular alterations and genetic backgrounds present in diseased cells, the signaling mechanisms cells use to communicate with their surrounding microenvironment, and the characteristic patterns of dissemination used by specific tumor cell types. Additionally, *in vitro* studies of brain tumor-derived cells have established the chemotactic potential of various cytokines and extracellular matrixes, evaluated the effectiveness of pharmaceutical cocktails on tumor growth, as well as enabled fundamental measurement of motility and directionality in tumor cell samples. While the majority of research efforts to date have focused on the origin and nature of tumorigenesis in glioma and medulloblastoma, the community is now beginning to examine the integrated role of cell migration in tumor growth and dissemination. Future research is needed to examine the existence and characteristics of tumor cell populations with highly motile phenotypes in order to establish cell migration as a viable therapeutic target, and start

Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S., Yang, W., Small, J.E.,

Herrlinger, U., Ourednik, V., Black, P.M., Breakefield, X.O., Snyder, E.Y., 2000. Neural stem cells display extensive tropism for pathology in adult brain: evidence

leptomeningeal dissemination of MBs.

(Gallo et al., 1996) has been displayed.

**7. Conclusion** 

**8. References** 

diffusive migration of tumor-derived cells into healthy brain.

designing treatment regimens based on cell migratory behaviors.

Subsequent microdevice designs now often generate soluble gradients by using passive diffusion. These systems can eliminate fluid flow near the surroundings of cells by using 3D hydrogels or high resistance channels so that transport occurs predominantly via diffusion (Beebe et al., 2002). In this configuration, two reservoirs are typically used to maintain given chemical concentration in a specified sink and source. Diffusion along adjoining microchannels then facilitates the formation of a concentration gradient between the reservoirs that enables cells to migrate along the defined gradient (Paguirigan and Beebe, 2008). While these systems eliminate the flow stresses imposed upon cells in flow-based devices, they require several hours to generate desired gradients, and rapid adjustment to the gradient profile is often difficult if not impractical. As a result, hybrid microsystems have been developed to enable the use diffusion with a minute level of convection to bolster formation of a desired gradient. For example, our group was able to generate steady-state gradients that were stable for 2-3 days using a bridge design and by exploiting the ultra-low bulk velocities generated by density differences between the reagents used (Figure 6B) (Kong et al., 2010).

The development of microfluidic platforms that incorporate real-time control of cell imaging and measurement of chemotactic concentration gradients is highly needed for understanding the dynamics of brain tumor interactions, an area which remains relatively unexplored when the majority of microfluidic studies focus on measurement of end-point cellular responses.

#### **6. Future prospects of anti-invasive brain tumor therapy**

The impetus for the development of anti-migratory therapeutic agents for brain tumors has been the desire to ease the manageability of the disease, by arresting tumor cells to their primary local environment. Such strategies can reduce the need to utilize the so-called "Search and Destroy" approach that is the currently suggested clinical necessity. Elucidation of possible mechanisms used by diffusely infiltrative glioma and MB cells will enable a better understanding of how to render these cells static, while providing targets for the development of pharmacological products capable of such a task.

More recently it has been suggested that enhancing the recruitment of endogenous progenitors toward tumor masses will aid in restoring the brain regions that have been resected or lost via necrosis (Cayre et al., 2009). Neural stem cells (NSCs) have aroused attention in the field of neurooncology as delivery vehicles of therapeutic genes. In addition to their multipotential capabilities that allow them to differentiate into neurons, astrocytes and oligodendrocytes, NSCs are also characterized by their remarkable capability to migrate through the brain (Gage, 2000; Yandava et al., 1999). The ability of implanted NSCs to distribute themselves throughout the tumors and follow invasive glioma cells has raised the idea of their therapeutic potential in targeting invasive glioma cells *in vivo* (Aboody et al., 2000; Staflin et al., 2009). Shimato and colleagues demonstrated *in vitro* that human NSCs exhibited extensive tropism for MB cells (Shimato et al., 2007). Using leptomeningeal dissemination mouse models, they confirmed *in vivo* that NSCs were able to distribute diffusely to MB cells that had spread throughout the entire spinal cord after implantation in the cisterna magna, and that genetically transformed NSCs functioned effectively in killing MB cells (Shimato et al., 2007). Similarly, genetically-modified NSCs were delivered intracranially and shown to target MBs (Kim et al., 2006). Recently, it was shown that human

Subsequent microdevice designs now often generate soluble gradients by using passive diffusion. These systems can eliminate fluid flow near the surroundings of cells by using 3D hydrogels or high resistance channels so that transport occurs predominantly via diffusion (Beebe et al., 2002). In this configuration, two reservoirs are typically used to maintain given chemical concentration in a specified sink and source. Diffusion along adjoining microchannels then facilitates the formation of a concentration gradient between the reservoirs that enables cells to migrate along the defined gradient (Paguirigan and Beebe, 2008). While these systems eliminate the flow stresses imposed upon cells in flow-based devices, they require several hours to generate desired gradients, and rapid adjustment to the gradient profile is often difficult if not impractical. As a result, hybrid microsystems have been developed to enable the use diffusion with a minute level of convection to bolster formation of a desired gradient. For example, our group was able to generate steady-state gradients that were stable for 2-3 days using a bridge design and by exploiting the ultra-low bulk velocities generated by density differences between the reagents used (Figure 6B)

The development of microfluidic platforms that incorporate real-time control of cell imaging and measurement of chemotactic concentration gradients is highly needed for understanding the dynamics of brain tumor interactions, an area which remains relatively unexplored when the majority of microfluidic studies focus on measurement of end-point

The impetus for the development of anti-migratory therapeutic agents for brain tumors has been the desire to ease the manageability of the disease, by arresting tumor cells to their primary local environment. Such strategies can reduce the need to utilize the so-called "Search and Destroy" approach that is the currently suggested clinical necessity. Elucidation of possible mechanisms used by diffusely infiltrative glioma and MB cells will enable a better understanding of how to render these cells static, while providing targets for the

More recently it has been suggested that enhancing the recruitment of endogenous progenitors toward tumor masses will aid in restoring the brain regions that have been resected or lost via necrosis (Cayre et al., 2009). Neural stem cells (NSCs) have aroused attention in the field of neurooncology as delivery vehicles of therapeutic genes. In addition to their multipotential capabilities that allow them to differentiate into neurons, astrocytes and oligodendrocytes, NSCs are also characterized by their remarkable capability to migrate through the brain (Gage, 2000; Yandava et al., 1999). The ability of implanted NSCs to distribute themselves throughout the tumors and follow invasive glioma cells has raised the idea of their therapeutic potential in targeting invasive glioma cells *in vivo* (Aboody et al., 2000; Staflin et al., 2009). Shimato and colleagues demonstrated *in vitro* that human NSCs exhibited extensive tropism for MB cells (Shimato et al., 2007). Using leptomeningeal dissemination mouse models, they confirmed *in vivo* that NSCs were able to distribute diffusely to MB cells that had spread throughout the entire spinal cord after implantation in the cisterna magna, and that genetically transformed NSCs functioned effectively in killing MB cells (Shimato et al., 2007). Similarly, genetically-modified NSCs were delivered intracranially and shown to target MBs (Kim et al., 2006). Recently, it was shown that human

**6. Future prospects of anti-invasive brain tumor therapy** 

development of pharmacological products capable of such a task.

(Kong et al., 2010).

cellular responses.

umbilical cord blood-derived stem cells can integrate into human MB after local delivery, and that MMP-2 expression by the tumor cells mediates this response through the SDF1/CXCR4 pathway (Bhoopathi et al., 2011). These results offer a new promising therapeutic modality that uses human stem cells for targeting intra-cranial as well as leptomeningeal dissemination of MBs.

Although significant results are being generated from the stem cell community, brain tumor researchers have only recently begun to reflect on the specifics of glial progenitor recruitment as a form of treatment for the disease. Glial progenitor cells have shown increased healing potential after supplementing the cultures with exogenous concentrations of VEGF D when compared to controls (Kranich et al., 2009). Using transwell assays, a dose dependent invasive response of murine neural stem cells towards human glioma conditioned media (Heese et al., 2005) and bi-potential O-2A progenitors toward PDGF (Gallo et al., 1996) has been displayed.

Ideally, as has been the case in previous years, the focus should be to target cytokines and their cognate receptors involved in glioma and MB chemotaxis signaling events. Moving forward, the community is in great need of technologies and strategies that can both approximate the chemical microenvironment present in the *in vivo* brain, and replicate these environments *in vitro*. In so doing, migration strategies can be developed that examine how combinations of cytokine and/or pharmacological cocktails can be used to limit the diffusive migration of tumor-derived cells into healthy brain.

#### **7. Conclusion**

The migration of glioma and medulloblastoma tumor cells into healthy brain tissue is a critical, yet poorly-understood, component of the tumor invasion and metastasis that contributes to poor patient prognosis. Extensive *in vivo* studies of brain tumors have generated invaluable data to elucidate the molecular alterations and genetic backgrounds present in diseased cells, the signaling mechanisms cells use to communicate with their surrounding microenvironment, and the characteristic patterns of dissemination used by specific tumor cell types. Additionally, *in vitro* studies of brain tumor-derived cells have established the chemotactic potential of various cytokines and extracellular matrixes, evaluated the effectiveness of pharmaceutical cocktails on tumor growth, as well as enabled fundamental measurement of motility and directionality in tumor cell samples. While the majority of research efforts to date have focused on the origin and nature of tumorigenesis in glioma and medulloblastoma, the community is now beginning to examine the integrated role of cell migration in tumor growth and dissemination. Future research is needed to examine the existence and characteristics of tumor cell populations with highly motile phenotypes in order to establish cell migration as a viable therapeutic target, and start designing treatment regimens based on cell migratory behaviors.

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**12** 

*USA* 

**Extracellular Matrix Microenvironment** 

Marzenna Wiranowska1 and Mumtaz V. Rojiani2

 *College of Medicine, University of South Florida, Tampa, Florida* 

*2Departments of Medicine and Pathology, GHSU Cancer Center Augusta, Georgia* 

Malignant gliomas are primary brain tumors, which are highly invasive but not known to metastasize outside the central nervous system (CNS). The median survival time of patients with glioma is only 6 months to 2 years depending on various patient, tumor and treatment parameters (Louis et al. 2007). The highly aggressive character of gliomas with glioblastoma multiforme (GBM) being the most aggressive subtype are characterized by their diffuse infiltration into the normal brain parenchyma and interaction with the extracellular matrix (ECM) components in the brain. Standard brain tumor therapies, which include surgery followed by chemotherapy and radiation are not effective in eradicating single glioma cells that migrated into the normal brain establishing new tumor foci. Glioma cells are locally invasive and when migrating through the ECM within several millimeters or centimeters from the main lesion they initiate recurrent tumors often distant to the primary lesion (Bolteus et al. 2001). The infiltrative path of glioma into the normal brain parenchyma involves the basement membrane of blood vessels and myelinated nerve fibers of white

The pattern of glioma cell invasion is related to the unique composition of the cerebral ECM microenvironment, which is remodeled during invasion by activated matrix metalloproteinases (MMPs) (reviewed by Rojiani et al. 2011). In addition, new ECM molecules are secreted and receptor adhesion molecules are expressed by glioma promoting the glioma cell-ECM interaction and signaling. Some of the secreted ECM molecules such as tenascin-C are known to be associated with cell motility and angiogenesis which are both essential for tumor development. Another important microenvironment component affecting glioma development was found to be mechanical force determined by ECM rigidity. More rigid ECM promotes glioma migration and proliferation and lower rigidity of ECM (similar to that of normal brain) would have an opposite effect (Ulrich et al. 2009). The recent sequencing data presented by the Cancer Genome Atlas Research Network (2008) revealed genomic abnormalities in GBM that relate to several signaling pathways such as Epidermal Growth Factor Receptor (EGFR) /Ras /PI3K known to be associated with ECM-related signaling (Ulrich et al. 2009). In addition, the recent integrated genomic analysis identified clinically relevant subtypes of GBM with its characteristic abnormalities

**1. Introduction** 

matter tracts (Rao 2003, Lefranc et al. 2005).

**in Glioma Progression** 

*1Department of Pathology and Cell Biology* 

tumors with standardized quantification of marker gene expression and clinical variables. Biomarker insights. 5, 153-68.


## **Extracellular Matrix Microenvironment in Glioma Progression**

Marzenna Wiranowska1 and Mumtaz V. Rojiani2

*1Department of Pathology and Cell Biology College of Medicine, University of South Florida, Tampa, Florida 2Departments of Medicine and Pathology, GHSU Cancer Center Augusta, Georgia USA* 

#### **1. Introduction**

256 Glioma – Exploring Its Biology and Practical Relevance

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inhibitors in cancer treatment. Oncogene. 19, 6642-50.

tumors with standardized quantification of marker gene expression and clinical

Malignant gliomas are primary brain tumors, which are highly invasive but not known to metastasize outside the central nervous system (CNS). The median survival time of patients with glioma is only 6 months to 2 years depending on various patient, tumor and treatment parameters (Louis et al. 2007). The highly aggressive character of gliomas with glioblastoma multiforme (GBM) being the most aggressive subtype are characterized by their diffuse infiltration into the normal brain parenchyma and interaction with the extracellular matrix (ECM) components in the brain. Standard brain tumor therapies, which include surgery followed by chemotherapy and radiation are not effective in eradicating single glioma cells that migrated into the normal brain establishing new tumor foci. Glioma cells are locally invasive and when migrating through the ECM within several millimeters or centimeters from the main lesion they initiate recurrent tumors often distant to the primary lesion (Bolteus et al. 2001). The infiltrative path of glioma into the normal brain parenchyma involves the basement membrane of blood vessels and myelinated nerve fibers of white matter tracts (Rao 2003, Lefranc et al. 2005).

The pattern of glioma cell invasion is related to the unique composition of the cerebral ECM microenvironment, which is remodeled during invasion by activated matrix metalloproteinases (MMPs) (reviewed by Rojiani et al. 2011). In addition, new ECM molecules are secreted and receptor adhesion molecules are expressed by glioma promoting the glioma cell-ECM interaction and signaling. Some of the secreted ECM molecules such as tenascin-C are known to be associated with cell motility and angiogenesis which are both essential for tumor development. Another important microenvironment component affecting glioma development was found to be mechanical force determined by ECM rigidity. More rigid ECM promotes glioma migration and proliferation and lower rigidity of ECM (similar to that of normal brain) would have an opposite effect (Ulrich et al. 2009).

The recent sequencing data presented by the Cancer Genome Atlas Research Network (2008) revealed genomic abnormalities in GBM that relate to several signaling pathways such as Epidermal Growth Factor Receptor (EGFR) /Ras /PI3K known to be associated with ECM-related signaling (Ulrich et al. 2009). In addition, the recent integrated genomic analysis identified clinically relevant subtypes of GBM with its characteristic abnormalities

Extracellular Matrix Microenvironment in Glioma Progression 259

lecticans contain lectin and HA-binding domains and within that group there are molecules such as aggrecan, versican, neurocan, and brain enriched hyaluronic acid binding protein/brevican (BEHAB/brevican) that act as linkers to ECM components. They bind to HA as well as to cell-surface receptors regulating many processes within the CNS during development, e.g., cell motility, axonal navigation etc. (Sim et al. 2009). Some of these molecules such as versican are known to be produced by glial cells and neural stem cells (Abaskharoun et al. 2010). Another member of CSPGs is phosphocan, an astroglial proteoglycan that binds to neural cell adhesion molecules and tenascin-C. Neuroglial protein-2 (NG2), also a CSPG proteoglycan, which is known as a characteristic marker of oligodendrocyte progenitor cells and pericytes in developing vasculature, is expressed by many gliomas. The NG2 positive cells have been suggested to be the originating cells for

Tenascins (C and R), a family of glycoproteins exist in the ECM as assemblies of several subunits expressed in zones of proliferation, migration, and morphogenesis and are known to play an important role in the developing CNS. For example, tenascin-C was found highly expressed in the subventricular zone and essential for neural stem cell development (reviewed by Wiranowska and Plaas 2008). Galectins (Gal) , mannose- binding lectins, are glycan-binding proteins found inside and outside the cells. Gal-1 is highly represented in the CNS and takes part in the development of neural and non-neural networks and Gal-3 interacts with other neural tissue derived glycoproteins and is expressed by astrocytes and endothelial cells (Quirico-Santos et al. 2010). The role of many ECM components of the

In the normal brain, the ECM complexes containing HA and PGs such as versican, brevican, neurocan, aggrecan, phosphacan and tenascin-C, tenascin-R, and link proteins form the ECM domains called perineuronal nets first described by Camillo Golgi in 1893. These perineuronal net aggregates enwrap the neuronal cell bodies and proximal dendrites of certain neurons and fill the space between neurons and glial processes. More recently, it was proposed that perineuronal nets within the brain are more heterogenous and include structures called "interstitial clefts" (Brightman 2002). As described by Brightman (Brightman 2002), interstitial clefts comprised of astrocytic walls, basal lamina and ECM molecules may vary in size , shape and content depending on the brain region. In addition, the size and the content of interstitial clefts was found to be different in the mature brain by being narrower with limited capacity for cell movement compared to that in the fetal brain. Here, in the fetal brain, the size and the content of interstitial clefts permit cell migration and outgrowth of neurites while in the mature brain cell migration in the interstitial clefts could

normal brain described above, are altered dramatically in glioma.

only occur after enzymatic degradation of the ECM (Brightman 2002).

**2.1 Extracellular matrix in the brain as a cytokine and growth factor depot** 

In the normal brain some regions are especially rich in ECM. These brain regions include subarachnoid space, supependymal packets, circumventricular organs (CVOs) supplied by fenestrated capillaries without blood-brain barrier (BBB), and perivascular space around arterioles and venules. These vessels are associated with stromal connective tissue space and lined by basal laminae containing heparan sulfate proteoglycans (HSPGs). HSPGs which are also components of ependymal, astroglial, and endothelial interfaces in the CNS (including interstitial clefts) have been suggested to serve as a storage site of growth factors and cytokines (Brightman and Kaya 2000). A large number of growth factors, for example,

glioma (Stallcup and Huang 2008).

in platelet derived growth factor receptor A (PDGFRA), isocitrate dehydrogenase 1 (IDH1), neurofibromin 1 (NF1), and confirmed EGFR mutations across all newly defined subtypes of GBM such as classical, proneural, neural and mesenchymal (Verhaak et al. 2010). The most recent studies by Holland (2011) in PDGF-driven mouse models of proneural GBMs with a focus on the biology, therapeutic response and the complexity of the microenvironment showed that some of the genes found in mice are predictive of the survival of patients with this proneural subtype of GBM. Interestingly, many of these genes are rather expressed in the stroma of the tumor than by the tumor cells themselves.

In this chapter, the most recent information pertaining to the glioma extracellular microenvironment and the possible biological targets within ECM for anti-glioma therapy will be reviewed.

#### **2. Extracellular matrix molecules in the normal brain**

In the central nervous system (CNS) approximately only 15-25% of the CNS volume is taken up by the extracellular space, while the majority of the CNS volume consists of cellular elements such as neurons, glia, astrocytic processes and blood vessels (Sykova 2002, Quirico-Santos et al. 2010). The components found within the extracellular space include various ions, metabolites, neurohormones, peptides and ECM molecules produced by neurons and glia. The ECM environment of the normal brain contains high levels of spacefilling carbohydrate molecules unbound to proteins such as the large glycosaminoglycan (GAG) hyaluronan (HA). HA binds to specific cell surface receptors such as cluster determinant 44 (CD44) adhesion molecule and receptor for hyaluronate mediated motility (RHAMM) regulating properties of ECM and tissue, e.g., proliferation, adhesion, motility etc. Protein-bound carbohydrate molecules, which are present in the normal brain at high levels, include sulfated proteoglycans such as chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPGs). In addition, fibrous proteins associated with the basement membranes of the brain's vasculature include collagens, fibronectin, and laminin (Wiranowska and Plaas 2008). The levels of these fibrous proteins in the normal brain are low compared to the connective tissue outside the central nervous system (Bellail et al. 2004, Quirico-Santos et al. 2010). However, the ECM microenvironment of glioma differs from the normal brain and varies depending on the grade of glioma, e.g., with the highly aggressive GBM producing collagen, fibronectin or laminin (Mahesparan et al. 2003). Several classes of ECM molecules play an important role in the normal CNS development but have altered functions in glioma are reviewed below.

The main classes of ECM components in the normal brain are GAG hyaluronan (HA), also called hyaluronic acid, and proteoglycans (PGs), which consist of a core protein attached to GAG chain. HA plays multiple roles in providing an organization of the pericellular matrix. There is a high diversity of PGs due to various core proteins, and variations in GAG side chains. Two classes of transmembrane PGs, glypicans and syndecans, which contain heparan sulphate (HS) side chains called HSPGs are found at high levels in the CNS.

An important class of PGs are chondroitin sulfate PGs (CSPGs), which are expressed at high levels in the regions of the developing fetal brain and later in mature brain in astrocytes and neurons (Rao 2003, Quirico-Santos et al. 2010). CSPGs, and especially the subclass of lecticans, are one of the major families of HA binding matrix glycoproteins in the CNS. A second family of PGs that bind HA in the CNS are HA- and proteoglycan-link proteins (HAPLNs) also called "link proteins", which bind both HA and lecticans. The PGs called

in platelet derived growth factor receptor A (PDGFRA), isocitrate dehydrogenase 1 (IDH1), neurofibromin 1 (NF1), and confirmed EGFR mutations across all newly defined subtypes of GBM such as classical, proneural, neural and mesenchymal (Verhaak et al. 2010). The most recent studies by Holland (2011) in PDGF-driven mouse models of proneural GBMs with a focus on the biology, therapeutic response and the complexity of the microenvironment showed that some of the genes found in mice are predictive of the survival of patients with this proneural subtype of GBM. Interestingly, many of these genes are rather expressed in

In this chapter, the most recent information pertaining to the glioma extracellular microenvironment and the possible biological targets within ECM for anti-glioma therapy

In the central nervous system (CNS) approximately only 15-25% of the CNS volume is taken up by the extracellular space, while the majority of the CNS volume consists of cellular elements such as neurons, glia, astrocytic processes and blood vessels (Sykova 2002, Quirico-Santos et al. 2010). The components found within the extracellular space include various ions, metabolites, neurohormones, peptides and ECM molecules produced by neurons and glia. The ECM environment of the normal brain contains high levels of spacefilling carbohydrate molecules unbound to proteins such as the large glycosaminoglycan (GAG) hyaluronan (HA). HA binds to specific cell surface receptors such as cluster determinant 44 (CD44) adhesion molecule and receptor for hyaluronate mediated motility (RHAMM) regulating properties of ECM and tissue, e.g., proliferation, adhesion, motility etc. Protein-bound carbohydrate molecules, which are present in the normal brain at high levels, include sulfated proteoglycans such as chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPGs). In addition, fibrous proteins associated with the basement membranes of the brain's vasculature include collagens, fibronectin, and laminin (Wiranowska and Plaas 2008). The levels of these fibrous proteins in the normal brain are low compared to the connective tissue outside the central nervous system (Bellail et al. 2004, Quirico-Santos et al. 2010). However, the ECM microenvironment of glioma differs from the normal brain and varies depending on the grade of glioma, e.g., with the highly aggressive GBM producing collagen, fibronectin or laminin (Mahesparan et al. 2003). Several classes of ECM molecules play an important role in the normal CNS development

The main classes of ECM components in the normal brain are GAG hyaluronan (HA), also called hyaluronic acid, and proteoglycans (PGs), which consist of a core protein attached to GAG chain. HA plays multiple roles in providing an organization of the pericellular matrix. There is a high diversity of PGs due to various core proteins, and variations in GAG side chains. Two classes of transmembrane PGs, glypicans and syndecans, which contain

An important class of PGs are chondroitin sulfate PGs (CSPGs), which are expressed at high levels in the regions of the developing fetal brain and later in mature brain in astrocytes and neurons (Rao 2003, Quirico-Santos et al. 2010). CSPGs, and especially the subclass of lecticans, are one of the major families of HA binding matrix glycoproteins in the CNS. A second family of PGs that bind HA in the CNS are HA- and proteoglycan-link proteins (HAPLNs) also called "link proteins", which bind both HA and lecticans. The PGs called

heparan sulphate (HS) side chains called HSPGs are found at high levels in the CNS.

the stroma of the tumor than by the tumor cells themselves.

**2. Extracellular matrix molecules in the normal brain** 

but have altered functions in glioma are reviewed below.

will be reviewed.

lecticans contain lectin and HA-binding domains and within that group there are molecules such as aggrecan, versican, neurocan, and brain enriched hyaluronic acid binding protein/brevican (BEHAB/brevican) that act as linkers to ECM components. They bind to HA as well as to cell-surface receptors regulating many processes within the CNS during development, e.g., cell motility, axonal navigation etc. (Sim et al. 2009). Some of these molecules such as versican are known to be produced by glial cells and neural stem cells (Abaskharoun et al. 2010). Another member of CSPGs is phosphocan, an astroglial proteoglycan that binds to neural cell adhesion molecules and tenascin-C. Neuroglial protein-2 (NG2), also a CSPG proteoglycan, which is known as a characteristic marker of oligodendrocyte progenitor cells and pericytes in developing vasculature, is expressed by many gliomas. The NG2 positive cells have been suggested to be the originating cells for glioma (Stallcup and Huang 2008).

Tenascins (C and R), a family of glycoproteins exist in the ECM as assemblies of several subunits expressed in zones of proliferation, migration, and morphogenesis and are known to play an important role in the developing CNS. For example, tenascin-C was found highly expressed in the subventricular zone and essential for neural stem cell development (reviewed by Wiranowska and Plaas 2008). Galectins (Gal) , mannose- binding lectins, are glycan-binding proteins found inside and outside the cells. Gal-1 is highly represented in the CNS and takes part in the development of neural and non-neural networks and Gal-3 interacts with other neural tissue derived glycoproteins and is expressed by astrocytes and endothelial cells (Quirico-Santos et al. 2010). The role of many ECM components of the normal brain described above, are altered dramatically in glioma.

In the normal brain, the ECM complexes containing HA and PGs such as versican, brevican, neurocan, aggrecan, phosphacan and tenascin-C, tenascin-R, and link proteins form the ECM domains called perineuronal nets first described by Camillo Golgi in 1893. These perineuronal net aggregates enwrap the neuronal cell bodies and proximal dendrites of certain neurons and fill the space between neurons and glial processes. More recently, it was proposed that perineuronal nets within the brain are more heterogenous and include structures called "interstitial clefts" (Brightman 2002). As described by Brightman (Brightman 2002), interstitial clefts comprised of astrocytic walls, basal lamina and ECM molecules may vary in size , shape and content depending on the brain region. In addition, the size and the content of interstitial clefts was found to be different in the mature brain by being narrower with limited capacity for cell movement compared to that in the fetal brain. Here, in the fetal brain, the size and the content of interstitial clefts permit cell migration and outgrowth of neurites while in the mature brain cell migration in the interstitial clefts could only occur after enzymatic degradation of the ECM (Brightman 2002).

#### **2.1 Extracellular matrix in the brain as a cytokine and growth factor depot**

In the normal brain some regions are especially rich in ECM. These brain regions include subarachnoid space, supependymal packets, circumventricular organs (CVOs) supplied by fenestrated capillaries without blood-brain barrier (BBB), and perivascular space around arterioles and venules. These vessels are associated with stromal connective tissue space and lined by basal laminae containing heparan sulfate proteoglycans (HSPGs). HSPGs which are also components of ependymal, astroglial, and endothelial interfaces in the CNS (including interstitial clefts) have been suggested to serve as a storage site of growth factors and cytokines (Brightman and Kaya 2000). A large number of growth factors, for example,

Extracellular Matrix Microenvironment in Glioma Progression 261

**3.1 Role of ECM and MMP molecules in vasculogenic mimicry in glioma: Historical** 

It was observed previously by Maniotis et al. (Maniotis et al. 1999) that blood vessels of highly aggressive tumors such as uveal melanoma originated from tumor cells, rather than from endothelial cells as it was originally expected. This phenomenon named vasculogenic mimicry (VM) was reported later also for other tumors including glioma (Yue and Chen 2005). Although the mechanism of VM could not be explained at that time, many studies evaluated MMPs and ECM interactions in search for clues. It was suggested that several components of the tumor microenvironment may be contributing to the development of VM. For example, consideration was given to MMPs' cleavage of laminin, VE-cadherinpromoted adherence of newly formed vascular channels to tumor cells, and dedifferentiation of tumor cells (Zhang et al. 2007). Three main factors were suspected to play a role in VM: 1) plasticity of malignant tumor cells, 2) remodeling of the ECM by MMPs secreted by tumor cells to obtain space for VM, and 3) the connection of newly formed VM channels with existing blood vessels to acquire blood from the host (Zhang et al. 2007). It was proposed by Maniotis et al. (Maniotis et al. 1999) that the level of the VM channel formation was directly proportional to the level of tumor aggressiveness and influenced by interstitial fluid pressure (IFP), a microenviromental factor known to affect angiogenesis. Tumors that proliferate rapidly have high IFP and compromised blood circulation. In addition, there is a limited blood supply from the host due to decreased endothelial cell sprouting and decreased formation of endothelium-lined blood vessels. Therefore, tumor cells that form VM channels obtain a sufficient blood supply to sustain tumor growth. It was observed that the blood vessels formed as a result of VM had a different structure than normal endothelial-lined blood vessels. VM channels were found to be lined by highly aggressive and poorly differentiated tumor cells that could degrade the base membrane of blood vessels by releasing proteases and migrate into the normal tissue. Recent data (Inoue et al. 2010) support this observation by showing that GBM cancer stem cells express MMP-

Anti-vascular and anti-angiogenic therapies that used molecules such as angiostatin or endostatin that target endothelial cells, showed no effect on tumors with VM. To overcome the lack of understanding of the molecular mechanisms underlying VM, several *in vitro*  studies were initiated in search of new therapeutic approaches based on the concept of ECM involvement in VM formation. For example, laminin was targeted *in vitro* showing that an anti-laminin antibody was able to inhibit VM channel formation by tumor cells (Sanz et al. 2003). Other *in vitro* studies targeted and inhibited MMP-2 and MMP-9 involved in VM by using doxcyline (Zhang et al. 2007). Also, it was shown that the Cox-2 inhibitor, celecoxib, inhibited *in vitro* VM formation in a dose-dependent manner (Basu et al. 2005). Although, these results were only obtained *in vitro*, there may be a recent indirect *in vivo* confirmation*.*  Interestingly, recent *in vivo* studies using a glioma mouse model, showed that non-steroidal anti-inflammatory drugs (NSAID) such as Cox-2 inhibitors suppress gliomagenesis (Fujita et al. 2011). Although the primary conclusion of this study was that gliomagenesis was suppressed due to inhibition of prostaglandin E2-dependent accumulation of myeloid derived suppressor cells in the tumor microenvironment, a secondary effect of Cox-2

**3. Extracellular matrix in glioma** 

13 responsible for invasion and migration of these cells.

inhibitors on VM may be involved as addressed in section 6.2.2.

**perspective** 

insulin-like growth factor (IGF), transforming growth factor–beta (TGF-beta), hepatocyte growth factor (HGF) were found to bind to HSPG (Folkman 1998). A similar observation was made for certain cytokines (reviewed by Wiranowska and Plaas 2008). It was suggested by Mercier et al. (Mercier et al. 2003) that cytokines and growth factors secreted by cells of connective tissue may accumulate in the basal lamina, interact with ECM proteins and affect biological processes including cytogenesis of stem cells in the CNS.

#### **2.2 Extracellular matrix stem cell niche in the brain**

In the CNS, the ability of normal stem cells to self-renew and to differentiate into specific cell types is controlled by the microenvironment of a CNS area in which these cells reside and which is called niche. Similarly, in other tissues and organs, stem cells are found in the protective microenvironment of niches, which are composed of ECM molecules and various differentiated cell types that release regulatory factors and provide direct contact with stem cells maintaining their quiescence. The CNS microenvironment of the neural stem cells (NSC) niche is also called vascular niche, because stem cells concentrate near blood vessels. The NSC niche consists of several ECM components, and includes the basal lamina and endothelial cells of vasculature (Doetsch 2003). These mature, differentiated vascular endothelial cells have an intimate association with stem cells and play a regulatory role in the NSC niche through secreted soluble factors. These factors were shown to promote activation of Notch, a neural precursor receptor, resulting in self-renewal of neural stem cells (Shen et al. 2004). In addition, the basement membrane (also known as basal lamina) contributes to the microenvironment and provides a substrate for stem cells' movement. The subventricular zone, a highly neurogenic area in the CNS, contains transmembrane HSPGs bound to the supependymal basal lamina located in proximity to the stem cells. As mentioned earlier (Section 2.1), HSPGs have the capacity to bind and to store a number of growth factors and cytokines thereby serving as a cytokine and growth factor depot. The growth factors and cytokines can diffuse quickly, and because of close proximity, they can reach high concentrations near the stem cells and regulate their development (Kearns et al. 2003) . For example, EGF and basic fibroblast growth factor (bFGF) stored in the ECM of the subventricular zone can have a stimulatory effect on stem cells by enhancing their proliferation. The growth factors and cytokines can be stored in the ECM throughout life. As mentioned earlier, one of ECM molecules, tenascin-C, is highly expressed in the subventricular zone and essential for neural stem cell development (Wiranowska and Plaas 2008). Tenascin-C plays a key role in the regulation of the developmental program of oligodendrocyte precursor cells (OPCs) and therefore confirming the importance of tenascin-C as an ECM component of the niche (Scadden 2006). Other ECM molecules, such

as laminin and fibronectin, stimulate motility of stem/progenitor cells while CSPGs have an inhibitory effect (Kearns et al. 2003). It was also observed that upon activation of MMPs by proinflammatory cytokines, the neural progenitor cells were stimulated to migrate to the site of injury (Ben-Hur et al. 2006). In summary, many modulatory molecules were described within the ECM of the stem cell niche and, interestingly, many of them were found in glioma, but at higher levels than in the normal brain. In addition, not only the levels but also the functions of many of these molecules differ between the normal brain and glioma such as CSPGs, which are inhibitory for stem cells migration within the niche of the normal brain but stimulatory for glioma cell migration (Kearns et al. 2003, Sim et al. 2009).

#### **3. Extracellular matrix in glioma**

260 Glioma – Exploring Its Biology and Practical Relevance

insulin-like growth factor (IGF), transforming growth factor–beta (TGF-beta), hepatocyte growth factor (HGF) were found to bind to HSPG (Folkman 1998). A similar observation was made for certain cytokines (reviewed by Wiranowska and Plaas 2008). It was suggested by Mercier et al. (Mercier et al. 2003) that cytokines and growth factors secreted by cells of connective tissue may accumulate in the basal lamina, interact with ECM proteins and affect

In the CNS, the ability of normal stem cells to self-renew and to differentiate into specific cell types is controlled by the microenvironment of a CNS area in which these cells reside and which is called niche. Similarly, in other tissues and organs, stem cells are found in the protective microenvironment of niches, which are composed of ECM molecules and various differentiated cell types that release regulatory factors and provide direct contact with stem cells maintaining their quiescence. The CNS microenvironment of the neural stem cells (NSC) niche is also called vascular niche, because stem cells concentrate near blood vessels. The NSC niche consists of several ECM components, and includes the basal lamina and endothelial cells of vasculature (Doetsch 2003). These mature, differentiated vascular endothelial cells have an intimate association with stem cells and play a regulatory role in the NSC niche through secreted soluble factors. These factors were shown to promote activation of Notch, a neural precursor receptor, resulting in self-renewal of neural stem cells (Shen et al. 2004). In addition, the basement membrane (also known as basal lamina) contributes to the microenvironment and provides a substrate for stem cells' movement. The subventricular zone, a highly neurogenic area in the CNS, contains transmembrane HSPGs bound to the supependymal basal lamina located in proximity to the stem cells. As mentioned earlier (Section 2.1), HSPGs have the capacity to bind and to store a number of growth factors and cytokines thereby serving as a cytokine and growth factor depot. The growth factors and cytokines can diffuse quickly, and because of close proximity, they can reach high concentrations near the stem cells and regulate their development (Kearns et al. 2003) . For example, EGF and basic fibroblast growth factor (bFGF) stored in the ECM of the subventricular zone can have a stimulatory effect on stem cells by enhancing their proliferation. The growth factors and cytokines can be stored in the ECM throughout life. As mentioned earlier, one of ECM molecules, tenascin-C, is highly expressed in the subventricular zone and essential for neural stem cell development (Wiranowska and Plaas 2008). Tenascin-C plays a key role in the regulation of the developmental program of oligodendrocyte precursor cells (OPCs) and therefore confirming the importance of tenascin-C as an ECM component of the niche (Scadden 2006). Other ECM molecules, such as laminin and fibronectin, stimulate motility of stem/progenitor cells while CSPGs have an inhibitory effect (Kearns et al. 2003). It was also observed that upon activation of MMPs by proinflammatory cytokines, the neural progenitor cells were stimulated to migrate to the site of injury (Ben-Hur et al. 2006). In summary, many modulatory molecules were described within the ECM of the stem cell niche and, interestingly, many of them were found in glioma, but at higher levels than in the normal brain. In addition, not only the levels but also the functions of many of these molecules differ between the normal brain and glioma such as CSPGs, which are inhibitory for stem cells migration within the niche of the normal brain but stimulatory for glioma cell migration (Kearns et al. 2003, Sim et al. 2009).

biological processes including cytogenesis of stem cells in the CNS.

**2.2 Extracellular matrix stem cell niche in the brain** 

#### **3.1 Role of ECM and MMP molecules in vasculogenic mimicry in glioma: Historical perspective**

It was observed previously by Maniotis et al. (Maniotis et al. 1999) that blood vessels of highly aggressive tumors such as uveal melanoma originated from tumor cells, rather than from endothelial cells as it was originally expected. This phenomenon named vasculogenic mimicry (VM) was reported later also for other tumors including glioma (Yue and Chen 2005). Although the mechanism of VM could not be explained at that time, many studies evaluated MMPs and ECM interactions in search for clues. It was suggested that several components of the tumor microenvironment may be contributing to the development of VM. For example, consideration was given to MMPs' cleavage of laminin, VE-cadherinpromoted adherence of newly formed vascular channels to tumor cells, and dedifferentiation of tumor cells (Zhang et al. 2007). Three main factors were suspected to play a role in VM: 1) plasticity of malignant tumor cells, 2) remodeling of the ECM by MMPs secreted by tumor cells to obtain space for VM, and 3) the connection of newly formed VM channels with existing blood vessels to acquire blood from the host (Zhang et al. 2007). It was proposed by Maniotis et al. (Maniotis et al. 1999) that the level of the VM channel formation was directly proportional to the level of tumor aggressiveness and influenced by interstitial fluid pressure (IFP), a microenviromental factor known to affect angiogenesis. Tumors that proliferate rapidly have high IFP and compromised blood circulation. In addition, there is a limited blood supply from the host due to decreased endothelial cell sprouting and decreased formation of endothelium-lined blood vessels. Therefore, tumor cells that form VM channels obtain a sufficient blood supply to sustain tumor growth. It was observed that the blood vessels formed as a result of VM had a different structure than normal endothelial-lined blood vessels. VM channels were found to be lined by highly aggressive and poorly differentiated tumor cells that could degrade the base membrane of blood vessels by releasing proteases and migrate into the normal tissue. Recent data (Inoue et al. 2010) support this observation by showing that GBM cancer stem cells express MMP-13 responsible for invasion and migration of these cells.

Anti-vascular and anti-angiogenic therapies that used molecules such as angiostatin or endostatin that target endothelial cells, showed no effect on tumors with VM. To overcome the lack of understanding of the molecular mechanisms underlying VM, several *in vitro*  studies were initiated in search of new therapeutic approaches based on the concept of ECM involvement in VM formation. For example, laminin was targeted *in vitro* showing that an anti-laminin antibody was able to inhibit VM channel formation by tumor cells (Sanz et al. 2003). Other *in vitro* studies targeted and inhibited MMP-2 and MMP-9 involved in VM by using doxcyline (Zhang et al. 2007). Also, it was shown that the Cox-2 inhibitor, celecoxib, inhibited *in vitro* VM formation in a dose-dependent manner (Basu et al. 2005). Although, these results were only obtained *in vitro*, there may be a recent indirect *in vivo* confirmation*.*  Interestingly, recent *in vivo* studies using a glioma mouse model, showed that non-steroidal anti-inflammatory drugs (NSAID) such as Cox-2 inhibitors suppress gliomagenesis (Fujita et al. 2011). Although the primary conclusion of this study was that gliomagenesis was suppressed due to inhibition of prostaglandin E2-dependent accumulation of myeloid derived suppressor cells in the tumor microenvironment, a secondary effect of Cox-2 inhibitors on VM may be involved as addressed in section 6.2.2.

Extracellular Matrix Microenvironment in Glioma Progression 263

for glioblastoma where changes in ECM rigidity can both increase and decrease cell motility and the extent of the effect was cell–type dependent (Thomas and DiMilla 2000). It was found that high ECM stiffness enhanced the expression of contractility-mediating proteins such as Rho (Paszek et al. 2005). ECM components have been found to be the main regulators of cell motility in the brain. For example, previous studies showed a stimulatory effect of ECM proteins such as fibronectin, collagen, laminin and others on glioma cell migration (Mahesparan et al. 2003). Ulrich et al. (Ulrich et al. 2009) had shown that glioma cells cultured on fibronectin-coated polymeric ECM with varied but defined mechanical rigidity exhibited altered cell morphology and cytoskeletal organization. These authors showed that glioma cells cultured on softer substrates showed a decreased spreading area, disappearing stress fibers and focal adhesions. Interestingly, all evaluated glioma cell lines cultured on the softest substrates were rounded but viable with cortical rings of F-actin and punctuate vinculin-positive focal complexes, and with no indication of apoptosis (Ulrich et al. 2009). The rigidity of the soft substrates used in that study was comparable to the ECM rigidity of normal brain parenchyma while an increased stiffness was characteristic for

In addition, it was shown that increasing ECM rigidity resulted in increased cell spreading, motility and proliferation. It was suggested previously that glioma cells actively remodel their microenvironment changing it from normal brain ECM to rigid tumor-like ECM (Nakada et al. 2007). Therefore, it was suggested that glioma cells modify their ECM through proteolytic degradation of the normal brain matrix and secretion of new ECM components, thereby providing for a stiffer and more rigid microenvironment which in turn sends mechanobiological signals that support glioma cell invasion (Ulrich et al. 2009). This was observed previously also for invading breast cancer cells (Provenzano et al. 2008). By targeting either the signaling pathways for mechanotaxis or mechanical remodeling itself, new therapeutic approaches could be developed for the treatment of glioma which would

Glioma cells constitutively produce HA and its production is increased during cell proliferation (Wiranowska and Naidu 1994, Wiranowska et al. 2010) promoting glioma invasion (Park et al. 2008). HA is synthesized at the plasma membrane by HA-synthases and the synthesis can be enhanced by various growth factors, e.g., epidermal growth factor (Knudson and Knudson 1993) Interestingly, the content of HA in glioma resembles that of embryonic brain cells (Delpech et al. 1993). HA binds to the HA-binding proteins called hyaladherins which include the CD44 surface receptor. CD44 is a transmembrane glycoprotein expressed by many cell types and by glioma. CD44 serves as a surface receptor

CD44 receptor is overexpressed in glioma cells *in vitro* (Wiranowska et al. 2000, Yu et al. 2010) and found *in vivo* at the leading edge of glioma at the brain-tumor interface (Wiranowska et al. 2006). The HA-CD44 interaction and CD44 shedding from the cell surface were found to be associated with glioma cell motility, migration, and infiltration into the normal brain parenchyma (Annabi et al. 2005). These authors also described that CD44 shedding was mediated by HA and accompanied by up-regulation of MT1-MMP

glioma and its surrounding stroma.

affect glioma invasion and proliferation.

expression.

**3.4 Extracellular matrix molecules in glioma** 

**3.4.1 Glycosaminoglycan hyaluronan and CD44 adhesion molecule** 

for ECM molecules such as HA and CSPGs (Ranuncolo et al. 2002).

