**4. Human glioma neurospheres**

254 Advances in the Biology, Imaging and Therapies for Glioblastoma

high-grade gliomas via inducing vascular alterations. U87 pcDNA3 and U87 IRE1 DN human glioma cells were selected as malignant glioma models that form highly versus poorly vascularized tumors, respectively (Drogat *et al*., 2007; Wehbe *et al*., 2010). GL261 cells give rise to quickly growing, and diffusively invasive intracranial tumors in C57BL/6 mice (Szatmori *et al*., 2006). RG2 and F98 glioma cell lines were both obtained from chemical induction as a result of administering ethylnitrosourea (ENU) to pregnant rats, where the progeny developed brain tumors that were isolated, and propagated and cloned in cell culture (Barth and Kaur, 2009). Human U87 cells are of high interest for angiogenesis studies (Cheng *et al*., 1996). The immunogenicity issue of the 9L/Lacz model can be

Xenograft models, induced by orthotopic (into native tumor sites) injection of primary tumor cells or tumor cell lines, represent the most frequently used *in vivo* cancer model systems for glioma research (Waerzeggers *et al*., 2010). Both cell culture and xenograft model systems lack the stepwise genetic alterations that are thought to occur during tumor progression, and often do not represent the genetic and cellular heterogeneity of primary tumors, as well as the complex tumor-stroma interaction (Waerzeggers *et al*., 2010). Genetically engineered mouse models (discussed below in the "Transgenic Mouse Models" section) better represent the causal genetic events and subsequent *in situ* molecular evolution, the tumor-stroma interactions, and consist of cellular subpopulations such as cancer stem cells (discussed further in the "Human Glioma Neurospheres" and "Viral-Induced Glioma Models" sections below), that occur in native tumors (Waerzeggers *et al*.,

Slow-growing, low- and high-grade, spontaneous gliomas can be generated with a chemically-induced model from the administration of ENU (Kish *et al*., 2001; Koestner, 1990). Transplacental ENU exposure of a pregnant female a day before gestation, results in the formation of low-grade oligodendrogliomas and mixed gliomas, with a tumor incidence approaching 100%, in rat pups at approximately 3-6 months of age (Koestner, 1990). In addition to oligodendrogliomas and mixed gliomas, unfortunately the ENU-induced model also results in the formation of meningiomas (Koestner *et al*., 1971), spinal cord tumors (Koestner *et al*., 1971) and other primitive neuroectodermal tumors (Vaquero *et al*., 1992), decreasing its potential as a reproducible model. In addition to the isolation of RG2 and F98 rat glioma cells from ENU induction, A15A5 neoplastic astrocytes have also been cloned

As we are beginning to understand the genetic mutations associated with gliomas, it is possible to generate transgenic mouse models that have these genetic mutations. Recent findings suggest that brain tumors originate from neural stem or progenitor cells. Some examples of transgenic mutations include deletions of gene combinations, such as Rb/p53, Rb/p53/PTEN or PTEN/p53 (Jacques *et al.*, 2010). pRb is a retinoblastoma protein, which is a tumor suppressor protein that is dysfunctional in many cancers. Rb controls excessive cell growth by inhibiting cell cycle progression until the cell is ready to divide (Chinnam and Goodrich, 2011; Lohmann, 2010). p53 which is also known as protein 53 is a tumor suppressor protein responsible for regulating the cell cycle (Kim et al., 2011; Maclaine and

resolved by using non-immunogenic models (e.g. RG2).

2010).

**3.2 Chemical-induced model** 

(Davaki and Lantos, 1980).

**3.3 Transgenic mouse models** 

GBM cancer-initiating cells have been found to mediate resistance to chemotherapy and radiation treatment, both used as follow-up therapies following surgical resection of the main tumor mass (Wei *et al*., 2010). Cells isolated from GBM that possess the capacity for self-renewal following radiation and chemotherapy, can form neurospheres when cultured *in vitro* (Wei *et al*., 2010). The glioma-associated cancer-initiating cells were found to express MHC-I (major histocompatibility I) but not MHC-II, CD-40 or CD80, which induces T-cell immune deficiency, and express the costimulatory inhibitory molecule, B7-H1, which plays a role in mediating immune resistance in gliomas and induces T-cell apoptosis (Wei *et al*., 2010). These neurospheres can be intracerebrally implanted into immune-compromised rodents to develop tumors *in vivo,* and therefore provide an experimental model that more closely resembles recurrent human GBM (radiation and chemotherapeutic resistant and induce immunosuppression) to evaluate new therapies. Another approach that takes into consideration the role of tumor-initiating stem cells, is to orthotopically implant tiny fragments of surgically-resected tumors, containing brain tumor stem cells within the glioblastoma tissue, into immunocompromised mice (xenograft model) brains with the use of a trocar system (Fei *et al*., 2010).