#### **3.2 Microenvironment of glioma stem cell vascular niche: New theory of vascular mimicry**

Recently, a new concept of cancer progenitor cells, also known as cancer-initiating cells or cancer stem cells (CSCs), was proposed. These self-renewing, multipotent CSCs are highly tumorigenic and resistant to conventional therapies (Lakka and Rao 2008). Glioblastoma CSCs resemble the normal NSC and express the markers Nestin+/ CD133+ found in the neural stem cell population. Also, glioma CSCs, similar to NSCs, concentrate around blood vessels in the vascular niches with easy access to nutrients, signaling molecules, and the vasculature itself as a substrate for migration (Calabrese et al. 2007, Denysenko et al. 2010). However, CSCs differ from NSCs in their distribution in the brain and their capacity to proliferate. For example, the normal NSCs, which proliferate at a low rate are found only in specific CNS regions such as hippocampus and subventricular zone. In contrast, highly proliferative glioma CSCs can be found distributed across in all regions of cerebrum and cerebellum within the tumors. It was proposed that the main difference between the normal NSCs and glioma CSCs may be the way in which these cells are modulated by the microenvironment of the vasculature within the niche (Calabrese et al. 2007). The vascular niche in brain tumors is abnormal in such that it contributes to the propagation of CSCs thereby enhancing tumor growth. Furthermore, the endothelial cells from this abnormal vascular niche can interact with brain tumor CSCs, as shown *in vitro,* providing certain extracellular regulatory factors and maintaining the self-renewal capability and undifferentiated state of these cells (Calabrese et al. 2007). In that way the glioma vasculature establishes a microenvironment of the niche in which CSCs can transmit and receive signals from the ECM. For example, it was shown that upon stimulation of CSCs by ECM of the vascular niche, the CSCs can secrete vascular endothelial growth factor (VEGF) promoting angiogenesis and thereby enhancing tumor growth (Bao et al. 2006). In addition, the vascular niche was shown to interfere with radiation and chemotherapy by shielding the CSCs and contributing to the resistance to treatments (Denysenko et al. 2010). It was also suggested that the microenvironment of the niche may play a role in tumor initiation based on the observation that non-tumorigenic cell populations may become tumorigenic depending on a certain microenvironment (Rosen and Jordan 2009). Recent reports show that GBM stem cells have similar capabilities as the normal NSCs and undergo differentiation into endothelial cells forming the majority of new blood vessels in gliomas (El Hallani et al. 2010, Ricci-Vitiani et al. 2010, Wang et al. 2010). Blocking VEGF or silencing VEGF receptor 2 inhibits the maturation of tumor endothelial progenitors into endothelium but does not stop the differentiation of CSCs (CD133+ cells) into endothelial cells. However, silencing of Notch (neural precursor receptor as mentioned in section 2.2) blocks the transition of CSCs (CD133+ cells) into endothelial progenitors (Wang et al. 2010). Further studies of the microenvironmental components within the brain tumor vascular niche could lead to new therapeutic targets for treatments of glioma.

#### **3.3 Extracellular matrix and mechanical rigidity in glioma**

There are anatomic variations in stiffness in the normal brain parenchyma (Elkin et al. 2007) with basement membrane of blood vessels and the myelinated fiber tracts of white matter exhibiting a higher mechanical rigidity (Lefranc et al. 2005) and both serving as an infiltrative path for glioma invasion (Rao 2003, Ulrich et al. 2009, Kumar 2009). It was first observed *in vitro* that directed migration of fibroblasts occurs from soft to stiff areas of the ECM, a phenomenon named mechanotaxis (Lo et al. 2000). A similar observation was made

Recently, a new concept of cancer progenitor cells, also known as cancer-initiating cells or cancer stem cells (CSCs), was proposed. These self-renewing, multipotent CSCs are highly tumorigenic and resistant to conventional therapies (Lakka and Rao 2008). Glioblastoma CSCs resemble the normal NSC and express the markers Nestin+/ CD133+ found in the neural stem cell population. Also, glioma CSCs, similar to NSCs, concentrate around blood vessels in the vascular niches with easy access to nutrients, signaling molecules, and the vasculature itself as a substrate for migration (Calabrese et al. 2007, Denysenko et al. 2010). However, CSCs differ from NSCs in their distribution in the brain and their capacity to proliferate. For example, the normal NSCs, which proliferate at a low rate are found only in specific CNS regions such as hippocampus and subventricular zone. In contrast, highly proliferative glioma CSCs can be found distributed across in all regions of cerebrum and cerebellum within the tumors. It was proposed that the main difference between the normal NSCs and glioma CSCs may be the way in which these cells are modulated by the microenvironment of the vasculature within the niche (Calabrese et al. 2007). The vascular niche in brain tumors is abnormal in such that it contributes to the propagation of CSCs thereby enhancing tumor growth. Furthermore, the endothelial cells from this abnormal vascular niche can interact with brain tumor CSCs, as shown *in vitro,* providing certain extracellular regulatory factors and maintaining the self-renewal capability and undifferentiated state of these cells (Calabrese et al. 2007). In that way the glioma vasculature establishes a microenvironment of the niche in which CSCs can transmit and receive signals from the ECM. For example, it was shown that upon stimulation of CSCs by ECM of the vascular niche, the CSCs can secrete vascular endothelial growth factor (VEGF) promoting angiogenesis and thereby enhancing tumor growth (Bao et al. 2006). In addition, the vascular niche was shown to interfere with radiation and chemotherapy by shielding the CSCs and contributing to the resistance to treatments (Denysenko et al. 2010). It was also suggested that the microenvironment of the niche may play a role in tumor initiation based on the observation that non-tumorigenic cell populations may become tumorigenic depending on a certain microenvironment (Rosen and Jordan 2009). Recent reports show that GBM stem cells have similar capabilities as the normal NSCs and undergo differentiation into endothelial cells forming the majority of new blood vessels in gliomas (El Hallani et al. 2010, Ricci-Vitiani et al. 2010, Wang et al. 2010). Blocking VEGF or silencing VEGF receptor 2 inhibits the maturation of tumor endothelial progenitors into endothelium but does not stop the differentiation of CSCs (CD133+ cells) into endothelial cells. However, silencing of Notch (neural precursor receptor as mentioned in section 2.2) blocks the transition of CSCs (CD133+ cells) into endothelial progenitors (Wang et al. 2010). Further studies of the microenvironmental components within the brain tumor vascular niche could

**3.2 Microenvironment of glioma stem cell vascular niche: New theory of vascular** 

lead to new therapeutic targets for treatments of glioma.

**3.3 Extracellular matrix and mechanical rigidity in glioma** 

There are anatomic variations in stiffness in the normal brain parenchyma (Elkin et al. 2007) with basement membrane of blood vessels and the myelinated fiber tracts of white matter exhibiting a higher mechanical rigidity (Lefranc et al. 2005) and both serving as an infiltrative path for glioma invasion (Rao 2003, Ulrich et al. 2009, Kumar 2009). It was first observed *in vitro* that directed migration of fibroblasts occurs from soft to stiff areas of the ECM, a phenomenon named mechanotaxis (Lo et al. 2000). A similar observation was made

**mimicry** 

for glioblastoma where changes in ECM rigidity can both increase and decrease cell motility and the extent of the effect was cell–type dependent (Thomas and DiMilla 2000). It was found that high ECM stiffness enhanced the expression of contractility-mediating proteins such as Rho (Paszek et al. 2005). ECM components have been found to be the main regulators of cell motility in the brain. For example, previous studies showed a stimulatory effect of ECM proteins such as fibronectin, collagen, laminin and others on glioma cell migration (Mahesparan et al. 2003). Ulrich et al. (Ulrich et al. 2009) had shown that glioma cells cultured on fibronectin-coated polymeric ECM with varied but defined mechanical rigidity exhibited altered cell morphology and cytoskeletal organization. These authors showed that glioma cells cultured on softer substrates showed a decreased spreading area, disappearing stress fibers and focal adhesions. Interestingly, all evaluated glioma cell lines cultured on the softest substrates were rounded but viable with cortical rings of F-actin and punctuate vinculin-positive focal complexes, and with no indication of apoptosis (Ulrich et al. 2009). The rigidity of the soft substrates used in that study was comparable to the ECM rigidity of normal brain parenchyma while an increased stiffness was characteristic for glioma and its surrounding stroma.

In addition, it was shown that increasing ECM rigidity resulted in increased cell spreading, motility and proliferation. It was suggested previously that glioma cells actively remodel their microenvironment changing it from normal brain ECM to rigid tumor-like ECM (Nakada et al. 2007). Therefore, it was suggested that glioma cells modify their ECM through proteolytic degradation of the normal brain matrix and secretion of new ECM components, thereby providing for a stiffer and more rigid microenvironment which in turn sends mechanobiological signals that support glioma cell invasion (Ulrich et al. 2009). This was observed previously also for invading breast cancer cells (Provenzano et al. 2008). By targeting either the signaling pathways for mechanotaxis or mechanical remodeling itself, new therapeutic approaches could be developed for the treatment of glioma which would affect glioma invasion and proliferation.

#### **3.4 Extracellular matrix molecules in glioma**

#### **3.4.1 Glycosaminoglycan hyaluronan and CD44 adhesion molecule**

Glioma cells constitutively produce HA and its production is increased during cell proliferation (Wiranowska and Naidu 1994, Wiranowska et al. 2010) promoting glioma invasion (Park et al. 2008). HA is synthesized at the plasma membrane by HA-synthases and the synthesis can be enhanced by various growth factors, e.g., epidermal growth factor (Knudson and Knudson 1993) Interestingly, the content of HA in glioma resembles that of embryonic brain cells (Delpech et al. 1993). HA binds to the HA-binding proteins called hyaladherins which include the CD44 surface receptor. CD44 is a transmembrane glycoprotein expressed by many cell types and by glioma. CD44 serves as a surface receptor for ECM molecules such as HA and CSPGs (Ranuncolo et al. 2002).

CD44 receptor is overexpressed in glioma cells *in vitro* (Wiranowska et al. 2000, Yu et al. 2010) and found *in vivo* at the leading edge of glioma at the brain-tumor interface (Wiranowska et al. 2006). The HA-CD44 interaction and CD44 shedding from the cell surface were found to be associated with glioma cell motility, migration, and infiltration into the normal brain parenchyma (Annabi et al. 2005). These authors also described that CD44 shedding was mediated by HA and accompanied by up-regulation of MT1-MMP expression.

Extracellular Matrix Microenvironment in Glioma Progression 265

by oligodendrocyte progenitor cells (section 2). NG2 expressed by glioma cells has a strong association with ECM ligands such as collagen VI and cellular ligands such as CD44. It has been implicated in the invasive behavior of glioma and found to be expressed *in vitro* and *in vivo* by highly migratory glioma cells while not found in non migratory cells (Lin et al. 1996, Galli et al. 2004, Wiranowska et al. 2006, Stallcup and Huang 2008). NG2 is not only expressed by oligodendrocyte progenitor cells and glioma cells but also by pericytes, which are associated with microvasculature and may play a role in the development of glioma vasculature (Stallcup and Huang 2008). Therefore, NG2 may be considered as one of the

The basement membrane of the cerebral vasculature contains collagens (type IV and V), fibronectin, laminin, vitronectin and HSPGs such as glypicans and syndecans. Some HSPGs, e.g., syndecan-2 were reported to be increased in brain tumors (Theocharis et al. 2010). Laminin, collagen and fibronectin were also shown to be expressed by normal brain tissue bordering with glioma cells in spheroids (Knott et al. 1998). In addition, some of these molecules are also expressed by cells of highly aggressive gliomas. For example, it was found that fibronectin was expressed by GBM *in vitro* and in gliomesenchymal junctions in tumors and their blood vessels (Rao 2003). Another molecule, vitronectin, was found to be expressed in late stage GBM while it was absent in normal brain and early stage of glioma (Yamamoto et al. 1994). Laminins, which were found in blood vessels and in the glial limitants externa in glioma, were also shown to be expressed by human glioma cells positive for glial fibrillary astrocytic protein (GFAP) (Tysnes et al. 1999). An active site on laminin which was capable of binding to CD44 was identified (Hibino et al. 2004). In addition, Ljubimova et al. (Ljubimova et al. 2004) found that highly invasive GBMs overexpressed laminin-8, a member of the subset of laminins characterized by containing the alpha4 chain. Moreover, these authors also found that laminin-8 not only facilitated tumor invasion *in vitro*, but was involved in tumor regrowth after completion of a therapy. On the contrary, a different isoform, laminin-9, was found in lower grade gliomas, astrocytomas, and at low levels in benign brain tumors and in normal brain tissue. Therefore, many of these ECM molecules originally known to be associated with vasculature and now found at various levels expressed by glioma cells could be considered as biomarkers of glioma progression. Tenascin –C , a proteoglycan synthesized by glial and neural crest cells is highly expressed in the subventricular zone and essential for the development of neural stem cells (as described in sections 2 & 2.2 ). Tenascin-C, which is believed to be produced by endothelial cells, was found around blood vessels in astrocytoma and its expression correlated with angiogenesis and tumor progression from grade II to grade III (Zagzag et al. 1995, Quirico-Santos et al. 2010). Tenascin-C was found overexpressed in invasive glioma both *in vitro* and *in vivo* (Mahesparan et al. 2003) thus confirming its significance as an ECM molecule in glioma pathology. Also, galectins are upregulated in glioma and shown to be involved in glioma cell migration and angiogenesis. While high levels of Gal-1 are correlated with aggressiveness of many tumors, the expression of Gal-3 by astrocytes and endothelial cells can be used diagnostically to differentiate GBM from other, less malignant types of glioma (Quirico-Santos et al. 2010). The schematic representation of ECM glioma microenvironment and the summary of representative ECM molecules and their functional significance are

main CSPGs involved in glioma progression.

shown in Figure 1 and Table1.

**3.4.3 Vasculature-associated ECM molecules expressed by gliomas** 

After binding to the CD44 receptor, HA can be endocytosed, transported into lysosomes and degraded by hyaluronidases into small oligosaccharides shown to have glioma - stimulatory activity (Novak et al. 1999). It was previously reported that while small HA fragments were found in the tumor tissues, native HA of high molecular mass was found in the normal and benign tissue (Rooney et al. 1995). Both the high levels of full length polymeric HA and its low molecular weight degradation products, HA fragments, known as oligosaccharides support glioma growth (Novak et al. 1999). The HA oligosaccharides, e.g., hexamer oligoHA-6 (HA-6) or decamer oligoHA-10 (HA-10) are able to displace full length HA via competition for CD44 receptor binding. HA can be effectively displaced by HA decasaccharides, such as HA-10, but not by HA oligosaccharides that are shorter than 10-mer (Tammi et al. 1998). It was observed that full length, large size HA had an antiangiogenic property, whereas smaller oligosaccharides after degradation (3-10 disaccharide units) were no longer anti-angiogenic (Deed et al. 1997). We found that small size oligosaccharide, decamer HA-10, exogenously added to the cell culture stimulated HA production by glioma cells (Wiranowska et al. 2010), as previously described for normal human fibroblasts (Luke and Prehm 1999). These authors found that displacement of nascent HA from the receptors by HA oligosaccharides led to stimulation of HA synthesis (Luke and Prehm 1999). Further studies of HA and the role of HA-CD44 interaction in glioma growth and invasiveness may provide new therapeutic targets for the treatment of glioma. Recent therapeutic approaches targeting HA-CD44 interaction are discussed in the Section 5.2.

#### **3.4.2 Chondroitin sulfate proteoglycans (CSPGs)**

CSPGs are expressed at elevated levels in the developing brain (as described in sections: 2 & 2.2). In the normal brain, they are known for their inhibitory effect on stem cell migration (section 2.2). In glioma however, CSPGs are upregulated and stimulate glioma cell migration (Kearns et al. 2003, Sim et al. 2009). The two members of the CSPG subclass of lecticans (described in section 2) such as versican and BEHAB/brevican are expressed at a higher level in glioma than in the normal brain tissue. In addition, it was reported that the VO/VI versican isoform expressed by migratory glioma cells interacts with surface receptors e.g., EGFR activating the ERK signaling pathway involved in tumor promotion (Ricciardelli et al. 2009). In addition, versican and brevican can form complexes with mesenchymal matrix proteins found in the ECM of glioma, but not in the ECM of the normal brain (Sim et al. 2009). Gliomas of various grades, e.g., astrocytoma and GBM secrete high levels of BEHAB/brevican. The CSPG lectican has an N-terminal HA-binding domain that interacts with fibronectin, thereby further stimulating glioma progression (Viapiano and Matthews 2006). Several other ECM molecules such as HA, CD44, tenascin and transforming growth factor beta2 (TGFbeta2) also interact with versican and promote brain tumor cell invasion. Recently, the link proteins HAPLN4 and HAPLN2 were shown to be reduced in malignant gliomas and it was suggested that this reduction may be associated with matrix remodeling by glioma. Therefore, in contrast to the normal brain tissue where CSPGs lecticans associated to HAPLNs serve as inhibitors of cell motility, in glioma this stabilizing role of link proteins may be reduced or lost resulting in proinvasive activity of CSPGs in glioma (Sim et al. 2009).

Another member of CSPGs family, neuroglial protein 2 (NG2), is also overexpressed in glioma (Schrappe et al. 1991, Wiranowska et al. 2006). NG2 was first found to be expressed

After binding to the CD44 receptor, HA can be endocytosed, transported into lysosomes and degraded by hyaluronidases into small oligosaccharides shown to have glioma - stimulatory activity (Novak et al. 1999). It was previously reported that while small HA fragments were found in the tumor tissues, native HA of high molecular mass was found in the normal and benign tissue (Rooney et al. 1995). Both the high levels of full length polymeric HA and its low molecular weight degradation products, HA fragments, known as oligosaccharides support glioma growth (Novak et al. 1999). The HA oligosaccharides, e.g., hexamer oligoHA-6 (HA-6) or decamer oligoHA-10 (HA-10) are able to displace full length HA via competition for CD44 receptor binding. HA can be effectively displaced by HA decasaccharides, such as HA-10, but not by HA oligosaccharides that are shorter than 10-mer (Tammi et al. 1998). It was observed that full length, large size HA had an antiangiogenic property, whereas smaller oligosaccharides after degradation (3-10 disaccharide units) were no longer anti-angiogenic (Deed et al. 1997). We found that small size oligosaccharide, decamer HA-10, exogenously added to the cell culture stimulated HA production by glioma cells (Wiranowska et al. 2010), as previously described for normal human fibroblasts (Luke and Prehm 1999). These authors found that displacement of nascent HA from the receptors by HA oligosaccharides led to stimulation of HA synthesis (Luke and Prehm 1999). Further studies of HA and the role of HA-CD44 interaction in glioma growth and invasiveness may provide new therapeutic targets for the treatment of glioma. Recent therapeutic approaches targeting HA-CD44 interaction are discussed in the

CSPGs are expressed at elevated levels in the developing brain (as described in sections: 2 & 2.2). In the normal brain, they are known for their inhibitory effect on stem cell migration (section 2.2). In glioma however, CSPGs are upregulated and stimulate glioma cell migration (Kearns et al. 2003, Sim et al. 2009). The two members of the CSPG subclass of lecticans (described in section 2) such as versican and BEHAB/brevican are expressed at a higher level in glioma than in the normal brain tissue. In addition, it was reported that the VO/VI versican isoform expressed by migratory glioma cells interacts with surface receptors e.g., EGFR activating the ERK signaling pathway involved in tumor promotion (Ricciardelli et al. 2009). In addition, versican and brevican can form complexes with mesenchymal matrix proteins found in the ECM of glioma, but not in the ECM of the normal brain (Sim et al. 2009). Gliomas of various grades, e.g., astrocytoma and GBM secrete high levels of BEHAB/brevican. The CSPG lectican has an N-terminal HA-binding domain that interacts with fibronectin, thereby further stimulating glioma progression (Viapiano and Matthews 2006). Several other ECM molecules such as HA, CD44, tenascin and transforming growth factor beta2 (TGFbeta2) also interact with versican and promote brain tumor cell invasion. Recently, the link proteins HAPLN4 and HAPLN2 were shown to be reduced in malignant gliomas and it was suggested that this reduction may be associated with matrix remodeling by glioma. Therefore, in contrast to the normal brain tissue where CSPGs lecticans associated to HAPLNs serve as inhibitors of cell motility, in glioma this stabilizing role of link proteins may be reduced or lost resulting in proinvasive activity of

Another member of CSPGs family, neuroglial protein 2 (NG2), is also overexpressed in glioma (Schrappe et al. 1991, Wiranowska et al. 2006). NG2 was first found to be expressed

Section 5.2.

**3.4.2 Chondroitin sulfate proteoglycans (CSPGs)** 

CSPGs in glioma (Sim et al. 2009).

by oligodendrocyte progenitor cells (section 2). NG2 expressed by glioma cells has a strong association with ECM ligands such as collagen VI and cellular ligands such as CD44. It has been implicated in the invasive behavior of glioma and found to be expressed *in vitro* and *in vivo* by highly migratory glioma cells while not found in non migratory cells (Lin et al. 1996, Galli et al. 2004, Wiranowska et al. 2006, Stallcup and Huang 2008). NG2 is not only expressed by oligodendrocyte progenitor cells and glioma cells but also by pericytes, which are associated with microvasculature and may play a role in the development of glioma vasculature (Stallcup and Huang 2008). Therefore, NG2 may be considered as one of the main CSPGs involved in glioma progression.

#### **3.4.3 Vasculature-associated ECM molecules expressed by gliomas**

The basement membrane of the cerebral vasculature contains collagens (type IV and V), fibronectin, laminin, vitronectin and HSPGs such as glypicans and syndecans. Some HSPGs, e.g., syndecan-2 were reported to be increased in brain tumors (Theocharis et al. 2010). Laminin, collagen and fibronectin were also shown to be expressed by normal brain tissue bordering with glioma cells in spheroids (Knott et al. 1998). In addition, some of these molecules are also expressed by cells of highly aggressive gliomas. For example, it was found that fibronectin was expressed by GBM *in vitro* and in gliomesenchymal junctions in tumors and their blood vessels (Rao 2003). Another molecule, vitronectin, was found to be expressed in late stage GBM while it was absent in normal brain and early stage of glioma (Yamamoto et al. 1994). Laminins, which were found in blood vessels and in the glial limitants externa in glioma, were also shown to be expressed by human glioma cells positive for glial fibrillary astrocytic protein (GFAP) (Tysnes et al. 1999). An active site on laminin which was capable of binding to CD44 was identified (Hibino et al. 2004). In addition, Ljubimova et al. (Ljubimova et al. 2004) found that highly invasive GBMs overexpressed laminin-8, a member of the subset of laminins characterized by containing the alpha4 chain. Moreover, these authors also found that laminin-8 not only facilitated tumor invasion *in vitro*, but was involved in tumor regrowth after completion of a therapy. On the contrary, a different isoform, laminin-9, was found in lower grade gliomas, astrocytomas, and at low levels in benign brain tumors and in normal brain tissue. Therefore, many of these ECM molecules originally known to be associated with vasculature and now found at various levels expressed by glioma cells could be considered as biomarkers of glioma progression.

Tenascin –C , a proteoglycan synthesized by glial and neural crest cells is highly expressed in the subventricular zone and essential for the development of neural stem cells (as described in sections 2 & 2.2 ). Tenascin-C, which is believed to be produced by endothelial cells, was found around blood vessels in astrocytoma and its expression correlated with angiogenesis and tumor progression from grade II to grade III (Zagzag et al. 1995, Quirico-Santos et al. 2010). Tenascin-C was found overexpressed in invasive glioma both *in vitro* and *in vivo* (Mahesparan et al. 2003) thus confirming its significance as an ECM molecule in glioma pathology. Also, galectins are upregulated in glioma and shown to be involved in glioma cell migration and angiogenesis. While high levels of Gal-1 are correlated with aggressiveness of many tumors, the expression of Gal-3 by astrocytes and endothelial cells can be used diagnostically to differentiate GBM from other, less malignant types of glioma (Quirico-Santos et al. 2010). The schematic representation of ECM glioma microenvironment and the summary of representative ECM molecules and their functional significance are shown in Figure 1 and Table1.

Extracellular Matrix Microenvironment in Glioma Progression 267

a logical target for anti-cancer therapy. Supporting evidence was generated using transgenic

The local infiltration of neoplastic cells into healthy CNS parenchyma is the hallmark of gliomas. In this context it is relevant to note that the brain extracellular matrix differs in its composition from other such matrices and that glioma cells have the ability to exploit this environment for invasion. Glioma cells aggressively disseminate as single cells through this unique ECM of the central nervous system. They infiltrate along the periphery of blood vessels or along the longitudinal white matter tracts utilizing different proteolytic enzymes, to achieve their goal of both invasion and metastasis. Serine, cysteine and metalloproteinases are employed to breakdown connective tissue barriers, induce angiogenesis and penetrate normal brain tissue thereby achieving the invasive phenotype (reviewed by Rao 2003). The urokinase plasminogen activator (uPA)/plasmin system of the serine protease family have been shown to be up-regulated in gliomas (Lakka and Rao 2008) and high uPA levels are associated with poor prognosis. Cathepsin B is a lysosomal cysteine protease shown to be secreted at increased levels in gliomas, with expression being significantly higher in glioblastoma than in low-grade glioma and normal brain (Rao 2003,

mouse models overexpressing MMPs (Ha et al. 2001).

Table 1. ECM molecules and their functional significance

**4.1 Proteases in glioma** 

Lakka and Rao 2008).

Fig. 1. Schematic representation of the extracellular matrix (ECM) microenvironment of invasive glioma with necrotic centers and associated brain parenchyma. Hyaluronic acid (HA), a long space-filling molecule composed of a carbohydrate chain is shown either unbound or bound to proteoglycans (PGs) via link proteins (LPs) or bound to CD44 receptor. Also shown are two glioma associated ECM molecules (both chondroitin sulfate proteoglycans): brain enriched hyaluronic acid binding protein/brevican (BEHAB/brevican) and neuroglial protein-2 (NG2) with the latter expressed by glioma cells and pericytes of blood vessels. In addition, glycoprotein tenascin-C, and matrix metalloproteinases (MMPs) are shown. Blood vessels (BV) shown in the glioma and the associated brain parenchyma contain fibrous proteins such as collagens, laminins etc. and heparan sulfate proteoglycans (HSPGs) associated with the BV basement membrane.

#### **4. Proteases, Matrix Metalloproteinases (MMPs), Their Inhibitors (TIMPS) and remodeling of ECM in glioma**

Matrix Metalloproteinases (MMPs) are a class of enzymes known to be involved in normal tissue remodeling, but also produced by glioma cells (Wiranowska et al. 2000) and involved in modification of glioma ECM. In the past, MMPs were considered a potential target for anti-cancer therapies. The result of ECM degradation by MMPs is the release and diffusion of cytokines and growth factors stored in the ECM with subsequent further activation of MMPs by these factors (Wiranowska and Plaas 2008). Since upregulation of MMPs was traditionally associated with inflammation and cancer progression, MMPs were considered

Fig. 1. Schematic representation of the extracellular matrix (ECM) microenvironment of invasive glioma with necrotic centers and associated brain parenchyma. Hyaluronic acid (HA), a long space-filling molecule composed of a carbohydrate chain is shown either unbound or bound to proteoglycans (PGs) via link proteins (LPs) or bound to CD44 receptor. Also shown are two glioma associated ECM molecules (both chondroitin sulfate

(BEHAB/brevican) and neuroglial protein-2 (NG2) with the latter expressed by glioma cells

**4. Proteases, Matrix Metalloproteinases (MMPs), Their Inhibitors (TIMPS) and** 

Matrix Metalloproteinases (MMPs) are a class of enzymes known to be involved in normal tissue remodeling, but also produced by glioma cells (Wiranowska et al. 2000) and involved in modification of glioma ECM. In the past, MMPs were considered a potential target for anti-cancer therapies. The result of ECM degradation by MMPs is the release and diffusion of cytokines and growth factors stored in the ECM with subsequent further activation of MMPs by these factors (Wiranowska and Plaas 2008). Since upregulation of MMPs was traditionally associated with inflammation and cancer progression, MMPs were considered

metalloproteinases (MMPs) are shown. Blood vessels (BV) shown in the glioma and the associated brain parenchyma contain fibrous proteins such as collagens, laminins etc. and heparan sulfate proteoglycans (HSPGs) associated with the BV basement membrane.

proteoglycans): brain enriched hyaluronic acid binding protein/brevican

**remodeling of ECM in glioma** 

and pericytes of blood vessels. In addition, glycoprotein tenascin-C, and matrix

a logical target for anti-cancer therapy. Supporting evidence was generated using transgenic mouse models overexpressing MMPs (Ha et al. 2001).


Table 1. ECM molecules and their functional significance

#### **4.1 Proteases in glioma**

The local infiltration of neoplastic cells into healthy CNS parenchyma is the hallmark of gliomas. In this context it is relevant to note that the brain extracellular matrix differs in its composition from other such matrices and that glioma cells have the ability to exploit this environment for invasion. Glioma cells aggressively disseminate as single cells through this unique ECM of the central nervous system. They infiltrate along the periphery of blood vessels or along the longitudinal white matter tracts utilizing different proteolytic enzymes, to achieve their goal of both invasion and metastasis. Serine, cysteine and metalloproteinases are employed to breakdown connective tissue barriers, induce angiogenesis and penetrate normal brain tissue thereby achieving the invasive phenotype (reviewed by Rao 2003). The urokinase plasminogen activator (uPA)/plasmin system of the serine protease family have been shown to be up-regulated in gliomas (Lakka and Rao 2008) and high uPA levels are associated with poor prognosis. Cathepsin B is a lysosomal cysteine protease shown to be secreted at increased levels in gliomas, with expression being significantly higher in glioblastoma than in low-grade glioma and normal brain (Rao 2003, Lakka and Rao 2008).

Extracellular Matrix Microenvironment in Glioma Progression 269

Tissue inhibitors of metalloproteinases (TIMPs) are specific endogenous inhibitors of MMPs

MMP up-regulation has been implicated in several broad disease categories including inflammation, vascular pathologies, and cancer. Analysis of MMP expression in cancer patients show strong correlation between increased expression of many MMPs and tumor progression in a wide range of malignancies including gliomas. Within the tumor, MMPs are secreted by tumor cells, as well as by stromal cells of the tumor (Rojiani et al. 2010). It appears that tumor cells produce a potent factor called extracellular matrix metalloproteinase inducer (EMMPRIN) a cell surface glycoprotein of the immunoglobulin superfamily. EMMRPIN stimulates MMP expression in stromal cells and also in tumor cells

Several studies have documented overexpression of MMPs in gliomas compared to normal brain tissue. However the MMPs involved in gliomas have almost exclusively been the

The glioma vasculature as well as infiltrating inflammatory cells, which form a portion of the glioma mass have been implicated in MMP expression (VanMeter et al. 2001). Strong gelatinase expression correlates with tumor grade (Forsyth et al. 1999, Wang et al. 2003). Intracranial implantation of glioblastoma cells in nude mice resulted in increased levels of MMP-9 during growth (Sawaya et al. 1998, Chintala et al. 1999). Raithatha et al. (Raithatha et al. 2000) carried out an RNA and protein localization study for gelatinases in a set of human gliomas with varied malignancy. They found that MMP-2 expression was most prominent in tumor cells whereas MMP-9 expression was seen in tumor cell but was more strongly expressed in the vasculature. Nakagawa et al. (Nakagawa et al. 1994) reported increased MMP-9 levels in blood vessels at proliferating margins. Recently Zhang et al. (Zhang et al. 2009) showed that knockdown of Akt2 resulted in decreased MMP-9 expression with concomitant decrease in glioma invasion *in vitro* and *in vivo.* It should be pointed out that EMMPRIN levels have been shown to increase in glioma and correlate with tumor grade (Sameshima et al. 2000). EMMPRIN has also been shown to increase hyaluronan and colocalizes with its receptor CD44 (Toole and Slomiany 2008). Given that Hyaluronan and CD44 are important players in the CNS and in gliomas (see section 3.4.1) EMMPRIN may

*In vitro* studies also manifest a strong correlation between the expression of gelatinases and glioma cell invasion (reviewed by Bellail et al. 2004). Using matrigel assay, it was shown that the most invasive GBM cell line produced the highest level of gelatinases (Uhm et al. 1996, Abe et al. 1994). Besides the gelatinases, there are a number of studies documenting the role of MT1-MMP as well as reports of other MMPs involved in gliomas. Lampert et al. (Lampert et al. 1998) found increased levels of gelatinases as well as MT1-MMP and MT2-MMP in brain tumors. Yamamoto et al. (Yamamoto et al. 1996) found that increased MT1-MMP expression is associated with the expression of activated form of MMP-2 which in turn correlated with malignant glioma progression *in vivo*. Overexpression of MT1-MMP in glioma cell lines leads to activation of pro-MMP-2 (Nakada et al. 2001, Deryugina et al. 1997). Additionally, other studies show that MT1-MMP is increased in glioma-associated microglia and that glioma-released factors trigger this expression by microglia. The MT1- MMP then activates glioma-derived proMMP-2 and promotes glioma expansion (Markovic et al. 2009). The known classical function of MT1-MMP is activation of proMMP-2 in

that have been correlated both positively and negatively in glioma invasion.

gelatinases MMP-2 and MMP-9 (reviewed by Rao 2003).

play a significant role in glioma invasion.

**4.2.1 MMPs in glioma** 

(Jodele et al. 2006).

The third and the most widely studied protease system implicated in gliomas are the matrix metalloproteinases (MMPs). MMPs are a diverse family of endopeptidases that utilize zinc at their active site and encompass a broad spectrum of substrates. Common structural features of MMPs include a signal peptide, a catalytic domain which harbors the conserved zinc-binding site and a hemopexin-like domain. The proteolytic activity of MMPs affects diverse cellular functions such as cell proliferation, adhesion, migration, angiogenesis, bone development, wound healing and mammary involution, among others, by virtue of cleavage of ECM constituents, pro-growth factors, growth factor receptors and cell adhesion molecules. Within the tumor microenvironment, MMPs have been well documented to play a critical role in metastasis and angiogenesis (Kessenbrock et al. 2010).

The family of Metzincin proteinases to which the MMPs belong also includes ADAM (a disintegrin and metalloproteinase) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs). The ADAMs are mostly cell associated, and responsible for cleavage of other proteins like amyloid precursor protein and Notch and hence are also called "sheddases". On the other hand the ADAMTS are secreted (Murphy 2008). The role of ADAMs family members in nervous system development has been documented (Yang et al. 2006).Though these two subfamilies have not been extensively studied in glioma, there is some documentation of their role in these tumors. In this context, it has been shown that ADAM 8 and 19 are overexpressed in glioma correlating with invasion. ADAMTS 4 and 5 cleave brevican, a component of the ECM in the normal brain and have been shown to be upregulated in glioma cells (Rivera et al. 2010).

#### **4.2 MMPs and their inhibitors (TIMPs) in glioma**

MMPs span a wide range of subtypes within this family of endopeptidases that utilize zinc at their active site and interact with different targets. The proteolytic activity of MMPs affects diverse cellular functions as mentioned above, particularly impacting cell proliferation, adhesion, migration and angiogenesis. Thus they are important effectors of tissue remodeling, acting at various levels. The human MMP family comprises of over 23 members and cleaves every component of the ECM. They are classified as follows:


The MT-MMPs are covalently linked to the cell surface, however secreted ones can also attach to the cell membrane by either binding to integrins or to CD44. MMPs are produced in cells as zymogens where cysteine from the pro-domain is bound to zinc at the catalytic site and require proteolytic cleavage for activation. Activation of MMPs often requires cleavage by other MMPs, or serine proteases outside the cell. However some, including the membrane-type MMPs, are activated intracellularly (Egeblad and Werb 2002). Besides activation of pro-enzymes, MMP activity is also regulated by gene expression, compartmentalization and inhibition of active enzymes by their specific tissue inhibitors. Tissue inhibitors of metalloproteinases (TIMPs) are specific endogenous inhibitors of MMPs that have been correlated both positively and negatively in glioma invasion.

#### **4.2.1 MMPs in glioma**

268 Glioma – Exploring Its Biology and Practical Relevance

The third and the most widely studied protease system implicated in gliomas are the matrix metalloproteinases (MMPs). MMPs are a diverse family of endopeptidases that utilize zinc at their active site and encompass a broad spectrum of substrates. Common structural features of MMPs include a signal peptide, a catalytic domain which harbors the conserved zinc-binding site and a hemopexin-like domain. The proteolytic activity of MMPs affects diverse cellular functions such as cell proliferation, adhesion, migration, angiogenesis, bone development, wound healing and mammary involution, among others, by virtue of cleavage of ECM constituents, pro-growth factors, growth factor receptors and cell adhesion molecules. Within the tumor microenvironment, MMPs have been well documented to play

The family of Metzincin proteinases to which the MMPs belong also includes ADAM (a disintegrin and metalloproteinase) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs). The ADAMs are mostly cell associated, and responsible for cleavage of other proteins like amyloid precursor protein and Notch and hence are also called "sheddases". On the other hand the ADAMTS are secreted (Murphy 2008). The role of ADAMs family members in nervous system development has been documented (Yang et al. 2006).Though these two subfamilies have not been extensively studied in glioma, there is some documentation of their role in these tumors. In this context, it has been shown that ADAM 8 and 19 are overexpressed in glioma correlating with invasion. ADAMTS 4 and 5 cleave brevican, a component of the ECM in the normal brain and have been shown to be

MMPs span a wide range of subtypes within this family of endopeptidases that utilize zinc at their active site and interact with different targets. The proteolytic activity of MMPs affects diverse cellular functions as mentioned above, particularly impacting cell proliferation, adhesion, migration and angiogenesis. Thus they are important effectors of tissue remodeling, acting at various levels. The human MMP family comprises of over 23

1. The archetypal MMPs: these include the collagenases i.e. MMP-1, MMP-8 and MMP-13; Stromelysins include MMP-3, MMP-10, MMP-11; Other archetypal MMPs e.g. the metalloelastase i.e MMP-12, also includes MMP-12, MMP-19, MMP-20 and MMP-27

4. Membran-type (MT)-MMPs include MMP-14, MMP-15, MMP-16, MMP-17, MMP-24 and MMP-25 A subgroup of glycosylphosphatidylinositol (GPI) MT-MMP includes

5. Type II transmembrane MMPs include MMP-23A and MMP-23B-identical proteins

The MT-MMPs are covalently linked to the cell surface, however secreted ones can also attach to the cell membrane by either binding to integrins or to CD44. MMPs are produced in cells as zymogens where cysteine from the pro-domain is bound to zinc at the catalytic site and require proteolytic cleavage for activation. Activation of MMPs often requires cleavage by other MMPs, or serine proteases outside the cell. However some, including the membrane-type MMPs, are activated intracellularly (Egeblad and Werb 2002). Besides activation of pro-enzymes, MMP activity is also regulated by gene expression, compartmentalization and inhibition of active enzymes by their specific tissue inhibitors.

members and cleaves every component of the ECM. They are classified as follows:

a critical role in metastasis and angiogenesis (Kessenbrock et al. 2010).

upregulated in glioma cells (Rivera et al. 2010).

2. Matrilysins include MMP-7 and MMP-26 3. Gelatinases include MMP-2 and MMP-9

MMP-17 and MMP-25.

encoded by distinct genes.

**4.2 MMPs and their inhibitors (TIMPs) in glioma** 

MMP up-regulation has been implicated in several broad disease categories including inflammation, vascular pathologies, and cancer. Analysis of MMP expression in cancer patients show strong correlation between increased expression of many MMPs and tumor progression in a wide range of malignancies including gliomas. Within the tumor, MMPs are secreted by tumor cells, as well as by stromal cells of the tumor (Rojiani et al. 2010). It appears that tumor cells produce a potent factor called extracellular matrix metalloproteinase inducer (EMMPRIN) a cell surface glycoprotein of the immunoglobulin superfamily. EMMRPIN stimulates MMP expression in stromal cells and also in tumor cells (Jodele et al. 2006).

Several studies have documented overexpression of MMPs in gliomas compared to normal brain tissue. However the MMPs involved in gliomas have almost exclusively been the gelatinases MMP-2 and MMP-9 (reviewed by Rao 2003).

The glioma vasculature as well as infiltrating inflammatory cells, which form a portion of the glioma mass have been implicated in MMP expression (VanMeter et al. 2001). Strong gelatinase expression correlates with tumor grade (Forsyth et al. 1999, Wang et al. 2003). Intracranial implantation of glioblastoma cells in nude mice resulted in increased levels of MMP-9 during growth (Sawaya et al. 1998, Chintala et al. 1999). Raithatha et al. (Raithatha et al. 2000) carried out an RNA and protein localization study for gelatinases in a set of human gliomas with varied malignancy. They found that MMP-2 expression was most prominent in tumor cells whereas MMP-9 expression was seen in tumor cell but was more strongly expressed in the vasculature. Nakagawa et al. (Nakagawa et al. 1994) reported increased MMP-9 levels in blood vessels at proliferating margins. Recently Zhang et al. (Zhang et al. 2009) showed that knockdown of Akt2 resulted in decreased MMP-9 expression with concomitant decrease in glioma invasion *in vitro* and *in vivo.* It should be pointed out that EMMPRIN levels have been shown to increase in glioma and correlate with tumor grade (Sameshima et al. 2000). EMMPRIN has also been shown to increase hyaluronan and colocalizes with its receptor CD44 (Toole and Slomiany 2008). Given that Hyaluronan and CD44 are important players in the CNS and in gliomas (see section 3.4.1) EMMPRIN may play a significant role in glioma invasion.

*In vitro* studies also manifest a strong correlation between the expression of gelatinases and glioma cell invasion (reviewed by Bellail et al. 2004). Using matrigel assay, it was shown that the most invasive GBM cell line produced the highest level of gelatinases (Uhm et al. 1996, Abe et al. 1994). Besides the gelatinases, there are a number of studies documenting the role of MT1-MMP as well as reports of other MMPs involved in gliomas. Lampert et al. (Lampert et al. 1998) found increased levels of gelatinases as well as MT1-MMP and MT2-MMP in brain tumors. Yamamoto et al. (Yamamoto et al. 1996) found that increased MT1-MMP expression is associated with the expression of activated form of MMP-2 which in turn correlated with malignant glioma progression *in vivo*. Overexpression of MT1-MMP in glioma cell lines leads to activation of pro-MMP-2 (Nakada et al. 2001, Deryugina et al. 1997). Additionally, other studies show that MT1-MMP is increased in glioma-associated microglia and that glioma-released factors trigger this expression by microglia. The MT1- MMP then activates glioma-derived proMMP-2 and promotes glioma expansion (Markovic et al. 2009). The known classical function of MT1-MMP is activation of proMMP-2 in

Extracellular Matrix Microenvironment in Glioma Progression 271

inhibit MMP activity. TIMP-1, -2 and -4 are secreted, whereas TIMP-3 is associated with the extracellular matrix. They are differentially regulated i.e. TIMP-1 expression is inducible,

The MMP-independent activity of TIMPs includes promotion of cell growth as exhibited by TIMP-1 and TIMP-2, apoptosis, angiogenesis as well as a role in cell signaling. These roles of TIMPs surfaced when overexpression of these molecules gave conflicting results. There are several earlier studies showing an inhibitory role of TIMPs in tumor growth and metastasis; however, a number of studies have also demonstrated a tumor-promoting function and serum levels of TIMP correlated with poor prognosis as well (reviewed by Rojiani et al.

This paradoxical tumor promoting and tumor inhibiting role of TIMPs extends to gliomas as well. In the normal murine brain TIMP-2, -3 and -4 are strongly expressed whereas there is very little TIMP-1 expression (reviewed by Crocker et al. 2004) . Studies demonstrating the classical role of TIMP show, for example, that adding TIMP-2 to cultured glioblastoma cells reduces their invasion (Rao et al. 1994). Likewise, use of recombinant TIMP-1 on glioma cells showed reduced glioma invasion (VanMeter et al. 2001). Also, TIMP-1 was shown to cause significant reduction in brain metastasis of implanted fibrosarcoma cells (Kruger et al. 1998) and a decrease in TIMP-2 levels in glioblastomas has been noted as well (Lampert et al. 1998). TIMP-3 overexpression suppresses glioma cell infiltration (Baker et al. 1999). Interestingly, *TIMP-3* gene is one of the most highly methylated genes found in brain

Nakada et al. (Nakada et al. 2001) found that TIMP-1 but not TIMP-2 levels were significantly higher in glioblastoma multiforme compared to other glioma grades when using sandwich enzyme immunoassays. The same study developed stable transfectants of MT1-MMP and found that invasion and gelatinase activity of these transfectants could be totally inhibited by recombinant TIMP-2 but not recombinant TIMP-1. Lampert et al. (Lampert et al. 1998) have demonstrated a significant increase in TIMP-1 levels in glioblastomas compared to low grade tumors. Pagenstecher et al. (Pagenstecher et al. 2001) investigated the expression profiles of 9 MMPs and all TIMPs in different gliomas and found that TIMP-1 expression was the highest in GBMs and grade I gliomas with expression being confined to walls of neovessels. Groft et al. (Groft et al. 2001) carried out an extensive study looking at the expression and localization of all four TIMPs in normal human brains and gliomas. A detailed analysis of the expression of mRNA and protein levels showed that TIMP-2 and TIMP-3 expression pattern did not alter with tumor grade. However TIMP-1 levels correlated positively with glioma malignancy, whereas TIMP-4 correlated negatively. TIMP-1 transcript expression was localized to tumor cell and the surrounding tumor vasculature while TIMP-4 transcripts were found mainly in tumor cells with minor expression seen in vessels. These authors also showed that in an *in vitro* assay, recombinant TIMP-4 reduced invasion of U251 glioma cells through Matrigel. Thus, although TIMPs have clearly been shown to play a significant role in the invasive and growth aspects of glioma, their precise roles remain elusive. However, TIMPs deserve further consideration in

MMPs play a crucial role in tumor growth, metastasis and angiogenesis and therefore have been the targets of antitumor therapy. Due to highly pleiotropic activities of MMPs the

whereas TIMP-2 expression is constitutive (Gomez et al. 1997, Nagase et al. 1999).

tumors (Esteller et al. 2001) and has been referred to as a tumor suppressor.

2010).

the search for targeted therapies.

**5.1 Targeting metalloproteinases** 

**5. Therapeutic targeting of ECM molecules in glioma** 

conjunction with TIMP-2. A complex of MT1-MMP and TIMP-2 interacts with proMMP-2 thus resulting in cleavage of the pro-domain from MMP-2 (Murphy et al. 1999). Hence, it is not surprising that many studies on glioma define activation of proMMP-2.