#### **4.1 Viral-induced glioma models**

Glial progenitor cells in the white matter and subventricular zone within the central nervous system were recently found to be the likely candidates for glioma-initiating cells (Assanah *et* 

Assessment of Rodent Glioma Models Using Magnetic Resonance Imaging Techniques 257

vessel morphology (Waerzeggers *et al*., 2010), particularly regarding the capillary bed. Magnetic resonance angiography (MRA) is also used to provide information on tumor vasculature associated with angiogenesis, however it tends to be restricted to major blood vessels >50 microns in diameter (Doblas *et al*., 2010). DWI has been used in cancer imaging to evaluate tumor cellularity and infiltration, as well as monitor therapeutic response (Kauppinen, 2002). Metabolic information can be obtained by monitoring tumor metabolites by a method called MR spectroscopy (MRS), or variations thereof, such as MR spectroscopic imaging (MRSI) or chemical shift imaging (CSI). Molecular alterations can be assessed with the use of targeting MR contrast agents which can specifically indicate levels of cancer biomarkers that may be elevated in malignant tumors. The development of targeted imaging ligands attached to MRI contrast agents allows the *in vivo* evaluation of tumor biology, such as tumor cell apoptosis, angiogenic blood vessels or the expression of specific

MRI is obtained on small animal MR imaging systems (7 - 11.7 Tesla), that can accommodate rodents such as mice and rats. MR images are obtained in multiple slices (0.5-1 mm thick) to visualize an entire tumor (Gartesier *et al*., 2010). Examples of rodent tumor models for gliomas (e.g. rat C6 and RG2 models, and mouse GL261 model) are shown in Figure 1, depicting heterogeneous tumors (right cerebral cortex, upper regions) following intracerebral (orthotopic) implantation of rat or mouse glioma cells (Doblas *et* 

From the multiple image slices through a tumor, tumor volumes can be measured, and the growth rate can be calculated from multiple imaging sessions over several days, weeks or months (as shown in Figure 2). Tumor areas are traced in multiple slices to calculate tumor volumes, which can be used to determine tumor growth and doubling times (Doblas *et al*., 2008, 2010; Garteiser *et al.*, 2010). Robust tumor volume determinations can be made by using manual or automated segmentation techniques, which can be used to delineate tumor margins on the basis of signal intensity differences from surrounding brain tissue (Waldman

Fig. 1. MR images of rodent gliomas. T2-weighted images of the rat C6 (A; 18 days following intracerebral implantation of cells) and RG2 (B; 13 days following cell implantation), and the mouse GL261 (C; 26 days following cell implantation) glioma models. Tumors appear as

heterogeneous regions in the upper right area of the cerebral cortex regions.

tumor antigens or signaling pathways (Waerzeggers *et al*., 2010).

**5.1 Tumor morphology** 

*al*., 2010).

*et al*., 2009).

*al*., 2006, 2009; Masui *et al*., 2010). Intracerebral implantation of PDGFB-green fluorescent protein (GFP)-expressing retroviruses into rodents were found to induce tumors that closely resembled diffuse human malignant gliomas which have been challenging to treat (Assanah *et al*., 2006; Masui *et al*., 2010). This model involves the use of a viral vector that stimulates neuronal stem cells to become glioma cells by expressing PDGF, which is involved in generating tumor cells (Masui *et al*., 2010). These studies demonstrate that both adult white matter and glial progenitors generate gliomas, as well as recruit resident progenitors to proliferate within the mitogenic environment of a tumor, and therefore contributing to the heterogeneous mass of cells that make up a malignant glioma (Assanah *et al*., 2006; Masui *et al*., 2010). It was previously demonstrated that PDGF-B could play a dose-dependent role in glial tumorigenesis, where PDGFR (PDGF receptor) signaling via elevating levels of PDGF-B chain expression quantitatively regulates tumor grade, and that PDGF-B expression is required to sustain high-grade oligodendrogliomas (Shih *et al*., 2004). PDGF-B expression in tumor cells was elevated by removing inhibitory regulatory elements in the *PDGFB* mRNA and a retroviral delivery system (Shih *et al*., 2004). To generate tumors, DF1 cells transfected with RCAS (repeat with splice acceptor) retroviral vectors, generating a culture of virusproducing cells, were injected intracranially into N-tva transgenic mice (Shih *et al*., 2004). By inhibiting PDGFR activity, it was possible to convert tumors from high to low grade (Shih *et al*., 2004).

Another recent study involved intracranial injection of lentiviral vectors with GFAP (glial fibrially acidic protein) or CMV (cytomegalovirus) vectors into compound *LoxP*-conditional mice, which resulted in K-Rasv12 expression and loss of p16Ink4a/p19Arf, with or without concomitant loss of p53 or Pten (de Vries *et al*., 2010). Like GFAP, CMV is a promoter (de Vries *et al*., 2010). CMV-Cre injection into *p53;Ink4a/Arf;K-Rasv12* mice was particularly found to result in the formation of high-grade gliomas within 2-3 weeks that had invasiveness and blood-brain barrier functionality characteristics that are found in human high-grade gliomas (de Vries *et al*., 2010).