The gelatinases MMP-2 and MMP-9 as well as the membrane-type protease MT1-MMP have been well documented to play pivotal roles in invasion and angiogenesis (Handsley and Edwards 2005). Vascular basement membrane components are well recognized substrates of MMP-2 and MMP-9. MMP-9 is a known component of the angiogenic switch regulating the bioavailability of VEGF (Bergers et al. 2000). MMP-2 expression has been correlated with the degree of vascularization of tumor nodules (Fang et al. 2000). MT1-MMP deficient mice have provided convincing evidence for its role in angiogenesis (reviewed by Handsley and Edwards 2005). These same MMPs are found at the invasive front of the tumor. Invadopodia are actin-rich protrusions of tumor cells with proteolytic activity. The gelatinases and the MT1-MMP localize to or become activated at the invadopodia (Stylli et al. 2008).

Within the realm of glioma angiogenesis, the gelatinases remain crucial. MMP-2 and MMP-9 showed positive correlation with glioma invasion and angiogenesis (Wang et al. 2003). In a mouse model, glioma growth required host MMP-2 to support angiogenesis (Takahashi et al. 2002). Small interferring RNA (siRNA)-mediated targeting of MMP-9 inhibits glioma angiogenesis in *in vitro* and *in vivo* models (Lakka et al. 2005). Hypoxia-inducible factor-1 α (HIF1α) was shown to induce recruitment of CD45+ cells amongst other cellular components, in a murine glioblastoma model. MMP-9 activity of these bone marrow derived CD45+ cells was essential and sufficient to initiate angiogenesis by increasing VEGF bioavailability (Du et al. 2008).

With regard to other MMPs, Lettau et al. (Lettau et al. 2010) have found that MMP-19 is strongly expressed in astroglial tumors and is also responsible for the invasion of glioma cells *in vitro*. In a study using the glioma cell line U251, Deng et al. (Deng et al. 2010) found that MMP-26 promoted cell invasion *in vitro* and *in vivo*. Stojic et al. (Stojic et al. 2008) have shown enhanced expression of MMP-1, MMP-11 and MMP-19 in glioblastoma multiforme in comparison to low grade astrocytomas and normal brain. Tenascin-C is an ECM protein of the brain parenchyma and its synthesis is known to be up-regulated in glioma. MMP-12 was implicated in the invasion of glioma cell lines using tenascin-C in a three-dimensional matrix model (Sarkar et al. 2006).

Hence, it appears that MMPs play a pivotal role in glioma aggressiveness which would appear to make them potential targets for therapy. However, it should be pointed out that MMPs, by virtue of their degradation capacity also generate endogenous angiogenesis inhibitors. Proteolytic cleavage of plasminogen by several MMPs generates angiostatin and endostatin is generated from the C-terminal fragment of collagen type XVIII. MMP-9 is involved in the release of Tumstatin, another inhibitor of angiogenesis. MMPs as potential targets are further discussed below.

#### **4.2.2 Tissue Inhibitors of Matrix Metalloproteinases (TIMPS) in glioma**

As mentioned above, MMP regulation occurs at four different levels i.e. transcription, zymogen activation, compartmentalization and natural endogenous inhibition. Inhibition by α2-macroglobulin and tissue inhibitors of metalloproteinases (TIMPs) occurs in the liquid phase and in tissues, respectively (Nagase et al. 1999, Brew et al. 2000). Although TIMPs have been known for their primary function of inhibiting MMPs, it has now been widely recognized that TIMPs exhibit additional biological activities independent of their MMP inhibitory function. There are four TIMP members: TIMP-1, TIMP-2, TIMP-3 and TIMP-4, all of which

conjunction with TIMP-2. A complex of MT1-MMP and TIMP-2 interacts with proMMP-2 thus resulting in cleavage of the pro-domain from MMP-2 (Murphy et al. 1999). Hence, it is

The gelatinases MMP-2 and MMP-9 as well as the membrane-type protease MT1-MMP have been well documented to play pivotal roles in invasion and angiogenesis (Handsley and Edwards 2005). Vascular basement membrane components are well recognized substrates of MMP-2 and MMP-9. MMP-9 is a known component of the angiogenic switch regulating the bioavailability of VEGF (Bergers et al. 2000). MMP-2 expression has been correlated with the degree of vascularization of tumor nodules (Fang et al. 2000). MT1-MMP deficient mice have provided convincing evidence for its role in angiogenesis (reviewed by Handsley and Edwards 2005). These same MMPs are found at the invasive front of the tumor. Invadopodia are actin-rich protrusions of tumor cells with proteolytic activity. The gelatinases and the

Within the realm of glioma angiogenesis, the gelatinases remain crucial. MMP-2 and MMP-9 showed positive correlation with glioma invasion and angiogenesis (Wang et al. 2003). In a mouse model, glioma growth required host MMP-2 to support angiogenesis (Takahashi et al. 2002). Small interferring RNA (siRNA)-mediated targeting of MMP-9 inhibits glioma angiogenesis in *in vitro* and *in vivo* models (Lakka et al. 2005). Hypoxia-inducible factor-1 α (HIF1α) was shown to induce recruitment of CD45+ cells amongst other cellular components, in a murine glioblastoma model. MMP-9 activity of these bone marrow derived CD45+ cells was essential and sufficient to initiate angiogenesis by increasing VEGF

With regard to other MMPs, Lettau et al. (Lettau et al. 2010) have found that MMP-19 is strongly expressed in astroglial tumors and is also responsible for the invasion of glioma cells *in vitro*. In a study using the glioma cell line U251, Deng et al. (Deng et al. 2010) found that MMP-26 promoted cell invasion *in vitro* and *in vivo*. Stojic et al. (Stojic et al. 2008) have shown enhanced expression of MMP-1, MMP-11 and MMP-19 in glioblastoma multiforme in comparison to low grade astrocytomas and normal brain. Tenascin-C is an ECM protein of the brain parenchyma and its synthesis is known to be up-regulated in glioma. MMP-12 was implicated in the invasion of glioma cell lines using tenascin-C in a three-dimensional

Hence, it appears that MMPs play a pivotal role in glioma aggressiveness which would appear to make them potential targets for therapy. However, it should be pointed out that MMPs, by virtue of their degradation capacity also generate endogenous angiogenesis inhibitors. Proteolytic cleavage of plasminogen by several MMPs generates angiostatin and endostatin is generated from the C-terminal fragment of collagen type XVIII. MMP-9 is involved in the release of Tumstatin, another inhibitor of angiogenesis. MMPs as potential

As mentioned above, MMP regulation occurs at four different levels i.e. transcription, zymogen activation, compartmentalization and natural endogenous inhibition. Inhibition by α2-macroglobulin and tissue inhibitors of metalloproteinases (TIMPs) occurs in the liquid phase and in tissues, respectively (Nagase et al. 1999, Brew et al. 2000). Although TIMPs have been known for their primary function of inhibiting MMPs, it has now been widely recognized that TIMPs exhibit additional biological activities independent of their MMP inhibitory function. There are four TIMP members: TIMP-1, TIMP-2, TIMP-3 and TIMP-4, all of which

**4.2.2 Tissue Inhibitors of Matrix Metalloproteinases (TIMPS) in glioma** 

not surprising that many studies on glioma define activation of proMMP-2.

MT1-MMP localize to or become activated at the invadopodia (Stylli et al. 2008).

bioavailability (Du et al. 2008).

matrix model (Sarkar et al. 2006).

targets are further discussed below.

inhibit MMP activity. TIMP-1, -2 and -4 are secreted, whereas TIMP-3 is associated with the extracellular matrix. They are differentially regulated i.e. TIMP-1 expression is inducible, whereas TIMP-2 expression is constitutive (Gomez et al. 1997, Nagase et al. 1999).

The MMP-independent activity of TIMPs includes promotion of cell growth as exhibited by TIMP-1 and TIMP-2, apoptosis, angiogenesis as well as a role in cell signaling. These roles of TIMPs surfaced when overexpression of these molecules gave conflicting results. There are several earlier studies showing an inhibitory role of TIMPs in tumor growth and metastasis; however, a number of studies have also demonstrated a tumor-promoting function and serum levels of TIMP correlated with poor prognosis as well (reviewed by Rojiani et al. 2010).

This paradoxical tumor promoting and tumor inhibiting role of TIMPs extends to gliomas as well. In the normal murine brain TIMP-2, -3 and -4 are strongly expressed whereas there is very little TIMP-1 expression (reviewed by Crocker et al. 2004) . Studies demonstrating the classical role of TIMP show, for example, that adding TIMP-2 to cultured glioblastoma cells reduces their invasion (Rao et al. 1994). Likewise, use of recombinant TIMP-1 on glioma cells showed reduced glioma invasion (VanMeter et al. 2001). Also, TIMP-1 was shown to cause significant reduction in brain metastasis of implanted fibrosarcoma cells (Kruger et al. 1998) and a decrease in TIMP-2 levels in glioblastomas has been noted as well (Lampert et al. 1998). TIMP-3 overexpression suppresses glioma cell infiltration (Baker et al. 1999). Interestingly, *TIMP-3* gene is one of the most highly methylated genes found in brain tumors (Esteller et al. 2001) and has been referred to as a tumor suppressor.

Nakada et al. (Nakada et al. 2001) found that TIMP-1 but not TIMP-2 levels were significantly higher in glioblastoma multiforme compared to other glioma grades when using sandwich enzyme immunoassays. The same study developed stable transfectants of MT1-MMP and found that invasion and gelatinase activity of these transfectants could be totally inhibited by recombinant TIMP-2 but not recombinant TIMP-1. Lampert et al. (Lampert et al. 1998) have demonstrated a significant increase in TIMP-1 levels in glioblastomas compared to low grade tumors. Pagenstecher et al. (Pagenstecher et al. 2001) investigated the expression profiles of 9 MMPs and all TIMPs in different gliomas and found that TIMP-1 expression was the highest in GBMs and grade I gliomas with expression being confined to walls of neovessels. Groft et al. (Groft et al. 2001) carried out an extensive study looking at the expression and localization of all four TIMPs in normal human brains and gliomas. A detailed analysis of the expression of mRNA and protein levels showed that TIMP-2 and TIMP-3 expression pattern did not alter with tumor grade. However TIMP-1 levels correlated positively with glioma malignancy, whereas TIMP-4 correlated negatively. TIMP-1 transcript expression was localized to tumor cell and the surrounding tumor vasculature while TIMP-4 transcripts were found mainly in tumor cells with minor expression seen in vessels. These authors also showed that in an *in vitro* assay, recombinant TIMP-4 reduced invasion of U251 glioma cells through Matrigel. Thus, although TIMPs have clearly been shown to play a significant role in the invasive and growth aspects of glioma, their precise roles remain elusive. However, TIMPs deserve further consideration in the search for targeted therapies.

#### **5. Therapeutic targeting of ECM molecules in glioma**

#### **5.1 Targeting metalloproteinases**

MMPs play a crucial role in tumor growth, metastasis and angiogenesis and therefore have been the targets of antitumor therapy. Due to highly pleiotropic activities of MMPs the

Extracellular Matrix Microenvironment in Glioma Progression 273

The modulation of the ECM microenvironment of the stem cell niche may be a promising approach especially in light of the finding that many of the genes and their products prognostic for the faith of proneural GBM were identified in the stroma of the brain tumor but not in the brain tumor cells themselves (as mentioned in Section 1: Holland 2011). Therapies targeting solely the brain tumors and their CSCs population may not be successful due to the high complexity and heterogeneity of brain tumors as demonstrated by the existence of various subtypes of GBMs. In addition, the genetic instability in expressing markers for undifferentiated cells within these tumors makes it difficult to assign the correct differentiation status of the tumor cells (Denysenko et al. 2010). Therefore, other therapeutic options should be pursued including therapies targeting the ECM of the CSCs vascular niches which would disrupt the microenvironment protective and supportive of

One of the stimulatory pro-angiogenic molecules released within the CSCs niche which enhances the survival of neural stem cells is VEGF (section 3.2). It has been shown that targeting VEGF and disruption of the niches by anti-VEGF treatment *in vivo* resulted in CSCs depletion and tumor growth inhibition (Calabrese et al. 2007). Some other studies however, showed that blocking VEGF *in vivo* resulted in growth of satellite tumors (Rubenstein et al. 2000). The early data obtained from clinical trials of GBM patients using VEGF-specific inhibitors such as bevacizumab combined with chemotherapeutic drug CPT-11, showed promising results (Calabrese et al. 2007). However, recent finding by Ricci-Vitiani et al. (Ricci-Vitiani et al. 2010) and Wang et al. (Wang et al. 2010) showed that newly formed blood vessels originating from the GBM stem cells that differentiated into endothelial cells were not responsive to anti-VEGF therapy (section 3.2). Most recently, it has been shown (di Tomaso et al. 2011) that patients treated with anti-VEGF therapy still contained Nestin + cells, a characteristic marker of the CSCs, despite a decreased vascularization of the brain tumors. The phase II results from anti-VEGF therapy in combination with chemotherapeutic CPT-11 failed to show prolonged survival of the GBM patients (Lai et al. 2011). Similar results were obtained from phase III clinical studies with recurrent GBM patients treated with cediranib, an inhibitor of VEGF alone or in combination with chemotherapeutic lomustine (Batchelor, 2010). In addition, it was shown recently by Takano et al. (Takano et al. 2010) that failure of bevacizumab treatment was associated with the high incidence of infiltrative tumors and MMP activity in the samples of urine. Therefore, based on the current state of knowledge, other approaches targeting ECM

There is evidence that some ECM molecules could serve as prognostic markers of antiangiogenic therapy of glioma indicative of the fate of the therapy. For example, Takano et al. (Takano et al. 2010) found that failure of bevacizumab (anti-VEGF antibody) treatment was associated with the high incidence of infiltrative tumors and levels of MMP activity in urine. Therefore, early detection and measurement of urine MMPs activity in samples from patients could be indicative of progressive disease. The detection of this biomarker could allow for earlier therapeutic intervention or alteration of a given anti-glioma therapy. Another ECM prognostic factor of potential therapeutic value could be presence of soluble collagen IV in the blood. It was shown by Sorensen et al. (Sorensen et al. 2009) that in patients treated with anti-angiogenic therapy using cediranib (pan-VEGF receptor tyrosine

molecules of the vascular niche of CSCs need to be developed.

**6.2 Glioma possible new prognostic factors and new targets** 

**6.2.1 ECM prognostic factors** 

the CSCs' self-renewal.

outcomes of the clinical studies targeting these molecules have been disappointing, resulting often in increased tumor growth (reviewed by Rojiani et al. 2011). The initial use of broad spectrum MMP inhibitors interfering with the function of many of these enzymes in clinical trials had unforeseen consequences and resulted in early termination of these studies (reviewed by Coussens et al. 2002). These disappointing results led to the realization that the experimental data had to be reevaluated. For example, in animal studies the inhibitors were administered in early or intermediate stages of cancer whereas in humans they were administered in advanced stages. Besides, it has now been well documented that a number of MMPs play a protective role and their elimination can have adverse consequences (Martin and Matrisian 2007).

However, given their significant contribution to tumor progression, MMPs still remain strong potential target candidates for therapeutic interventions. Therefore, it is not surprising that there are a number of MMP inhibitors (MMPI) in clinical trials (reviewed by Roy et al. 2009). Their effectiveness has yet to be proven. Also, the timing of delivery has been reconsidered since drugs given at earlier stages of cancer appear to be more effective than when given in advanced stages (Roy et al. 2009).

Despite the adaptation of clinical trials and because of the pleiotropic activities of MMPs, a prediction of outcome is still difficult. Therefore, attention is now also given to other ECM molecules in the glioma microenvironment. New classes of targets need to be identified, eg. ECM molecules found within the cancer stem cell niche. Examples of other ECM targets are discussed below (sections 5.2, 6.1 and 6.2).

#### **5.2 Targeting HA and CD44 adhesion molecule**

We showed previously that blocking CD44 and interfering with HA-CD44 ligand-receptor interaction resulted in inhibition of glioma cell invasion, decreased HA production and led to glioma cell apoptosis (Wiranowska et al. 1998, Wiranowska et al. 2010). In addition, recent data by Xu et al. (Xu et al. 2010) showed that CD44 attenuates activation of the Hippo signaling pathway and that knockdown of CD44 expression resulted in the inhibition of glioblastoma. It was suggested that CD44 is a prime therapeutic target for treatment of glioblastoma (Xu et al. 2010). Therefore, further studies of this promising biological target molecule in glioma are warranted. The soluble recombinant CD44-HA-binding domain (CD44-HABD) inhibited proliferation of endothelial cells *in vitro* , blocked angiogenesis *in vivo* and inhibited growth of various tumors (Pall et al. 2004) providing hope for a new therapeutic approach to glioma. In addition, HA was recently used *in vivo* as a delivery carrier for chemotherapeutic paclitaxel (HA-paclitaxel) targeting CD44 positive ovarian carcinoma (Auzenne et al. 2007). Also, recently cisplatin carrying HA nanoparticles were evaluated as potential treatment for cancer (Jeong et al. 2008).

#### **6. Future therapies of glioma**

#### **6.1 ECM therapeutic targets: Hopes and disappointments**

Current therapies like surgery, radiotherapy and chemotherapy are aimed at debulking of the brain tumor mass as well as targeting and eradicating proliferating tumor cells. However, these therapies do not address the quiescent population of the cancer stem cells. These cells, which are nourished and protected from therapeutic interventions by the microenvironment of the CSCs vascular niche can repopulate and initiate new tumor foci in the brain (Denysenko et al. 2010)

outcomes of the clinical studies targeting these molecules have been disappointing, resulting often in increased tumor growth (reviewed by Rojiani et al. 2011). The initial use of broad spectrum MMP inhibitors interfering with the function of many of these enzymes in clinical trials had unforeseen consequences and resulted in early termination of these studies (reviewed by Coussens et al. 2002). These disappointing results led to the realization that the experimental data had to be reevaluated. For example, in animal studies the inhibitors were administered in early or intermediate stages of cancer whereas in humans they were administered in advanced stages. Besides, it has now been well documented that a number of MMPs play a protective role and their elimination can have adverse consequences

However, given their significant contribution to tumor progression, MMPs still remain strong potential target candidates for therapeutic interventions. Therefore, it is not surprising that there are a number of MMP inhibitors (MMPI) in clinical trials (reviewed by Roy et al. 2009). Their effectiveness has yet to be proven. Also, the timing of delivery has been reconsidered since drugs given at earlier stages of cancer appear to be more effective

Despite the adaptation of clinical trials and because of the pleiotropic activities of MMPs, a prediction of outcome is still difficult. Therefore, attention is now also given to other ECM molecules in the glioma microenvironment. New classes of targets need to be identified, eg. ECM molecules found within the cancer stem cell niche. Examples of other ECM targets are

We showed previously that blocking CD44 and interfering with HA-CD44 ligand-receptor interaction resulted in inhibition of glioma cell invasion, decreased HA production and led to glioma cell apoptosis (Wiranowska et al. 1998, Wiranowska et al. 2010). In addition, recent data by Xu et al. (Xu et al. 2010) showed that CD44 attenuates activation of the Hippo signaling pathway and that knockdown of CD44 expression resulted in the inhibition of glioblastoma. It was suggested that CD44 is a prime therapeutic target for treatment of glioblastoma (Xu et al. 2010). Therefore, further studies of this promising biological target molecule in glioma are warranted. The soluble recombinant CD44-HA-binding domain (CD44-HABD) inhibited proliferation of endothelial cells *in vitro* , blocked angiogenesis *in vivo* and inhibited growth of various tumors (Pall et al. 2004) providing hope for a new therapeutic approach to glioma. In addition, HA was recently used *in vivo* as a delivery carrier for chemotherapeutic paclitaxel (HA-paclitaxel) targeting CD44 positive ovarian carcinoma (Auzenne et al. 2007). Also, recently cisplatin carrying HA nanoparticles were

Current therapies like surgery, radiotherapy and chemotherapy are aimed at debulking of the brain tumor mass as well as targeting and eradicating proliferating tumor cells. However, these therapies do not address the quiescent population of the cancer stem cells. These cells, which are nourished and protected from therapeutic interventions by the microenvironment of the CSCs vascular niche can repopulate and initiate new tumor foci in

(Martin and Matrisian 2007).

than when given in advanced stages (Roy et al. 2009).

discussed below (sections 5.2, 6.1 and 6.2).

**6. Future therapies of glioma** 

the brain (Denysenko et al. 2010)

**5.2 Targeting HA and CD44 adhesion molecule** 

evaluated as potential treatment for cancer (Jeong et al. 2008).

**6.1 ECM therapeutic targets: Hopes and disappointments** 

The modulation of the ECM microenvironment of the stem cell niche may be a promising approach especially in light of the finding that many of the genes and their products prognostic for the faith of proneural GBM were identified in the stroma of the brain tumor but not in the brain tumor cells themselves (as mentioned in Section 1: Holland 2011). Therapies targeting solely the brain tumors and their CSCs population may not be successful due to the high complexity and heterogeneity of brain tumors as demonstrated by the existence of various subtypes of GBMs. In addition, the genetic instability in expressing markers for undifferentiated cells within these tumors makes it difficult to assign the correct differentiation status of the tumor cells (Denysenko et al. 2010). Therefore, other therapeutic options should be pursued including therapies targeting the ECM of the CSCs vascular niches which would disrupt the microenvironment protective and supportive of the CSCs' self-renewal.

One of the stimulatory pro-angiogenic molecules released within the CSCs niche which enhances the survival of neural stem cells is VEGF (section 3.2). It has been shown that targeting VEGF and disruption of the niches by anti-VEGF treatment *in vivo* resulted in CSCs depletion and tumor growth inhibition (Calabrese et al. 2007). Some other studies however, showed that blocking VEGF *in vivo* resulted in growth of satellite tumors (Rubenstein et al. 2000). The early data obtained from clinical trials of GBM patients using VEGF-specific inhibitors such as bevacizumab combined with chemotherapeutic drug CPT-11, showed promising results (Calabrese et al. 2007). However, recent finding by Ricci-Vitiani et al. (Ricci-Vitiani et al. 2010) and Wang et al. (Wang et al. 2010) showed that newly formed blood vessels originating from the GBM stem cells that differentiated into endothelial cells were not responsive to anti-VEGF therapy (section 3.2). Most recently, it has been shown (di Tomaso et al. 2011) that patients treated with anti-VEGF therapy still contained Nestin + cells, a characteristic marker of the CSCs, despite a decreased vascularization of the brain tumors. The phase II results from anti-VEGF therapy in combination with chemotherapeutic CPT-11 failed to show prolonged survival of the GBM patients (Lai et al. 2011). Similar results were obtained from phase III clinical studies with recurrent GBM patients treated with cediranib, an inhibitor of VEGF alone or in combination with chemotherapeutic lomustine (Batchelor, 2010). In addition, it was shown recently by Takano et al. (Takano et al. 2010) that failure of bevacizumab treatment was associated with the high incidence of infiltrative tumors and MMP activity in the samples of urine. Therefore, based on the current state of knowledge, other approaches targeting ECM molecules of the vascular niche of CSCs need to be developed.

#### **6.2 Glioma possible new prognostic factors and new targets 6.2.1 ECM prognostic factors**

There is evidence that some ECM molecules could serve as prognostic markers of antiangiogenic therapy of glioma indicative of the fate of the therapy. For example, Takano et al. (Takano et al. 2010) found that failure of bevacizumab (anti-VEGF antibody) treatment was associated with the high incidence of infiltrative tumors and levels of MMP activity in urine. Therefore, early detection and measurement of urine MMPs activity in samples from patients could be indicative of progressive disease. The detection of this biomarker could allow for earlier therapeutic intervention or alteration of a given anti-glioma therapy. Another ECM prognostic factor of potential therapeutic value could be presence of soluble collagen IV in the blood. It was shown by Sorensen et al. (Sorensen et al. 2009) that in patients treated with anti-angiogenic therapy using cediranib (pan-VEGF receptor tyrosine

Extracellular Matrix Microenvironment in Glioma Progression 275

ECM and the surrounding stroma play an essential role in glioma progression but more studies need to be done in order to find proper targets within the ECM to slow or inhibit glioma growth. This is supported by most recent finding by Holland (Holland 2011) that genes expressed in the microenvironment of the stroma rather than in the glioma itself may be predictive of glioma patient survival. This chapter provides a review of recent information relating to ECM targets for anti-glioma therapy. Consideration was given to various ECM molecules within the normal brain, in glioma and in the vascular niche harboring glioma stem cells. Consideration was also given to ECM rigidity and its effect on glioma progression. In addition to discussing some pertinent ECM molecules in glioma progression, also new emerging ECM targets and new prognostic markers candidates were discussed. All in all, ECM molecules are of great importance in the development of new therapeutic strategies and the information compiled in this chapter summarizing their role should be suitable to give guidance for the search and the development of new ECM anti-

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8.

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kinase inhibitor) increased levels of collagen IV were found. This increase in circulating collagen IV was explained as the result of "blood vessels normalization" involving thinning of the abnormally thick tumor associated basement membrane of blood vessels. Increased levels of circulating collagen IV were associated with progression-free and overall patient survival.

#### **6.2.2 New ECM targets**

Recent data by Inoue et al. (Inoue et al. 2010) showed that cancer stem-like cells of GBM express MMP-13 responsible for invasion and migration of these cells suggesting that, MMP-13 might be a potential new therapeutic target for glioblastomas. Blocking VEGF or silencing VEGF receptor 2 inhibits the maturation of tumor endothelial progenitors into endothelium but does not stop the differentiation of CSCs (CD133+ cells) into endothelial cells. However, silencing of Notch (neural precursor receptor as mentioned in section 2.2) blocks the transition of CSCs (CD133+ cells) into endothelial progenitors (Wang et al. 2010). Therefore, Notch may be another potential new target in the stem cell niche. Fujita et al. (Fujita et al. 2011) showed that non-steroidal anti-inflammatory drugs (NSAID) such as Cox-2 inhibitors suppressed gliomagenesis via inhibition of prostaglandin E2 mediated accumulation of myeloid derived suppressor cells. In addition, as mentioned earlier (section 3.1) the Cox-2 inhibitor, celecoxib, inhibited *in vitro* microvascular channel formation associated with VEGF down regulation (Basu et al. 2005). The effect was more pronounced and prostaglandin E2-independent in a highly invasive breast cancer cell line when compared to a less invasive cell line. Based on these findings, it could be hypothesized that the NSAID class may have potential application in highly invasive glioma by targeting of the inflammatory molecules and immune cells as well as VM channel formation in the ECM.

#### **7. Summary and conclusions**

Despite years of research, glioma therapy remains a challenge due to very limited therapeutic options and short survival of glioma patients (as described in section 1). Until recently the focus of research evaluating possible target molecules for treatment was mainly on the cancer cell itself and its genetic characterization. Therefore, routine anti-glioma therapies such as chemotherapy or radiation are solely focused on targeting proliferating glioma cells by interfering with their cell cycle and resulting in glioma cell death. Very little attention was given to evaluation and validation of therapeutic targets in the extracellular matrix in glioma, its vasculature and brain stroma outside the tumor. Recent clinical trials targeting glioma vasculature with anti-angiogenic molecules initially provided encouraging results but later failed to be effective in slowing glioma progression. On the contrary, some of the anti-angiogenic treatments despite achieving blood vessel "normalization" within the tumor later resulted in the development of new glioma foci in the brain parenchyma away from the main lesion. It became known recently that a population of glioma CSCs present within the tumors was capable of escaping this anti-angiogenic therapy giving rise to tumor endothelium which had no markers for anti-angiogenic therapy.

Another attempt to stop glioma cell invasion into the normal brain parenchyma involved targeting various MMPs responsible for remodeling ECM during glioma progression. Although there were high expectations, the results of these studies were disappointing again. It was found that many of the targeted MMPs had anti-tumor as well as tumorigenic activities and blocking or eliminating their activity lead to glioma regrowth. Clearly, the ECM and the surrounding stroma play an essential role in glioma progression but more studies need to be done in order to find proper targets within the ECM to slow or inhibit glioma growth. This is supported by most recent finding by Holland (Holland 2011) that genes expressed in the microenvironment of the stroma rather than in the glioma itself may be predictive of glioma patient survival. This chapter provides a review of recent information relating to ECM targets for anti-glioma therapy. Consideration was given to various ECM molecules within the normal brain, in glioma and in the vascular niche harboring glioma stem cells. Consideration was also given to ECM rigidity and its effect on glioma progression. In addition to discussing some pertinent ECM molecules in glioma progression, also new emerging ECM targets and new prognostic markers candidates were discussed. All in all, ECM molecules are of great importance in the development of new therapeutic strategies and the information compiled in this chapter summarizing their role should be suitable to give guidance for the search and the development of new ECM antiglioma targets.

#### **8. References**

274 Glioma – Exploring Its Biology and Practical Relevance

kinase inhibitor) increased levels of collagen IV were found. This increase in circulating collagen IV was explained as the result of "blood vessels normalization" involving thinning of the abnormally thick tumor associated basement membrane of blood vessels. Increased levels of circulating collagen IV were associated with progression-free and overall patient

Recent data by Inoue et al. (Inoue et al. 2010) showed that cancer stem-like cells of GBM express MMP-13 responsible for invasion and migration of these cells suggesting that, MMP-13 might be a potential new therapeutic target for glioblastomas. Blocking VEGF or silencing VEGF receptor 2 inhibits the maturation of tumor endothelial progenitors into endothelium but does not stop the differentiation of CSCs (CD133+ cells) into endothelial cells. However, silencing of Notch (neural precursor receptor as mentioned in section 2.2) blocks the transition of CSCs (CD133+ cells) into endothelial progenitors (Wang et al. 2010). Therefore, Notch may be another potential new target in the stem cell niche. Fujita et al. (Fujita et al. 2011) showed that non-steroidal anti-inflammatory drugs (NSAID) such as Cox-2 inhibitors suppressed gliomagenesis via inhibition of prostaglandin E2 mediated accumulation of myeloid derived suppressor cells. In addition, as mentioned earlier (section 3.1) the Cox-2 inhibitor, celecoxib, inhibited *in vitro* microvascular channel formation associated with VEGF down regulation (Basu et al. 2005). The effect was more pronounced and prostaglandin E2-independent in a highly invasive breast cancer cell line when compared to a less invasive cell line. Based on these findings, it could be hypothesized that the NSAID class may have potential application in highly invasive glioma by targeting of the inflammatory molecules and immune cells as well as VM channel formation in the ECM.

Despite years of research, glioma therapy remains a challenge due to very limited therapeutic options and short survival of glioma patients (as described in section 1). Until recently the focus of research evaluating possible target molecules for treatment was mainly on the cancer cell itself and its genetic characterization. Therefore, routine anti-glioma therapies such as chemotherapy or radiation are solely focused on targeting proliferating glioma cells by interfering with their cell cycle and resulting in glioma cell death. Very little attention was given to evaluation and validation of therapeutic targets in the extracellular matrix in glioma, its vasculature and brain stroma outside the tumor. Recent clinical trials targeting glioma vasculature with anti-angiogenic molecules initially provided encouraging results but later failed to be effective in slowing glioma progression. On the contrary, some of the anti-angiogenic treatments despite achieving blood vessel "normalization" within the tumor later resulted in the development of new glioma foci in the brain parenchyma away from the main lesion. It became known recently that a population of glioma CSCs present within the tumors was capable of escaping this anti-angiogenic therapy giving rise to tumor

Another attempt to stop glioma cell invasion into the normal brain parenchyma involved targeting various MMPs responsible for remodeling ECM during glioma progression. Although there were high expectations, the results of these studies were disappointing again. It was found that many of the targeted MMPs had anti-tumor as well as tumorigenic activities and blocking or eliminating their activity lead to glioma regrowth. Clearly, the

endothelium which had no markers for anti-angiogenic therapy.

survival.

**6.2.2 New ECM targets** 

**7. Summary and conclusions** 


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**13** 

*2USA* 

Xiao-hong Yao et al.\*

*1People's Republic of China* 

**The Role of Chemoattractant Receptors** 

Chemoattractant receptors are a superfamily of G-protein coupled seven transmembrane cell surface receptors (GPCRs), which transduce extracellular signals into intracellular effector pathways through the activation of heterotrimeric G proteins. This superfamily includes GPCRs for classical chemoattractants such as formyl peptides (fMLF) produced by Gram negative bacteria and host cell mitochondria, the complement cleavage components, leukotriene B4 (LTB4), and platelet activating factor (PAF) as well as GPCRs for chemokines

Chemoattractant GPCRs have the ability to mediate directional migration of cells along a gradient of a chemoattractant. Initially, these receptors were identified mainly on leukocytes, where they play an important role in the trafficking of such cells to sites of inflammation and to lymphoid organs in immune responses (Le et al., 2004). However, during the past few years, both hematopoietic and nonhematopoietic cells have been found to express various chemoattractant GPCRs and are capable of migrating in response to agonists produced in tissue microenvironment. The interaction of chemoattractant GPCRs with their agonists participates in a variety of essential pathophysiological processes including immune responses, inflammation, host defense against microbial infection,

Chemoattractants and their GPCRs are widely expressed in the brain by neurons, glial and microglia cells. They are involved not only in cell migration during development and inflammation, but also act as regulators of neuronal survival, neurotransmission and cellcell communications (Ambrosini and Aloiso, 2004), as the third major transmitter system in the brain (Adler and Rogers, 2005). In addition, chemoattractants and their GPCRs are disregulated in neurodegenerative diseases, multiple sclerosis and brain tumors (Balkwill, 2004; Ransohoff et al., 2007). A number of chemoattractant GPCRs have been detected in

hematopoiesis as well as cancer progression and metastasis (Huang et al., 2008).

glioma cells including FPR1 and chemokine GPCRs (Table 1).

\*Ying Liu, Jian Huang, Ye Zhou, Keqiang Chen, Wanghua Gong,

Mingyong Liu, Xiu-wu Bian and Ji Ming Wang *Third Military Medical University, Chongqing, China* 

*National Cancer Institute at Frederick, Frederick, MD, USA* 

*Fudan University, Shanghai, China* 

**1. Introduction** 

(Le et al., 2002).

**in the Progression of Glioma** 

*1Third Military Medical University, Chongqing, 2National Cancer Institute at Frederick, Frederick, MD* 


## **The Role of Chemoattractant Receptors in the Progression of Glioma**

#### Xiao-hong Yao et al.\*

*1Third Military Medical University, Chongqing, 2National Cancer Institute at Frederick, Frederick, MD 1People's Republic of China 2USA* 

#### **1. Introduction**

284 Glioma – Exploring Its Biology and Practical Relevance

Zhang, B., Gu, F., She, C., Guo, H., Li, W., Niu, R., Fu, L., Zhang, N. and Ma, Y. (2009).

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125(3): 585-95.

"Reduction of Akt2 inhibits migration and invasion of glioma cells." Int J Cancer

Chemoattractant receptors are a superfamily of G-protein coupled seven transmembrane cell surface receptors (GPCRs), which transduce extracellular signals into intracellular effector pathways through the activation of heterotrimeric G proteins. This superfamily includes GPCRs for classical chemoattractants such as formyl peptides (fMLF) produced by Gram negative bacteria and host cell mitochondria, the complement cleavage components, leukotriene B4 (LTB4), and platelet activating factor (PAF) as well as GPCRs for chemokines (Le et al., 2002).

Chemoattractant GPCRs have the ability to mediate directional migration of cells along a gradient of a chemoattractant. Initially, these receptors were identified mainly on leukocytes, where they play an important role in the trafficking of such cells to sites of inflammation and to lymphoid organs in immune responses (Le et al., 2004). However, during the past few years, both hematopoietic and nonhematopoietic cells have been found to express various chemoattractant GPCRs and are capable of migrating in response to agonists produced in tissue microenvironment. The interaction of chemoattractant GPCRs with their agonists participates in a variety of essential pathophysiological processes including immune responses, inflammation, host defense against microbial infection, hematopoiesis as well as cancer progression and metastasis (Huang et al., 2008).

Chemoattractants and their GPCRs are widely expressed in the brain by neurons, glial and microglia cells. They are involved not only in cell migration during development and inflammation, but also act as regulators of neuronal survival, neurotransmission and cellcell communications (Ambrosini and Aloiso, 2004), as the third major transmitter system in the brain (Adler and Rogers, 2005). In addition, chemoattractants and their GPCRs are disregulated in neurodegenerative diseases, multiple sclerosis and brain tumors (Balkwill, 2004; Ransohoff et al., 2007). A number of chemoattractant GPCRs have been detected in glioma cells including FPR1 and chemokine GPCRs (Table 1).

Mingyong Liu, Xiu-wu Bian and Ji Ming Wang

<sup>\*</sup>Ying Liu, Jian Huang, Ye Zhou, Keqiang Chen, Wanghua Gong,

*Third Military Medical University, Chongqing, China* 

*Fudan University, Shanghai, China* 

*National Cancer Institute at Frederick, Frederick, MD, USA* 

The Role of Chemoattractant Receptors in the Progression of Glioma 287

2008; Ping et al., 2011), suggesting the important role of these GPCRs in glioma initiation. In this article, we will review the contribution of chemoattractant GPCRs in glioma progression

**2. The role of the classical chemoattractant GPCR, FPR1, in the progression** 

Human FPR1 (originally named FPR) was detected in 1976 on the surface of human neutrophils, and was cloned in 1990 from a myeloid leukemia-cell line. FPR1 binds Nformyl-methionyl-leucyl-phenylalanine (fMLF), a product of the Gram negative bacteria, as well as mitochondria formylated peptide, and elicits a cascade of signal transduction events mediated by pertussis toxin-sensitive G proteins of the Gi subtype and controlled by phospholipase C (PLC) and phosphoinositide (PI) 3 kinases (Pan et al., 2000). Human myeloid cells activated by FPR1 agonist peptides undergo rapid shape change, showing increased adhesion, chemotaxis, phagocytosis and release of bactericidal and proinflammatory mediators. These functions of FPR1 enable myeloid cells to have proinflammatory and antimicrobial activities. In fact, depletion of the human FPR1 counterpart mFPR1 from mice decreased their resistance to infection by Listeria monocytogenes. Although FPR1 has been shown to be a GPCR that mediates host defense against bacterial infection by phagocytic leukocytes, we found that FPR1 was also selectively expressed by tumor cells in more highly malignant human glioma specimens (Zhou et al., 2005). These findings prompted us to use established human glioma cell lines to investigate the relationship between FPR1 expression and the biological behavior of the tumor cells. For example, the human GBM cell line U87 expresses higher levels of FPR1 and forms more rapidly growing tumors in nude mice than glioma cell lines derived from low grade gliomas, which do not express FPR1 (Zhou et al., 2005). Therefore, observations with glioma cell lines lead us to hypothesize that FPR1 is selectively expressed by more highly malignant

The function of FPR1 in GBM cells was extensively examined by using the prototype chemotactic agonist peptide, bacterial fMLF as a stimulator. In addition to inducing robust chemotaxis and calcium mobilization of GBM cells by fMLF, FPR1 exhibited several unique properties that are closely related to tumor progression. For instance, activation of FPR1 in GBM cells under suboptimal culture conditions (i.e. at low fetal calf serum (FCS) concentration) supports the survival of tumor cells in association with increased intracellular levels of the anti-apoptotic protein Bcl-2. In addition, FPR1 agonist peptide activated two important transcription factors, namely NF-κB and STAT3 in GBM cells. Increased NF-κB translocation has been observed as a consequence of FPR1 signaling pathway also in phagocytic leukocytes (Huang et al., 2001); FPR1 signaling in GBM cells stimulated the phosphorylation of STAT3 at Ser-727 and Tyr-105 residues, while only Ser-727 was phosphorylated in human monocytes. Another transcription factor hypoxia inducible factor-1α (HIF-1α), which induces the adaptation to hypoxic microenvironment by regulating the gene transcription in several processes such as cell oxygen uptake, glucose metabolism, angiogenesis, cell survival and apoptosis, was also activated by FPR1 agonists

and discuss the potential for GPCRs as therapeutic targets in glioma.

glioma cells and may play a role in promoting tumor growth.

**2.2 Function of FPR1 in GBM cells** 

in GBM cells (Zhou et al., 2005).

**of GBM** 

**2.1 Identification of FPR1 in GBM** 


GIMs: glioma infiltrating macrophages; Treg: regulatory T cells.

Table 1. The expression of chemoattractant GPCRs in glioma

Glioma is the most common tumor type in human brain. Nearly two-thirds of human gliomas are highly malignant with rapid progression, high invasiveness, vigorous angiogenesis and resistance to chemotherapy and radiation treatment (Bar, 2011). Glioblastoma (GBM), the most aggressive form of malignant glioma, is characterized by extensive infiltration into the surrounding normal brain tissues and multifocal necrosis. Despite multiple therapeutic regimens (Jahraus and Friedman, 2010), the 2-year survival rate of patients with GBM is less than 30% and has not changed over the past two decades. Because of the increasing incidence of GBM and very poor prognosis, a better understanding of GBM initiation and progression is crucial for the development of more effective therapeutic approaches. GBM cells utilize the normal physiological functions of chemoattractant GPCRs to promote their growth by sensing cognate ligands produced in the microenvironment that enhance tumor cell proliferation, invasion and the production of angiogenic factors such as vascular endothelial cell growth factor (VEGF) and the chemokine CXCL8 (IL-8) (Yao et al., 2008; Ping et al., 2007). Recently, the chemoattractant GPCRs FPR1 and CXCR4 were also found to be expressed by glioma stem-like cells (GSLCs) and to mediate GSLC chemotaxis and the production of VEGF (Ping et al., 2007; Yao et al., 2008; Ping et al., 2011), suggesting the important role of these GPCRs in glioma initiation. In this article, we will review the contribution of chemoattractant GPCRs in glioma progression and discuss the potential for GPCRs as therapeutic targets in glioma.

#### **2. The role of the classical chemoattractant GPCR, FPR1, in the progression of GBM**

#### **2.1 Identification of FPR1 in GBM**

286 Glioma – Exploring Its Biology and Practical Relevance

Ligand (cell sources) Major effects on glioma References

Growth; Invasion; Angiogenesis

CXCL8 (glioma cells) Invasion Raychaudhuri et al.,

CXCL8 (glioma cells) Angiogenesis Brat DJ et al., 2005

Growth; Angiogenesis;

CXCL13 (glioma cells) Not clear Bajetto et al., 2006

CCL2 (glioma cells) Migration Liang Y et al., 2008

anti-invasion based on whether CX3CL1 is soluble or membrane

Migration

CCR3 (glioma cells) CCL3L1 (glioma cells) Proliferation Kouno et al., 2004 CCR4 (Treg cells) CCL22 (glioma cells) Treg infiltration Jacobs et al., 2010 CCR5 (glioma cells) CCL3L1 (glioma cells) Proliferation Kouno et al., 2004

CX3CL1 (glioma cells) Tumorigenesis; Pro-or

bound.

Glioma is the most common tumor type in human brain. Nearly two-thirds of human gliomas are highly malignant with rapid progression, high invasiveness, vigorous angiogenesis and resistance to chemotherapy and radiation treatment (Bar, 2011). Glioblastoma (GBM), the most aggressive form of malignant glioma, is characterized by extensive infiltration into the surrounding normal brain tissues and multifocal necrosis. Despite multiple therapeutic regimens (Jahraus and Friedman, 2010), the 2-year survival rate of patients with GBM is less than 30% and has not changed over the past two decades. Because of the increasing incidence of GBM and very poor prognosis, a better understanding of GBM initiation and progression is crucial for the development of more effective therapeutic approaches. GBM cells utilize the normal physiological functions of chemoattractant GPCRs to promote their growth by sensing cognate ligands produced in the microenvironment that enhance tumor cell proliferation, invasion and the production of angiogenic factors such as vascular endothelial cell growth factor (VEGF) and the chemokine CXCL8 (IL-8) (Yao et al., 2008; Ping et al., 2007). Recently, the chemoattractant GPCRs FPR1 and CXCR4 were also found to be expressed by glioma stem-like cells (GSLCs) and to mediate GSLC chemotaxis and the production of VEGF (Ping et al., 2007; Yao et al.,

Zhou et al., 2005; Huang et al., 2007, 2008, and 2010

Maru et al., 2008

Liu et al., 2008

Ping et al., 2007 and

2011

2011

Anti-apoptosis Hattermann et al., 2010

Proliferation; Growth Liu et al., 2010;

Chemoattractant

(expressing cells)

FPR1 (glioma cells) fMLF (bacteria);

Annexin1(necrotic glioma cells)

CXCL10 (glioma cells) ; CXCL9 (glioma cells)

CXCL12 (glioma cells and stromal cells)

CXCL12 (glioma cells and stromal cells)

GIMs: glioma infiltrating macrophages; Treg: regulatory T cells. Table 1. The expression of chemoattractant GPCRs in glioma

GPCRs

CXCR1 (glioma cells)

CXCR2 (glioma cells)

CXCR3 (glioma cells)

CXCR4 (glioma cells)

CXCR5 (glioma cells)

CXCR7 (glioma cells)

CCR2A (glioma cells)

CX3CR1

GIMs)

(glioma cells and

Human FPR1 (originally named FPR) was detected in 1976 on the surface of human neutrophils, and was cloned in 1990 from a myeloid leukemia-cell line. FPR1 binds Nformyl-methionyl-leucyl-phenylalanine (fMLF), a product of the Gram negative bacteria, as well as mitochondria formylated peptide, and elicits a cascade of signal transduction events mediated by pertussis toxin-sensitive G proteins of the Gi subtype and controlled by phospholipase C (PLC) and phosphoinositide (PI) 3 kinases (Pan et al., 2000). Human myeloid cells activated by FPR1 agonist peptides undergo rapid shape change, showing increased adhesion, chemotaxis, phagocytosis and release of bactericidal and proinflammatory mediators. These functions of FPR1 enable myeloid cells to have proinflammatory and antimicrobial activities. In fact, depletion of the human FPR1 counterpart mFPR1 from mice decreased their resistance to infection by Listeria monocytogenes. Although FPR1 has been shown to be a GPCR that mediates host defense against bacterial infection by phagocytic leukocytes, we found that FPR1 was also selectively expressed by tumor cells in more highly malignant human glioma specimens (Zhou et al., 2005). These findings prompted us to use established human glioma cell lines to investigate the relationship between FPR1 expression and the biological behavior of the tumor cells. For example, the human GBM cell line U87 expresses higher levels of FPR1 and forms more rapidly growing tumors in nude mice than glioma cell lines derived from low grade gliomas, which do not express FPR1 (Zhou et al., 2005). Therefore, observations with glioma cell lines lead us to hypothesize that FPR1 is selectively expressed by more highly malignant glioma cells and may play a role in promoting tumor growth.

#### **2.2 Function of FPR1 in GBM cells**

The function of FPR1 in GBM cells was extensively examined by using the prototype chemotactic agonist peptide, bacterial fMLF as a stimulator. In addition to inducing robust chemotaxis and calcium mobilization of GBM cells by fMLF, FPR1 exhibited several unique properties that are closely related to tumor progression. For instance, activation of FPR1 in GBM cells under suboptimal culture conditions (i.e. at low fetal calf serum (FCS) concentration) supports the survival of tumor cells in association with increased intracellular levels of the anti-apoptotic protein Bcl-2. In addition, FPR1 agonist peptide activated two important transcription factors, namely NF-κB and STAT3 in GBM cells. Increased NF-κB translocation has been observed as a consequence of FPR1 signaling pathway also in phagocytic leukocytes (Huang et al., 2001); FPR1 signaling in GBM cells stimulated the phosphorylation of STAT3 at Ser-727 and Tyr-105 residues, while only Ser-727 was phosphorylated in human monocytes. Another transcription factor hypoxia inducible factor-1α (HIF-1α), which induces the adaptation to hypoxic microenvironment by regulating the gene transcription in several processes such as cell oxygen uptake, glucose metabolism, angiogenesis, cell survival and apoptosis, was also activated by FPR1 agonists in GBM cells (Zhou et al., 2005).

The Role of Chemoattractant Receptors in the Progression of Glioma 289

and PKC, which are activated by FPR1 agonist in GBM cells. Thus stimulation of FPR1 activates MMPs in GBM cells and increases proteolytic processes in the tumor

Despite extensive characterization of FPR1 function in GBM cells, whether host-derived agonists are present in the tumor microenvironment remains unknown. We tested GBM cell responses to the neutrophil granule protein cathepsin G, which is an endogenous agonist for FPR1 and induces the migration of myloid cells. We determinated that cathepsin G is capable of inducing the migration of GBM cells expressing FPR1 (Sun et al., 2004). However, cathepsin G is unlikely to be present in brain unless substantial tissue damage compromises the blood brain barrier and results in the release of this FPR1 agonist into the brain by neutrophils. We therefore examined other possible sources of potential FPR1 agonists that may act on GBM cells. Since mitochondrial peptides are also potential endogenous FPR1 agonists and GBMs frequently contain necrotic foci in the rapidly growing tumor mass that may release mitochondrial components, we examined the presence of FPR1 agonists in supernatants of necrotic tumor cells. Indeed, supernatants of necrotic GBM cells and tumors formed by GBM cells in nude mice induced potent chemotaxis of live GBM cells as well as a rat basophil leukemia-cell line transfected to express human FPR1 (ETFR cells). The chemotactic activity released by necrotic GBM cells and tumor tissues was blocked by an anti-FPR1 antibody and by a FPR-specific antagonist tBoc-MLF (Zhou et al., 2005). The robust intracellular Ca2+ mobilization induced in GBM cells by necrotic GBM cell supernatant attenuated the subsequent cell response to fMLF, suggesting that agonist contained in the supernatants of necrotic tumor cells share a receptor with fMLF (Zhou et al., 2005). Further evidence to support the release of FPR1 agonists by necrotic GBM cells was provided by the observation that the tumor cell supernatant down-regulated FPR1 expressed on the surface of human monocytes and FPR1 expressing ETFR cells. These observations confirm that FPR1 expressed on GBM cells is able to recognize agonist activity released in the tumor microenvironment in a paracrine and/or autocrine loop (Zhou et al., 2005). Our recent effort to characterize the biochemical nature of the FPR1 agonist activity released by necrotic GBM cells revealed that the glucocorticoid binding protein annexin1 (AnxA1), which has been reported to be an agonist for FPR1 and its variant receptor FPR2, can promote tumor cell invasion and angiogenesis. AnxA1 accounts for the majority of the FPR1 agonist activity released by necrotic GBM cells because depletion of AnxA1 from the necrotic tumor supernatant markedly reduced its capacity to stimulate FPR1 on viable GBM cells (Yang et al., unpublished observation). We therefore established a paradigm for the role of FPR1 in GBM progression in which FPR1 in GBM cells by responding to necrotic tumor cell-released agonist such as AnxA1 transactivates EGFR and the two receptors cooperate to promote the growth, invasion, angiogenesis and progression of GBM (Fig 1A).

**2.4 Identification of endogenous FPR1 agonist released by necrotic GBM cells** 

**3. The role of the chemokine GPCR CXCR4 in glioma progression** 

CXCR4 selectively binds the CXC chemokine stromal cell-derived factor 1 (SDF-1), also known as CXCL12 (Furusato et al., 2010). CXCR4 is normally expressed in a wide variety of cells and tissues. The CXCR4 agonist CXCL12 was first cloned from a murine bone marrow stromal cell line, and was produced in high quantity by marrow stromal cells. In addition to

**3.1 CXCR4 and its ligand CXCL12** 

microenvironment (Huang et al., 2010).

Since both STAT3 and HIF-1α are implicated in the transcriptional activation of the gene coding for VEGF, we investigated the effect of activating FPR1 on the production of VEGF by tumor cells. We found that supernatants from fMLF-stimulated GBM cells induced the migration and tubule formation of human vascular endothelial cells (EC) (Zhou et al., 2005). This property of the tumor cell supernatant was abolished by a neutralizing anti-human VEGF antibody (Zhou et al., 2005), suggesting VEGF was released by FPR1 agoniststimulated GBM cells. FPR1 in GBM cells was subsequently shown to promote the release of another angiogenic factor, the chemokine CXCL8 (IL-8) (Yao et al., 2008). The contribution of FPR1 to GBM progression was then tested in vivo in nude mice. Tumor cells containing small interference (si) RNA targeting FPR1 mRNA yielded tumors in nude mice with markedly reduced rate of growth as compared to control cells transfected with random siRNA (Zhou et al., 2005). Thus, the functional studies provide strong evidence for the involvement of FPR1 in supporting the rapid progression of GBM.

Crosstalk between GPCRs and growth factor receptors plays an important role in orchestrating the interaction of intracellular signaling molecules implicated in tumor growth, angiogenesis and metastasis (Lappano and Maggiolini, 2011). The crosstalk between GPCRs and the receptor for epidermal growth factor (EGFR) has been shown to promote the progression of colon, lung, breast, ovarian, prostate, and head and neck carcinomas (Hart et al., 2005). Like many malignant tumors of human and mouse origin, human GBM cells express high levels of EGFR and stimulation with EGF increases tumor cell chemotaxis and proliferation with rapid phosphorylation of at least 4 tyrosine residues in the C-terminal domain of EGFR (Huang et al., 2007). When GBM cells were stimulated with the FPR1 agonist fMLF, EGFR was also rapidly phosphorylated but with restriction to a single tyrosine residue Tyr992. This transactivation of EGFR by FPR1 agonist peptide accounted for approximately 40% of the biological activity of FPR1 in GBM cells and was dependent on a Src kinase pathway (Huang et al., 2007). Moreover, GBM cells depleted of either FPR1 or EGFR grew more slowly as compared with parental cells and depletion of both receptors further reduced the tumorigenicity of the GBM cells (Huang et al., 2007). Thus, FPR1 aberrantly expressed in GBM cells is capable of exploiting the function of EGFR to exacerbate the malignant behavior of the tumor cells. Since interference with both receptors additionally reduced tumor growth, FPR1 and EGFR also had non-redundant functions (Huang et al., 2008).

#### **2.3 The involvement of FPR1 in GBM cell invasion**

The ability of GBM cells to invade into surrounding brain tissue is a critical pathological event in the progression of GBM. In the human GBM cell line U87, there are FPR1+ and FPR1- subpopulations which could be isolated and cloned. FPR1+ cells showed a more "motile" phenotype in vitro as compared with cells lacking FPR1 expression (Huang et al., 2010). Moreover, although both FPR1+ and FPR- GBM cells implanted subcutaneously into nude mice developed tumors, only tumors formed by FPR1+ cells invaded the surrounding connective tissues. In addition, FPR1- cells transfected with FPR1 showed enhanced mobility in vitro and the in vivo capacity to form more rapidly growing and invasive tumors in mice. Tumor invasion depends not only on tumor cell mobility, but also on the capacity of tumor cells to secrete metal matrimetalloproteases (MMPs) that degrade extracellular matrix (ECM) and facilitate the detachment of highly motive tumor cells. Stimulation of GBM cells with FPR1 agonist peptide up-regulates the expression of MMP2 and MMP9 and increases the release of pro-MMP2. As reported in the literature, regulation of MMPs is controlled by AP1 transcription factor complex through MAP kinase pathways

Since both STAT3 and HIF-1α are implicated in the transcriptional activation of the gene coding for VEGF, we investigated the effect of activating FPR1 on the production of VEGF by tumor cells. We found that supernatants from fMLF-stimulated GBM cells induced the migration and tubule formation of human vascular endothelial cells (EC) (Zhou et al., 2005). This property of the tumor cell supernatant was abolished by a neutralizing anti-human VEGF antibody (Zhou et al., 2005), suggesting VEGF was released by FPR1 agoniststimulated GBM cells. FPR1 in GBM cells was subsequently shown to promote the release of another angiogenic factor, the chemokine CXCL8 (IL-8) (Yao et al., 2008). The contribution of FPR1 to GBM progression was then tested in vivo in nude mice. Tumor cells containing small interference (si) RNA targeting FPR1 mRNA yielded tumors in nude mice with markedly reduced rate of growth as compared to control cells transfected with random siRNA (Zhou et al., 2005). Thus, the functional studies provide strong evidence for the

Crosstalk between GPCRs and growth factor receptors plays an important role in orchestrating the interaction of intracellular signaling molecules implicated in tumor growth, angiogenesis and metastasis (Lappano and Maggiolini, 2011). The crosstalk between GPCRs and the receptor for epidermal growth factor (EGFR) has been shown to promote the progression of colon, lung, breast, ovarian, prostate, and head and neck carcinomas (Hart et al., 2005). Like many malignant tumors of human and mouse origin, human GBM cells express high levels of EGFR and stimulation with EGF increases tumor cell chemotaxis and proliferation with rapid phosphorylation of at least 4 tyrosine residues in the C-terminal domain of EGFR (Huang et al., 2007). When GBM cells were stimulated with the FPR1 agonist fMLF, EGFR was also rapidly phosphorylated but with restriction to a single tyrosine residue Tyr992. This transactivation of EGFR by FPR1 agonist peptide accounted for approximately 40% of the biological activity of FPR1 in GBM cells and was dependent on a Src kinase pathway (Huang et al., 2007). Moreover, GBM cells depleted of either FPR1 or EGFR grew more slowly as compared with parental cells and depletion of both receptors further reduced the tumorigenicity of the GBM cells (Huang et al., 2007). Thus, FPR1 aberrantly expressed in GBM cells is capable of exploiting the function of EGFR to exacerbate the malignant behavior of the tumor cells. Since interference with both receptors additionally reduced tumor growth, FPR1

The ability of GBM cells to invade into surrounding brain tissue is a critical pathological event in the progression of GBM. In the human GBM cell line U87, there are FPR1+ and FPR1- subpopulations which could be isolated and cloned. FPR1+ cells showed a more "motile" phenotype in vitro as compared with cells lacking FPR1 expression (Huang et al., 2010). Moreover, although both FPR1+ and FPR- GBM cells implanted subcutaneously into nude mice developed tumors, only tumors formed by FPR1+ cells invaded the surrounding connective tissues. In addition, FPR1- cells transfected with FPR1 showed enhanced mobility in vitro and the in vivo capacity to form more rapidly growing and invasive tumors in mice. Tumor invasion depends not only on tumor cell mobility, but also on the capacity of tumor cells to secrete metal matrimetalloproteases (MMPs) that degrade extracellular matrix (ECM) and facilitate the detachment of highly motive tumor cells. Stimulation of GBM cells with FPR1 agonist peptide up-regulates the expression of MMP2 and MMP9 and increases the release of pro-MMP2. As reported in the literature, regulation of MMPs is controlled by AP1 transcription factor complex through MAP kinase pathways

involvement of FPR1 in supporting the rapid progression of GBM.

and EGFR also had non-redundant functions (Huang et al., 2008).

**2.3 The involvement of FPR1 in GBM cell invasion** 

and PKC, which are activated by FPR1 agonist in GBM cells. Thus stimulation of FPR1 activates MMPs in GBM cells and increases proteolytic processes in the tumor microenvironment (Huang et al., 2010).

#### **2.4 Identification of endogenous FPR1 agonist released by necrotic GBM cells**

Despite extensive characterization of FPR1 function in GBM cells, whether host-derived agonists are present in the tumor microenvironment remains unknown. We tested GBM cell responses to the neutrophil granule protein cathepsin G, which is an endogenous agonist for FPR1 and induces the migration of myloid cells. We determinated that cathepsin G is capable of inducing the migration of GBM cells expressing FPR1 (Sun et al., 2004). However, cathepsin G is unlikely to be present in brain unless substantial tissue damage compromises the blood brain barrier and results in the release of this FPR1 agonist into the brain by neutrophils. We therefore examined other possible sources of potential FPR1 agonists that may act on GBM cells. Since mitochondrial peptides are also potential endogenous FPR1 agonists and GBMs frequently contain necrotic foci in the rapidly growing tumor mass that may release mitochondrial components, we examined the presence of FPR1 agonists in supernatants of necrotic tumor cells. Indeed, supernatants of necrotic GBM cells and tumors formed by GBM cells in nude mice induced potent chemotaxis of live GBM cells as well as a rat basophil leukemia-cell line transfected to express human FPR1 (ETFR cells). The chemotactic activity released by necrotic GBM cells and tumor tissues was blocked by an anti-FPR1 antibody and by a FPR-specific antagonist tBoc-MLF (Zhou et al., 2005). The robust intracellular Ca2+ mobilization induced in GBM cells by necrotic GBM cell supernatant attenuated the subsequent cell response to fMLF, suggesting that agonist contained in the supernatants of necrotic tumor cells share a receptor with fMLF (Zhou et al., 2005). Further evidence to support the release of FPR1 agonists by necrotic GBM cells was provided by the observation that the tumor cell supernatant down-regulated FPR1 expressed on the surface of human monocytes and FPR1 expressing ETFR cells. These observations confirm that FPR1 expressed on GBM cells is able to recognize agonist activity released in the tumor microenvironment in a paracrine and/or autocrine loop (Zhou et al., 2005). Our recent effort to characterize the biochemical nature of the FPR1 agonist activity released by necrotic GBM cells revealed that the glucocorticoid binding protein annexin1 (AnxA1), which has been reported to be an agonist for FPR1 and its variant receptor FPR2, can promote tumor cell invasion and angiogenesis. AnxA1 accounts for the majority of the FPR1 agonist activity released by necrotic GBM cells because depletion of AnxA1 from the necrotic tumor supernatant markedly reduced its capacity to stimulate FPR1 on viable GBM cells (Yang et al., unpublished observation). We therefore established a paradigm for the role of FPR1 in GBM progression in which FPR1 in GBM cells by responding to necrotic tumor cell-released agonist such as AnxA1 transactivates EGFR and the two receptors cooperate to promote the growth, invasion, angiogenesis and progression of GBM (Fig 1A).

#### **3. The role of the chemokine GPCR CXCR4 in glioma progression**

#### **3.1 CXCR4 and its ligand CXCL12**

CXCR4 selectively binds the CXC chemokine stromal cell-derived factor 1 (SDF-1), also known as CXCL12 (Furusato et al., 2010). CXCR4 is normally expressed in a wide variety of cells and tissues. The CXCR4 agonist CXCL12 was first cloned from a murine bone marrow stromal cell line, and was produced in high quantity by marrow stromal cells. In addition to

The Role of Chemoattractant Receptors in the Progression of Glioma 291

activation of CXCR4 promotes the proliferation of GBM cell lines based on the activation of ERK1/2 and PI3K/Akt (Bian et al., 2007). In agreement with data obtained from GBM cell lines, 80% of clinical GBM samples express high levels of phosphorylated Akt (Hambardzumyan et al., 2008). CXCL12 induces the proliferation of primary GBM cells expressing CXCR4 by significantly increasing DNA synthesis in tumor cells (do Carmo et al., 2010). CXCR4-mediated tumor cell proliferation may also be amplified by EGFR signaling, since stimulation of CXCR4 has been reported to transactivate EGFR in many tumors of the epithelial lineage (Dolce et al., 2011). In fact, as discussed earlier, EGFR in GBM cells is transactivated by another chemoattractant GPCR FPR1, and the two receptors co-operate to promote the growth of GBM (Huang et al., 2007). The role of CXCR4 in promoting glioma growth was further supported by the use of a small molecule CXCR4 antagonist, AMD3100, which significantly inhibited tumor cell proliferation in vitro and

Another important property of CXCR4 is to increase GBM cell resistance to apoptosis. Blockade of CXCR4 in glioma cells by the antagonist AMD3100 increased the rate of apoptosis, confirming the ability of CXCR4 to support tumor cell survival (do Carmo et al., 2010). This anti-apoptotic effect is associated with the activation of PI3K/Akt (do Carmo et al., 2010), an observation consistent with results obtained from a variety of tumors in which CXCR4 actively contributes to the resistance of tumor cells to apoptosis. Stimulation of CXCR4 activates NF-κB, which in turn inhibits radiation-induced TNF-α production by glioma cells and increases tumor cell survival. In addition to directly protecting tumor cells from radiation-induced apoptosis, CXCR4 indirectly promotes cell survival by increasing their adherence. For example, stimulation of CXCR4 promotes the adhesion of glioma cells to vitronectin, a glioma-derived extracellular matrix protein, and prevents tumor cell death (do Carmo et al., 2010). Taken together, published results support the conclusion that CXCR4 plays an important role in promoting the proliferation and survival of glioma cells.

tumorigenicity in nude mice (do Carmo et al., 2010; and Dolce et al., 2011).

**3.3.2 CXCR4 promotes the production of angiogenic factors by glioma cells** 

ECs are associated with increased cell survival (Salmaggi et al., 2004).

The requirement of CXCR4 and CXCL12 for angiogenesis was revealed by the prenatal lethal phenotype of both CXCR4 and CXCL12 knockout mice due to defects in the vascular development of gastrointestinal tract and cardiogenesis (Tachibana et al., 1998). In vitro, activation of CXCR4 in ECs stimulates the formation of capillary-like tubules (Salvatore et al., 2010). ECs in gliomas have been shown to be genetically and functionally distinct from normal ECs, and exhibit higher expression of CXCR4 and its ligand CXCL12. Proliferating ECs in GBM are positive for CXCR4 and its ligand CXCL12, while ECs that form a single layer in the capillaries of the anaplastic astrocytoma appeared to be negative for these two molecules. The lower levels of CXCR4/CXCL12 expression in anaplastic astrocytoma may contribute to the lower density of proliferating microvasculature. Consistent with these observations, CXCR4 and CXCL12 are detected in both malignant glioma cells and vascular

Interestingly, elevated CXCL12 levels by themselves in gliomas failed to induce significant vascularization. This was associated with the co-presence of low levels of VEGF, suggesting synergism of these angiogenic factors (Kryczek et al., 2005). In fact, although a major angiogenic factor in GBM, VEGF was detected only in a few cells or not at all in low-grade astrocytomas or in the normal brain tissue (Takano et al., 2010). Clinical and experimental evidence indicates that CXCR4 activation induces the production of VEGF in human glioma

mediating cell chemotaxis in response to CXCL12, CXCR4 also acts as a co-receptor for CD4 cell entry of T tropic HIV. In mouse models, deletion of CXCL12 or CXCR4 results in embryonic death with defects in the development of cardiac and central nervous systems as well as reduction in hematopoietic stem-cell homing (Zou et al., 1998). CXCR4 is upregulated in more than 20 different types of malignant tumors (Kryczek et al., 2007). Further studies show that CXCR4 regulates tumor progression by mediating tumor cell proliferation and metastasis as well as angiogenesis.

#### **3.2 The effect of CXCR4 on glioma invasion and metastasis**

CXCR4 expression was detected in primary human glioma specimens and the level of CXCR4 was correlated with the degree of malignancy of the tumors. In vitro, CXCR4+ malignant glioma cells secrete its ligand CXCL12, suggesting that two molecules may exert paracrine and autocrine regulation of glioma progression (Bajetto et al., 2006). Studies performed in human GBM specimens demonstrated that tumor cells infiltrating into surrounding brain tissues express higher levels of CXCR4, suggesting CXCR4 expression may define more highly invasive tumor cells. This is corroborated by in vitro experiments showing that invasive human glioma cells overexpress CXCR4 as compared with noninvasive tumor cells (Ehtesham et al., 2006). Invasive cells isolated from rat C6 glioma cell line express both CXCR4 and CXCL12 at high levels (Ehtesham et al., 2006). Moreover, application of CXCR4 antagonist or siRNA targeting CXCR4 in vivo inhibited the invasion of tumors formed by invasive C6 glioma cells.

The invasion process of GBM requires the detachment of invading cells from tumor mass, attachment of tumor cells to ECM components, ECM degradation, and subsequent cell infiltration into surrounding brain tissues. Attachment of tumor cells to ECM components is an essential phase of invasion mediated by integrins that are overexpressed on both glioma cells and tumor vasculature. Recognition of CXCL12 by CXCR4 activates tumor-associated integrins, such as α2, α4, α5, and β1 to promote tumor dissemination (Hartman et al., 2004, and 2005). Inhibition of integrin function disrupts GBM cell migration. In vitro, interference of CXCR4 with the urokinase-receptor (uPAR) reduces the adhesion of CXCL12-mediated CXCR4+ GBM cells to collagen, the main component of ECM (Montuori et al., 2010).

ECM degradation by MMPs enhances tumor invasion. In vitro, glioma cells with lower production of MMP-9 show diminished migration and invasion and such cells no longer form tumors following intracranial injection into nude mice. MMP-2 and -9 have been identified as MMPs in high grade gliomas and their level of expression directly correlates with the grade of glioma malignancy (Stojic et al., 2008). Similar to FPR1, CXCR4 mediated glioma invasion in vivo was also associated with its capacity to activate MMPs (Kryczek et al., 2007). It has been reported that CXCR4/ERK/NF-κ B signaling pathway induces the upregulation of MMPs in glioma cells. Activation of CXCR4 by its ligand CXCL12 also promotes tumor invasion by release of MMP-9.

#### **3.3 CXCR4 in glioma growth and angiogenesis**

#### **3.3.1 Role of CXCR4/CXCL12 in malignant glioma growth and survival**

The CXCR4 ligand CXCL12 produced by tumor and stromal cells interacting with CXCR4 on tumor cells results in the activation of several downstream pathways, including MAPK/ERK1/2, PI3k and Akt, as well as NF-κB. These pathways are known to participate in the regulation of cell proliferation and survival in normal or malignant glial cells. In vitro

mediating cell chemotaxis in response to CXCL12, CXCR4 also acts as a co-receptor for CD4 cell entry of T tropic HIV. In mouse models, deletion of CXCL12 or CXCR4 results in embryonic death with defects in the development of cardiac and central nervous systems as well as reduction in hematopoietic stem-cell homing (Zou et al., 1998). CXCR4 is upregulated in more than 20 different types of malignant tumors (Kryczek et al., 2007). Further studies show that CXCR4 regulates tumor progression by mediating tumor cell proliferation

CXCR4 expression was detected in primary human glioma specimens and the level of CXCR4 was correlated with the degree of malignancy of the tumors. In vitro, CXCR4+ malignant glioma cells secrete its ligand CXCL12, suggesting that two molecules may exert paracrine and autocrine regulation of glioma progression (Bajetto et al., 2006). Studies performed in human GBM specimens demonstrated that tumor cells infiltrating into surrounding brain tissues express higher levels of CXCR4, suggesting CXCR4 expression may define more highly invasive tumor cells. This is corroborated by in vitro experiments showing that invasive human glioma cells overexpress CXCR4 as compared with noninvasive tumor cells (Ehtesham et al., 2006). Invasive cells isolated from rat C6 glioma cell line express both CXCR4 and CXCL12 at high levels (Ehtesham et al., 2006). Moreover, application of CXCR4 antagonist or siRNA targeting CXCR4 in vivo inhibited the invasion

The invasion process of GBM requires the detachment of invading cells from tumor mass, attachment of tumor cells to ECM components, ECM degradation, and subsequent cell infiltration into surrounding brain tissues. Attachment of tumor cells to ECM components is an essential phase of invasion mediated by integrins that are overexpressed on both glioma cells and tumor vasculature. Recognition of CXCL12 by CXCR4 activates tumor-associated integrins, such as α2, α4, α5, and β1 to promote tumor dissemination (Hartman et al., 2004, and 2005). Inhibition of integrin function disrupts GBM cell migration. In vitro, interference of CXCR4 with the urokinase-receptor (uPAR) reduces the adhesion of CXCL12-mediated

CXCR4+ GBM cells to collagen, the main component of ECM (Montuori et al., 2010).

**3.3.1 Role of CXCR4/CXCL12 in malignant glioma growth and survival** 

ECM degradation by MMPs enhances tumor invasion. In vitro, glioma cells with lower production of MMP-9 show diminished migration and invasion and such cells no longer form tumors following intracranial injection into nude mice. MMP-2 and -9 have been identified as MMPs in high grade gliomas and their level of expression directly correlates with the grade of glioma malignancy (Stojic et al., 2008). Similar to FPR1, CXCR4 mediated glioma invasion in vivo was also associated with its capacity to activate MMPs (Kryczek et al., 2007). It has been reported that CXCR4/ERK/NF-κ B signaling pathway induces the upregulation of MMPs in glioma cells. Activation of CXCR4 by its ligand CXCL12 also

The CXCR4 ligand CXCL12 produced by tumor and stromal cells interacting with CXCR4 on tumor cells results in the activation of several downstream pathways, including MAPK/ERK1/2, PI3k and Akt, as well as NF-κB. These pathways are known to participate in the regulation of cell proliferation and survival in normal or malignant glial cells. In vitro

and metastasis as well as angiogenesis.

of tumors formed by invasive C6 glioma cells.

promotes tumor invasion by release of MMP-9.

**3.3 CXCR4 in glioma growth and angiogenesis** 

**3.2 The effect of CXCR4 on glioma invasion and metastasis** 

activation of CXCR4 promotes the proliferation of GBM cell lines based on the activation of ERK1/2 and PI3K/Akt (Bian et al., 2007). In agreement with data obtained from GBM cell lines, 80% of clinical GBM samples express high levels of phosphorylated Akt (Hambardzumyan et al., 2008). CXCL12 induces the proliferation of primary GBM cells expressing CXCR4 by significantly increasing DNA synthesis in tumor cells (do Carmo et al., 2010). CXCR4-mediated tumor cell proliferation may also be amplified by EGFR signaling, since stimulation of CXCR4 has been reported to transactivate EGFR in many tumors of the epithelial lineage (Dolce et al., 2011). In fact, as discussed earlier, EGFR in GBM cells is transactivated by another chemoattractant GPCR FPR1, and the two receptors co-operate to promote the growth of GBM (Huang et al., 2007). The role of CXCR4 in promoting glioma growth was further supported by the use of a small molecule CXCR4 antagonist, AMD3100, which significantly inhibited tumor cell proliferation in vitro and tumorigenicity in nude mice (do Carmo et al., 2010; and Dolce et al., 2011).

Another important property of CXCR4 is to increase GBM cell resistance to apoptosis. Blockade of CXCR4 in glioma cells by the antagonist AMD3100 increased the rate of apoptosis, confirming the ability of CXCR4 to support tumor cell survival (do Carmo et al., 2010). This anti-apoptotic effect is associated with the activation of PI3K/Akt (do Carmo et al., 2010), an observation consistent with results obtained from a variety of tumors in which CXCR4 actively contributes to the resistance of tumor cells to apoptosis. Stimulation of CXCR4 activates NF-κB, which in turn inhibits radiation-induced TNF-α production by glioma cells and increases tumor cell survival. In addition to directly protecting tumor cells from radiation-induced apoptosis, CXCR4 indirectly promotes cell survival by increasing their adherence. For example, stimulation of CXCR4 promotes the adhesion of glioma cells to vitronectin, a glioma-derived extracellular matrix protein, and prevents tumor cell death (do Carmo et al., 2010). Taken together, published results support the conclusion that CXCR4 plays an important role in promoting the proliferation and survival of glioma cells.

#### **3.3.2 CXCR4 promotes the production of angiogenic factors by glioma cells**

The requirement of CXCR4 and CXCL12 for angiogenesis was revealed by the prenatal lethal phenotype of both CXCR4 and CXCL12 knockout mice due to defects in the vascular development of gastrointestinal tract and cardiogenesis (Tachibana et al., 1998). In vitro, activation of CXCR4 in ECs stimulates the formation of capillary-like tubules (Salvatore et al., 2010). ECs in gliomas have been shown to be genetically and functionally distinct from normal ECs, and exhibit higher expression of CXCR4 and its ligand CXCL12. Proliferating ECs in GBM are positive for CXCR4 and its ligand CXCL12, while ECs that form a single layer in the capillaries of the anaplastic astrocytoma appeared to be negative for these two molecules. The lower levels of CXCR4/CXCL12 expression in anaplastic astrocytoma may contribute to the lower density of proliferating microvasculature. Consistent with these observations, CXCR4 and CXCL12 are detected in both malignant glioma cells and vascular ECs are associated with increased cell survival (Salmaggi et al., 2004).

Interestingly, elevated CXCL12 levels by themselves in gliomas failed to induce significant vascularization. This was associated with the co-presence of low levels of VEGF, suggesting synergism of these angiogenic factors (Kryczek et al., 2005). In fact, although a major angiogenic factor in GBM, VEGF was detected only in a few cells or not at all in low-grade astrocytomas or in the normal brain tissue (Takano et al., 2010). Clinical and experimental evidence indicates that CXCR4 activation induces the production of VEGF in human glioma

The Role of Chemoattractant Receptors in the Progression of Glioma 293

CXCL12 and for another chemokine e.g. interferon-inducible T cell α chemoattractant (I-TAC; also known as CXCL11). CXCR7 is expressed in several tumors and plays an important role in preventing tumor cell apoptosis and promoting tumor cell adhesion to ECs, a key step for the development of blood-borne metastasis (Burns et al., 2006). In glioma specimens, CXCR7 is widely distributed in tumor cells, microglia and ECs. In contrast, CXCR4 seems to be restricted to certain subsets of glioma cells and tumor stem-like cell populations. While the CXCR4 level is significantly higher in GBM than in lower grade gliomas, no distribution difference was detected for CXCR7 (Hattermann et al., 2010). One study reported that in eight glioma cell lines tested, only one expresses CXCR4. However, CXCR7 is highly expressed in all glioma cell lines (Hattermann et al., 2010). Interestingly, tumor stem-like cells derived from GBM cell line express CXCR4, but not CXCR7. In addition, differentiated glioma cells often are found to express CXCR7, but not CXCR4. These observations suggest that there is a difference between the role of CXCR4 and CXCR7 in the function of glioma cells. In some tumors, CXCR7 and CXCL12 are co-localized and

Initially, CXCR7 was regarded as a decoy receptor that recognizes CXCL12 or a coreceptor that may form a heterodimeric complex with CXCR4 to enhance CXCL12 signaling in embryonic cells. Subsequently, CXCR7 was demonstrated to be functionally active in glioma cells. CXCR7 activation by CXCL12 stimulates a transient phosphorylation of ERK1/2 and inhibits the apoptosis of glioma cells induced by camptothecin and temozolomide, but did not increase tumor cell proliferation and migration (Hattermann et al., 2010). CXCR7 activation also did not elicit calcium mobilization in tumor cells, but increases their adhesion (Burns et al., 2006). The absence of ligand-induced calcium influx and cell migration distinguishes the CXCR7 signaling pathway from CXCR4 and other typical chemokine GPCRs. In cells transiently transfected with human CXCR7 and rat cells expressing CXCR7, the signaling of CXCR7 is not mediated by Gαi protein, but by β-arrestins associated with the phosphorylation of MAP kinases (Rajagopal et al., 2010). Based on these properties of CXCR7, it is assumed that some of the previously reported effects of CXCL12 on glioma cells, such as phosphorylation of kinases and prevention of apoptosis might be partially mediated by CXCR7. Since ECs isolated from GBM express high levels of CXCR7 mRNA, it is postulated that CXCR7 may be involved in the formation of glioma vasculature (Takano et al., 2010). Indeed, in many CXCR7+ tumors, VEGF and CXCL8 (IL-8) are up-regulated. Therefore, CXCR7 is a novel chemokine GPCR that promotes glioma progression by

Accumulating evidence suggests that CXCR7 and CXCR4 interact with each other in malignant tumors. In human rhabdomyosarcomas (Grymula et al., 2010), downregulation of CXCR7 expression by hypoxia was thought to increase CXCL12 signaling through CXCR4 thus rendered rhabdomyosarcoma cells more motile and prone to detach from the primary tumor. Confocal microscopy shows that in glioma cell lines, CXCR7 is mainly localized in the space between the plasma membrane and endosomal compartment, whereas CXCR4 is mostly present on the cell surface of membrane (Calatozzolo et al., 2011). The biological significance of the distinct pattern of CXCR7 and CXCR4 expression in glioma cells is not clear. However, in somatic cells, CXCR7 facilitates CXCR4-mediated migration of

potentially cooperate in tumor progression (Hattermann et al., 2010).

supporting tumor cell survival, adhesion and possibly vessel formation.

**4.3 Potential interactions between CXCR7 and CXCR4** 

**4.2 CXCR7 may mediate glioma progression** 

cells and glioma stem-like cells (Ping et al., 2007 and 2011). Therefore, CXCR4 may contribute to the production of VEGF by malignant glioma cells and the two pro-angiogenic factors synergistically promote angiogenesis in tumor. In addition to VEGF, the activation of CXCR4 in gliomas also is associated with increases the secretion of an angiogenic chemokine, CXCL8 (IL-8) (Ping et al., 2007). Interestingly, VEGF binds the receptors on ECs and leads to the up-regulation of the anti-apoptotic molecule Bcl-2 as well as the release of CXCL8 from ECs (Nör et al., 2001). CXCL8 then is capable of maintaining the angiogenic phenotype of ECs in an autocrine and paracrine manner (Nör et al., 2001; Heidemann et al., 2003). In addition, the activation of CXCR4 also results in NF-κB translocation in glioma cells, which elicits the production of other angiogenic chemokines, such as CXCL1, CXCL2, and CXCL5 (Richmond et al., 2002). Therefore, glioma angiogenesis is the result of a wellcoordinated process participated in by multiple angiogenic factors among which CXCR4 appears to be an upstream initiator.

#### **3.3.3 CXCR4/CXCL12 mediates vasculogenesis by mobilizing bone marrow derived progenitor cells**

In addition to tumor angiogenesis, which is thought to be established by the sprouting of blood vessels through the division of normal differentiated host ECs present in the tissue adjacent to tumor, another way to generate tumor vessels is through the process of vasculogenesis, which is formed by the recruitment of circulating EC precursor cells or bone marrow-derived cells (BMDCs) (Garcia-Barros et al., 2003). Circulating EC progenitor cells mobilized from the bone marrow are normally present in the peripheral blood of several species and participate in the neovascularization in tumor and in ischemic tissues (Spaeth et al., 2009). CXCR4 has been demonstrated to guide prime stem cells to the sites of rapid vascular expansion during embryonic organogenesis (Napoli et al., 2010). The pivotal role of CXCR4 and its ligand CXCL12 in vasculogenesis has been demonstrated in gene deletion mice as discussed earlier.

Similar to the development of embryonic vessels, CXCR4 mediates tumor neovascularization by switching from angiogenesis in the recurrent malignant glioma to vasculogenesis. For instance, tumor growth supported mainly by angiogenesis from nearby normal vessels is abrogated by irradiation (Kioi et al., 2010). As a consequence, the growth of new tumor vasculature in irradiation animals will rely mainly on circulating blood EC progenitor cells from the bone marrow. Studies have demonstrated that CXCR4 is a key factor for the influx of BMDCs into the recurrent tumor after irradiation, since both the CXCR4 inhibitor AMD3100 and antibodies against CXCR4 are able to block the recruitment of BMDCs into tumor and prevent the restoration of the vasculature (Kioi et al., 2010). Hypoxia also mediates tumor vasculogenensis through CXCR4 in animal models. Irradiation results in a hypoxic microenvironment in the tumor resulting in the upregulation of the transcription factor HIF-1 (Ahn and Brown, 2008) and enhanced production of CXCL12 and VEGF. CXCL12 then induces the homing of CD11b+ BMDCs into the tumor site to initiate the formation of new vasculature (Kioi et al., 2010).

#### **4. CXCR7/CXCL12**

#### **4.1 CXCR7 expression in glioma**

Although it was believed that CXCL12 uses CXCR4 as a sole receptor, recent studies have shown that CXCR7, a newly identified chemokine GPCR, acts as an alternate receptor for

cells and glioma stem-like cells (Ping et al., 2007 and 2011). Therefore, CXCR4 may contribute to the production of VEGF by malignant glioma cells and the two pro-angiogenic factors synergistically promote angiogenesis in tumor. In addition to VEGF, the activation of CXCR4 in gliomas also is associated with increases the secretion of an angiogenic chemokine, CXCL8 (IL-8) (Ping et al., 2007). Interestingly, VEGF binds the receptors on ECs and leads to the up-regulation of the anti-apoptotic molecule Bcl-2 as well as the release of CXCL8 from ECs (Nör et al., 2001). CXCL8 then is capable of maintaining the angiogenic phenotype of ECs in an autocrine and paracrine manner (Nör et al., 2001; Heidemann et al., 2003). In addition, the activation of CXCR4 also results in NF-κB translocation in glioma cells, which elicits the production of other angiogenic chemokines, such as CXCL1, CXCL2, and CXCL5 (Richmond et al., 2002). Therefore, glioma angiogenesis is the result of a wellcoordinated process participated in by multiple angiogenic factors among which CXCR4

**3.3.3 CXCR4/CXCL12 mediates vasculogenesis by mobilizing bone marrow derived** 

In addition to tumor angiogenesis, which is thought to be established by the sprouting of blood vessels through the division of normal differentiated host ECs present in the tissue adjacent to tumor, another way to generate tumor vessels is through the process of vasculogenesis, which is formed by the recruitment of circulating EC precursor cells or bone marrow-derived cells (BMDCs) (Garcia-Barros et al., 2003). Circulating EC progenitor cells mobilized from the bone marrow are normally present in the peripheral blood of several species and participate in the neovascularization in tumor and in ischemic tissues (Spaeth et al., 2009). CXCR4 has been demonstrated to guide prime stem cells to the sites of rapid vascular expansion during embryonic organogenesis (Napoli et al., 2010). The pivotal role of CXCR4 and its ligand CXCL12 in vasculogenesis has been demonstrated in gene deletion

Similar to the development of embryonic vessels, CXCR4 mediates tumor neovascularization by switching from angiogenesis in the recurrent malignant glioma to vasculogenesis. For instance, tumor growth supported mainly by angiogenesis from nearby normal vessels is abrogated by irradiation (Kioi et al., 2010). As a consequence, the growth of new tumor vasculature in irradiation animals will rely mainly on circulating blood EC progenitor cells from the bone marrow. Studies have demonstrated that CXCR4 is a key factor for the influx of BMDCs into the recurrent tumor after irradiation, since both the CXCR4 inhibitor AMD3100 and antibodies against CXCR4 are able to block the recruitment of BMDCs into tumor and prevent the restoration of the vasculature (Kioi et al., 2010). Hypoxia also mediates tumor vasculogenensis through CXCR4 in animal models. Irradiation results in a hypoxic microenvironment in the tumor resulting in the upregulation of the transcription factor HIF-1 (Ahn and Brown, 2008) and enhanced production of CXCL12 and VEGF. CXCL12 then induces the homing of CD11b+ BMDCs

into the tumor site to initiate the formation of new vasculature (Kioi et al., 2010).

Although it was believed that CXCL12 uses CXCR4 as a sole receptor, recent studies have shown that CXCR7, a newly identified chemokine GPCR, acts as an alternate receptor for

appears to be an upstream initiator.

**progenitor cells** 

mice as discussed earlier.

**4. CXCR7/CXCL12** 

**4.1 CXCR7 expression in glioma** 

CXCL12 and for another chemokine e.g. interferon-inducible T cell α chemoattractant (I-TAC; also known as CXCL11). CXCR7 is expressed in several tumors and plays an important role in preventing tumor cell apoptosis and promoting tumor cell adhesion to ECs, a key step for the development of blood-borne metastasis (Burns et al., 2006). In glioma specimens, CXCR7 is widely distributed in tumor cells, microglia and ECs. In contrast, CXCR4 seems to be restricted to certain subsets of glioma cells and tumor stem-like cell populations. While the CXCR4 level is significantly higher in GBM than in lower grade gliomas, no distribution difference was detected for CXCR7 (Hattermann et al., 2010). One study reported that in eight glioma cell lines tested, only one expresses CXCR4. However, CXCR7 is highly expressed in all glioma cell lines (Hattermann et al., 2010). Interestingly, tumor stem-like cells derived from GBM cell line express CXCR4, but not CXCR7. In addition, differentiated glioma cells often are found to express CXCR7, but not CXCR4. These observations suggest that there is a difference between the role of CXCR4 and CXCR7 in the function of glioma cells. In some tumors, CXCR7 and CXCL12 are co-localized and potentially cooperate in tumor progression (Hattermann et al., 2010).

#### **4.2 CXCR7 may mediate glioma progression**

Initially, CXCR7 was regarded as a decoy receptor that recognizes CXCL12 or a coreceptor that may form a heterodimeric complex with CXCR4 to enhance CXCL12 signaling in embryonic cells. Subsequently, CXCR7 was demonstrated to be functionally active in glioma cells. CXCR7 activation by CXCL12 stimulates a transient phosphorylation of ERK1/2 and inhibits the apoptosis of glioma cells induced by camptothecin and temozolomide, but did not increase tumor cell proliferation and migration (Hattermann et al., 2010). CXCR7 activation also did not elicit calcium mobilization in tumor cells, but increases their adhesion (Burns et al., 2006). The absence of ligand-induced calcium influx and cell migration distinguishes the CXCR7 signaling pathway from CXCR4 and other typical chemokine GPCRs. In cells transiently transfected with human CXCR7 and rat cells expressing CXCR7, the signaling of CXCR7 is not mediated by Gαi protein, but by β-arrestins associated with the phosphorylation of MAP kinases (Rajagopal et al., 2010). Based on these properties of CXCR7, it is assumed that some of the previously reported effects of CXCL12 on glioma cells, such as phosphorylation of kinases and prevention of apoptosis might be partially mediated by CXCR7. Since ECs isolated from GBM express high levels of CXCR7 mRNA, it is postulated that CXCR7 may be involved in the formation of glioma vasculature (Takano et al., 2010). Indeed, in many CXCR7+ tumors, VEGF and CXCL8 (IL-8) are up-regulated. Therefore, CXCR7 is a novel chemokine GPCR that promotes glioma progression by supporting tumor cell survival, adhesion and possibly vessel formation.

#### **4.3 Potential interactions between CXCR7 and CXCR4**

Accumulating evidence suggests that CXCR7 and CXCR4 interact with each other in malignant tumors. In human rhabdomyosarcomas (Grymula et al., 2010), downregulation of CXCR7 expression by hypoxia was thought to increase CXCL12 signaling through CXCR4 thus rendered rhabdomyosarcoma cells more motile and prone to detach from the primary tumor. Confocal microscopy shows that in glioma cell lines, CXCR7 is mainly localized in the space between the plasma membrane and endosomal compartment, whereas CXCR4 is mostly present on the cell surface of membrane (Calatozzolo et al., 2011). The biological significance of the distinct pattern of CXCR7 and CXCR4 expression in glioma cells is not clear. However, in somatic cells, CXCR7 facilitates CXCR4-mediated migration of

The Role of Chemoattractant Receptors in the Progression of Glioma 295

Since CX3CL1 and CX3CR1 are co-expressed by glioma cells, they are hypothesized to play a role in glioma growth in an autocrine loop. However, the interaction of CX3CR1 with

**VEGF CXCL8**

**CXCR4**

**Integrins MMPs**

**Angiogenesis Invasion Proliferation**

**Membrane anchored CX3CL1**

**CX3CR1+ Microglia/Macrophages**

**Cleaved CX3CL1**

**CXCL12 Stromal cells**

**GBM cells**

**CXCR7**

**PI3K MAPK**

**5.2 The direct effect of CX3CR1 on glioma cells** 

**A B**

**Necrotic GBM cells**

**Src EGFR** <sup>p</sup>

**Invasion**

CX3CL1 that increases the invasiveness of the individual tumor cells.

Fig. 1. The role of chemoattractant GPCRs in glioma progression. A. FPR1 and EGFR cooperate to exacerbate the progression of GBM. FPR1 in GBM cells was activated by agonists released by necrotic tumor cells to promote GBM cell survival, invasion and angiogenesis. The FPR1 function in GBM cells is mediated in part by transactivation of EGFR through a Src kinase pathway. B. Interaction of CXCR4 with CXCL12 produced by glioma cells and stromal cells promotes the proliferation, invasion and angiogenesis of tumor. The activity of CXCL12 may be partially mediated by another CXCL12 receptor CXCR7. C. CX3CL1 secreted by glioma cells increases the infiltration of microglia

/macrophages expressing CX3CR1 and promotes tumor progression. Interaction of CX3CR1 with CX3CL1 produced by glioma cells increases cell-cell adhesion in tumor that inhibits the invasion of tumor cells. However tumor cells activated by CXCR4 ligand CXCL12 cleave

CX3CL1 has been shown to inhibit glioma cell invasion in vitro (Sciumè et al., 2010). This activity of CX3CR1 may be attributed to the peculiar structure of the agonist CX3CL1 and may account for its ability to directly promote cell-cell adhesion when expressed as a

**CX3CR1 CXCL12**

**Invasion inhibition** 

**Tumorigenesis**

**Agonists**

**PKC**

**NF-**κ**B STAT3 HIF-1**α

**VEGF CXCL8**

**CX3CR1**

**Cleaved CX3CL1**

**FPR1**

**PI3K MAPK**

**Invasion Survival Angiogenesis** 

**Bcl-2**

**C Microglia/Macrophages**

**CX3CL1 CX3CL1**

**Glioma cell Glioma cell**

**Glioma cell Glioma cell**

**CX3CR1**

**MMPs**

primordial germ cells by controlling the level of CXCL12 in the microenvironment to form a chemotactic gradient (Boldajipour et al., 2008). In HeLa cells, CXCR7 acts as a scavenger receptor for CXCL12, which results in the internalization of CXCL12 and the subsequent reduction of CXCR4 activity (Naumann et al., 2010). An alternative mechanism by which CXCR7 regulates CXCR4 activity may be its potential to form heterodimer with CXCR4. In fact, some studies have shown changes in CXCR4 signaling by heterodimerization with CXCR7. Although the precise mechanisms of interaction between CXCR7 and CXCR4 and the consequences in glioma progression remain to be determined, the available results suggest an important role for CXCR7 in regulating the activity of the more ubiquitously expressed CXCR4 in gliomas (Fig. 1B).

#### **5. CX3CR1/CX3CL1 in glioma progression**

Another chemoattractant GPCR CX3CR1 and its agonist CX3CL1 have also been reported to play a role in glioma progression. CX3CL1 is one of the most highly expressed chemokines in the brain (Bazan et al., 1997) and is a peculiar member of the chemokine family which can mediate both chemotaxis and adhesion of inflammatory cells via its highly selective receptor CX3CR1. CX3CR1 is overexpressed in gliomas at both mRNA and protein levels, regardless of tumor classification and clinical severity, while CX3CL1 expression is correlated with glioma grade and overall patient survival (Locatelli et al., 2010). CX3CL1 is more highly expressed in tumor area near sites of necrosis suggesting that necrosis may directly enhance CX3CL1 transcription in tumor cells, or indirectly via inflammatory cytokines released by necrotic cells, including TNFα, which is a potent stimulant of CX3CL1 transcription (Marchesi et al., 2010). The increased expression of CX3CL1 in higher grade gliomas implies the involvement of CX3CL1 and its receptor CX3CR1 in tumor progression. CX3CR1 and CX3CL1 contribute to glioma progression in two ways: (1) by affecting the host defense mediated by immune cells and (2) by directly promoting tumor cell proliferation.

#### **5.1 The role of CX3CR1 in immune cell activation in the brain**

In colorectal cancer patients, high expression of CX3CR1 in tumor tissue is correlated with increased density of tumor infiltrating lymphocytes, which is associated with more favorable prognosis (Dimberg et al., 2007). CX3CR1 deficient mice bearing B16 melanoma are reported to show increased lung tumor metastasis and cachexia as well as reduced recruitment of monocytes and NK cells into the tumor (Yu et al., 2007). Thus, CX3CR1 may promote the infiltration of immune cells with antitumor activity.

Glioma-infiltrating microglia/macrophages (GIMs) are the major component in the stroma of glioma tumors and these cells express CX3CR1. In vitro, activation of CX3CR1 in GIMs isolated from human glioma specimens increases these cell adhesion and migration in response to CX3CL1 (Held-Feindt et al., 2010). Blocking CX3CR1 by a specific antibody reduced the migration of GIMs in response to the conditioned medium containing CX3CL1 secreted by human GBM cell lines (Held-Feindt et al., 2010). However, GIMs in glioma stroma did not mediate antiglioma immune responses (Liu et al., 2008). In fact, GIMs are characterized by a phenotype that may potentially promote tumorigenesis, i.e., more likely functioning as type Ⅱ macrophages. Also, CX3CR1 activation increases the expression of MMP2, 9 and 14 in GIMs, which may not only favor the migration and adhesion of GIMs, but also the infiltration of normal brain tissue by tumor cells (Markovic et al., 2005).

#### **5.2 The direct effect of CX3CR1 on glioma cells**

**Glioma cell Glioma cell**

294 Glioma – Exploring Its Biology and Practical Relevance

primordial germ cells by controlling the level of CXCL12 in the microenvironment to form a chemotactic gradient (Boldajipour et al., 2008). In HeLa cells, CXCR7 acts as a scavenger receptor for CXCL12, which results in the internalization of CXCL12 and the subsequent reduction of CXCR4 activity (Naumann et al., 2010). An alternative mechanism by which CXCR7 regulates CXCR4 activity may be its potential to form heterodimer with CXCR4. In fact, some studies have shown changes in CXCR4 signaling by heterodimerization with CXCR7. Although the precise mechanisms of interaction between CXCR7 and CXCR4 and the consequences in glioma progression remain to be determined, the available results suggest an important role for CXCR7 in regulating the activity of the more ubiquitously

Another chemoattractant GPCR CX3CR1 and its agonist CX3CL1 have also been reported to play a role in glioma progression. CX3CL1 is one of the most highly expressed chemokines in the brain (Bazan et al., 1997) and is a peculiar member of the chemokine family which can mediate both chemotaxis and adhesion of inflammatory cells via its highly selective receptor CX3CR1. CX3CR1 is overexpressed in gliomas at both mRNA and protein levels, regardless of tumor classification and clinical severity, while CX3CL1 expression is correlated with glioma grade and overall patient survival (Locatelli et al., 2010). CX3CL1 is more highly expressed in tumor area near sites of necrosis suggesting that necrosis may directly enhance CX3CL1 transcription in tumor cells, or indirectly via inflammatory cytokines released by necrotic cells, including TNFα, which is a potent stimulant of CX3CL1 transcription (Marchesi et al., 2010). The increased expression of CX3CL1 in higher grade gliomas implies the involvement of CX3CL1 and its receptor CX3CR1 in tumor progression. CX3CR1 and CX3CL1 contribute to glioma progression in two ways: (1) by affecting the host defense

mediated by immune cells and (2) by directly promoting tumor cell proliferation.

In colorectal cancer patients, high expression of CX3CR1 in tumor tissue is correlated with increased density of tumor infiltrating lymphocytes, which is associated with more favorable prognosis (Dimberg et al., 2007). CX3CR1 deficient mice bearing B16 melanoma are reported to show increased lung tumor metastasis and cachexia as well as reduced recruitment of monocytes and NK cells into the tumor (Yu et al., 2007). Thus, CX3CR1 may

Glioma-infiltrating microglia/macrophages (GIMs) are the major component in the stroma of glioma tumors and these cells express CX3CR1. In vitro, activation of CX3CR1 in GIMs isolated from human glioma specimens increases these cell adhesion and migration in response to CX3CL1 (Held-Feindt et al., 2010). Blocking CX3CR1 by a specific antibody reduced the migration of GIMs in response to the conditioned medium containing CX3CL1 secreted by human GBM cell lines (Held-Feindt et al., 2010). However, GIMs in glioma stroma did not mediate antiglioma immune responses (Liu et al., 2008). In fact, GIMs are characterized by a phenotype that may potentially promote tumorigenesis, i.e., more likely functioning as type Ⅱ macrophages. Also, CX3CR1 activation increases the expression of MMP2, 9 and 14 in GIMs, which may not only favor the migration and adhesion of GIMs,

but also the infiltration of normal brain tissue by tumor cells (Markovic et al., 2005).

**5.1 The role of CX3CR1 in immune cell activation in the brain** 

promote the infiltration of immune cells with antitumor activity.

expressed CXCR4 in gliomas (Fig. 1B).

**5. CX3CR1/CX3CL1 in glioma progression** 

Since CX3CL1 and CX3CR1 are co-expressed by glioma cells, they are hypothesized to play a role in glioma growth in an autocrine loop. However, the interaction of CX3CR1 with

Fig. 1. The role of chemoattractant GPCRs in glioma progression. A. FPR1 and EGFR cooperate to exacerbate the progression of GBM. FPR1 in GBM cells was activated by agonists released by necrotic tumor cells to promote GBM cell survival, invasion and angiogenesis. The FPR1 function in GBM cells is mediated in part by transactivation of EGFR through a Src kinase pathway. B. Interaction of CXCR4 with CXCL12 produced by glioma cells and stromal cells promotes the proliferation, invasion and angiogenesis of tumor. The activity of CXCL12 may be partially mediated by another CXCL12 receptor CXCR7. C. CX3CL1 secreted by glioma cells increases the infiltration of microglia /macrophages expressing CX3CR1 and promotes tumor progression. Interaction of CX3CR1 with CX3CL1 produced by glioma cells increases cell-cell adhesion in tumor that inhibits the invasion of tumor cells. However tumor cells activated by CXCR4 ligand CXCL12 cleave CX3CL1 that increases the invasiveness of the individual tumor cells.

CX3CL1 has been shown to inhibit glioma cell invasion in vitro (Sciumè et al., 2010). This activity of CX3CR1 may be attributed to the peculiar structure of the agonist CX3CL1 and may account for its ability to directly promote cell-cell adhesion when expressed as a

The Role of Chemoattractant Receptors in the Progression of Glioma 297

xenograft tumors formed by human GBM cells transplanted intracranially into mice, with

Studies have also revealed the potential benefit of a combination of CXCR4 inhibitor with chemotherapy and radiotherapy in malignant glioma patients. In tests on a variety of GBM cell lines, a conventional cytotoxic chemotherapeutic agent, BCNU, in combination with the CXCR4 antagonist AMD3100 exhibits synergistic inhibition of tumor cell growth in vitro. In vivo in animal models, subtherapeutic doses of BCNU and AMD3100 also result in tumor regression, which is attributed to increased tumor cell apoptosis and decreased proliferation (Redjal et al., 2006). These effects of AMD3100 in conjunction with its capacity to reduce the recruitment of bone marrow EPCs to recurrent tumors post irradiation, suggest that targeting CXCR4 may not only directly inhibit tumor cell proliferation, but also indirectly

Considering targeting CXCR4 as a means of inhibiting glioma, the ability of the CXCR4 agonist CXCL12 to activate CXCR7 casts doubts about whether blockage of CXCR4 alone is sufficient without simultaneously inhibiting CXCR7. In fact, inhibition of CXCR4 only partially decreases the responsiveness of tumor cells to CXCL12 in several animal models. Studies have found that GSLCs express high levels of CXCR4 and low levels of CXCR7 (Hattermann et al., 2010). In contrast, differentiation of GSLCs markedly decreased CXCR4 expression but up-regulated CXCR7. It is therefore postulated that CXCR4 may mediate GSLC chemotaxis and survival, whereas differentiated glioma cells are protected from apoptosis by CXCR7 in response to CXCL12. It is therefore important to design strategies that target one or both CXCL12 receptors based on the stages of glioma cell differentiation. Small molecule natural compounds constitute another source of inhibition of chemoattractant GPCRs with therapeutic potential for gliomas (Ping et al., 2007). One of such compounds is Nordy, a chiral mimetic of a natural lipoxygenase inhibitor nordihydroguaiaretic acid. Nordy has been shown to exhibit a broad inhibitory activity on chemoattractant GPCRs such as CXCR4 and FPR1 on GBM cells by downregulating receptor expression, interfering with their signal transduction pathways and reducing tumor cell production of angiogenic factors VEGF and the chemokine CXCL8 (Ping et al., 2007; Chen et al., 2006 and 2007). In addition, Nordy has been found to inhibit GBM cell proliferation and to promote tumor cell differentiation into a lesser malignant phenotype. Recently, Nordy was found to inhibit the self-renewal of glioma stem cells and growth of xenografts generated by the stem cells (Wang et al., 2011). However, the effect of Nordy may not be specific by targeting only chemoattractant GPCRs on GBM cells. Further studies are required to identify more specific receptor targeting natural compounds with minimal side

There is now mounting evidence that chemoattractant GPCRs play multiple roles in the progression of malignant gliomas, by mediating the tumor cell growth, invasion and angiogenesis (Table 1). However, further molecular epidemiologic and genetic studies are required to obtain a better understanding of the mechanisms of the function of these receptors in glioma cells. It is especially important not to single out a given receptor to study glioma biology, but rather, studies should consider the complex host environment in which many factors may drive the aberrant expression of chemoattractant GPCRs and ligands. In addition, the interaction of chemoattractant GPCRs such as CXCR4 and FPR1 with other

increased apoptosis of the transplanted GBM cells (Rubin et al., 2003).

abrogates neovascularization in GBMs (Kioi et al., 2010).

effects on key physiological cell processes.

**8. Conclusions** 

transmembrane protein therefore impeding cell motility. The effect of this CX3CR1 and CX3CL1 interaction was reduced by TGF-β1 (Sciumè et al., 2010), which is also produced by glioma cells and downregulates CX3CL1 expression. The in vivo role of CX3CR1 in glioma growth is more complex. CXCL12 constitutively expressed in the central nervous system (CNS) activates CXCR4 in glioma cells to promote the cleavage of CX3CL1 into a soluble form that reduces the intercellular adhesion and results in the dissemination of glioma cells (Cook et al., 2010). Thus, it is postulated that CX3CR1 in the CNS may favor the invasion of glioma cells into neighboring tissues. In support of this assumption, CX3CR1 and CX3CL1 have been reported to drive the neurotropic cancer cells to disseminate to peripheral nerves (Marchesi et al., 2010), a distinct but largely under appreciated route of metastasis, which has been shown in several tumors, including tumors of the brain, prostate, stomach, pancreas, bladder, and colorectum, as well as head and neck carcinoma. Thus, the balance between the transmembrane and soluble form plays an important role in the activity of CX3CL1 to either prevent or promote glioma progression (Fig. 1C).

#### **6. Involvement of chemoattractant GPCRs in infiltration of gliomas by regulatory T cells (Tregs)**

Tregs have been recognized as one of the major immune cell components that suppress host anti-tumor responses. Recruitment of Tregs into tumors contributes to tolerance by suppressing autoreactive T cells. It has been shown that Tregs infiltrate human brain tumors (Tran Tang et al., 2010) and preferentially accumulate in high grade malignant gliomas such as GBM. The importance of Tregs in the control of anti-tumor immune responses in experimental mouse glioma models is demonstrated by the observation that transient Treg depletion markedly augments the anti-tumor immunity (Tran Tang et al., 2010). Treg trafficking in vivo is facilitated by chemokine receptors. For instance, Treg accumulation in ovarian carcinoma is mediated by the chemokine receptor CCR4, which binds the ligand CCL22 produced in the tumor where specific T cell immunity is compromised (Curiel et al., 2004). Analysis of lymphocyte subsets in GBM from patients shows that tumor infiltrating Tregs highly express CCR4 (Jacobs et al., 2010) and the ligand CCL22 is produced by GBM cells. But unlike ovarian carcinoma in which Treg accumulation clearly correlates with reduced patient survival, there is no correlation between Tregs and overall survival of GBM patients. Regardless, post-surgical immunotherapy has been proposed as a potentially valid method to eliminate residual GBM cells while preserving surrounding healthy brain cells.

#### **7. Chemoattractant GPCRs in gliomas as potential therapeutic targets**

Given the broad range of functions of chemoattractant GPCRs in malignant glioma development, progression, invasion and angiogenesis, blockage of these receptors is considered a novel therapeutic approach in conjunction with conventional surgical resection, irradiation and chemotherapy. Based on the association of CXCR4 with the malignant behavior of glioma, anti-CXCR4 monoclonal antibody and specific low-molecular weight antagonist for CXCR4 have been tested for their effects on tumor cell growth in vitro and in vivo. As predicted, anti-CXCR4 monoclonal antibody is able to attenuate the migration and proliferation of human GBM cells induced by CXCL12 (Cheng et al., 2009). In addition, administration of the CXCR4 antagonist AMD3100 suppressed the growth of

transmembrane protein therefore impeding cell motility. The effect of this CX3CR1 and CX3CL1 interaction was reduced by TGF-β1 (Sciumè et al., 2010), which is also produced by glioma cells and downregulates CX3CL1 expression. The in vivo role of CX3CR1 in glioma growth is more complex. CXCL12 constitutively expressed in the central nervous system (CNS) activates CXCR4 in glioma cells to promote the cleavage of CX3CL1 into a soluble form that reduces the intercellular adhesion and results in the dissemination of glioma cells (Cook et al., 2010). Thus, it is postulated that CX3CR1 in the CNS may favor the invasion of glioma cells into neighboring tissues. In support of this assumption, CX3CR1 and CX3CL1 have been reported to drive the neurotropic cancer cells to disseminate to peripheral nerves (Marchesi et al., 2010), a distinct but largely under appreciated route of metastasis, which has been shown in several tumors, including tumors of the brain, prostate, stomach, pancreas, bladder, and colorectum, as well as head and neck carcinoma. Thus, the balance between the transmembrane and soluble form plays an important role in the activity of

CX3CL1 to either prevent or promote glioma progression (Fig. 1C).

**regulatory T cells (Tregs)** 

**6. Involvement of chemoattractant GPCRs in infiltration of gliomas by** 

**7. Chemoattractant GPCRs in gliomas as potential therapeutic targets** 

Given the broad range of functions of chemoattractant GPCRs in malignant glioma development, progression, invasion and angiogenesis, blockage of these receptors is considered a novel therapeutic approach in conjunction with conventional surgical resection, irradiation and chemotherapy. Based on the association of CXCR4 with the malignant behavior of glioma, anti-CXCR4 monoclonal antibody and specific low-molecular weight antagonist for CXCR4 have been tested for their effects on tumor cell growth in vitro and in vivo. As predicted, anti-CXCR4 monoclonal antibody is able to attenuate the migration and proliferation of human GBM cells induced by CXCL12 (Cheng et al., 2009). In addition, administration of the CXCR4 antagonist AMD3100 suppressed the growth of

Tregs have been recognized as one of the major immune cell components that suppress host anti-tumor responses. Recruitment of Tregs into tumors contributes to tolerance by suppressing autoreactive T cells. It has been shown that Tregs infiltrate human brain tumors (Tran Tang et al., 2010) and preferentially accumulate in high grade malignant gliomas such as GBM. The importance of Tregs in the control of anti-tumor immune responses in experimental mouse glioma models is demonstrated by the observation that transient Treg depletion markedly augments the anti-tumor immunity (Tran Tang et al., 2010). Treg trafficking in vivo is facilitated by chemokine receptors. For instance, Treg accumulation in ovarian carcinoma is mediated by the chemokine receptor CCR4, which binds the ligand CCL22 produced in the tumor where specific T cell immunity is compromised (Curiel et al., 2004). Analysis of lymphocyte subsets in GBM from patients shows that tumor infiltrating Tregs highly express CCR4 (Jacobs et al., 2010) and the ligand CCL22 is produced by GBM cells. But unlike ovarian carcinoma in which Treg accumulation clearly correlates with reduced patient survival, there is no correlation between Tregs and overall survival of GBM patients. Regardless, post-surgical immunotherapy has been proposed as a potentially valid method to eliminate residual GBM cells while preserving surrounding healthy brain cells.

xenograft tumors formed by human GBM cells transplanted intracranially into mice, with increased apoptosis of the transplanted GBM cells (Rubin et al., 2003).

Studies have also revealed the potential benefit of a combination of CXCR4 inhibitor with chemotherapy and radiotherapy in malignant glioma patients. In tests on a variety of GBM cell lines, a conventional cytotoxic chemotherapeutic agent, BCNU, in combination with the CXCR4 antagonist AMD3100 exhibits synergistic inhibition of tumor cell growth in vitro. In vivo in animal models, subtherapeutic doses of BCNU and AMD3100 also result in tumor regression, which is attributed to increased tumor cell apoptosis and decreased proliferation (Redjal et al., 2006). These effects of AMD3100 in conjunction with its capacity to reduce the recruitment of bone marrow EPCs to recurrent tumors post irradiation, suggest that targeting CXCR4 may not only directly inhibit tumor cell proliferation, but also indirectly abrogates neovascularization in GBMs (Kioi et al., 2010).

Considering targeting CXCR4 as a means of inhibiting glioma, the ability of the CXCR4 agonist CXCL12 to activate CXCR7 casts doubts about whether blockage of CXCR4 alone is sufficient without simultaneously inhibiting CXCR7. In fact, inhibition of CXCR4 only partially decreases the responsiveness of tumor cells to CXCL12 in several animal models. Studies have found that GSLCs express high levels of CXCR4 and low levels of CXCR7 (Hattermann et al., 2010). In contrast, differentiation of GSLCs markedly decreased CXCR4 expression but up-regulated CXCR7. It is therefore postulated that CXCR4 may mediate GSLC chemotaxis and survival, whereas differentiated glioma cells are protected from apoptosis by CXCR7 in response to CXCL12. It is therefore important to design strategies that target one or both CXCL12 receptors based on the stages of glioma cell differentiation.

Small molecule natural compounds constitute another source of inhibition of chemoattractant GPCRs with therapeutic potential for gliomas (Ping et al., 2007). One of such compounds is Nordy, a chiral mimetic of a natural lipoxygenase inhibitor nordihydroguaiaretic acid. Nordy has been shown to exhibit a broad inhibitory activity on chemoattractant GPCRs such as CXCR4 and FPR1 on GBM cells by downregulating receptor expression, interfering with their signal transduction pathways and reducing tumor cell production of angiogenic factors VEGF and the chemokine CXCL8 (Ping et al., 2007; Chen et al., 2006 and 2007). In addition, Nordy has been found to inhibit GBM cell proliferation and to promote tumor cell differentiation into a lesser malignant phenotype. Recently, Nordy was found to inhibit the self-renewal of glioma stem cells and growth of xenografts generated by the stem cells (Wang et al., 2011). However, the effect of Nordy may not be specific by targeting only chemoattractant GPCRs on GBM cells. Further studies are required to identify more specific receptor targeting natural compounds with minimal side effects on key physiological cell processes.

#### **8. Conclusions**

There is now mounting evidence that chemoattractant GPCRs play multiple roles in the progression of malignant gliomas, by mediating the tumor cell growth, invasion and angiogenesis (Table 1). However, further molecular epidemiologic and genetic studies are required to obtain a better understanding of the mechanisms of the function of these receptors in glioma cells. It is especially important not to single out a given receptor to study glioma biology, but rather, studies should consider the complex host environment in which many factors may drive the aberrant expression of chemoattractant GPCRs and ligands. In addition, the interaction of chemoattractant GPCRs such as CXCR4 and FPR1 with other

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#### **9. Acknowledgments**

The author thanks Dr Joost J. Oppenheim of the National Cancer Institute, NIH, USA, for reviewing the manuscript. This project was supported in part by the National Basic Research Program of China (973 Program, No. 2010CB529403), the National Natural Science Foundation of China (NSFC, No. 30800421) and the Natural Science Foundation Project of CQ (CSTC, 2008BB5136). This project was also funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E and was supported in part by the Intramural Research Program of the NCI, NIH.

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**Part 4** 

**Glioma Immunology** 


**Part 4** 

**Glioma Immunology** 

302 Glioma – Exploring Its Biology and Practical Relevance

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chemokine receptor CXCR4 in haematopoiesis and in cerebellar development.

**14** 

*India* 

Anirban Ghosh

**Immune Connection in Glioma:** 

*Panihati Mahavidyalaya (West Bengal State University), West Bengal,* 

After the hypothesis of 'immune surveillance' in tumor proposed in 1970, several investigations showed the evidences of its existence where immune system is able to recognize the defects due to tumor onset (MacFarlan Burnet, 1970). But how far this surveillance is effective in the compartmentalized brain in case of glioma still remains a big uncertainty. Glioma is one of the deadliest types of cancer for its rapid growth, invasiveness and short life expectancy of the victim. So, figuring out of the extent of host immune efficiency in glioma is crucial. As glioma is able to create a hostile environment for the immune cells by releasing different soluble factors, expressing death receptors and by receptor camouflaging etc, the working situation for the host immunity becomes more difficult. Therefore, proper assessment of the role of brain immune connection in glioma is crucial to explore the probable level of support that can be extended by the immune defense mechanism against glioma. This immune resistance is also vital to support the present

therapeutic modalities including adjuvants used for treating glioma.

**2. Death of 'Privilege' myth: Immunocytes do not spare brain from their** 

At the beginning of 20th Century, brain was thought to be a separate organ mostly abandoned by the immune system. The initial evidences of immune compromise of the brain compartment were observed from 1920s with the tumor tissue transplantation studies. Rat sarcoma, when transplanted in mouse brain parenchyma, was found to grow better in comparison to its subcutaneous and intramuscular (systemic) transplantations (Shirai, 1921). On contrary, when portions of recipient spleen were co-transferred with tumor in brain parenchyma, inhibition in tumor growth occurred (Murphy and Sturm, 1923). Thus a weak or less efficient immune intervention in brain was conceptualized and the term 'immune

With this, another set of observations in late 19th century and afterwards developed a concept of existence of a barrier between blood and CNS tissue. Basically, Paul Ehrlich's observation with the intravenous administration of vital dye in experimental animals

**2.1 'Immune privilege' of brain: A notion that prevailed more than 5 decades** 

privilege' was proposed by Billingham and Boswell (Billingham & Boswell, 1953).

**1. Introduction** 

**vigilance** 

**Fiction, Fact and Option** 

*Immunobiology Lab, Department of Zoology* 

## **Immune Connection in Glioma: Fiction, Fact and Option**

Anirban Ghosh

*Immunobiology Lab, Department of Zoology Panihati Mahavidyalaya (West Bengal State University), West Bengal, India* 

#### **1. Introduction**

After the hypothesis of 'immune surveillance' in tumor proposed in 1970, several investigations showed the evidences of its existence where immune system is able to recognize the defects due to tumor onset (MacFarlan Burnet, 1970). But how far this surveillance is effective in the compartmentalized brain in case of glioma still remains a big uncertainty. Glioma is one of the deadliest types of cancer for its rapid growth, invasiveness and short life expectancy of the victim. So, figuring out of the extent of host immune efficiency in glioma is crucial. As glioma is able to create a hostile environment for the immune cells by releasing different soluble factors, expressing death receptors and by receptor camouflaging etc, the working situation for the host immunity becomes more difficult. Therefore, proper assessment of the role of brain immune connection in glioma is crucial to explore the probable level of support that can be extended by the immune defense mechanism against glioma. This immune resistance is also vital to support the present therapeutic modalities including adjuvants used for treating glioma.

#### **2. Death of 'Privilege' myth: Immunocytes do not spare brain from their vigilance**

#### **2.1 'Immune privilege' of brain: A notion that prevailed more than 5 decades**

At the beginning of 20th Century, brain was thought to be a separate organ mostly abandoned by the immune system. The initial evidences of immune compromise of the brain compartment were observed from 1920s with the tumor tissue transplantation studies. Rat sarcoma, when transplanted in mouse brain parenchyma, was found to grow better in comparison to its subcutaneous and intramuscular (systemic) transplantations (Shirai, 1921). On contrary, when portions of recipient spleen were co-transferred with tumor in brain parenchyma, inhibition in tumor growth occurred (Murphy and Sturm, 1923). Thus a weak or less efficient immune intervention in brain was conceptualized and the term 'immune privilege' was proposed by Billingham and Boswell (Billingham & Boswell, 1953).

With this, another set of observations in late 19th century and afterwards developed a concept of existence of a barrier between blood and CNS tissue. Basically, Paul Ehrlich's observation with the intravenous administration of vital dye in experimental animals

Immune Connection in Glioma: Fiction, Fact and Option 307

Fig. 1. This figure represents routes of CNS antigen escape and immune cell connection from brain to peripheral circulation. T cells initially are primed by the CNS antigen leaked from brain to the cervical lymph node or olfactory bulb or nasal mucosa, become activated and reach to the blood vessel, subarachnoid area, Virchow-Robin space or perivascular space and CSF in brain. There also a repriming of the glioma antigen specific lymphocytes occurs by the APC circulating or residing at those spaces. Choroid plexus is also a very important route of this CNS antigen escape and site of antigen presentation to lymphocytes by the local APCs. These specific glioma antigen primed activated lymphocytes enter into the brain

parenchyma and invade towards glioma. [The box has been elaborated in Figure – 4]

Early experiments showed that though graft rejection is comparatively slow in brain, once the graft is familiar to the immune system outside brain, the rejection occurs rapidly (Mason *et al*, 1986; Sedgwick, 1995). Simultaneously, Wekerle and colleagues demonstrated that activated or antigen primed T cell from the periphery can cross the BBB nonspecifically (Wekerle *et al*, 1986). Following experiments supported the fact when it was found that CD4+ T cell blasts of any specificity injected intravenous to experimental animals can pass into CNS tissue, although myelin antigen specific T cells are found to remain longer (Hickey *et al*, 1991). This delay of the myelin antigen specific T cells suggests some mechanism that

**2.3 Lymphocytes assess CNS antigen and enter into neuropil 2.3.1 T cells can pass into neuropil and interact with brain APCs** 

showed infiltration of the dye in other organs except brain. That led him to propose a barrier between brain and blood stream (Ehrlich, 1885 & 1904). Goldmann's study showed that tracers injected in blood do not enter into the parenchyma proper in brain, but accumulated in the choroid plexus, perivascular space or lymphatic clefts (Obersteiner, 1870; Goldmann, 1913). The 'no entry' status of blood immunocytes was further fueled with the xeno- and allogenic tissue transplantation studies (Medawar, 1948; Barker & Billingham, 1977). In the following decades ultrastructural studies of the blood capillaries in brain showed the distinct cellular organization present in the interphase of blood and brain that prevent the flow of blood immunocytes and large molecular weight solvents into brain (Reese & Karnovsky, 1967; Engelhardt & Wolburg, 2002). Thus blood-brain-barrier (BBB) encapsulates the brain and seems to maintain the 'immune privilege' status. Till 1980s no direct lymphatic drainage from nervous system was detected. Negligible expression of MHC and undetected dendritic cells (DC) in brain indicated the inefficiency of antigen presentation in the organ (Sedgwick, 1995; Perry, 1998).

#### **2.2 Detection of secret routes connecting brain with systemic circulation**

But in last two decades a paradigm shift has occurred in this 'immune privilege' rank of brain. Basically, three obstacles that maintain the privilege are – i) lack of drainage of CNS antigens at least to cervical lymph node, ii) hindrance to easy access of T cells in the CNS parenchyma and iii) T cells require antigen presentation in the reaction site by the APCs which were thought to be scarce in brain. Initial experiments suggested that CNS antigens can drip outside passively by a different route along the olfactory nerves on to the cribriform plate which is connected to the lymphatics of nasal submucosa and finally to the cervical lymph node (Cserr & Knopf, 1992; Sedgwick, 1995). Tracer studies indicated CSF drainage to cervical lymph node (Boulton *et al*, 1999). CSF circulates from ventricles through subarachnoid spaces (a space between arachnoid and pial membrane filled with CSF and surround the brain and spinal cord) and it has access to Virchow-Robin space that surrounds blood vessels when they enter brain parenchyma [Figure – 1]. Ependymal lining of the ventricles lack 'tight junctions' and in other specialized perivascular spaces including Virchow-Robin's that porosity is also present, which help in clearance of interstitial fluid from brain parenchyma (Ransohoff *et al*, 2003; Piccio *et al*, 2002). So the protein antigens have the probable, though slightly difficult than other organs, access to the lymphoid tissue through CSF. This generates opportunity for the passage of immunocytes.

The afferent arm of CNS immune response is initiated with the antigen leakage from brain parenchyma to CSF; whereas the efferent arm is largely progressed with the migration of leukocytes to CNS into different routes. Ransohoff and colleagues identified three distinct routes which are – i) cells from blood extravasate through choroid plexus to the CSF, ii) leukocytes flowing through internal carotid artery cross the post capillary venules in the subarachnoid space and Virchow-Robin perivascular space and iii) finally leukocytes may cross the BBB deep into the brain to enter directly into the brain parenchyma (Ransohoff *et al*, 2003). Precisely speaking, BBB is a metaphor that describes the property of brain vasculatures restricting the entry of large molecules and cells (Bechmann *et al*, 2007). The perivascular spaces exist in the pre- and post-capillary segments in brain where a heterogeneous assembly of lymphocytic and monocytic cells are observed, more during inflammation. Both in the perivascular spaces and after entering into parenchyma they encounter antigen presenting cells (APCs) to continue the immune response in brain.

showed infiltration of the dye in other organs except brain. That led him to propose a barrier between brain and blood stream (Ehrlich, 1885 & 1904). Goldmann's study showed that tracers injected in blood do not enter into the parenchyma proper in brain, but accumulated in the choroid plexus, perivascular space or lymphatic clefts (Obersteiner, 1870; Goldmann, 1913). The 'no entry' status of blood immunocytes was further fueled with the xeno- and allogenic tissue transplantation studies (Medawar, 1948; Barker & Billingham, 1977). In the following decades ultrastructural studies of the blood capillaries in brain showed the distinct cellular organization present in the interphase of blood and brain that prevent the flow of blood immunocytes and large molecular weight solvents into brain (Reese & Karnovsky, 1967; Engelhardt & Wolburg, 2002). Thus blood-brain-barrier (BBB) encapsulates the brain and seems to maintain the 'immune privilege' status. Till 1980s no direct lymphatic drainage from nervous system was detected. Negligible expression of MHC and undetected dendritic cells (DC) in brain indicated the inefficiency of antigen

presentation in the organ (Sedgwick, 1995; Perry, 1998).

**2.2 Detection of secret routes connecting brain with systemic circulation** 

through CSF. This generates opportunity for the passage of immunocytes.

But in last two decades a paradigm shift has occurred in this 'immune privilege' rank of brain. Basically, three obstacles that maintain the privilege are – i) lack of drainage of CNS antigens at least to cervical lymph node, ii) hindrance to easy access of T cells in the CNS parenchyma and iii) T cells require antigen presentation in the reaction site by the APCs which were thought to be scarce in brain. Initial experiments suggested that CNS antigens can drip outside passively by a different route along the olfactory nerves on to the cribriform plate which is connected to the lymphatics of nasal submucosa and finally to the cervical lymph node (Cserr & Knopf, 1992; Sedgwick, 1995). Tracer studies indicated CSF drainage to cervical lymph node (Boulton *et al*, 1999). CSF circulates from ventricles through subarachnoid spaces (a space between arachnoid and pial membrane filled with CSF and surround the brain and spinal cord) and it has access to Virchow-Robin space that surrounds blood vessels when they enter brain parenchyma [Figure – 1]. Ependymal lining of the ventricles lack 'tight junctions' and in other specialized perivascular spaces including Virchow-Robin's that porosity is also present, which help in clearance of interstitial fluid from brain parenchyma (Ransohoff *et al*, 2003; Piccio *et al*, 2002). So the protein antigens have the probable, though slightly difficult than other organs, access to the lymphoid tissue

The afferent arm of CNS immune response is initiated with the antigen leakage from brain parenchyma to CSF; whereas the efferent arm is largely progressed with the migration of leukocytes to CNS into different routes. Ransohoff and colleagues identified three distinct routes which are – i) cells from blood extravasate through choroid plexus to the CSF, ii) leukocytes flowing through internal carotid artery cross the post capillary venules in the subarachnoid space and Virchow-Robin perivascular space and iii) finally leukocytes may cross the BBB deep into the brain to enter directly into the brain parenchyma (Ransohoff *et al*, 2003). Precisely speaking, BBB is a metaphor that describes the property of brain vasculatures restricting the entry of large molecules and cells (Bechmann *et al*, 2007). The perivascular spaces exist in the pre- and post-capillary segments in brain where a heterogeneous assembly of lymphocytic and monocytic cells are observed, more during inflammation. Both in the perivascular spaces and after entering into parenchyma they

encounter antigen presenting cells (APCs) to continue the immune response in brain.

Fig. 1. This figure represents routes of CNS antigen escape and immune cell connection from brain to peripheral circulation. T cells initially are primed by the CNS antigen leaked from brain to the cervical lymph node or olfactory bulb or nasal mucosa, become activated and reach to the blood vessel, subarachnoid area, Virchow-Robin space or perivascular space and CSF in brain. There also a repriming of the glioma antigen specific lymphocytes occurs by the APC circulating or residing at those spaces. Choroid plexus is also a very important route of this CNS antigen escape and site of antigen presentation to lymphocytes by the local APCs. These specific glioma antigen primed activated lymphocytes enter into the brain parenchyma and invade towards glioma. [The box has been elaborated in Figure – 4]

#### **2.3 Lymphocytes assess CNS antigen and enter into neuropil 2.3.1 T cells can pass into neuropil and interact with brain APCs**

Early experiments showed that though graft rejection is comparatively slow in brain, once the graft is familiar to the immune system outside brain, the rejection occurs rapidly (Mason *et al*, 1986; Sedgwick, 1995). Simultaneously, Wekerle and colleagues demonstrated that activated or antigen primed T cell from the periphery can cross the BBB nonspecifically (Wekerle *et al*, 1986). Following experiments supported the fact when it was found that CD4+ T cell blasts of any specificity injected intravenous to experimental animals can pass into CNS tissue, although myelin antigen specific T cells are found to remain longer (Hickey *et al*, 1991). This delay of the myelin antigen specific T cells suggests some mechanism that

Immune Connection in Glioma: Fiction, Fact and Option 309

layer is crucial (Tran *et al*, 1998; Greter *et al*, 2005) [Figure – 2]. Then the infiltrated lymphocytes may come across the APC present in the brain parenchyma or at the site of pathogenesis. The T cell-microglia interactions in CNS autoimmune encephalomyelitis (EAE) or brain tumor; T cell secretion of Th1 cytokines to mature microglia as functional APC and resulting restimulation of leukocytes by them; counter regulation of this inflammation by Th2 induction by microglia etc had been detailed mostly on the functional

But a new generation of cell tracking experiments and lebelling methods now provide us more direct evidences of the events occurring beneath the skull. GFP-labelled unspecifically activated CD4 T lymphocytes when injected into cortex and ventricle of mice brain, there path through the cross-section of entire head-neck region was monitored. Irrespective of the sites, it was visualized that they pass through the cribroid plate, reach to nasal mucosa and accumulate in the cervical lymph node (Goldmann *et al*, 2006). Shifting the focus on CD8+ T cells, it was recently found that selective traffic of antigen specific CD8 T cells occurs in brain. Using immunofluroscence and confocal micrographs it was found that the process is dependent on luminal expression of MHC class I by cerebral endothelium in response to intracerebral antigen injection. Significantly, the process is quite independent of perivascular macrophages and different from CD4+ T cell entry (Galea *et al*, 2007). After visualizing the entry and exit of T cells from brain their activities in the brain needs a close

Despite the efforts of host immune system, which Burnet and Thomas described as 'immune surveillance', malignant glioma can evade and overcome this defense to grow. There are several generalized strategies for the tumor cells to bypass the immune resistance. They can

a. Making the immune system ignorant about the tumor growth by lacking the tumor antigens in lymphoid organs, growing in immune privileged position, creating physical

b. Actively impairing and suppressing the immune system by down-regulating the expression of MHC genes or imposing defects in antigen processing, secreting suppressive cytokines like TGF-β, IL-10 etc and other factors like prostaglandins. c. Inducing tolerance to immune system by minimizing costimulation that results into anergy and central tolerance to the tumor antigen as many of them produce selfantigens or mimic them. Regulatory T cell mediated inhibition of DC maturation and T cell activation in the tumor environment plays a crucial role for dampening the immune

d. Counter attacking the immune system by expressing different death receptor ligands like CD95L, decoy receptors, TRAIL family etc and expressing anti-apoptotic molecules

Glioma adapt most of these mechanisms successfully with their additional advantage to grow in a position which has been visited or monitored by the peripheral immune cells less

barriers by stoma, lacking adhesion molecules for cellular interactions etc.

studies committed so far (Aloisi *et al*, 2000; Ghosh & Chaudhuri, 2010).

**2.3.2 Cell tracking experiments visualize leukocyte access in brain** 

**3. But glioma makes the immune system puzzled** 

be simply categorized as follows –

resistance.

for themselves.

frequently and less aggressively.

watching.

holds them to process and react in brain parenchyma. The answer is the cross-talk between them and microglia (or brain APC). Microglia is found *ex-vivo* to induce IFN-γ and TNF production from CD4 T cells as their effector activation, but do not support proliferation by IL-2 and induce apoptosis. Interestingly, perivascular macrophages show activation with IL-2 mediated proliferation and survival of CD4+ T cells (Ford *et al*, 1996).

Fig. 2. This section of brain parenchyma shows a blood capillary containing leukocytes. Many of them are at the margin of the capillary, tethering the endothelium and extravaseting at the perivascular spaces. Perivascular microglia/macrophage are visible. Few infiltrated leukocytes are found scattered in brain parenchyma. At least one rod shaped ramified microglia is detectable in the parenchyma. A simple H/E staining section of brain furnishes these visual evidences of neuro-immune connection. (Magnification 1000X, oil immersion in Olympus CH20*i* Microscope and photographed by Olympus DSC)

The reverse is also visible in the GvHD affected CNS model where CD4+αβTCR+CD2+T cells infiltrat and scatter deep into neuropil or brain parenchyma. The microglial cells show activation with many fold increase in their CD11b/c, CD45 and MHC class II expression and cluster with intimate association with these T cells *in situ* that lead to microglial activation, proliferation and expansion (Sedgwick *et al*, 1998). Thus both microglia and infiltrated lymphocytes influence each other for their maturation and effector function in brain.

With the citations of entry of lymphocytes in brain, the role of CNS APCs started to come into surface. They are subdivided as microglia and perivascular macrophages based on their position, morphology, immunophenotype and functional priority (Sedgwick *et al*, 1991; Bechmann *et al*, 2001). Therefore entry of lymphocytes into neuropil through pre- and postcapillary vessels needs a two step process. Crossing the vessel endothelium, muscle layers and basement membranes lymphocytes and blood borne monocytes reside at the perivascular space encapsulated with glial limitans and pericytes. Next step is more restricted where the leukocytes cross the layer of glial limitans and step in to neuropil (Bechmann *et al*, 2007). To proceed for this step, brain APC associated with this limiting

holds them to process and react in brain parenchyma. The answer is the cross-talk between them and microglia (or brain APC). Microglia is found *ex-vivo* to induce IFN-γ and TNF production from CD4 T cells as their effector activation, but do not support proliferation by IL-2 and induce apoptosis. Interestingly, perivascular macrophages show activation with IL-

Fig. 2. This section of brain parenchyma shows a blood capillary containing leukocytes.

extravaseting at the perivascular spaces. Perivascular microglia/macrophage are visible. Few infiltrated leukocytes are found scattered in brain parenchyma. At least one rod shaped ramified microglia is detectable in the parenchyma. A simple H/E staining section of brain furnishes these visual evidences of neuro-immune connection. (Magnification 1000X, oil immersion in Olympus CH20*i* Microscope and photographed by Olympus DSC)

The reverse is also visible in the GvHD affected CNS model where CD4+αβTCR+CD2+T cells infiltrat and scatter deep into neuropil or brain parenchyma. The microglial cells show activation with many fold increase in their CD11b/c, CD45 and MHC class II expression and cluster with intimate association with these T cells *in situ* that lead to microglial activation, proliferation and expansion (Sedgwick *et al*, 1998). Thus both microglia and infiltrated

With the citations of entry of lymphocytes in brain, the role of CNS APCs started to come into surface. They are subdivided as microglia and perivascular macrophages based on their position, morphology, immunophenotype and functional priority (Sedgwick *et al*, 1991; Bechmann *et al*, 2001). Therefore entry of lymphocytes into neuropil through pre- and postcapillary vessels needs a two step process. Crossing the vessel endothelium, muscle layers and basement membranes lymphocytes and blood borne monocytes reside at the perivascular space encapsulated with glial limitans and pericytes. Next step is more restricted where the leukocytes cross the layer of glial limitans and step in to neuropil (Bechmann *et al*, 2007). To proceed for this step, brain APC associated with this limiting

lymphocytes influence each other for their maturation and effector function in brain.

Many of them are at the margin of the capillary, tethering the endothelium and

2 mediated proliferation and survival of CD4+ T cells (Ford *et al*, 1996).

layer is crucial (Tran *et al*, 1998; Greter *et al*, 2005) [Figure – 2]. Then the infiltrated lymphocytes may come across the APC present in the brain parenchyma or at the site of pathogenesis. The T cell-microglia interactions in CNS autoimmune encephalomyelitis (EAE) or brain tumor; T cell secretion of Th1 cytokines to mature microglia as functional APC and resulting restimulation of leukocytes by them; counter regulation of this inflammation by Th2 induction by microglia etc had been detailed mostly on the functional studies committed so far (Aloisi *et al*, 2000; Ghosh & Chaudhuri, 2010).

#### **2.3.2 Cell tracking experiments visualize leukocyte access in brain**

But a new generation of cell tracking experiments and lebelling methods now provide us more direct evidences of the events occurring beneath the skull. GFP-labelled unspecifically activated CD4 T lymphocytes when injected into cortex and ventricle of mice brain, there path through the cross-section of entire head-neck region was monitored. Irrespective of the sites, it was visualized that they pass through the cribroid plate, reach to nasal mucosa and accumulate in the cervical lymph node (Goldmann *et al*, 2006). Shifting the focus on CD8+ T cells, it was recently found that selective traffic of antigen specific CD8 T cells occurs in brain. Using immunofluroscence and confocal micrographs it was found that the process is dependent on luminal expression of MHC class I by cerebral endothelium in response to intracerebral antigen injection. Significantly, the process is quite independent of perivascular macrophages and different from CD4+ T cell entry (Galea *et al*, 2007). After visualizing the entry and exit of T cells from brain their activities in the brain needs a close watching.

#### **3. But glioma makes the immune system puzzled**

Despite the efforts of host immune system, which Burnet and Thomas described as 'immune surveillance', malignant glioma can evade and overcome this defense to grow. There are several generalized strategies for the tumor cells to bypass the immune resistance. They can be simply categorized as follows –


Glioma adapt most of these mechanisms successfully with their additional advantage to grow in a position which has been visited or monitored by the peripheral immune cells less frequently and less aggressively.

Immune Connection in Glioma: Fiction, Fact and Option 311

their repeated exposure to immune selection act as the key to develop glioma cell variants

Glioma cells are capable to secret copious factors that influence the immune system negatively. Cyclooxygenase enzyme COX-2 derived prostaglandin E2 (PGE2) bind with its receptor EPI-4 on glioma cells and encourage them to invade by increasing motility. PGE2 downregulate Th1 cytokines like IL-2, IFNγ and TNFα, and upregulate Th2 cytokines like IL-4, IL-10 and IL-6 (Wang & Dubois, 2006). Glioma cells secrete IL-10 which inhibit IL-2 induced T cell proliferation, DC and macrophage activation (Grutz, 2005). IL-10 is expressed by Treg cells present in glioma vicinity (Sakaguchi, 2005). TGFβ with its three isoform (TGFβ1,2,3) is involved in regulating inflammation, angiogenesis and proliferation (Govinden and Bhoola, 2003). TGFβ is the dominant isoform expressed by glioblastoma. They inhibit maturation of professional APCs, obstruct the synthesis of cytotoxic molecules including perforin, granzymes, FasL in activated CTL (Thomas and Massague, 2005). This cytokine may also efficiently recruit T reg cells in glioma. Glioma shows a considerable level of resistance against Fas induced apoptosis. Decoy receptor 3 (DcR3) is expressed in brain tumor and prevents Fas mediated apoptosis as well as decreases infiltration of CD4 and CD8 T cells (Roth *et al*, 2001). Apoptosis inhibitory proteins (IAPs) are active in glioma which inhibit caspase activity (Gomez & Kruse, 2006). Some of the glioma cells express FasL

As cell to cell contact plays an important role to deliver the immune assault, glioma cells take the strategy to minimize or impair these adhesions. Cell adhesion interaction between glioma and immune cells was found to be prevented by a thick glycosaminoglycan coating and protect the neoplastic cells from CTL action (Dick *et al*, 1983). In glioma condition, ICAM-1/LFA-1 interaction is interrupted which inhibit target cell lysis by tumor specific T and NK cells (Schiltz *et al*, 2002; Fiore *et al*, 2002). The aberrant HLA class I expression in glioma helps them to evade T cell detection of transformed cells and subsequent cytotoxicity (Rosenberg *et al*, 2003). In glioma, B7-H1 (B7-homologue 1, a costimulatory molecule) inhibits allogenic T cell activation and associated cytokine secretion (Wilmotte *et al*, 2005). Some other factors like Indoleamine 2,3-dioxygenase (IDO) expression in glioma cells cause T cells to starve for tryptophan, cell cycle arrest and tolerance (Uyttenhove *et al*, 2003). Interestingly, IFNγ stimulate IDO production in glioma, create a local tryptophan shortage and T cell tolerance (Shirey *et al*, 2006). The activation of STAT-3 is another trick for glioma. STAT-3 regulates the anti-apoptotic proteins like Bcl-2, Bcl-XL, Mcl-1, cFLIP, surviving etc in glioma (Rahaman *et al*, 2005; Akasaki *et al*, 2006). Glioma uses various factors including chemokines and matrix degrading enzymes secreted from the brain macrophage/microglia

population for their migration and spread in brain (these will be discussed later).

**4. Undaunted immune effort continues to resist transformed cells** 

**brain tumor** 

**4.1 T cells mature to effector state after local interaction with APCs in CNS, even in** 

The experimental evidences furnished by Ford and his colleagues in 1996 revealed that the resident antigen presenting cell of brain i.e., microglia, interacts with the T cells to induce final effector function (Ford *et al*, 1996). Years' later studies with GFP-labeled encephalitogenic T cells specific for MBP (TMBP-GFP cells) showed that, with the onset of the disease, huge number of CD4+ effector cells infiltrate in CNS with upregulated chemokine receptors. But these infiltrated TMBP-GFP cells when recovered after 24 hrs from brain

with enhanced capacity to evade immune defense.

to counteract with the immunocytes (Husain *et al*, 1998).

#### **3.1 Glioma drastically reduces immune efficiency**

Different studies on a number of patients harboring glioma revealed that they suffered from impaired cell mediated immunity (Elliott *et al*, 1984). *In vitro* studies showed that peripheral blood lymphocytes (PBL) obtained from patients with gliomas proliferated poorly in response to mitogen and/or antigen stimulation *in vitro* and unresponsiveness to T-cell mitogens concanavalin A (ConA), phytohemagglutinin (PHA) and anti-CD3 mAb etc (McVicar *et al*, 1992). A number of potential mechanisms explaining the observed immunesuppression including qualitative or quantitative alterations in cell surface marker expression on T-cells, elevated suppressor cell activity or T-cell lymphopenia were explored. T-cells obtained from glioma patients have intrinsic defects, which synthesize and secrete less than normal levels of interleuken-2 (IL-2) required for T-cell proliferation. IL-2 mRNA synthesis is impaired with less production of IL-2 receptor (IL-2R), and they also are unable to enter G1 phase of cell cycle (Elliott *et al*, 1990). Additionally, the numbers of CD4+ T-cells obtained from patients are reduced to a great extent than CD8+ T-cells, which predominately infiltrate glioma, but are deprived from CD4+ help. Based on their inability to produce and respond to IL-2 and lack of CD4+ help, T-cells obtained from glioma patients appear to be anergic (Elliott *et al*, 1990; Giometto *et al*, 1996).

Wide level of T-cell signaling defects are observed in glioma patient derived T-cells. T-cells from glioma patients show reduced tyrosine phosphorylation compared to normal T cells, which is mostly reduced in PLCγ1. Additionally, both PLCγ1 and p56lck protein levels are found reduced dramatically and thus it causes the overall impairment of TCR/CD3 mediated signaling. Reduced p56lck and associated signals also resists the cells to make sufficient contact with APCs, reduced their appropriate stimuli and movements (Marford *et al*, 1997, Dix *et al*, 1999). Severe T-cells lymphopenia i.e. rapid depletion of the cells is an important feature of glioma patients. As CD4+ T-cells are reduced in number and less responsive to mitogens and antigens, IL-2 and IFNγ production decreases further. Because both these cytokines are important for generation of LAK cells and CTL activity, they are responsible for impaired generation of antigen-stimulated, MHC-unrestricted cytotoxicity observed in glioma patients (Urbani *et al*, 1996; Dix *et al*, 1999). Even glioma condition facilitates to increase Th2 type IL-10 production and inhibit Th1 type IL-12 and TIL secrete predominantly Th2 type cytokines underscoring the Th1 effect. Glioma has been shown to synthesize and secrete multiple factors including TGFβ, PGE2, IL-10 and gangliosides (Zou *et al*, 1999, Huettner *et al*, 1997). Gliomas synthesize and secrete TGFβ1, 2, and 3 which downregulate monocyte surface marker expression, cytokine secretion, cytotoxicity and T-cell responsiveness. Gangliosides (GANGs) are components of human plasma with GM3 and GD3 being major constituents, and can bind to both plasma proteins and lipoproteins. The highest concentrations of GANGs are found in brain and mainly include GM1, GD1a, GD1b and GI1b. These are highly immunosuppressive by inhibiting T-cell proliferation, CD4 expression, generation of CTL and NK cell activity. In addition, GANGs may also suppress Ca2+ flux in T-cells (Ladisch *et al*, 1992; Zou *et al*, 1999; Dix *et al*, 1999).

#### **3.2 The mechanism behind glioma immune evasion**

Like any other tumors immune selective pressure on the glioma cells is also working to eradicate abnormal cells. Though the initial intensity is less and additional time is required to recognize and react, the precision is much higher against the neoplastic cells in brain (which will be discussed in the following sections). The genetic instability of glioma and

Different studies on a number of patients harboring glioma revealed that they suffered from impaired cell mediated immunity (Elliott *et al*, 1984). *In vitro* studies showed that peripheral blood lymphocytes (PBL) obtained from patients with gliomas proliferated poorly in response to mitogen and/or antigen stimulation *in vitro* and unresponsiveness to T-cell mitogens concanavalin A (ConA), phytohemagglutinin (PHA) and anti-CD3 mAb etc (McVicar *et al*, 1992). A number of potential mechanisms explaining the observed immunesuppression including qualitative or quantitative alterations in cell surface marker expression on T-cells, elevated suppressor cell activity or T-cell lymphopenia were explored. T-cells obtained from glioma patients have intrinsic defects, which synthesize and secrete less than normal levels of interleuken-2 (IL-2) required for T-cell proliferation. IL-2 mRNA synthesis is impaired with less production of IL-2 receptor (IL-2R), and they also are unable to enter G1 phase of cell cycle (Elliott *et al*, 1990). Additionally, the numbers of CD4+ T-cells obtained from patients are reduced to a great extent than CD8+ T-cells, which predominately infiltrate glioma, but are deprived from CD4+ help. Based on their inability to produce and respond to IL-2 and lack of CD4+ help, T-cells obtained from glioma patients appear to be

Wide level of T-cell signaling defects are observed in glioma patient derived T-cells. T-cells from glioma patients show reduced tyrosine phosphorylation compared to normal T cells, which is mostly reduced in PLCγ1. Additionally, both PLCγ1 and p56lck protein levels are found reduced dramatically and thus it causes the overall impairment of TCR/CD3 mediated signaling. Reduced p56lck and associated signals also resists the cells to make sufficient contact with APCs, reduced their appropriate stimuli and movements (Marford *et al*, 1997, Dix *et al*, 1999). Severe T-cells lymphopenia i.e. rapid depletion of the cells is an important feature of glioma patients. As CD4+ T-cells are reduced in number and less responsive to mitogens and antigens, IL-2 and IFNγ production decreases further. Because both these cytokines are important for generation of LAK cells and CTL activity, they are responsible for impaired generation of antigen-stimulated, MHC-unrestricted cytotoxicity observed in glioma patients (Urbani *et al*, 1996; Dix *et al*, 1999). Even glioma condition facilitates to increase Th2 type IL-10 production and inhibit Th1 type IL-12 and TIL secrete predominantly Th2 type cytokines underscoring the Th1 effect. Glioma has been shown to synthesize and secrete multiple factors including TGFβ, PGE2, IL-10 and gangliosides (Zou *et al*, 1999, Huettner *et al*, 1997). Gliomas synthesize and secrete TGFβ1, 2, and 3 which downregulate monocyte surface marker expression, cytokine secretion, cytotoxicity and T-cell responsiveness. Gangliosides (GANGs) are components of human plasma with GM3 and GD3 being major constituents, and can bind to both plasma proteins and lipoproteins. The highest concentrations of GANGs are found in brain and mainly include GM1, GD1a, GD1b and GI1b. These are highly immunosuppressive by inhibiting T-cell proliferation, CD4 expression, generation of CTL and NK cell activity. In addition, GANGs may also suppress

Ca2+ flux in T-cells (Ladisch *et al*, 1992; Zou *et al*, 1999; Dix *et al*, 1999).

Like any other tumors immune selective pressure on the glioma cells is also working to eradicate abnormal cells. Though the initial intensity is less and additional time is required to recognize and react, the precision is much higher against the neoplastic cells in brain (which will be discussed in the following sections). The genetic instability of glioma and

**3.2 The mechanism behind glioma immune evasion** 

**3.1 Glioma drastically reduces immune efficiency** 

anergic (Elliott *et al*, 1990; Giometto *et al*, 1996).

their repeated exposure to immune selection act as the key to develop glioma cell variants with enhanced capacity to evade immune defense.

Glioma cells are capable to secret copious factors that influence the immune system negatively. Cyclooxygenase enzyme COX-2 derived prostaglandin E2 (PGE2) bind with its receptor EPI-4 on glioma cells and encourage them to invade by increasing motility. PGE2 downregulate Th1 cytokines like IL-2, IFNγ and TNFα, and upregulate Th2 cytokines like IL-4, IL-10 and IL-6 (Wang & Dubois, 2006). Glioma cells secrete IL-10 which inhibit IL-2 induced T cell proliferation, DC and macrophage activation (Grutz, 2005). IL-10 is expressed by Treg cells present in glioma vicinity (Sakaguchi, 2005). TGFβ with its three isoform (TGFβ1,2,3) is involved in regulating inflammation, angiogenesis and proliferation (Govinden and Bhoola, 2003). TGFβ is the dominant isoform expressed by glioblastoma. They inhibit maturation of professional APCs, obstruct the synthesis of cytotoxic molecules including perforin, granzymes, FasL in activated CTL (Thomas and Massague, 2005). This cytokine may also efficiently recruit T reg cells in glioma. Glioma shows a considerable level of resistance against Fas induced apoptosis. Decoy receptor 3 (DcR3) is expressed in brain tumor and prevents Fas mediated apoptosis as well as decreases infiltration of CD4 and CD8 T cells (Roth *et al*, 2001). Apoptosis inhibitory proteins (IAPs) are active in glioma which inhibit caspase activity (Gomez & Kruse, 2006). Some of the glioma cells express FasL to counteract with the immunocytes (Husain *et al*, 1998).

As cell to cell contact plays an important role to deliver the immune assault, glioma cells take the strategy to minimize or impair these adhesions. Cell adhesion interaction between glioma and immune cells was found to be prevented by a thick glycosaminoglycan coating and protect the neoplastic cells from CTL action (Dick *et al*, 1983). In glioma condition, ICAM-1/LFA-1 interaction is interrupted which inhibit target cell lysis by tumor specific T and NK cells (Schiltz *et al*, 2002; Fiore *et al*, 2002). The aberrant HLA class I expression in glioma helps them to evade T cell detection of transformed cells and subsequent cytotoxicity (Rosenberg *et al*, 2003). In glioma, B7-H1 (B7-homologue 1, a costimulatory molecule) inhibits allogenic T cell activation and associated cytokine secretion (Wilmotte *et al*, 2005).

Some other factors like Indoleamine 2,3-dioxygenase (IDO) expression in glioma cells cause T cells to starve for tryptophan, cell cycle arrest and tolerance (Uyttenhove *et al*, 2003). Interestingly, IFNγ stimulate IDO production in glioma, create a local tryptophan shortage and T cell tolerance (Shirey *et al*, 2006). The activation of STAT-3 is another trick for glioma. STAT-3 regulates the anti-apoptotic proteins like Bcl-2, Bcl-XL, Mcl-1, cFLIP, surviving etc in glioma (Rahaman *et al*, 2005; Akasaki *et al*, 2006). Glioma uses various factors including chemokines and matrix degrading enzymes secreted from the brain macrophage/microglia population for their migration and spread in brain (these will be discussed later).

#### **4. Undaunted immune effort continues to resist transformed cells**

#### **4.1 T cells mature to effector state after local interaction with APCs in CNS, even in brain tumor**

The experimental evidences furnished by Ford and his colleagues in 1996 revealed that the resident antigen presenting cell of brain i.e., microglia, interacts with the T cells to induce final effector function (Ford *et al*, 1996). Years' later studies with GFP-labeled encephalitogenic T cells specific for MBP (TMBP-GFP cells) showed that, with the onset of the disease, huge number of CD4+ effector cells infiltrate in CNS with upregulated chemokine receptors. But these infiltrated TMBP-GFP cells when recovered after 24 hrs from brain

Immune Connection in Glioma: Fiction, Fact and Option 313

Microglia is designated as local or resident antigen presenting cell throughout the brain parenchyma or neuropil and its margin. Most emphatic efforts of microglial research in the last two decades were invested to explore its functional relevance in the brain tissue. The striking feature of microglia is its versatile ability to respond according to the CNS microenvironment. Functionally speaking, microglia is a hybrid between white blood cells and glial cells, which is intended to protect and support the neuronal environment in brain. Microglia can respond against an extensive list of biochemical factors ranging as diverse as

Nearly two decade of studies demonstrated the causative effects of chemokines in glioma microenvironment for macrophage/microglia infiltration. The C-C family chemokines, viz, monocyte chemotactic protein 1 (MCP-1) was first purified from glioma and Leung *et al*, 1997 found that with increased MCP-1 the macrophage/microglia infiltrates in glioma. Astrocytoma cells were found capable of producing MCP-1, and complementary receptor CCR2 was present and expressed on activated microglia (Leung *et al*, 1997). In 2003, evidence showed that, MCP-1 promoted the microglial migration in glioma and microglia infested glioma grew rapidly (Platten *et al*, 2003). The involvement of PI-3K/Akt pathway was assumed in the secretion of microglia derived factors that mediate glioma invasiveness (Joy *et al*, 2003; Pu *et al*, 2004). CSF-1 (colony stimulating factor 1), which acted as chemotactic and mitogenic factors for myeloid lineage cells, and its receptor, were long been detected in glioma, whereas microglia also possessed the option of secreting and receiving the factor from self and neighboring cells. Eventually from glioma G-CSF/GM-CSF (granulocytes and granulocytes macrophage colony stimulating factor) were secreted and influenced the differentiation and maturation process of microglia like other myeloid lineage cells. Badie *et al*, 1999, *in vitro* demonstrated the specific microglia attracting capacity of glioma by the hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF signals the spectrum of mesenchymal cells for mitogenic stimulation, invasion and extravasation. Microglia possesses its receptor Met and can produce HGF/SF, whereas the glioma cells are capable of doing the same (Badie & Schartner, 2001). Thus, the balancing ratios of the factors in glioma microenvironment act as the determinant in the migration process of the cells. In 2002, the 15.3 KDa heparin-binding peptide Pleiotrophin (PTN) was found to appear in adult human glioma, normally a mitogenic/angiogenic factor in embryonic stage. Uniquely, PTN did not help to proliferate the glioma cells by its own, rather influenced microglial accumulation by acting as strong chemotactic and mitogenic agent. Its action thus passively helped glioma growth by targeting endothelial and microglial cells (Mentlein & Held-Feindt, 2002). The question arose that what would be the interest of glioma to include microglia in its vicinity? In fact, the role of infiltrating macrophage/microglia in the process of angiogenesis in glioma was hinted previously when macrophage associated hemeoxygenase-1 (HO-1) enzyme was found to be correlated with the vascular density of human glioma (Nishie *et al* 1999). Another enzyme cyclooxygenase (COX)-2 was found to be produced in higher amount in microglia isolated from intracranial glioma which increased the prostaglandin E2 (PGE2) production. The study suggested that glioma infiltrating microglia contributed in developing fatal cerebral edema in glioma through COX-2 dependent pathway. It was reported that PGE2 increased the permeability of vascular endothelium by cytoskeletal rearrangement where TNFα acted as positive inducer. In that case, microglia was the source of both COX-2 and TNFα probably playing a role in its own migration, leukocyte trafficking and in parallel, glioma invasiveness (Badie *et al*, 2003).

**4.2 Microglia, the local/resident APCs of brain accumulate in glioma** 

glycoconjugates, neurotransmitters or cytokines/chemokines (Nakamura, 2002).

parenchyma or neuropil they showed fresh sign of reactivation with upregulation of OX-40 and IL-2R, and upregulated expression of IFNγ, TNFα, TGFβ, IL-2, IL-10, CD3 mRNA expression (Flugel *et al*, 2001). These observations suggested the importance of brain APC at the site of proper functioning of the recruited cells.

Fig. 3. The section shows that infiltrated lymphocytes in brain parenchyma interact with transformed oligodendroglial cells in a murine oligodendroglioma model and offer them the 'kiss of death'. One oligodendroglioma cell nucleus is protruded out of the cytoplasm and for the other cell, the lymphocyte is overlapping with it. One oligodendroglioma cell, one astrocyte and a free lymphocyte is visible in the field. (Magnification 1000X, oil immersion in Olympus CH20i Microscope and photographed by Olympus DSC)

The function of APC is now becoming more substantial in CNS as some new experimental evidences indicate that they are important to reshape and retain Ag-specific CTLs in the site of neuropathogenesis. In a series of experiments Walker and team addressed the issue with precision. Whether CNS retention of tumor specific MHC class I restricted CD8+ T cells also require recognition of local APC cross-presenting tumor Ag was the problem under scrutiny. They used murine glioma transfected with cDNA encoding HLA-CW3 implanted in mice model. The observation was a massive expansion of H2Kd/CW3170-179-specific CTL using BV10TCR after immunization with CW3. In that animal model the endogenous presentation was tactically avoided and due to the absence of MHC class II on MT-CW3 transfect, the CD4 arm is also avoided. Therefore any expansion of H2Kd restricted CW3 specific CTL suggests the involvement of hosts APC at the tumor site. Detection of the localization of specific CTL, their cytotoxic efficacy and retention of effector functions in an antigen dependent manner speaks the importance of local APC within CNS (Calzascia *et al*, 2003). They are activated macrophage/microglia and cells with DC phenotype found infiltrated heavily in tumor. In subsequent studies they also provide support for the fact that T cell homing at the specific CNS tumor site is defined by the cross-presentating APCs there and not predetermined in the lymph nodes where initial priming occurs (Calzascia *et al*, 2005). In extended studies it was further found that Ag-experienced CD8+ T cells further differentiate at the intracerebral tumor site with enhanced IFNγ and Granzyme-B expression and induction of αEβ7 integrin that facilitate their retention in brain (Masson *et al*, 2007). Thus cytotoxic activity of lymphocytes on glioma is not rare [Figure – 3].

parenchyma or neuropil they showed fresh sign of reactivation with upregulation of OX-40 and IL-2R, and upregulated expression of IFNγ, TNFα, TGFβ, IL-2, IL-10, CD3 mRNA expression (Flugel *et al*, 2001). These observations suggested the importance of brain APC at

Fig. 3. The section shows that infiltrated lymphocytes in brain parenchyma interact with transformed oligodendroglial cells in a murine oligodendroglioma model and offer them the 'kiss of death'. One oligodendroglioma cell nucleus is protruded out of the cytoplasm and for the other cell, the lymphocyte is overlapping with it. One oligodendroglioma cell, one astrocyte and a free lymphocyte is visible in the field. (Magnification 1000X, oil immersion

The function of APC is now becoming more substantial in CNS as some new experimental evidences indicate that they are important to reshape and retain Ag-specific CTLs in the site of neuropathogenesis. In a series of experiments Walker and team addressed the issue with precision. Whether CNS retention of tumor specific MHC class I restricted CD8+ T cells also require recognition of local APC cross-presenting tumor Ag was the problem under scrutiny. They used murine glioma transfected with cDNA encoding HLA-CW3 implanted in mice model. The observation was a massive expansion of H2Kd/CW3170-179-specific CTL using BV10TCR after immunization with CW3. In that animal model the endogenous presentation was tactically avoided and due to the absence of MHC class II on MT-CW3 transfect, the CD4 arm is also avoided. Therefore any expansion of H2Kd restricted CW3 specific CTL suggests the involvement of hosts APC at the tumor site. Detection of the localization of specific CTL, their cytotoxic efficacy and retention of effector functions in an antigen dependent manner speaks the importance of local APC within CNS (Calzascia *et al*, 2003). They are activated macrophage/microglia and cells with DC phenotype found infiltrated heavily in tumor. In subsequent studies they also provide support for the fact that T cell homing at the specific CNS tumor site is defined by the cross-presentating APCs there and not predetermined in the lymph nodes where initial priming occurs (Calzascia *et al*, 2005). In extended studies it was further found that Ag-experienced CD8+ T cells further differentiate at the intracerebral tumor site with enhanced IFNγ and Granzyme-B expression and induction of αEβ7 integrin that facilitate their retention in brain (Masson *et al*, 2007).

in Olympus CH20i Microscope and photographed by Olympus DSC)

Thus cytotoxic activity of lymphocytes on glioma is not rare [Figure – 3].

the site of proper functioning of the recruited cells.

#### **4.2 Microglia, the local/resident APCs of brain accumulate in glioma**

Microglia is designated as local or resident antigen presenting cell throughout the brain parenchyma or neuropil and its margin. Most emphatic efforts of microglial research in the last two decades were invested to explore its functional relevance in the brain tissue. The striking feature of microglia is its versatile ability to respond according to the CNS microenvironment. Functionally speaking, microglia is a hybrid between white blood cells and glial cells, which is intended to protect and support the neuronal environment in brain. Microglia can respond against an extensive list of biochemical factors ranging as diverse as glycoconjugates, neurotransmitters or cytokines/chemokines (Nakamura, 2002).

Nearly two decade of studies demonstrated the causative effects of chemokines in glioma microenvironment for macrophage/microglia infiltration. The C-C family chemokines, viz, monocyte chemotactic protein 1 (MCP-1) was first purified from glioma and Leung *et al*, 1997 found that with increased MCP-1 the macrophage/microglia infiltrates in glioma. Astrocytoma cells were found capable of producing MCP-1, and complementary receptor CCR2 was present and expressed on activated microglia (Leung *et al*, 1997). In 2003, evidence showed that, MCP-1 promoted the microglial migration in glioma and microglia infested glioma grew rapidly (Platten *et al*, 2003). The involvement of PI-3K/Akt pathway was assumed in the secretion of microglia derived factors that mediate glioma invasiveness (Joy *et al*, 2003; Pu *et al*, 2004). CSF-1 (colony stimulating factor 1), which acted as chemotactic and mitogenic factors for myeloid lineage cells, and its receptor, were long been detected in glioma, whereas microglia also possessed the option of secreting and receiving the factor from self and neighboring cells. Eventually from glioma G-CSF/GM-CSF (granulocytes and granulocytes macrophage colony stimulating factor) were secreted and influenced the differentiation and maturation process of microglia like other myeloid lineage cells. Badie *et al*, 1999, *in vitro* demonstrated the specific microglia attracting capacity of glioma by the hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF signals the spectrum of mesenchymal cells for mitogenic stimulation, invasion and extravasation. Microglia possesses its receptor Met and can produce HGF/SF, whereas the glioma cells are capable of doing the same (Badie & Schartner, 2001). Thus, the balancing ratios of the factors in glioma microenvironment act as the determinant in the migration process of the cells.

In 2002, the 15.3 KDa heparin-binding peptide Pleiotrophin (PTN) was found to appear in adult human glioma, normally a mitogenic/angiogenic factor in embryonic stage. Uniquely, PTN did not help to proliferate the glioma cells by its own, rather influenced microglial accumulation by acting as strong chemotactic and mitogenic agent. Its action thus passively helped glioma growth by targeting endothelial and microglial cells (Mentlein & Held-Feindt, 2002). The question arose that what would be the interest of glioma to include microglia in its vicinity? In fact, the role of infiltrating macrophage/microglia in the process of angiogenesis in glioma was hinted previously when macrophage associated hemeoxygenase-1 (HO-1) enzyme was found to be correlated with the vascular density of human glioma (Nishie *et al* 1999). Another enzyme cyclooxygenase (COX)-2 was found to be produced in higher amount in microglia isolated from intracranial glioma which increased the prostaglandin E2 (PGE2) production. The study suggested that glioma infiltrating microglia contributed in developing fatal cerebral edema in glioma through COX-2 dependent pathway. It was reported that PGE2 increased the permeability of vascular endothelium by cytoskeletal rearrangement where TNFα acted as positive inducer. In that case, microglia was the source of both COX-2 and TNFα probably playing a role in its own migration, leukocyte trafficking and in parallel, glioma invasiveness (Badie *et al*, 2003).

Immune Connection in Glioma: Fiction, Fact and Option 315

Regarding the cytokine microenvironment, the role of TGF-β was hinted in migration (Milner & Campbell, 2002). The specific importance of the cytokine was demonstrated by RNAi mediated gene silencing of TGF-β in promoting growth and invasiveness of glioma by integrin family adhesion molecule (Wesolowska *et al*, 2008). Recently it was found that cyclosporin A (CsA), an inhibitor of calcineurin and immunosuppressive in effect, could inhibit microglia mediated glioma invasion and cause to change morphological structure of

In the present context, most of the studies demonstrated pro-tumerogenic action of microglia in glioma, which was facilitated by the secretary products, signaling molecules including cytokines, chemokines and receptors etc. In parallel, glioma cells favor microglial migration and encroachment in its vicinity. Though primarily macrophages/microglia are the cells to defend host tissue from faulty or malfunctioning cells or pathogens, their pro-glioma role leads to confusion. Remarkably, several findings also came with hopeful antagonistic results as already mentioned, where the roles of TNFα, TGFβ, A1AR dependent MMP-2 inhibition etc were focused (Villeneuve *et al*, 2005; Nakagawa *et al*, 2007; Synowitz *et al* 2006; Wesolowska *et al*, 2008). In 2007, Galarneau and team demonstrated that macrophage/microglia depletion helped in glioma growth (Galarneau *et al*, 2007). The study hinted for a separate anti-tumor potential of the cells. These contradicting results present microglia with a double agent stature.

To determine the antigen presenting role of microglia their MHC class II expression along with the co-stimulatory molecule like B7.1 (CD80) and B7.2 (CD86) had been evaluated. Badie and his team found the lower level of expression of these essential surface molecule for APC function in microglia freshly isolated from glioma invasive cells and that suggested suppressive effect on glioma microenvironment *in vivo* (Badie & Schartner, 2001). In a comparative study of different rodent glioma model viz., C6, 9L and RG2, the expression profile was found to vary significantly depending on the immunogenicity of the model. The costimulatory B7 molecule expression could be favored when microglia were rejuvenated by cytokines GM-CSF and IFN-γ *in vitro* (Badie *et al*, 2002). At the same time, Graeber with his colleagues scanned 97 glioma samples of different WHO grades and found no such simple relations of the MHC expression of the cell with tumor grades, rather found downregulation of MHC class II in tissue areas where dense glioblastoma cells were infested. According to them microglia were functional in astrocytic tumors, though might be subjected to suppression with T cell clonal anergy in that glioma microenvironment (Tran *et al*, 1998). The stimulatory effect of the novel glycoprotein T11TS/SLFA-3 on microglial MHC class II expression was found. The dose-time dependent efficient MHC expression was found on microglia in rodent bearing experimental glioma when treated with T11TS (Begum *et al*, 2004). Chaudhuri and her team also identified another important costimulatory molecule CD2 on microglia, which could also be regulated by the glycoprotein dose in glioma condition (Begum *et al*, 2004; Chaudhuri & Ghosh, 2006). A separate study by her team found simultaneous co-expression of MHC class II and CD2 on microglia in glioma where both expressed in low quantity (Sarkar *et al*, 2004). This observation with others supported the view of immunosuppressive milieu offered to microglia in glioma mostly by TGFβ, IL-10, PGE2, gangliosides etc (Zou *et al*, 1999; Graeber *et al*, 2002), which could also simultaneously cripple the infiltrated lymphocytes (Dix *et al*, 1999). In this regard, the fact that microglia was the source of that IL-10 in glioma, had been finally established by

microglia via MAPK signaling (Silwa *et al*, 2007).

**4.4 Glioma antigen presentation by microglia** 

Wagner and team (Wagner *et al*, 1999).

Contrary to the reports and assumptions, others demonstrated that TNF dependent action enhanced the macrophage/microglia recruitment in glioma where they form small cavities named microcysts and reduces the glioma growth (Villeneuve *et al*, 2005). A report stated that, the infiltrated macrophages (CD11b+CCR3-CD45high) caused TNF induced apoptosis in GL261 glioma cells where related microglial cells (CD11b+CCR3+CD45+) were negligible (Nakagawa *et al*, 2007). Actually the thin line of demarcation of cellular identity between macrophage and microglia could not exclude any of them from the function of TNF dependent glioma elimination. Opposing the recent believe of pro-tumoregenic role of macrophage population in different tumors, the reports raised question against application of anti-inflammatory drugs to suppress microglial action in glioma. To reestablish its role against glioma, the mechanism of their phagocytic recognition, killing machinery, and antigen presentation to CTL etc must have to be introspected more specifically.

#### **4.3 Microglia protects host brain as well as support glioma: A bi-edged sword**

Further findings showed that microglia helped glioma to invade by releasing matrix degrading enzymes. Even it was recently found that in rare Neurofibromatosis 1 (NF1), the heterozygote microglia had the role to promote glioma growth (Daginakatte & Gutmann, 2007). In 2005, it was found that metalloprotease-2 (MMP-2) activity was increased in microglia by the soluble factors released from glioma cells (Markovic *et al*, 2005). Thus glioma in turn influenced microglia to invade and migrate, which was utilized by neoplastic cells itself to spread and grow. Previously, a separate study hinted the process when the motility of GL261 mouse glioma cells was assessed in presence of microglia. Even the microglia stimulated with GM-CSF or LPS enhanced this migration (Bettinger *et al*, 2002). Adenosine mediated anti-inflammatory effects on macrophage cell lines by modulating the cytokine balance was observed. Additionally, macrophage and microglia both the cells were found to present adenosine receptors. In 2006, Synowtz and his team found the effect of nucleoside Adenosine on microglial cell and glioma. Deficiency of A1 adenosine receptor (A1AR) on microglia helped to grow the GL261 glioblastoma cells and increased number of A1AR expressing microglia in the site inhibited this growth. The mentioned study also hinted that adenosine signaling through the receptor depleted glioma influenced microglial MMP-2 release, which in turn restricted glioma growth and invasion (Synowitz *et al* 2006). Again, Kettenmann and colleagues observed that Microglia express membrane type 1 metalloprotease (MT1-MMP) in glioma condition, which helps to activate glioma released pro-MMP2 and thus promotes the spread of glioma in brain parenchyma (Markovic *et al*, 2009). Their most recent finding is that the antibiotic minocycline attenuates microglial MT1- MMP expression in glioma and as a result neoplastic cell expansion is reduced in glioma (Markovic *et al*, 2011).

Plasticity, its migration to the site of injury or inflammation, response and then departure from the site required a plausible explanation mostly for its movement. Microglia was found to express α6β1 integrin, which was the receptor for laminin expressed on the extracellular matrix constituent projections of astrocytes. This particular adhesion was for migration and under strict control of cytokine milieu (Milner & Campbell, 2002). It was found recently that another integrin α5β1 expressed both on glioma and microglial cells were capable of inhibiting glioma growth when attenuated. Remarkably it was found that, the attenuation process and resulting depletion of glioma required the presence of microglial cells (Färber *et al*, 2008). It might be probable that microglia secreting products had control over this integrin-laminin adhesion and migration of cell itself and invasive migration of glioma cells.

Contrary to the reports and assumptions, others demonstrated that TNF dependent action enhanced the macrophage/microglia recruitment in glioma where they form small cavities named microcysts and reduces the glioma growth (Villeneuve *et al*, 2005). A report stated that, the infiltrated macrophages (CD11b+CCR3-CD45high) caused TNF induced apoptosis in GL261 glioma cells where related microglial cells (CD11b+CCR3+CD45+) were negligible (Nakagawa *et al*, 2007). Actually the thin line of demarcation of cellular identity between macrophage and microglia could not exclude any of them from the function of TNF dependent glioma elimination. Opposing the recent believe of pro-tumoregenic role of macrophage population in different tumors, the reports raised question against application of anti-inflammatory drugs to suppress microglial action in glioma. To reestablish its role against glioma, the mechanism of their phagocytic recognition, killing machinery, and

antigen presentation to CTL etc must have to be introspected more specifically.

(Markovic *et al*, 2011).

**4.3 Microglia protects host brain as well as support glioma: A bi-edged sword** 

Further findings showed that microglia helped glioma to invade by releasing matrix degrading enzymes. Even it was recently found that in rare Neurofibromatosis 1 (NF1), the heterozygote microglia had the role to promote glioma growth (Daginakatte & Gutmann, 2007). In 2005, it was found that metalloprotease-2 (MMP-2) activity was increased in microglia by the soluble factors released from glioma cells (Markovic *et al*, 2005). Thus glioma in turn influenced microglia to invade and migrate, which was utilized by neoplastic cells itself to spread and grow. Previously, a separate study hinted the process when the motility of GL261 mouse glioma cells was assessed in presence of microglia. Even the microglia stimulated with GM-CSF or LPS enhanced this migration (Bettinger *et al*, 2002). Adenosine mediated anti-inflammatory effects on macrophage cell lines by modulating the cytokine balance was observed. Additionally, macrophage and microglia both the cells were found to present adenosine receptors. In 2006, Synowtz and his team found the effect of nucleoside Adenosine on microglial cell and glioma. Deficiency of A1 adenosine receptor (A1AR) on microglia helped to grow the GL261 glioblastoma cells and increased number of A1AR expressing microglia in the site inhibited this growth. The mentioned study also hinted that adenosine signaling through the receptor depleted glioma influenced microglial MMP-2 release, which in turn restricted glioma growth and invasion (Synowitz *et al* 2006). Again, Kettenmann and colleagues observed that Microglia express membrane type 1 metalloprotease (MT1-MMP) in glioma condition, which helps to activate glioma released pro-MMP2 and thus promotes the spread of glioma in brain parenchyma (Markovic *et al*, 2009). Their most recent finding is that the antibiotic minocycline attenuates microglial MT1- MMP expression in glioma and as a result neoplastic cell expansion is reduced in glioma

Plasticity, its migration to the site of injury or inflammation, response and then departure from the site required a plausible explanation mostly for its movement. Microglia was found to express α6β1 integrin, which was the receptor for laminin expressed on the extracellular matrix constituent projections of astrocytes. This particular adhesion was for migration and under strict control of cytokine milieu (Milner & Campbell, 2002). It was found recently that another integrin α5β1 expressed both on glioma and microglial cells were capable of inhibiting glioma growth when attenuated. Remarkably it was found that, the attenuation process and resulting depletion of glioma required the presence of microglial cells (Färber *et al*, 2008). It might be probable that microglia secreting products had control over this integrin-laminin adhesion and migration of cell itself and invasive migration of glioma cells. Regarding the cytokine microenvironment, the role of TGF-β was hinted in migration (Milner & Campbell, 2002). The specific importance of the cytokine was demonstrated by RNAi mediated gene silencing of TGF-β in promoting growth and invasiveness of glioma by integrin family adhesion molecule (Wesolowska *et al*, 2008). Recently it was found that cyclosporin A (CsA), an inhibitor of calcineurin and immunosuppressive in effect, could inhibit microglia mediated glioma invasion and cause to change morphological structure of microglia via MAPK signaling (Silwa *et al*, 2007).

In the present context, most of the studies demonstrated pro-tumerogenic action of microglia in glioma, which was facilitated by the secretary products, signaling molecules including cytokines, chemokines and receptors etc. In parallel, glioma cells favor microglial migration and encroachment in its vicinity. Though primarily macrophages/microglia are the cells to defend host tissue from faulty or malfunctioning cells or pathogens, their pro-glioma role leads to confusion. Remarkably, several findings also came with hopeful antagonistic results as already mentioned, where the roles of TNFα, TGFβ, A1AR dependent MMP-2 inhibition etc were focused (Villeneuve *et al*, 2005; Nakagawa *et al*, 2007; Synowitz *et al* 2006; Wesolowska *et al*, 2008). In 2007, Galarneau and team demonstrated that macrophage/microglia depletion helped in glioma growth (Galarneau *et al*, 2007). The study hinted for a separate anti-tumor potential of the cells. These contradicting results present microglia with a double agent stature.

#### **4.4 Glioma antigen presentation by microglia**

To determine the antigen presenting role of microglia their MHC class II expression along with the co-stimulatory molecule like B7.1 (CD80) and B7.2 (CD86) had been evaluated. Badie and his team found the lower level of expression of these essential surface molecule for APC function in microglia freshly isolated from glioma invasive cells and that suggested suppressive effect on glioma microenvironment *in vivo* (Badie & Schartner, 2001). In a comparative study of different rodent glioma model viz., C6, 9L and RG2, the expression profile was found to vary significantly depending on the immunogenicity of the model. The costimulatory B7 molecule expression could be favored when microglia were rejuvenated by cytokines GM-CSF and IFN-γ *in vitro* (Badie *et al*, 2002). At the same time, Graeber with his colleagues scanned 97 glioma samples of different WHO grades and found no such simple relations of the MHC expression of the cell with tumor grades, rather found downregulation of MHC class II in tissue areas where dense glioblastoma cells were infested. According to them microglia were functional in astrocytic tumors, though might be subjected to suppression with T cell clonal anergy in that glioma microenvironment (Tran *et al*, 1998).

The stimulatory effect of the novel glycoprotein T11TS/SLFA-3 on microglial MHC class II expression was found. The dose-time dependent efficient MHC expression was found on microglia in rodent bearing experimental glioma when treated with T11TS (Begum *et al*, 2004). Chaudhuri and her team also identified another important costimulatory molecule CD2 on microglia, which could also be regulated by the glycoprotein dose in glioma condition (Begum *et al*, 2004; Chaudhuri & Ghosh, 2006). A separate study by her team found simultaneous co-expression of MHC class II and CD2 on microglia in glioma where both expressed in low quantity (Sarkar *et al*, 2004). This observation with others supported the view of immunosuppressive milieu offered to microglia in glioma mostly by TGFβ, IL-10, PGE2, gangliosides etc (Zou *et al*, 1999; Graeber *et al*, 2002), which could also simultaneously cripple the infiltrated lymphocytes (Dix *et al*, 1999). In this regard, the fact that microglia was the source of that IL-10 in glioma, had been finally established by Wagner and team (Wagner *et al*, 1999).

Fig. 4. This schematic diagram shows the activities of immune components in brain during glioma. After activated glioma antigen specific T cells enter into brain parenchyma they progressed towards glioma through parenchyma. Simultaneously, during glioma pathogenesis resident resting microglia get activated, upregulate their receptors, enhance

Immune Connection in Glioma: Fiction, Fact and Option 317

Hemiberger and colleagues studied the myeloid lineage cells in post-operative tissue samples in human glioma. Accepting the cellular identity crisis, these workers found macrophage/microglia and dendritic cell populations within the tumor tissue. In their higher grade glioma samples microglial population though found to express MHC class II, lacked co-stimulatory CD80, CD86 and CD40, crucial for T cell functioning. Activation of microglia via Toll-like receptors (TLRs) were also insignificant to augment tumoricidal activity (Hussain *et al*, 2006a). Particularly, proinflammatory cytokines including IL-1β, IL-6, TNF α could not be sufficiently released to launch substantial innate immune function against glioma, however their phagocytic function was not impaired and also exhibited low level of non-specific cytotoxicity (Hussain *et al*, 2006b). In rodent glioma model lack of proinflammatory cytokines IFN γ, IL-12 and IL-6, and conversely cumulative dominance of IL-4 and IL-10 favoring the suppression of immune response was recently observed (Ghosh *et al*, 2010). Microglial activity stature was also reflected in their morphometry in scanning electron microscopy (SEM), where their filapodial extensions, sizes and shapes had shown

The cytotoxic effector function is an important part of CNS microglia/macrophage population which mainly dependents on its phagocytic mode of action. For the purpose super oxide anion production is the major function of phagocytic cells, however microglia generate sufficient endogenous NO in addition (Beyer *et al*, 2000). When microglial effector function in rat glioma model was studied, it was found that microglia mostly depends on NO production than ROS generation for exerting effector activity, whereas peripheral macrophages mainly depends on ROS for their normal phagocytic functions (Ghosh *et al*, 2007). With tumoricidal actions NO plays certain role in complex signaling network of cytokine production, angiogenesis etc in brain microenvironment. Even microglia was found to release iNOS/NO from astrocytoma cells in contact. This was by IL-1β production of activated microglia and probably via p38 MAPK and NF-κB signaling pathway (Kim *et al*,

their local antigen presenting capacity and move towards glioma vicinity where astrocytic projections may assist this movement by providing the pavement for integrin-laminin interaction. At the glioma site, microglia locally represent antigens, produce cytokines and adhesion molecules to dialogue with lymphocytes which help them to mature and attain final effector function. At this point, a triangular complex interaction circuit become active between infiltrated lymphocytes, microglia and glioma cells when glioma tries to cripple the immune attack by applying many of its tricks (as discussed in the text). Overall cytokine, chemokine, growth factor and immunosuppressive factor become crucial to determine the success of immune defense or glioma. At the next step, cytotoxic T cells exert perforin, granzyme, FasL and other cytotoxic means to kill glioma and microglia uses its reactive oxygen and nitrogen intermediates to damage the abnormal or neoplastic cells. Target cells, intact, damaged or dead debris are scavenged by microglia. If the immune system manages to overcome the situation for a certain period they return to homeostasis. But aggressive glioma, after the initial arrest, overrules the immune defense by its rapid proliferation rate, immune evasion strategies and diverse modes for bypassing the attack. Gaining dominance during the triangular interaction phase marks the success of the party in the succeeding

phases (Adopted from *Chaudhuri & Ghosh, 2006, CNSAMC*).

noticeable alterations (Begum *et al*, 2003).

**4.5 Microglia can destroy glioma cells** 

2006).

Fig. 4. This schematic diagram shows the activities of immune components in brain during glioma. After activated glioma antigen specific T cells enter into brain parenchyma they progressed towards glioma through parenchyma. Simultaneously, during glioma

pathogenesis resident resting microglia get activated, upregulate their receptors, enhance

their local antigen presenting capacity and move towards glioma vicinity where astrocytic projections may assist this movement by providing the pavement for integrin-laminin interaction. At the glioma site, microglia locally represent antigens, produce cytokines and adhesion molecules to dialogue with lymphocytes which help them to mature and attain final effector function. At this point, a triangular complex interaction circuit become active between infiltrated lymphocytes, microglia and glioma cells when glioma tries to cripple the immune attack by applying many of its tricks (as discussed in the text). Overall cytokine, chemokine, growth factor and immunosuppressive factor become crucial to determine the success of immune defense or glioma. At the next step, cytotoxic T cells exert perforin, granzyme, FasL and other cytotoxic means to kill glioma and microglia uses its reactive oxygen and nitrogen intermediates to damage the abnormal or neoplastic cells. Target cells, intact, damaged or dead debris are scavenged by microglia. If the immune system manages to overcome the situation for a certain period they return to homeostasis. But aggressive glioma, after the initial arrest, overrules the immune defense by its rapid proliferation rate, immune evasion strategies and diverse modes for bypassing the attack. Gaining dominance during the triangular interaction phase marks the success of the party in the succeeding phases (Adopted from *Chaudhuri & Ghosh, 2006, CNSAMC*).

Hemiberger and colleagues studied the myeloid lineage cells in post-operative tissue samples in human glioma. Accepting the cellular identity crisis, these workers found macrophage/microglia and dendritic cell populations within the tumor tissue. In their higher grade glioma samples microglial population though found to express MHC class II, lacked co-stimulatory CD80, CD86 and CD40, crucial for T cell functioning. Activation of microglia via Toll-like receptors (TLRs) were also insignificant to augment tumoricidal activity (Hussain *et al*, 2006a). Particularly, proinflammatory cytokines including IL-1β, IL-6, TNF α could not be sufficiently released to launch substantial innate immune function against glioma, however their phagocytic function was not impaired and also exhibited low level of non-specific cytotoxicity (Hussain *et al*, 2006b). In rodent glioma model lack of proinflammatory cytokines IFN γ, IL-12 and IL-6, and conversely cumulative dominance of IL-4 and IL-10 favoring the suppression of immune response was recently observed (Ghosh *et al*, 2010). Microglial activity stature was also reflected in their morphometry in scanning electron microscopy (SEM), where their filapodial extensions, sizes and shapes had shown noticeable alterations (Begum *et al*, 2003).

#### **4.5 Microglia can destroy glioma cells**

The cytotoxic effector function is an important part of CNS microglia/macrophage population which mainly dependents on its phagocytic mode of action. For the purpose super oxide anion production is the major function of phagocytic cells, however microglia generate sufficient endogenous NO in addition (Beyer *et al*, 2000). When microglial effector function in rat glioma model was studied, it was found that microglia mostly depends on NO production than ROS generation for exerting effector activity, whereas peripheral macrophages mainly depends on ROS for their normal phagocytic functions (Ghosh *et al*, 2007). With tumoricidal actions NO plays certain role in complex signaling network of cytokine production, angiogenesis etc in brain microenvironment. Even microglia was found to release iNOS/NO from astrocytoma cells in contact. This was by IL-1β production of activated microglia and probably via p38 MAPK and NF-κB signaling pathway (Kim *et al*, 2006).

Immune Connection in Glioma: Fiction, Fact and Option 319

2010). Even some of the approaches interestingly propose microglia as an effective vehicle for gene therapy and drug delivery by using its predifferentiated cellular status (Neumann, 2006). Present immunotherapeutic advancements and limitations will be discussed in other chapters of this book and beyond the scope of this article. In this essay, the basic immune mechanisms, which are active in glioma, has been detailed. Further development of effective therapy needs this fundamental background knowledge for setting up a new

I am thankful to Prof Swapna Chaudhuri of School of Tropical Medicine, Kolkata who initiated my drive in this field and constantly supported me with valuable suggestions. I also admit the cooperation of Debasis Das to develop the illustrations and support of my

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**6. Acknowledgement** 

**7. References** 

Expression of Fas ligand on a cell may cause damage to adjoining cells or infiltrated activated lymphocytes in the tissue by triggering the death pathway. In glioma, FasL expression was thought to be a means of immune escape, whereas, investigations found FasL expression also on microglial cells in glioma (Badie *et al*, 2001). Hence, it could be thought that microglia would play role in local immune suppression by limiting the lymphocyte populations; or it might express FasL to damage glioma cells, which in turn restricted or crippled the lymphocytes infiltrated locally. Microglia prefers to phagocytose the damaged cells rather than intact ones (Bohatschek *et al*, 2001). A study showed that after adoptive transfer of alloreactive cytotoxic T cells in rat 9L gliosarcoma infested brain, the CTL damaged glioma cells were removed by phagocytic scavenging activities of microglia, whereas undamaged ones were spared (Kulprathipanja & Kruse, 2004). In this process of either Fas or oxidative stress mediated cell death, externalization of phosphatidylserine was found to be crucial in corpse clearance by microglia. Experimental evidences revealed increasing PS externalization in correlation with cytotoxic effector functions of microglia and infiltrated lymphocytes. Additionally, the investigation showed that microglia population was more steady than infiltrated lymphocytes as Bcl-2 aided the cells to maintain that steady turnover rate and low apoptosis in brain microenvironment (Kagan *et al*, 2002 & Ghosh *et al*, 2007).

#### **5. Conclusion: Future immuno-therapeutics must capitalize this resident and infiltrated immunocyte liaison to combat glioma**

Now it can be clearly stated that lymphocytes can enter into the brain parenchyma. During their entry, they are checked by the antigen presenting cells for their glioma antigen specificity. APCs try to ensure this glioma specific T cell entry by restimulating the candidates presenting the glioma antigens mostly in perivascular space and allied blood brain interface (Engelherdt, 2010). After entering they migrate to glioma and again interact with the local APCs which help them to gain maturation and final effector function. In this process peripheral APC in brain and 'so called' resident microglia as well as DCs also play very important role. The role of local APC in glioma immunity has been detailed by Ghosh and Chaudhuri with a new outlook to explain the contradiction of glioma promoting role of microglia (Ghosh and Chaudhuri, 2010).These myeloid cell populations have the potential to act as chief immunomodulator in brain by surveillance in the tissue environment, guiding the leukocytes in CNS and simultaneously exerting effector function to neoplastic cells. The damaging activities of the cell are probably their own misfired 'goodness' or their potential that is misled by glioma cells. Thus, both the lymphocytes and local APC (predominantly microglia) are capable of exerting the immune effector function against glioma in spite of the immune-compromise in the brain and glioma immune evasion strategies [Figure – 4].

Now the success of immunotherapeutic approaches against glioma, largely as adjuvant therapeutic strategy, depends on the proper usages of this delicate immune defense against this detrimental threat. The battle becomes more interesting and challenging because the opponent is extremely clever. So development of immunotherapeutic strategies against glioma needs detailed and critical interpretation of the work-plan of immunity in glioma and its careful application. Basic findings are increasing the repertoire of information about immune activity deep into the brain during glioma which in turn provide us newer tactics or facilitate the amendment of old ones for better effects (Dietrich *et al*, 2010; Vauleon *et al*, 2010). Even some of the approaches interestingly propose microglia as an effective vehicle for gene therapy and drug delivery by using its predifferentiated cellular status (Neumann, 2006). Present immunotherapeutic advancements and limitations will be discussed in other chapters of this book and beyond the scope of this article. In this essay, the basic immune mechanisms, which are active in glioma, has been detailed. Further development of effective therapy needs this fundamental background knowledge for setting up a new immunotherapeutic intervention against glioma.

#### **6. Acknowledgement**

I am thankful to Prof Swapna Chaudhuri of School of Tropical Medicine, Kolkata who initiated my drive in this field and constantly supported me with valuable suggestions. I also admit the cooperation of Debasis Das to develop the illustrations and support of my colleagues and students to continue my work.

#### **7. References**

318 Glioma – Exploring Its Biology and Practical Relevance

Expression of Fas ligand on a cell may cause damage to adjoining cells or infiltrated activated lymphocytes in the tissue by triggering the death pathway. In glioma, FasL expression was thought to be a means of immune escape, whereas, investigations found FasL expression also on microglial cells in glioma (Badie *et al*, 2001). Hence, it could be thought that microglia would play role in local immune suppression by limiting the lymphocyte populations; or it might express FasL to damage glioma cells, which in turn restricted or crippled the lymphocytes infiltrated locally. Microglia prefers to phagocytose the damaged cells rather than intact ones (Bohatschek *et al*, 2001). A study showed that after adoptive transfer of alloreactive cytotoxic T cells in rat 9L gliosarcoma infested brain, the CTL damaged glioma cells were removed by phagocytic scavenging activities of microglia, whereas undamaged ones were spared (Kulprathipanja & Kruse, 2004). In this process of either Fas or oxidative stress mediated cell death, externalization of phosphatidylserine was found to be crucial in corpse clearance by microglia. Experimental evidences revealed increasing PS externalization in correlation with cytotoxic effector functions of microglia and infiltrated lymphocytes. Additionally, the investigation showed that microglia population was more steady than infiltrated lymphocytes as Bcl-2 aided the cells to maintain that steady turnover rate and low apoptosis in brain microenvironment (Kagan *et al*, 2002 &

**5. Conclusion: Future immuno-therapeutics must capitalize this resident and** 

Now it can be clearly stated that lymphocytes can enter into the brain parenchyma. During their entry, they are checked by the antigen presenting cells for their glioma antigen specificity. APCs try to ensure this glioma specific T cell entry by restimulating the candidates presenting the glioma antigens mostly in perivascular space and allied blood brain interface (Engelherdt, 2010). After entering they migrate to glioma and again interact with the local APCs which help them to gain maturation and final effector function. In this process peripheral APC in brain and 'so called' resident microglia as well as DCs also play very important role. The role of local APC in glioma immunity has been detailed by Ghosh and Chaudhuri with a new outlook to explain the contradiction of glioma promoting role of microglia (Ghosh and Chaudhuri, 2010).These myeloid cell populations have the potential to act as chief immunomodulator in brain by surveillance in the tissue environment, guiding the leukocytes in CNS and simultaneously exerting effector function to neoplastic cells. The damaging activities of the cell are probably their own misfired 'goodness' or their potential that is misled by glioma cells. Thus, both the lymphocytes and local APC (predominantly microglia) are capable of exerting the immune effector function against glioma in spite of the immune-compromise in the brain and glioma immune evasion strategies [Figure – 4]. Now the success of immunotherapeutic approaches against glioma, largely as adjuvant therapeutic strategy, depends on the proper usages of this delicate immune defense against this detrimental threat. The battle becomes more interesting and challenging because the opponent is extremely clever. So development of immunotherapeutic strategies against glioma needs detailed and critical interpretation of the work-plan of immunity in glioma and its careful application. Basic findings are increasing the repertoire of information about immune activity deep into the brain during glioma which in turn provide us newer tactics or facilitate the amendment of old ones for better effects (Dietrich *et al*, 2010; Vauleon *et al*,

**infiltrated immunocyte liaison to combat glioma** 

Ghosh *et al*, 2007).


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Nakamura Y (2002). Regulating factors for microglial activation. Biol Pharm Bull 25: 945-953.


**15** 

*Russia* 

Olga Leplina et al.\*

*Institute of Clinical Immunology SB RAMS* 

**Direct Antitumor Activity of Interferon-Induced** 

Dendritic cells (DCs) are well known for their capacity to induce adaptive antitumor immune response through their unique ability to uptake, processing and presenting antigens (Ags), and tumor-specific T cell activation. In addition, cytokines produced by dendritic cells are able to regulate the direction and strength of immune response, activate the cytotoxic cells (NK-, NKT-cells) and participate in the coordination of the humoral

An increasing number of reports evidenced that besides this role, DCs may display additional antitumor effects. Indeed, DCs in vitro can inhibit proliferation and provide a direct cytotoxic effect on tumor cells. In this, human monocyte-derived DCs might exert antitumor activity through multiple TNF family members (i.e. TNF-α, lymphotoxin-α1β2, FasL, TRAIL), as well as perforin and/or granzyme (Wesa & Storkus, 2008; Chauvin &

Direct tumor cell killing by DCs themselves appear to be highly important since involves immediate presentation of tumor-associated Ags in the context of MHC molecules for recognition by cognate T cells, inducing a specific immune response. Importantly, pleiotropy in DC mechanisms of cytotoxicity allows DCs to overcome the resistance of tumor cells that are heterogeneous with regard to their sensitivity to the various death pathways. A number of evidence suggests that the direct antitumor effect of DCs is not purely in vitro phenomenon, and is implemented in vivo. First, DCs are present in the tumors, and their higher content correlates with a more favorable prognosis (Becker 1999). Second, intra-tumoral injection of intact DC (not loaded with tumor antigen) has been shown to correlate with reduced tumor growth and even regression (Becker et al., 2001; Ehtesham et al., 2003). Finally, it is shown that the intra-tumoral introduction of DCs improves the effectiveness of chemotherapy, which may be due to synergistic effects of

Tamara Tyrinova1, Marina Tikhonova1, Ekaterina Shevela1, Vyacheslav Stupak2, Sergey Mishinov2,

Ivan Pendyurin2, Mikhail Sadovoy2, Alexander Ostanin1 and Elena Chernykh1

immune response (Melief, 2008; Banchereau et al., 2000).

cytostatics and DCs (Vanderheyde et al., 2004).

*1Institute of Clinical Immunology SB RAMS, Russia 2Institute of Traumatology and Orthopedics,Russia* 

**1. Introduction** 

Josien, 2008).

 \*

**Dendritic Cells of Healthy Donors** 

**and Patients with Primary Brain Tumors** 


## **Direct Antitumor Activity of Interferon-Induced Dendritic Cells of Healthy Donors and Patients with Primary Brain Tumors**

Olga Leplina et al.\* *Institute of Clinical Immunology SB RAMS Russia* 

#### **1. Introduction**

324 Glioma – Exploring Its Biology and Practical Relevance

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Uyttenhove C, Pilotte L, Theate I *et al* (2003). Evidence for a tumoral immune resistance

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Villeneuve J, Tremblay P and Vallières L (2005). Tumor necrosis factor reduces brain tumor

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resident microglial cells from the normal and inflamed central nervous system.

by proinflammatory cytokines influences IDO activation in epithelial cells. J Int Cyt

Dendritic cells (DCs) are well known for their capacity to induce adaptive antitumor immune response through their unique ability to uptake, processing and presenting antigens (Ags), and tumor-specific T cell activation. In addition, cytokines produced by dendritic cells are able to regulate the direction and strength of immune response, activate the cytotoxic cells (NK-, NKT-cells) and participate in the coordination of the humoral immune response (Melief, 2008; Banchereau et al., 2000).

An increasing number of reports evidenced that besides this role, DCs may display additional antitumor effects. Indeed, DCs in vitro can inhibit proliferation and provide a direct cytotoxic effect on tumor cells. In this, human monocyte-derived DCs might exert antitumor activity through multiple TNF family members (i.e. TNF-α, lymphotoxin-α1β2, FasL, TRAIL), as well as perforin and/or granzyme (Wesa & Storkus, 2008; Chauvin & Josien, 2008).

Direct tumor cell killing by DCs themselves appear to be highly important since involves immediate presentation of tumor-associated Ags in the context of MHC molecules for recognition by cognate T cells, inducing a specific immune response. Importantly, pleiotropy in DC mechanisms of cytotoxicity allows DCs to overcome the resistance of tumor cells that are heterogeneous with regard to their sensitivity to the various death pathways. A number of evidence suggests that the direct antitumor effect of DCs is not purely in vitro phenomenon, and is implemented in vivo. First, DCs are present in the tumors, and their higher content correlates with a more favorable prognosis (Becker 1999). Second, intra-tumoral injection of intact DC (not loaded with tumor antigen) has been shown to correlate with reduced tumor growth and even regression (Becker et al., 2001; Ehtesham et al., 2003). Finally, it is shown that the intra-tumoral introduction of DCs improves the effectiveness of chemotherapy, which may be due to synergistic effects of cytostatics and DCs (Vanderheyde et al., 2004).

*1Institute of Clinical Immunology SB RAMS, Russia* 

<sup>\*</sup> Tamara Tyrinova1, Marina Tikhonova1, Ekaterina Shevela1, Vyacheslav Stupak2, Sergey Mishinov2, Ivan Pendyurin2, Mikhail Sadovoy2, Alexander Ostanin1 and Elena Chernykh1

*<sup>2</sup>Institute of Traumatology and Orthopedics,Russia* 

Direct Antitumor Activity of Interferon-Induced

under standard cell culture conditions.

**2.1.4 Cytotoxicity assay** 

**2.1.3 Cell lines** 

the formula:

**2.1.5 Cytostatic assay** 

**2.1.6 Apoptosis detection** 

region studied.

Trypan blue exclusion was more than 93-95% in all cases.

Dendritic Cells of Healthy Donors and Patients with Primary Brain Tumors 327

and 48h, respectively. For some experiments DC supernatans generated from LPS-activated IFNα-DCs were collected. The viability of obtained IFNα–DCs or IL4–DCs determined by

Tumor cell lines used in this study included leukemia cell line Jurkat (T- lymphoblast cell leukemia) and solid tumor-derived cell lines: epithelial cells of human larynx carcinoma HEp-2 and glioblastoma U-87 were purchased from American Type Culture Collection (Manassas, VA). All cell lines were of human origin, mycoplasma free and were grown

Generated IFNα–DCs were tested for their cytotoxic activity against various tumor cell lines including Jurkat, HEp-2 and U-87. Before coculture, target cells were labeled with [3H]thymidine (1 µCi/well) for 18 h at 37°C, washed and placed at 104/well in 96-well tissue culture plates in RPMI-1640 medium containing 10% FCS. Cell-free supernatants from DC cultures (30%, v/v) or different numbers of effector cells (DCs) were added to tumor cells at effector:target (E:T) ratios of 10:1, 20:1 and 40:1. In some experiments DCs were preincubated for 1 h with the following fusion proteins: rhTNFR1/TNFRSF1A Fc chimera (10 μg/ml), rhFas/TNFRSF6/CD95 Fc chimera (10 μg/ml), and rhTRAIL R2/TNFRSF10B Fc chimera (10 μg/ml; all reagents from R & D Systems, USA). After 18 h of culture cells were harvested and thymidine incorporation was measured on a Liquid Scintillation beta-Counter (Packard Instrument, Meriden, CT). Percentage of cytotoxicity was calculated by

[1 - (cpm in cocultures of tumor and effector cells or DC supernatans/ cpm in tumor cell cultures)] x 100%.

Cytostatic activity of DCs was evaluated by their ability to suppress the proliferation of tumor line cells (HEp-2 and U-87). For this, the target cells (103/well) were incubated for 48 h in 96-well plates alone and in the presence of effector cells at E:T ratios about 10:1, 20:1 and 40:1. Cell proliferation was measured by [3H]thymidine incorporation (1 μCi/well for last 24

[1 - (cpm in cultures with effector cells / cpm in control cultures)] x 100%.

To determine the level of apoptosis, HEp-2 tumor cells were preliminary stained with vital dye CFSE (2 mM, Molecular probes, USA) for 15 min, then washed in RPMI-1640/10% FCS and incubated in 96-well tissue culture plates (104/well) in the presence of IFNα-DCs at a ratio 10:1 for 18 hours. The number of cell divisions was analyzed by flow cytometry (FACS Calibur, Becton Dickinson, USA) on channel FL1 (CFSE fluorescence) with the emission of 517 nm. The level of apoptosis was detected by DNA intercalating dye 7-AAD (Calboichem, Israel). Results were expressed as a percentage of positive cells to the total cell number in the

h). The percentage of cytostatic activity was calculated by the formula:

Most studies on antitumor activity of DCs in humans were performed with myeloid DCs isolated from peripheral blood or generate in vitro from peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Cytotoxic activity of these DCs was found to be stronger after treatment with type I interferons (IFN) or IFNγ (Liu et al., 2001; Fanger et al., 1999). More recently, monocytederived DCs generated in the presence of GM-CSF and IFNα instead of IL-4 were described. Is also known that IFNα-DCs are characterized by a higher expression of some molecules (TRAIL, FasL), which may mediate the cytotoxic/cytostatic activity of DCs (Chauvin & Josien, 2008). Indeed, we have demonstrated that LPS-activated IFNα-DCs inhibit the growth of tumor cell line HEp-2 more efficiently than IL4-DCs (Leplina et al., 2010). However, the antitumor potential of DCs generated in the presence of IFNα, remain virtually unexplored.

Of great relevance is also an issue on safety of DC cytotoxic activity in cancer settings. Recently, we found that in patients with malignant gliomas IFNα-DCs generated in vitro are able to activate Th1 cells and induce an antitumor immune response (Leplina et al., 2007a). Nevertheless, these DCs exhibit several phenotypic and functional features, such as the moderate delay of differentiation/maturation and low capacity to induce the IFNγproducing T-cells in mixt lymphocyte culture (MLC) (Leplina et al., 2007b). Given the data on the development of immune insufficiency and monocyte dysfunctions in patients with malignant brain tumors (Khonina et al., 2002) the study of various functions of DCs in this pathology is important not only in terms of understanding pathogenesis of the disease, but also to rationale the therapy with dendritic cells. In this article we investigated the cytotoxic potential of human monocyte-derived DCs generated under replacement of IL-4 with IFNα, and compared cytostatic/cytotoxic activities of IFNα-induced DCs in healthy donors and patients with brain tumors.

#### **2. The text of the article**

#### **2.1 Materials and methods**

#### **2.1.1 Patients**

The study was held in 32 healthy volunteers and 37 patients with brain tumors (21 men and 16 women; from 21 to 71 years; median age 37 years). Patients' group included 20 patients with histologically verified gliblastoma (Grade IV), 8 - with astrocytoma (Grade III) and 9 with angioreticuloma, fibrilyarno-protoplasmic astrocytoma or meningioma (Grade I-II). All studies were performed after receiving a written informed consent.

#### **2.1.2 In vitro differentiation and maturation of DCs**

Peripheral blood mononuclear cells (MNCs) were obtained by density gradient centrifugation (Ficoll-Paque, Sigma-Aldrich) of heparinized whole blood samples. Dendritic cells were generated by culturing of plastic-adherent MNC fraction in 6-well plates (Nunclon, Denmark) in RPMI-1640 medium (Sigma-Aldrich), supplemented with 0,3 mg/ml L-glutamine, 5 mM HEPES buffer, 100 µg/ml gentamicin and 5% fetal calf serum (FCS, Sigma-Aldrich), in the presence of recombinant human (rh) GM-CSF (40 ng/ml, Sigma-Aldrich) and rhIFN-α (Roferon-A, 1000 U/ml, Roche, Switzerland) for 4 days (IFNα-DCs) or with rhGM-CSF (40 ng/ml) and IL-4 (40 ng/ml, Sigma-Aldrich) for 5 days ( IL4- DCs). The resulting immature DCs were further exposed with 10 µg/ml lipopolysaccharide (LPS E.colli 0114: B4, Sigma-Aldrich) into IFNα-DC and IL4-DC cultures for additional 24h and 48h, respectively. For some experiments DC supernatans generated from LPS-activated IFNα-DCs were collected. The viability of obtained IFNα–DCs or IL4–DCs determined by Trypan blue exclusion was more than 93-95% in all cases.

#### **2.1.3 Cell lines**

326 Glioma – Exploring Its Biology and Practical Relevance

Most studies on antitumor activity of DCs in humans were performed with myeloid DCs isolated from peripheral blood or generate in vitro from peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Cytotoxic activity of these DCs was found to be stronger after treatment with type I interferons (IFN) or IFNγ (Liu et al., 2001; Fanger et al., 1999). More recently, monocytederived DCs generated in the presence of GM-CSF and IFNα instead of IL-4 were described. Is also known that IFNα-DCs are characterized by a higher expression of some molecules (TRAIL, FasL), which may mediate the cytotoxic/cytostatic activity of DCs (Chauvin & Josien, 2008). Indeed, we have demonstrated that LPS-activated IFNα-DCs inhibit the growth of tumor cell line HEp-2 more efficiently than IL4-DCs (Leplina et al., 2010). However, the antitumor potential of DCs generated in the presence of IFNα, remain

Of great relevance is also an issue on safety of DC cytotoxic activity in cancer settings. Recently, we found that in patients with malignant gliomas IFNα-DCs generated in vitro are able to activate Th1 cells and induce an antitumor immune response (Leplina et al., 2007a). Nevertheless, these DCs exhibit several phenotypic and functional features, such as the moderate delay of differentiation/maturation and low capacity to induce the IFNγproducing T-cells in mixt lymphocyte culture (MLC) (Leplina et al., 2007b). Given the data on the development of immune insufficiency and monocyte dysfunctions in patients with malignant brain tumors (Khonina et al., 2002) the study of various functions of DCs in this pathology is important not only in terms of understanding pathogenesis of the disease, but also to rationale the therapy with dendritic cells. In this article we investigated the cytotoxic potential of human monocyte-derived DCs generated under replacement of IL-4 with IFNα, and compared cytostatic/cytotoxic activities of IFNα-induced DCs in healthy donors and

The study was held in 32 healthy volunteers and 37 patients with brain tumors (21 men and 16 women; from 21 to 71 years; median age 37 years). Patients' group included 20 patients with histologically verified gliblastoma (Grade IV), 8 - with astrocytoma (Grade III) and 9 with angioreticuloma, fibrilyarno-protoplasmic astrocytoma or meningioma (Grade I-II). All

Peripheral blood mononuclear cells (MNCs) were obtained by density gradient centrifugation (Ficoll-Paque, Sigma-Aldrich) of heparinized whole blood samples. Dendritic cells were generated by culturing of plastic-adherent MNC fraction in 6-well plates (Nunclon, Denmark) in RPMI-1640 medium (Sigma-Aldrich), supplemented with 0,3 mg/ml L-glutamine, 5 mM HEPES buffer, 100 µg/ml gentamicin and 5% fetal calf serum (FCS, Sigma-Aldrich), in the presence of recombinant human (rh) GM-CSF (40 ng/ml, Sigma-Aldrich) and rhIFN-α (Roferon-A, 1000 U/ml, Roche, Switzerland) for 4 days (IFNα-DCs) or with rhGM-CSF (40 ng/ml) and IL-4 (40 ng/ml, Sigma-Aldrich) for 5 days ( IL4- DCs). The resulting immature DCs were further exposed with 10 µg/ml lipopolysaccharide (LPS E.colli 0114: B4, Sigma-Aldrich) into IFNα-DC and IL4-DC cultures for additional 24h

studies were performed after receiving a written informed consent.

**2.1.2 In vitro differentiation and maturation of DCs** 

virtually unexplored.

patients with brain tumors.

**2. The text of the article 2.1 Materials and methods** 

**2.1.1 Patients** 

Tumor cell lines used in this study included leukemia cell line Jurkat (T- lymphoblast cell leukemia) and solid tumor-derived cell lines: epithelial cells of human larynx carcinoma HEp-2 and glioblastoma U-87 were purchased from American Type Culture Collection (Manassas, VA). All cell lines were of human origin, mycoplasma free and were grown under standard cell culture conditions.

#### **2.1.4 Cytotoxicity assay**

Generated IFNα–DCs were tested for their cytotoxic activity against various tumor cell lines including Jurkat, HEp-2 and U-87. Before coculture, target cells were labeled with [3H]thymidine (1 µCi/well) for 18 h at 37°C, washed and placed at 104/well in 96-well tissue culture plates in RPMI-1640 medium containing 10% FCS. Cell-free supernatants from DC cultures (30%, v/v) or different numbers of effector cells (DCs) were added to tumor cells at effector:target (E:T) ratios of 10:1, 20:1 and 40:1. In some experiments DCs were preincubated for 1 h with the following fusion proteins: rhTNFR1/TNFRSF1A Fc chimera (10 μg/ml), rhFas/TNFRSF6/CD95 Fc chimera (10 μg/ml), and rhTRAIL R2/TNFRSF10B Fc chimera (10 μg/ml; all reagents from R & D Systems, USA). After 18 h of culture cells were harvested and thymidine incorporation was measured on a Liquid Scintillation beta-Counter (Packard Instrument, Meriden, CT). Percentage of cytotoxicity was calculated by the formula:

[1 - (cpm in cocultures of tumor and effector cells or DC supernatans/ cpm in tumor cell cultures)] x 100%.

#### **2.1.5 Cytostatic assay**

Cytostatic activity of DCs was evaluated by their ability to suppress the proliferation of tumor line cells (HEp-2 and U-87). For this, the target cells (103/well) were incubated for 48 h in 96-well plates alone and in the presence of effector cells at E:T ratios about 10:1, 20:1 and 40:1. Cell proliferation was measured by [3H]thymidine incorporation (1 μCi/well for last 24 h). The percentage of cytostatic activity was calculated by the formula:

[1 - (cpm in cultures with effector cells / cpm in control cultures)] x 100%.

#### **2.1.6 Apoptosis detection**

To determine the level of apoptosis, HEp-2 tumor cells were preliminary stained with vital dye CFSE (2 mM, Molecular probes, USA) for 15 min, then washed in RPMI-1640/10% FCS and incubated in 96-well tissue culture plates (104/well) in the presence of IFNα-DCs at a ratio 10:1 for 18 hours. The number of cell divisions was analyzed by flow cytometry (FACS Calibur, Becton Dickinson, USA) on channel FL1 (CFSE fluorescence) with the emission of 517 nm. The level of apoptosis was detected by DNA intercalating dye 7-AAD (Calboichem, Israel). Results were expressed as a percentage of positive cells to the total cell number in the region studied.

Direct Antitumor Activity of Interferon-Induced

molecules but not secreted proteins.

of tumor cells using an apoptotic mechanism.

**0**

**5**

**10**

**cytotoxic activity (%)**

**15**

**20**

Dendritic Cells of Healthy Donors and Patients with Primary Brain Tumors 329

To examine the possible mechanisms underlying the cytotoxic activity of IFNα-DCs against tumor cells, we evaluated the cytotoxic activity associated with DC culture-conditioned medium. Contrary to DCs themselves, IFNα-DC culture-conditioned medium lacked cytotoxic activity or had a low ability to lyse tumor cells. Indeed, supernatants of DC cultures added to targets at 30% (v/v) were unable to lyse HEp-2 cells, and had some cytotoxic activity against Jurkat cell line (13,0 ± 1,7%) (Fig.2). These results showed that mediators of DC-associated antitumor activity are more likely cell membrane-bound

To investigate whether DC killer activity involved the induction of apoptosis, we next analyzed cell cycle in tumor cells HEp-2 pre-labeled with vital dye CFSE (Fig.3). Coculturing of CFSE-labeled HEp-2 cells with IFNα-DCs resulted in significant increase in the level of apoptosis detected in tumor cells. In addition, the cultivation of tumor cells with IFNα-DCs was accompanied by a decrease in number of cycling tumor cells (S + G2M phases of the cell cycle). These results showed that DCs in vitro can efficiently induce death

**HEp-2 Jurkat**

Fig. 2. Cytotoxic activity of DC culture-conditioned medium against Jurkat and HEp-2. The figure represents individual values of cytotoxic activity mediated by supernatants from cultures of healthy donor IFNα-DCs against tumor cell lines HEp-2 (n = 17) and Jurkat (n = 6). Jurkat cells and HEp-2 cells (104cells/well) were labeled with [3H]thymidine and

These data result in the suggestion that the cytotoxic activity of IFNα-DCs is conditioned by induction of apoptosis in tumor cells and that, along with a cytotoxic effect, IFNα-DCs apparently could block cell cycle in tumor cells, thereby providing cytostatic effect. Indeed, analysis of IFNα-DCs impact on the proliferation of tumor cells (Fig. 4) revealed a

incubated with DC culture-conditioned medium (30%, v/v) for 18 hours.

**2.2.3 Growth inhibition effect of IFNα-DCs on tumor cell lines HEp-2 and U-87** 

pronounced antiproliferative effect exerted by donor DCs against the cell line HEp-2.

PU= 0,028

**2.2.2 Induction of apoptosis of tumor cell line HEp-2 by IFNα-DCs** 

#### **2.1.7 TNFα production**

DC-free supernatants collected as deascribe above were measured for soluble TNFα by ELISA using a commercial kit (R & D Systems, USA) according to the manufacturer`s recommendations.

#### **2.1.8 Statistical analysis**

The data were expressed as mean ± SE. Statistica 6.0 software for Windows (StatSoft Inc. USA) was used for analysis of data. Statistical comparisons were performed using the nonparametric Mann-Whitney U test. P-values < 0,05 indicate significant differences.

#### **2.2 Results**

#### **2.2.1 Cytotoxic activity of donor IFNα-DCs and DC supernatants against tumor cell lines**

First, we assessed whether in vitro generated mature IFNα-DCs could lyse tumor cell lines. IFNα-DCs in our study possessed significant dose-dependent cytotoxic activity against [3Н]thymidine-labeled tumor cell lines HЕp-2 and Jurkat. As illustrated in Fig.1A, Jurkat cells were lysed more efficiently at all E:T cell ratios. The most pronounced differences in cytotoxic activity of IFNα-DCs against Jurkat and HEp-2 were observed at a E:T ratio of 20:1 (35,17 ± 5,6% and 16,44 ± 4,01 %, respectively; PU=0,027). High cytotoxic activity of IFNα-DCs was also manifested when human glioblastoma U-87 cells were used as targets (Fig.1B). In this case, the cytotoxic potential of DCs at 20:1 was two-fold higher than with HEp-2 cells (38,6 ± 8,3%; PU=0,049). Taken together, these data indicate that i) mature IFNα-DCs mediated significant antitumor cytotoxic activity, effective at various E:T cell ratios, and ii) the cytotoxic activity of IFNα-DCs against tumor cell lines Jurkat and U-87 unlike HEp-2 cells was considerably higher.

Fig. 1. Cytotoxic activity of donor IFNα-DCs against Jurkat, Hep-2, and U-87. A) The average values of IFNα-DC cytotoxic activity against tumor cell lines Jurkat and HEp-2 are presented. Effector cells (DCs) and [3H]thymidine-labeled target cells (Jurkat and HEp-2) were cultured for 18 h at ratios indicated. B) Cytotoxic activity of IFNα-DCs against Jurkat, HEp-2 and U-87 tumor cells at E:T ratio of 20:1. Results are shown as mean ± SE of triplicate values. \* - PU <0,05 - between HEp-2 and Jurkat at E:T ratio of 20:1 (U - Wilcoxon's test, Mann-Whitney).

DC-free supernatants collected as deascribe above were measured for soluble TNFα by ELISA using a commercial kit (R & D Systems, USA) according to the manufacturer`s

The data were expressed as mean ± SE. Statistica 6.0 software for Windows (StatSoft Inc. USA) was used for analysis of data. Statistical comparisons were performed using the

nonparametric Mann-Whitney U test. P-values < 0,05 indicate significant differences.

Fig. 1. Cytotoxic activity of donor IFNα-DCs against Jurkat, Hep-2, and U-87. A) The average values of IFNα-DC cytotoxic activity against tumor cell lines Jurkat and HEp-2 are presented. Effector cells (DCs) and [3H]thymidine-labeled target cells (Jurkat and HEp-2) were cultured for 18 h at ratios indicated. B) Cytotoxic activity of IFNα-DCs against Jurkat, HEp-2 and U-87 tumor cells at E:T ratio of 20:1. Results are shown as mean ± SE of triplicate values. \* - PU <0,05 - between HEp-2 and Jurkat at E:T ratio of 20:1 (U - Wilcoxon's test,

**НEр-2(n=9) Jurkat (n=6)**

**B**

**cytotoxic activity (%)**

**Jurkat (n=6) HEp-2 (n=9) U-87 (n=5)**

PU= 0,027 PU= 0,049

**2.2.1 Cytotoxic activity of donor IFNα-DCs and DC supernatants against tumor cell** 

First, we assessed whether in vitro generated mature IFNα-DCs could lyse tumor cell lines. IFNα-DCs in our study possessed significant dose-dependent cytotoxic activity against [3Н]thymidine-labeled tumor cell lines HЕp-2 and Jurkat. As illustrated in Fig.1A, Jurkat cells were lysed more efficiently at all E:T cell ratios. The most pronounced differences in cytotoxic activity of IFNα-DCs against Jurkat and HEp-2 were observed at a E:T ratio of 20:1 (35,17 ± 5,6% and 16,44 ± 4,01 %, respectively; PU=0,027). High cytotoxic activity of IFNα-DCs was also manifested when human glioblastoma U-87 cells were used as targets (Fig.1B). In this case, the cytotoxic potential of DCs at 20:1 was two-fold higher than with HEp-2 cells (38,6 ± 8,3%; PU=0,049). Taken together, these data indicate that i) mature IFNα-DCs mediated significant antitumor cytotoxic activity, effective at various E:T cell ratios, and ii) the cytotoxic activity of IFNα-DCs against tumor cell lines Jurkat and U-87 unlike HEp-2

**2.1.7 TNFα production** 

**2.1.8 Statistical analysis** 

cells was considerably higher.

**\***

**40:1 20:1 10:1 effector:target**

Mann-Whitney).

**cytotoxic activity (%)**

**A**

recommendations.

**2.2 Results** 

**lines** 

To examine the possible mechanisms underlying the cytotoxic activity of IFNα-DCs against tumor cells, we evaluated the cytotoxic activity associated with DC culture-conditioned medium. Contrary to DCs themselves, IFNα-DC culture-conditioned medium lacked cytotoxic activity or had a low ability to lyse tumor cells. Indeed, supernatants of DC cultures added to targets at 30% (v/v) were unable to lyse HEp-2 cells, and had some cytotoxic activity against Jurkat cell line (13,0 ± 1,7%) (Fig.2). These results showed that mediators of DC-associated antitumor activity are more likely cell membrane-bound molecules but not secreted proteins.

#### **2.2.2 Induction of apoptosis of tumor cell line HEp-2 by IFNα-DCs**

To investigate whether DC killer activity involved the induction of apoptosis, we next analyzed cell cycle in tumor cells HEp-2 pre-labeled with vital dye CFSE (Fig.3). Coculturing of CFSE-labeled HEp-2 cells with IFNα-DCs resulted in significant increase in the level of apoptosis detected in tumor cells. In addition, the cultivation of tumor cells with IFNα-DCs was accompanied by a decrease in number of cycling tumor cells (S + G2M phases of the cell cycle). These results showed that DCs in vitro can efficiently induce death of tumor cells using an apoptotic mechanism.

Fig. 2. Cytotoxic activity of DC culture-conditioned medium against Jurkat and HEp-2. The figure represents individual values of cytotoxic activity mediated by supernatants from cultures of healthy donor IFNα-DCs against tumor cell lines HEp-2 (n = 17) and Jurkat (n = 6). Jurkat cells and HEp-2 cells (104cells/well) were labeled with [3H]thymidine and incubated with DC culture-conditioned medium (30%, v/v) for 18 hours.

#### **2.2.3 Growth inhibition effect of IFNα-DCs on tumor cell lines HEp-2 and U-87**

These data result in the suggestion that the cytotoxic activity of IFNα-DCs is conditioned by induction of apoptosis in tumor cells and that, along with a cytotoxic effect, IFNα-DCs apparently could block cell cycle in tumor cells, thereby providing cytostatic effect. Indeed, analysis of IFNα-DCs impact on the proliferation of tumor cells (Fig. 4) revealed a pronounced antiproliferative effect exerted by donor DCs against the cell line HEp-2.

Direct Antitumor Activity of Interferon-Induced

Wilcoxon's test, Mann-Whitney).

respectively, at E:T ratio of 10:1, PU <0,05).

activity.

Dendritic Cells of Healthy Donors and Patients with Primary Brain Tumors 331

To get inside into the mechanism that could be responsible for DC tumoricidal activity, we have investigated the role of key molecules involved in the apoptosis pathway. Cytotoxic activity of DCs is attributed to the expression of some proapoptotic molecules such as TRAIL, FasL, perforin, granzymes A and B, TNF-α, lymphotoxin-α1, β2 (Chauvin & Josien, 2008). To further characterized the molecular mechanisms by which HEp-2 cell death results from interaction with DCs, we studied the effect of some soluble receptors at the DCmediated cytotoxic activity. As evident from Fig. 5, pretreatment of IFNα-DCs with TNFR1: Fc resulted in almost complete neutralization of DC cytotoxic activity, whereas pretreatment with soluble forms of TRAIL-R2: Fc and Fas: Fc did not followed by suppression of DC killer

Fig. 5. Neutralization of DC cytotoxic function by soluble forms of R:Fc, specific for TNFfamily ligands. [3H]thymidine-labeled tumor cells HEp-2 (104 cells/well) were incubated for 18 hours with IFNα-DCs (at E:T ratio of 20:1) pre-treated for 1 h with TRAIL-R2: Fc fusion protein (10 μg/ml; n = 6), or TNFR1: Fc fusion protein (10 μg/ml; n = 6), or Fas: Fc fusion protein (10 μg/ml; n = 6). Data are presented as mean (M ± SE) of cytotoxic activity of IFNα-DCs *vs* HEp-2. \* - PU <0,01 - between intact DCs and DCs treated with TNFR1: Fc (U -

Thus, our data suggest that lysis of HEp-2 cells is not related with TRAIL- and FasL-mediated cytotoxicity but occurs with the involvement of TNFα molecules, since blocking of TNFα/TNFR1 binding leads to almost full suppression of DC killer activity. Apparently, the involvement only a single of three described mechanisms of DC cytotoxicity is due to resistance of tumor cells HEp-2 to TRAIL-and FasL-mediated apoptosis and determines relatively low cytotoxic activity of DCs against HEp-2 cells compared to Jurkat and U-87 which are sensitive to FasL-and TRAIL-mediated apoptosis (Röhn et al., 2001; Hoves et al., 2003).

Since we proposed IFNα-DCs may have a more pronounced antitumor activity than DCs generated with GM-CSF and IL-4, we then investigated whether cytotoxic and cytostatic activities of these two types of LPS-activated DC were distinct. As seen in Fig.6, IFNα-DCs possessed the higher ability to lyse leukemia cells Jurkat (Fig. 6A) and comparable cytotoxic activity in HEp-2 cultures (Fig.6B). However, IFNα-DCs were found to be more effective in suppressing the growth of tumor cell line HEp-2 than IL4-DCs (45 ± 6% *vs* 29 ± 7%,

**2.2.5 Cytotoxic activity of donor IL4-DCs in compared with IFNα-DCs** 

**2.2.4 Role of TNFα, FasL and TRAIL in cytotoxic activity of IFNα-DCs** 

Fig. 3. Effect of IFNα-DCs from healthy donors on the cell cycle in HEp-2. The figure shows the relative content (%) of CFSE-labeled HEp-2 cells in cell cycle phases in the absence of DCs (control; n= 4) and in co-cultures with IFNa-DCs (n= 4) for 18 hours at E:T ratio of 10:1. The data are presented as M ± SE (%). \* - PU<0,01 (U - Wilcoxon's test, Mann-Whitney).

Fig. 4. Tumor-inhibiting activity of IFNα-DCs from healthy donors against НЕр-2, Jurkat, and U-87. А) The graph shows the mean values (M ± SE) of cytostatic activity of IFNα-DCs against HEp-2 tumor cells (n = 8). Effector cells (DC) and target cells were cultured at ratios indicated for 24 hours, followed by the introduction of [3H]thymidine for 24 hours. **B)** Cytostatic activity rendered by IFNα-DCs against HEp-2 (n = 8), Jurkat (n = 7) and U-87 (n = 5) in E:T ratio of 20:1. \* - PU <0,05 - between the cytostatic activity of DCs *vs* HEp-2 and U-87 (U - Wilcoxon's test, Mann-Whitney).

Importantly, DCs mediated potent inhibitory activity (45,4 ± 6,24%) even at a low E:T cell ratio (10:1). Moreover, IFNα-DCs also suppressed the proliferation of glioblastoma cell line U-87. However, in this case inhibition was almost two-fold lower, accounting for 27,4 ± 4,4% at E:T ratio of 20:1 *vs* 52,4 ± 4,4% in HEp-2 cultures (p <0,05). Thus, in our study IFNα-DCs were found to be cytostatic for tumor cell lines. Comparative analysis of cytotoxic and cytostatic activity mediated by IFNα-DCs showed no correlations between the level of DC cytotoxicity and their ability to inhibit the proliferation of HEp2 (rS = 0,21; p = 0,7), U-87 (rS = 0,5; p = 0,28) and Jurkat (rS = 0,33; p = 0,5) tumor cell line. The lack of such a relationship was also indicated by the fact that in cultures of U-87 dendritic cells displayed the highest cytotoxic effect while their cytostatic effects were only moderate. Contrary, in cultures of HEp-2 DCs had a relatively low cytotoxic effect and pronounced anti-proliferative activity.

**+IFNα-DCs**

**\***

**HEp-2 Jurkat U-87**

**% control**

**apoptosis G0/G1 S+G2M**

Fig. 3. Effect of IFNα-DCs from healthy donors on the cell cycle in HEp-2. The figure shows the relative content (%) of CFSE-labeled HEp-2 cells in cell cycle phases in the absence of DCs (control; n= 4) and in co-cultures with IFNa-DCs (n= 4) for 18 hours at E:T ratio of 10:1. The data are presented as M ± SE (%). \* - PU<0,01 (U - Wilcoxon's test, Mann-Whitney).

Fig. 4. Tumor-inhibiting activity of IFNα-DCs from healthy donors against НЕр-2, Jurkat, and U-87. А) The graph shows the mean values (M ± SE) of cytostatic activity of IFNα-DCs against HEp-2 tumor cells (n = 8). Effector cells (DC) and target cells were cultured at ratios indicated for 24 hours, followed by the introduction of [3H]thymidine for 24 hours. **B)** Cytostatic activity rendered by IFNα-DCs against HEp-2 (n = 8), Jurkat (n = 7) and U-87 (n = 5) in E:T ratio of 20:1. \* - PU <0,05 - between the cytostatic activity of DCs *vs* HEp-2 and

**cytostatic activity, %**

**B**

Importantly, DCs mediated potent inhibitory activity (45,4 ± 6,24%) even at a low E:T cell ratio (10:1). Moreover, IFNα-DCs also suppressed the proliferation of glioblastoma cell line U-87. However, in this case inhibition was almost two-fold lower, accounting for 27,4 ± 4,4% at E:T ratio of 20:1 *vs* 52,4 ± 4,4% in HEp-2 cultures (p <0,05). Thus, in our study IFNα-DCs were found to be cytostatic for tumor cell lines. Comparative analysis of cytotoxic and cytostatic activity mediated by IFNα-DCs showed no correlations between the level of DC cytotoxicity and their ability to inhibit the proliferation of HEp2 (rS = 0,21; p = 0,7), U-87 (rS = 0,5; p = 0,28) and Jurkat (rS = 0,33; p = 0,5) tumor cell line. The lack of such a relationship was also indicated by the fact that in cultures of U-87 dendritic cells displayed the highest cytotoxic effect while their cytostatic effects were only moderate. Contrary, in cultures of HEp-2 DCs had a relatively low cytotoxic effect and pronounced

**\***

U-87 (U - Wilcoxon's test, Mann-Whitney).

**40:1 20:1 10:1 effector:target**

anti-proliferative activity.

**cytostatic activity, %**

**A**

#### **2.2.4 Role of TNFα, FasL and TRAIL in cytotoxic activity of IFNα-DCs**

To get inside into the mechanism that could be responsible for DC tumoricidal activity, we have investigated the role of key molecules involved in the apoptosis pathway. Cytotoxic activity of DCs is attributed to the expression of some proapoptotic molecules such as TRAIL, FasL, perforin, granzymes A and B, TNF-α, lymphotoxin-α1, β2 (Chauvin & Josien, 2008). To further characterized the molecular mechanisms by which HEp-2 cell death results from interaction with DCs, we studied the effect of some soluble receptors at the DCmediated cytotoxic activity. As evident from Fig. 5, pretreatment of IFNα-DCs with TNFR1: Fc resulted in almost complete neutralization of DC cytotoxic activity, whereas pretreatment with soluble forms of TRAIL-R2: Fc and Fas: Fc did not followed by suppression of DC killer activity.

Fig. 5. Neutralization of DC cytotoxic function by soluble forms of R:Fc, specific for TNFfamily ligands. [3H]thymidine-labeled tumor cells HEp-2 (104 cells/well) were incubated for 18 hours with IFNα-DCs (at E:T ratio of 20:1) pre-treated for 1 h with TRAIL-R2: Fc fusion protein (10 μg/ml; n = 6), or TNFR1: Fc fusion protein (10 μg/ml; n = 6), or Fas: Fc fusion protein (10 μg/ml; n = 6). Data are presented as mean (M ± SE) of cytotoxic activity of IFNα-DCs *vs* HEp-2. \* - PU <0,01 - between intact DCs and DCs treated with TNFR1: Fc (U - Wilcoxon's test, Mann-Whitney).

Thus, our data suggest that lysis of HEp-2 cells is not related with TRAIL- and FasL-mediated cytotoxicity but occurs with the involvement of TNFα molecules, since blocking of TNFα/TNFR1 binding leads to almost full suppression of DC killer activity. Apparently, the involvement only a single of three described mechanisms of DC cytotoxicity is due to resistance of tumor cells HEp-2 to TRAIL-and FasL-mediated apoptosis and determines relatively low cytotoxic activity of DCs against HEp-2 cells compared to Jurkat and U-87 which are sensitive to FasL-and TRAIL-mediated apoptosis (Röhn et al., 2001; Hoves et al., 2003).

#### **2.2.5 Cytotoxic activity of donor IL4-DCs in compared with IFNα-DCs**

Since we proposed IFNα-DCs may have a more pronounced antitumor activity than DCs generated with GM-CSF and IL-4, we then investigated whether cytotoxic and cytostatic activities of these two types of LPS-activated DC were distinct. As seen in Fig.6, IFNα-DCs possessed the higher ability to lyse leukemia cells Jurkat (Fig. 6A) and comparable cytotoxic activity in HEp-2 cultures (Fig.6B). However, IFNα-DCs were found to be more effective in suppressing the growth of tumor cell line HEp-2 than IL4-DCs (45 ± 6% *vs* 29 ± 7%, respectively, at E:T ratio of 10:1, PU <0,05).

Direct Antitumor Activity of Interferon-Induced

unaltered cytotoxic activity (Table 1).

activity are presented.

**activity vs HEp-2 cells** 

cells (LQ=10,3%).

Grade I-II (n=3) and III-IV (n=3) brain tumors.

Dendritic Cells of Healthy Donors and Patients with Primary Brain Tumors 333

Analysis of patients according to the degree of tumor malignancy demonstrated that the decrease in cytotoxic activity of DCs was typical for patients with high grade (III-IV) gliomas while patients with low grade (I-II) intracerebral gliomas were characterized by

Grade I-II (n=9) Grade III-IV (n=28)

E:T ratio = 20:1 Donors (n=22) Patients with brain tumors

Median 17,0 20,0 0

LQ-UQ 10,3- 32,0 13,0- 29,0 0- 4,0

M ± S.E 21,5 ± 2,6 22,56 ± 5,64 4,75 ± 1,95

Table 1. Cytotoxic activity of IFNα-DCs in patients with low and high grade glioma

Effector cells (IFNα-DCs) were generated from peripheral blood of healthy donors and patients with low grade (I-II) and high grade (III-IV) gliomas and cultured with [3H]thymidine-labeled target cells (HEp-2) for 18 h at E:T ratio of 20:1. The average values (M ± SE), Median and interquartile range (from low to upper quartile, LQ-UQ) of cytotoxic

Figure 8 shows the individual examples of cytotoxic activity of IFNα-DCs of patients with

Considering that the degree of malignancy is predictive factor of patient survival, we further questioned about the survival rates of patients with intact and reduced levels of DC cytotoxic activity against HEp-2 cells (Fig. 9). The criterion for division into such groups was the lower quartile range of cytotoxic activity mediated by donor IFNα-DCs against HEp-2

Patients with decreased cytotoxic activity of IFNα-DCs (< 10,3%, 1 patient with Grade II and 15 patients with Grade III-IV) differed by a lower survival rate compared with patients of the opposite group. For example, a median of survival in patients with low DC cytotoxic activity was about 13 months, and in the group with unchanged cytotoxic activity of DCs all patients (5 patients with Grade I-II and 4 patients with Grade III) were followed alive.

Next, we investigated whether DCs of patients with brain tumors could inhibit the growth of HEp-2 cells (Table 2). While donor DCs possessed the marked cytostatic activity, IFNα-DCs of patients with intracerebral gliomas were found to be incapable of suppressing the proliferation of HEp-2. Moreover, the addition of DCs led to 3-fold increased tumor cell proliferation. The index of DC impact ranged from 0,5 to 7,2, averaging about 3,06± 0,4. It should be noted that such a stimulatory effect of DCs on HEp-2 cell growth was detected both in patients with high grade III-IV (3,08 ± 0,65; n = 19) and low grade I-II (3,37 ± 1,49; n = 8) tumors, and unlike cytotoxic activity, was independent on the degree of malignancy.

**2.2.8 Growth inhibition effect of patient IFNα-DCs on tumor cell line HEp-2** 

**2.2.7 Survival rates of patients with intact and reduced levels of IFNα-DC cytotoxic** 

Fig. 6. Cytotoxic activity of IFNα-DCs and IL4-DCs of healthy donors against tumor lines Jurkat (A) and HEp-2 (B). Data are presented as mean (M ± SE) of cytotoxic activity. Effector cells (donor IFNα-DCs and IL4-DCs) were incubated with target cells ([3H]thymidinelabeled tumor cell lines Jurkat and HEp-2) at ratios indicated for 18 h. \*- РU<0,05 – between IFNα-DCs and IL4-DCs against Jurkat (U - Wilcoxon's test, Mann-Whitney).

#### **2.2.6 Cytotoxic activity of patient IFNα-DCs** *vs* **HEp-2**

While donor DCs were found to be tumoricidal, evaluation of the cytotoxic activity of DCs generated in vitro from peripheral blood of brain glioma patients revealed they were significantly less cytotoxic against HEp-2 cells (Fig.7A). The decrease of cytotoxic activity was manifested at all E:T ratios which were analyzed. At the same time assessment of patient DC killer activity at E:T ratio of 20:1 (n=37) allowed to reveal significant heterogeneity for DC cytotoxic potential in patients with brain tumors (Fig.7B). Indeed, in 25 patients (67%) cytotoxic activity was completely absent, whereas remained relatively unaltered in another 12 patients (32%).

Fig. 7. Cytotoxic activity of IFNα-DCs of patients with brain tumors against HEp-2. **A)** Effector cells (donor and patient IFNα-DCs) were cultured with [3H]thymidine-labeled target cells (HEp-2) for 18 h at ratios indicated. Results are shown as the mean ± SE of DC cytotoxic activity. \*- PU <0,01 - between donor and patient DCs (U - Wilcoxon's test, Mann-Whitney) at E:T ratio of 40:1 and 20:1. **B)** Individual values of cytotoxic activity mediated by IFNα-DCs of healthy donors and brain tumor patients against HEp-2 are presented. Effector cells (DC) were cultured with [3H]thymidine-labeled target cells (HEp-2) for 18 h at ratio of 20:1.

**IFNa-DCs IL4-DCs**

Fig. 6. Cytotoxic activity of IFNα-DCs and IL4-DCs of healthy donors against tumor lines Jurkat (A) and HEp-2 (B). Data are presented as mean (M ± SE) of cytotoxic activity. Effector cells (donor IFNα-DCs and IL4-DCs) were incubated with target cells ([3H]thymidinelabeled tumor cell lines Jurkat and HEp-2) at ratios indicated for 18 h. \*- РU<0,05 – between

**cytotoxic activity,%**

**B**

**40:1 20:1 10:1 effector:target**

**healthy donors patients**

**IL4-DCs IFNa-DCs**

While donor DCs were found to be tumoricidal, evaluation of the cytotoxic activity of DCs generated in vitro from peripheral blood of brain glioma patients revealed they were significantly less cytotoxic against HEp-2 cells (Fig.7A). The decrease of cytotoxic activity was manifested at all E:T ratios which were analyzed. At the same time assessment of patient DC killer activity at E:T ratio of 20:1 (n=37) allowed to reveal significant heterogeneity for DC cytotoxic potential in patients with brain tumors (Fig.7B). Indeed, in 25 patients (67%) cytotoxic activity was completely absent, whereas remained relatively

Fig. 7. Cytotoxic activity of IFNα-DCs of patients with brain tumors against HEp-2. **A)** Effector cells (donor and patient IFNα-DCs) were cultured with [3H]thymidine-labeled target cells (HEp-2) for 18 h at ratios indicated. Results are shown as the mean ± SE of DC cytotoxic activity. \*- PU <0,01 - between donor and patient DCs (U - Wilcoxon's test, Mann-Whitney) at E:T ratio of 40:1 and 20:1. **B)** Individual values of cytotoxic activity mediated by IFNα-DCs of healthy donors and brain tumor patients against HEp-2 are presented. Effector cells (DC) were

**B**

**cytotoxic activity(%)**

cultured with [3H]thymidine-labeled target cells (HEp-2) for 18 h at ratio of 20:1.

IFNα-DCs and IL4-DCs against Jurkat (U - Wilcoxon's test, Mann-Whitney).

**2.2.6 Cytotoxic activity of patient IFNα-DCs** *vs* **HEp-2** 

**healthy donors patients**

**40:1 20:1 10:1 effector:target**

unaltered in another 12 patients (32%).

**\* \***

**40:1 20:1 10:1 effector:target**

**cytotoxic activity(%)**

**A**

**\***

**cytotoxic activity,%**

**A**

Analysis of patients according to the degree of tumor malignancy demonstrated that the decrease in cytotoxic activity of DCs was typical for patients with high grade (III-IV) gliomas while patients with low grade (I-II) intracerebral gliomas were characterized by unaltered cytotoxic activity (Table 1).


Table 1. Cytotoxic activity of IFNα-DCs in patients with low and high grade glioma

Effector cells (IFNα-DCs) were generated from peripheral blood of healthy donors and patients with low grade (I-II) and high grade (III-IV) gliomas and cultured with [3H]thymidine-labeled target cells (HEp-2) for 18 h at E:T ratio of 20:1. The average values (M ± SE), Median and interquartile range (from low to upper quartile, LQ-UQ) of cytotoxic activity are presented.

Figure 8 shows the individual examples of cytotoxic activity of IFNα-DCs of patients with Grade I-II (n=3) and III-IV (n=3) brain tumors.

#### **2.2.7 Survival rates of patients with intact and reduced levels of IFNα-DC cytotoxic activity vs HEp-2 cells**

Considering that the degree of malignancy is predictive factor of patient survival, we further questioned about the survival rates of patients with intact and reduced levels of DC cytotoxic activity against HEp-2 cells (Fig. 9). The criterion for division into such groups was the lower quartile range of cytotoxic activity mediated by donor IFNα-DCs against HEp-2 cells (LQ=10,3%).

Patients with decreased cytotoxic activity of IFNα-DCs (< 10,3%, 1 patient with Grade II and 15 patients with Grade III-IV) differed by a lower survival rate compared with patients of the opposite group. For example, a median of survival in patients with low DC cytotoxic activity was about 13 months, and in the group with unchanged cytotoxic activity of DCs all patients (5 patients with Grade I-II and 4 patients with Grade III) were followed alive.

#### **2.2.8 Growth inhibition effect of patient IFNα-DCs on tumor cell line HEp-2**

Next, we investigated whether DCs of patients with brain tumors could inhibit the growth of HEp-2 cells (Table 2). While donor DCs possessed the marked cytostatic activity, IFNα-DCs of patients with intracerebral gliomas were found to be incapable of suppressing the proliferation of HEp-2. Moreover, the addition of DCs led to 3-fold increased tumor cell proliferation. The index of DC impact ranged from 0,5 to 7,2, averaging about 3,06± 0,4. It should be noted that such a stimulatory effect of DCs on HEp-2 cell growth was detected both in patients with high grade III-IV (3,08 ± 0,65; n = 19) and low grade I-II (3,37 ± 1,49; n = 8) tumors, and unlike cytotoxic activity, was independent on the degree of malignancy.

Direct Antitumor Activity of Interferon-Induced

Wilcoxon's test, Mann-Whitney).

Dendritic Cells of Healthy Donors and Patients with Primary Brain Tumors 335

The table represents the individual values of indexes of IFNα-DC impact on proliferation of tumor cell line HEp-2. For this, effector cells (DCs) and targets were cultured at 20:1 for 24 hours, followed by the introduction of [3H]thymidine for the next 24 hours. The index of DC impact was calculated by the formula: cpm in cultures with target and effector cells / cpm in control cultures without effector cells. \* - PU <0,01 between donors and patients (U -

**Patients Diagnosis DC cytostatic activity** 

P 1, female, 60 years Grade 1 5,6 Р 2, female, 71 years Grade 1 6,2 Р 3, male, 53 years Grade 1 0,9 P 4, female, 35 years Grade 2 7,2 Р 5, male, 42 years Grade 2 3,4 P 6, female, 38 years Grade 2 0,8 P 7, female, 36 years Grade 2 0,5 P 8, female, 35 years Grade 2 0,5

M±SE (n=8) 3,37 ± 1,49

 M±SE (n=19) 3,08 ± 0,645 **Patients** (n=27) 3,06 ± 0,4\* **Healthy donors** (n=14) 0,4 ± 0,04

Table 2. Effect of IFNα-DCs of patients with brain tumors on the proliferation of HEp-2 cells

Р 9, male, 56 years Grade 3 5,0 Р 10, male, 25 years Grade 3 3,5 Р 11, male, 46 years Grade 3 0,5 Р 12, male, 29 years Grade 3 2,3 P 13, female, 24 years Grade 3 3,2 Р 14, male, 32 years Grade 4 3,1 P 15, female, 54 years Grade 4 5,2 Р 16, male, 48 years Grade 4 3,2 Р 17, male, 53 years Grade 4 2,4 Р 18, male,41 years Grade 4 2,4 Р 19, male, 34 years Grade 4 1,8 Р 20, male, 24 years Grade 4 1,4 Р 21, male, 38 years Grade 4 0,8 Р 22, male,47 years Grade 4 2,3 P 23, female, 45 years Grade 4 6,9 P 24, female, 58 years Grade 4 6,5 P 25, female, 60 years Grade 4 6,9 P 26, female, 71 years Grade 4 0,8 P 27,female, 57 years Grade 4 2,0

**(Indexes of DC impact)** 

Fig. 8. Cytotoxic activity of IFNα-DCs of individual patients. Figure represents the individual values of cytotoxic activity of IFNα-DCs generated in vitro from peripheral blood of tumor patients against HEp-2. Effector cells (DCs) and [3H]thymidine-labeled HEp-2 cells were co-cultured for 18 h at ratios indicated. Percentage of cytotoxicity was calculated as follows: [1 - (cpm in cultures with target and effector cells / cpm in control cultures without effector cells)] x 100%.

Fig. 9. Survival rates of patients with brain tumors based on the level of DC cytotoxic activity. Dotted line: cytotoxic activity of DCs of patients is below (<10,3%) donor lower quartile values. Solid line: cytotoxic activity of patient DCs is above 10,3%.

**cytitixic activity (%)**

**cytotoxic activity (%)**

 **cytotoxic activity (%)**

**40/1 20/1 10/1 effector:target**

**40/1 20/1 10/1 effector:target**

**40/1 20/1 10/1 effector:target**

Fig. 8. Cytotoxic activity of IFNα-DCs of individual patients. Figure represents the individual values of cytotoxic activity of IFNα-DCs generated in vitro from peripheral blood of tumor patients against HEp-2. Effector cells (DCs) and [3H]thymidine-labeled HEp-2 cells were co-cultured for 18 h at ratios indicated. Percentage of cytotoxicity was calculated as follows: [1 - (cpm in cultures with target and effector cells / cpm in control cultures without

A. I-II GRADE B. III-IV GRADE

**<sup>150</sup> ct<10,3%**

**ct>10,3%**

**0 50 100**

Fig. 9. Survival rates of patients with brain tumors based on the level of DC cytotoxic activity. Dotted line: cytotoxic activity of DCs of patients is below (<10,3%) donor lower

quartile values. Solid line: cytotoxic activity of patient DCs is above 10,3%.

**month**

**0**

**50**

**100**

**Percent survival**

effector cells)] x 100%.

**cytotoxic activity (%)**

**cytotoxic activity (%)**

**cytotixic activity (%)**

**40/1 20/1 10/1 effector:target**

**40/1 20/1 10/1 effector:target**

**40/1 20/1 10/1 effector:target**

The table represents the individual values of indexes of IFNα-DC impact on proliferation of tumor cell line HEp-2. For this, effector cells (DCs) and targets were cultured at 20:1 for 24 hours, followed by the introduction of [3H]thymidine for the next 24 hours. The index of DC impact was calculated by the formula: cpm in cultures with target and effector cells / cpm in control cultures without effector cells. \* - PU <0,01 between donors and patients (U - Wilcoxon's test, Mann-Whitney).


Table 2. Effect of IFNα-DCs of patients with brain tumors on the proliferation of HEp-2 cells

Direct Antitumor Activity of Interferon-Induced

statistical significance.

**Patients**

**Healthy donors** 

**2.3 Discussion** 

malignant gliomas and healthy donors.

healthy donors and patients with brain tumors.

Dendritic Cells of Healthy Donors and Patients with Primary Brain Tumors 337

day cultures of LPS-activated IFNα-DCs. As follows from Table 4, supernatants from patient DC cultures differed little from healthy donor culture-conditioned medium by the level of TNFα production. A slight decrease in production of TNFα was a tendency, which had no

P 1, male, 39 years Grade 4 856 P 2, female, 46 years Grade 3 767 P 3, male, 35 years Grade 3 800 P 4, male, 46 years Grade 3 914 P 5, male, 24 years Grade 4 918 P 6, female, 43 years Grade 4 693 P 7, female, 38 years Grade 2 256 P 8, male, 52 years Grade 4 486 P 9, male, 68years Grade 3 710

(n=9) M ± SE 711 ± 82

(n=11) M ± SE 824 ± 59

Table 4. TNFα concentrations in cultures of donor and glioma patient IFNα-DCs. The table represents the individual and average values (M ± SE) of TNFα concentrations in culture supernatants of IFNα-DCs generated in vitro from peripheral blood of patients with

The ability of DCs generated in vitro to inhibit the growth of human tumor cell lines and lyse tumor cells was first demonstrated by Chapoval (Chapoval et al., 2000). Thereafter, spontaneous cytotoxicity mediated by DCs without any stimulation was also described by other authors (Vanderheyde et al. 2004; Yang et al., 2001; Manna & Mohanakumar, 2002; Joo et al., 2002 ; Janjic et al., 2002), which revealed that the tumoricidal potential of DCs generated in the presence of GM-CSF and IL-4 was mediated by effector molecules such as FasL (Yang et al., 2001), TNF (Manna & Mohanakumar, 2002; Joo et al., 2002), lymphotoxinα1, β2 (Lu et al., 2002) or TRAIL (Liu et al., 2001). The cytolytic properties of cultured human monocyte-derived DCs are enhanced by certain activation stimuli, such as LPS (Chapoval et al., 2000; Manna & Mohanakumar, 2002). While myeloid DCs being treated with IFN exhibited upregulation of intracellular TRAIL and increased cytotoxic potential (Liu et al., 2001), study of antitumor activity of DCs generated in the presence of IFNα were not performed previously. In this study, we reported the novel data on cytostatic/cytotoxic activities of LPS-activated IFNα-DCs generated in vitro from peripheral blood monocytes of

We report here that LPS-activated IFNα-DCs can lyse both NK-sensitive (Jurkat lymphoma cells) and NK-resistant (HEp-2, U-87) tumor cell lines. Such a cytotoxic effect requires cell contact, since the supernatants of IFNα-DCs either lack or possess the poor cytotoxic activity. Using HEp-2 tumor cells as targets, we revealed that DCs appear to promote the apoptosis and suppress cell cycle in tumor cells, thus having a cytostatic effect. Cytostatic

**Patients Diagnosis TNFa (pg/ml**)

#### **2.2.9 Cytotoxic activity of patient IFNα-DCs vs U-87**

In the next experiments, we investigated cytotoxic potential of patient IFNα-DCs towards TRAIL-sensitive tumor line U-87. Interestingly, we found no decrease in killer activity of patient DCs in this case. Furthermore, IFNα-DCs of patients with malignant tumors (Grade III-IV), unable to lyse HEp-2 cells, were highly cytotoxic against U-87 cells compared with healthy donors (Table 3). Further experiments demonstrated the ability of patient DCs to inhibit the proliferation of U-87 cell line which is also more expressed in patients than in donors (46,6 ± 7,5 and 27,4 ± 4,4%, respectively; РU< 0,01). In this, we found a strong positive correlation between cytotoxic and cytostatic activities (rS = 0,89; p = 0,001). Thus, impairment of cytotoxic and cytostatic activity of patient DCs was only revealed against HEp-2 cells.


Table 3. Cytotoxic/cytostatic activity of DCs in patients with brain tumors against U-87. The table represents the individual and average values (M ± SE) of cytotoxic and cytostatic activities of patient and donor DCs. Cytotoxicity was measured by coculturing of DCs and [3H]thymidine-labeled U-87 cells for 18 h at 20:1. For cytostatic activity evaluation, DCs and U-87 cells were cultured at 20:1 for 24 hours, followed by the introduction of [3H]thymidine for 24 hours. \* - РU <0,01 between donors and patients (U - Wilcoxon's test, Mann-Whitney).

#### **2.2.10 TNFα production by donor and patient IFNα-DCs**

Since the cytotoxic activity of DCs against HEp-2 cells was related with TNF-mediated apoptosis, we further compared the ability of DCs of patients with malignant gliomas (Grade III-IV) and donors to produce TNFα. The concentration of TNFα was evaluated in 4day cultures of LPS-activated IFNα-DCs. As follows from Table 4, supernatants from patient DC cultures differed little from healthy donor culture-conditioned medium by the level of TNFα production. A slight decrease in production of TNFα was a tendency, which had no statistical significance.


Table 4. TNFα concentrations in cultures of donor and glioma patient IFNα-DCs. The table represents the individual and average values (M ± SE) of TNFα concentrations in culture supernatants of IFNα-DCs generated in vitro from peripheral blood of patients with malignant gliomas and healthy donors.

#### **2.3 Discussion**

336 Glioma – Exploring Its Biology and Practical Relevance

In the next experiments, we investigated cytotoxic potential of patient IFNα-DCs towards TRAIL-sensitive tumor line U-87. Interestingly, we found no decrease in killer activity of patient DCs in this case. Furthermore, IFNα-DCs of patients with malignant tumors (Grade III-IV), unable to lyse HEp-2 cells, were highly cytotoxic against U-87 cells compared with healthy donors (Table 3). Further experiments demonstrated the ability of patient DCs to inhibit the proliferation of U-87 cell line which is also more expressed in patients than in donors (46,6 ± 7,5 and 27,4 ± 4,4%, respectively; РU< 0,01). In this, we found a strong positive correlation between cytotoxic and cytostatic activities (rS = 0,89; p = 0,001). Thus, impairment of cytotoxic and cytostatic activity of patient DCs was only revealed against

**activity (%)** 

**DC cytostatic activity** (%)

**2.2.9 Cytotoxic activity of patient IFNα-DCs vs U-87** 

**Patients Diagnosis DC cytotoxic** 

Р 1, male, 36 years Grade 4 76 54

Р 2, male, 24 years Grade 4 55 28

P 3, female,69 years Grade 4 39 40

P 4, female, 42 years Grade 3 60 44

P 5, female, 71 years Grade 3 57 40

P 6, female, 54 years Grade 4 60 43

P 7, female, 46 years Grade 4 56 12

Р 8, male, 48 years Grade 4 80 89

Р 9, male, 24 years Grade 3 69 69

**2.2.10 TNFα production by donor and patient IFNα-DCs** 

**Patients** (n=9) 61,3 ± 4,1\* 46,6 ± 7,5\*

**Healthy donors** (n=5) 38,6 ± 8,3 27,4 ± 4,4

Table 3. Cytotoxic/cytostatic activity of DCs in patients with brain tumors against U-87. The table represents the individual and average values (M ± SE) of cytotoxic and cytostatic activities of patient and donor DCs. Cytotoxicity was measured by coculturing of DCs and [3H]thymidine-labeled U-87 cells for 18 h at 20:1. For cytostatic activity evaluation, DCs and U-87 cells were cultured at 20:1 for 24 hours, followed by the introduction of [3H]thymidine for 24 hours. \* - РU <0,01 between donors and patients (U - Wilcoxon's test, Mann-Whitney).

Since the cytotoxic activity of DCs against HEp-2 cells was related with TNF-mediated apoptosis, we further compared the ability of DCs of patients with malignant gliomas (Grade III-IV) and donors to produce TNFα. The concentration of TNFα was evaluated in 4-

HEp-2 cells.

The ability of DCs generated in vitro to inhibit the growth of human tumor cell lines and lyse tumor cells was first demonstrated by Chapoval (Chapoval et al., 2000). Thereafter, spontaneous cytotoxicity mediated by DCs without any stimulation was also described by other authors (Vanderheyde et al. 2004; Yang et al., 2001; Manna & Mohanakumar, 2002; Joo et al., 2002 ; Janjic et al., 2002), which revealed that the tumoricidal potential of DCs generated in the presence of GM-CSF and IL-4 was mediated by effector molecules such as FasL (Yang et al., 2001), TNF (Manna & Mohanakumar, 2002; Joo et al., 2002), lymphotoxinα1, β2 (Lu et al., 2002) or TRAIL (Liu et al., 2001). The cytolytic properties of cultured human monocyte-derived DCs are enhanced by certain activation stimuli, such as LPS (Chapoval et al., 2000; Manna & Mohanakumar, 2002). While myeloid DCs being treated with IFN exhibited upregulation of intracellular TRAIL and increased cytotoxic potential (Liu et al., 2001), study of antitumor activity of DCs generated in the presence of IFNα were not performed previously. In this study, we reported the novel data on cytostatic/cytotoxic activities of LPS-activated IFNα-DCs generated in vitro from peripheral blood monocytes of healthy donors and patients with brain tumors.

We report here that LPS-activated IFNα-DCs can lyse both NK-sensitive (Jurkat lymphoma cells) and NK-resistant (HEp-2, U-87) tumor cell lines. Such a cytotoxic effect requires cell contact, since the supernatants of IFNα-DCs either lack or possess the poor cytotoxic activity. Using HEp-2 tumor cells as targets, we revealed that DCs appear to promote the apoptosis and suppress cell cycle in tumor cells, thus having a cytostatic effect. Cytostatic

Direct Antitumor Activity of Interferon-Induced

cytotoxicity in patients with malignant gliomas.

malignant brain tumors.

**3. Conclusion** 

Dendritic Cells of Healthy Donors and Patients with Primary Brain Tumors 339

apoptosis (Choi et al., 2004). As we have found, DC cytotoxicity on HEp-2 cells likely engaged TNF/TNFR1 pathway than TRAIL or FasL effects. Given these facts, the high cytotoxic activity of patient DCs against U-87 cells and dramatic decline of such activity against HEp-2 tumor cells could indicate a defect in TNFα-related mechanism of DC

Our facts seemed to be important in several aspects. First, the defect in cytotoxic activity of DCs may be of interest in diagnosis and prognosis, since the expression of cytotoxic activity is associated with tumor malignancy and survival rates. Second, this phenomenon is of interest from the pathogenetic point of view. Malignant brain tumors induce a weak antigen-specific response due to tumor-induced immunosuppression, as well as localization of the tumor in the immunologically privileged brain tissues (Parney et al., 2000). We have previously shown that patient IFNα-DCs are characterized by intact antigen-presenting function, capable of activating T cells to produce Th1 cytokines and induce proliferation of T lymphocytes to antigens of tumor cell lysates (Chernykh et al., 2009). Then impairment of DC effector functions may inhibit the early induction of antigen-specific immune response being yet another reason for infringement of anti-tumor protection in patients with

We can also assume that if tumoricidal potential of DCs is disturbed, cytoreductive therapy (radio-chemotherapy) becomes especially important, both in regard of direct elimination of tumor cells and release of tumor antigens required to start specific immune response. However, whether IFNα-DCs could effectively destroy primary glioma cells and does this ability is disrupted in patients with malignant gliomas remains to find. Most of glioblastoma cell lines and primary cells gliomas are absolutely resistant to cytotoxic effect of certain proapoptogenic molecules, such as TRAIL (Eramo et al., 2005) and TNF. At that, the effective destruction of tumor lines by DCs requires the interaction of separate molecules. Besides, it is shown that some proapoptogenic molecules could induce the expression of receptors for another mediators of apoptosis. Such as, TNFα can induce the expression of

Fas and sensitize glioma cells to FasL/Fas-mediated apoptosis (Weller et al., 1994).

immunotherapeutic approaches to the treatment of malignant brain gliomas.

The further elucidation of the DC cytotoxic/static activity mechanisms and the possible role of the defect of DC cytotoxic properties in patients with gliomas as weel as the studies on correlation between the antitumor activity of IFNα-DCs and clinical outcomes could probably explain the different sensitivity of cancer patients to the treatment, and justify new

The capacity of IFNα-DCs to lyse tumor cell lines and inhibit their proliferation has been investigated. LPS-activated IFNα-DCs of healthy donors were shown to have dosedependent cytotoxic and cytostatic activity against various tumor lines through the induction of apoptosis and arrest of cell cycle. DCs lysed both TRAIL-sensitive (Jurkat cells) and TRAIL-resistant (HEp-2) cells, and cytotoxic activity against HEp-2 line was mediated through the TNF-TNFR1 pathway. In contrast to healthy donors, DCs of patients with malignant glioma failed to inhibit growth, but stimulated proliferation of HEp-2 cells. In addition, patient DCs had significantly reduced cytotoxic activity against HEp-2 cells. Patients with decreased cytotoxic activity were characterized by significantly lower survival since defect of cytotoxic activity was associated with high-grade glioma. The defective cytotoxic activity of DCs noted against HEp-2 cells was not revealed against glioblastoma U-

activity was also confirmed by the tumor growth/proliferation inhibiting capacity realized by IFNα-DCs. When compare the cytotoxicity of IFNα-DCs and IL4-DCs, we revealed that IFNα-DCs expressed the higher ability to kill Jurkat tumor cells as well as comparable with IL4-DCs cytotoxic activity in HEp-2 cultures.

Since the highest cytotoxic activity of IFNα-DCs was manifested in U-87 and Jurkat tumor cell cultures, which are reported to be sensitive to TRAIL-induced apoptosis (Lee et al., 2003; Panner et al., 2005; Siegelin et al., 2009), it is reasonable to assume that this cytotoxicity could be due to the stimulative effect of IFNα on TRAIL expression (Riboldi et al., 2009). Indeed, as a further corroboration of the suggested cytotoxic capacity, our data demonstrated that LPS-activated IFNα-DCs were found to contain significantly higher amounts of cells expressing membrane-bound TRAIL compared with IL4-DCs (data not shown). Apparently, these data could also explain a higher cytotoxic activity of IFNα-DCs on Jurkat tumor cells.

There are a very few data on sensitivity of HEp-2 cells to the cytotoxic effect of DCs mediated by TNF family molecules. The tumoricidal activity was not mediated by FasL/Fas or TRAIL/TRAILR2 systems, whereas TNFα/TNFR1 blocking completely abolished the ability of DCs to lyse HEp-2 cells. Thus, tumor cell line HEp-2 cells can be considered as resistant to FasL- and TRAIL-induced apoptosis, but sensitive to cytotoxicity triggered by TNFα/TNFR1 pathway. At that, the fact that anti–TNFα antibody almost completely decreased cytotoxicity, while DC culture-conditioned medium containing quite high concentrations of TNFα lacked lytic activity (Leplina et al., 2007b), further implies that membrane-bound, not the soluble form, of TNFα partially contributes to the effect.

Our results are consistent with the reported data on TNFα expression by HEp-2 cells (Paland et al., 2008), as well as the resistance of this tumor cell line to FasL-induced apoptosis (Morton & Blaho, 2007). The lack of sensitivity HEp-2 cells to TRAIL/TRAILRIIinduced death explains the absence of distinction in the cytotoxic activity of IFNα-DCs and IL4-DCs against HEp-2 cells.

Since we revealed no correlations between cytotoxic and cytostatic effects of IFNα-DCs on HEp-2 cells, then one can believe that cytotoxicity and cytostasis could be mediated by distinct mechanisms. Similar tendency was observed for IL4-DCs (Vanderheyde et al., 2004), where the authors have shown that LPS-stimulated IL4-DCs possess TNFα-associated cytostatic activity unrelated with cytotoxicity. Indeed, IL4-DCs inhibited the growth of modified Jurkat cells deficient in caspase-8 or overexpressing Bcl-2. On the other hand, supernatants of IL4-DCs suppressed the proliferation of non-modified Jurkat cells, but showed no cytotoxic activity. Evidently, cytostatic and cytotoxic activities of IFNα-DCs are also implemented through independent mechanisms. This hypothesis could be confirmed by the fact that a higher cytotoxic activity of IFNα-DCs against U-87 and Jurkat cells was associated with less pronounced DC cytostatic activity on these lines compared with HEp-2. Investigation of the effector functions of IFNα-DCs in patients with intracerebral gliomas revealed impaired ability of these cells to lyse HEp-2 tumor cells. Importantly, such an impairment of DC cytotoxicity was identified mainly in patients with high grade (III-IV) brain tumors, while in low grade (I-II) tumors DCs were quite effective killers. Furthermore, patients with intact DC cytotoxic activity had a higher survival rate than patients with reduced killer activity. In addition, patient DCs regardless of tumor histology showed no cytotoxic activity against HEp-2 cells, whereas both cytotoxic and cytostatic activities of DCs against U-87 cells were found to be enhanced.

According to the data, glioblastoma cells U-87 are resistant to cytotoxicity mediated by TNFα (Sawada et al., 2004), and sensitive to TRAIL- (Knight et al., 2001) and Fas-induced apoptosis (Choi et al., 2004). As we have found, DC cytotoxicity on HEp-2 cells likely engaged TNF/TNFR1 pathway than TRAIL or FasL effects. Given these facts, the high cytotoxic activity of patient DCs against U-87 cells and dramatic decline of such activity against HEp-2 tumor cells could indicate a defect in TNFα-related mechanism of DC cytotoxicity in patients with malignant gliomas.

Our facts seemed to be important in several aspects. First, the defect in cytotoxic activity of DCs may be of interest in diagnosis and prognosis, since the expression of cytotoxic activity is associated with tumor malignancy and survival rates. Second, this phenomenon is of interest from the pathogenetic point of view. Malignant brain tumors induce a weak antigen-specific response due to tumor-induced immunosuppression, as well as localization of the tumor in the immunologically privileged brain tissues (Parney et al., 2000). We have previously shown that patient IFNα-DCs are characterized by intact antigen-presenting function, capable of activating T cells to produce Th1 cytokines and induce proliferation of T lymphocytes to antigens of tumor cell lysates (Chernykh et al., 2009). Then impairment of DC effector functions may inhibit the early induction of antigen-specific immune response being yet another reason for infringement of anti-tumor protection in patients with malignant brain tumors.

We can also assume that if tumoricidal potential of DCs is disturbed, cytoreductive therapy (radio-chemotherapy) becomes especially important, both in regard of direct elimination of tumor cells and release of tumor antigens required to start specific immune response.

However, whether IFNα-DCs could effectively destroy primary glioma cells and does this ability is disrupted in patients with malignant gliomas remains to find. Most of glioblastoma cell lines and primary cells gliomas are absolutely resistant to cytotoxic effect of certain proapoptogenic molecules, such as TRAIL (Eramo et al., 2005) and TNF. At that, the effective destruction of tumor lines by DCs requires the interaction of separate molecules. Besides, it is shown that some proapoptogenic molecules could induce the expression of receptors for another mediators of apoptosis. Such as, TNFα can induce the expression of Fas and sensitize glioma cells to FasL/Fas-mediated apoptosis (Weller et al., 1994).

The further elucidation of the DC cytotoxic/static activity mechanisms and the possible role of the defect of DC cytotoxic properties in patients with gliomas as weel as the studies on correlation between the antitumor activity of IFNα-DCs and clinical outcomes could probably explain the different sensitivity of cancer patients to the treatment, and justify new immunotherapeutic approaches to the treatment of malignant brain gliomas.

### **3. Conclusion**

338 Glioma – Exploring Its Biology and Practical Relevance

activity was also confirmed by the tumor growth/proliferation inhibiting capacity realized by IFNα-DCs. When compare the cytotoxicity of IFNα-DCs and IL4-DCs, we revealed that IFNα-DCs expressed the higher ability to kill Jurkat tumor cells as well as comparable with

Since the highest cytotoxic activity of IFNα-DCs was manifested in U-87 and Jurkat tumor cell cultures, which are reported to be sensitive to TRAIL-induced apoptosis (Lee et al., 2003; Panner et al., 2005; Siegelin et al., 2009), it is reasonable to assume that this cytotoxicity could be due to the stimulative effect of IFNα on TRAIL expression (Riboldi et al., 2009). Indeed, as a further corroboration of the suggested cytotoxic capacity, our data demonstrated that LPS-activated IFNα-DCs were found to contain significantly higher amounts of cells expressing membrane-bound TRAIL compared with IL4-DCs (data not shown). Apparently, these data could also explain a higher cytotoxic activity of IFNα-DCs on Jurkat tumor cells. There are a very few data on sensitivity of HEp-2 cells to the cytotoxic effect of DCs mediated by TNF family molecules. The tumoricidal activity was not mediated by FasL/Fas or TRAIL/TRAILR2 systems, whereas TNFα/TNFR1 blocking completely abolished the ability of DCs to lyse HEp-2 cells. Thus, tumor cell line HEp-2 cells can be considered as resistant to FasL- and TRAIL-induced apoptosis, but sensitive to cytotoxicity triggered by TNFα/TNFR1 pathway. At that, the fact that anti–TNFα antibody almost completely decreased cytotoxicity, while DC culture-conditioned medium containing quite high concentrations of TNFα lacked lytic activity (Leplina et al., 2007b), further implies that

membrane-bound, not the soluble form, of TNFα partially contributes to the effect.

Our results are consistent with the reported data on TNFα expression by HEp-2 cells (Paland et al., 2008), as well as the resistance of this tumor cell line to FasL-induced apoptosis (Morton & Blaho, 2007). The lack of sensitivity HEp-2 cells to TRAIL/TRAILRIIinduced death explains the absence of distinction in the cytotoxic activity of IFNα-DCs and

Since we revealed no correlations between cytotoxic and cytostatic effects of IFNα-DCs on HEp-2 cells, then one can believe that cytotoxicity and cytostasis could be mediated by distinct mechanisms. Similar tendency was observed for IL4-DCs (Vanderheyde et al., 2004), where the authors have shown that LPS-stimulated IL4-DCs possess TNFα-associated cytostatic activity unrelated with cytotoxicity. Indeed, IL4-DCs inhibited the growth of modified Jurkat cells deficient in caspase-8 or overexpressing Bcl-2. On the other hand, supernatants of IL4-DCs suppressed the proliferation of non-modified Jurkat cells, but showed no cytotoxic activity. Evidently, cytostatic and cytotoxic activities of IFNα-DCs are also implemented through independent mechanisms. This hypothesis could be confirmed by the fact that a higher cytotoxic activity of IFNα-DCs against U-87 and Jurkat cells was associated with less pronounced DC cytostatic activity on these lines compared with HEp-2. Investigation of the effector functions of IFNα-DCs in patients with intracerebral gliomas revealed impaired ability of these cells to lyse HEp-2 tumor cells. Importantly, such an impairment of DC cytotoxicity was identified mainly in patients with high grade (III-IV) brain tumors, while in low grade (I-II) tumors DCs were quite effective killers. Furthermore, patients with intact DC cytotoxic activity had a higher survival rate than patients with reduced killer activity. In addition, patient DCs regardless of tumor histology showed no cytotoxic activity against HEp-2 cells, whereas both cytotoxic and cytostatic activities of DCs

According to the data, glioblastoma cells U-87 are resistant to cytotoxicity mediated by TNFα (Sawada et al., 2004), and sensitive to TRAIL- (Knight et al., 2001) and Fas-induced

IL4-DCs cytotoxic activity in HEp-2 cultures.

IL4-DCs against HEp-2 cells.

against U-87 cells were found to be enhanced.

The capacity of IFNα-DCs to lyse tumor cell lines and inhibit their proliferation has been investigated. LPS-activated IFNα-DCs of healthy donors were shown to have dosedependent cytotoxic and cytostatic activity against various tumor lines through the induction of apoptosis and arrest of cell cycle. DCs lysed both TRAIL-sensitive (Jurkat cells) and TRAIL-resistant (HEp-2) cells, and cytotoxic activity against HEp-2 line was mediated through the TNF-TNFR1 pathway. In contrast to healthy donors, DCs of patients with malignant glioma failed to inhibit growth, but stimulated proliferation of HEp-2 cells. In addition, patient DCs had significantly reduced cytotoxic activity against HEp-2 cells. Patients with decreased cytotoxic activity were characterized by significantly lower survival since defect of cytotoxic activity was associated with high-grade glioma. The defective cytotoxic activity of DCs noted against HEp-2 cells was not revealed against glioblastoma U-

Direct Antitumor Activity of Interferon-Induced

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#### **4. Acknowledgment**

We are grateful to our patients for their courage and faith in us.

#### **5. References**


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**Part 5** 

**Glioma Model and Culture Systems** 

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