**Assessment of Rodent Glioma Models Using Magnetic Resonance Imaging Techniques**

Rheal A. Towner, Ting He, Sabrina Doblas and Nataliya Smith *Advanced Magnetic Resonance Center, Oklahoma Medical Research Foundation Oklahoma City U.S.A.* 

### **1. Introduction**

250 Advances in the Biology, Imaging and Therapies for Glioblastoma

Sawlani, R. N., J. Raizer, et al. "Glioblastoma: a method for predicting response to

Schaefer, P. W., P. E. Grant, et al. (2000). "Diffusion-weighted MR imaging of the brain."

Schluter, M., B. Stieltjes, et al. (2005). "Detection of tumour infiltration in axonal fibre

Stejskal, E. O. and J. E. Tanner (1965). "Spin Diffusion Measurements: Spin Echoes in the

Sugahara, T., Y. Korogi, et al. (1999). "Usefulness of diffusion-weighted MRI with echo-

Thomsen, C., O. Henriksen, et al. (1987). "In vivo measurement of water self diffusion in the human brain by magnetic resonance imaging." Acta Radiol 28(3): 353-61. Tien, R. D., G. J. Felsberg, et al. (1994). "MR imaging of high-grade cerebral gliomas: value of

Tofts, P. S. and A. G. Kermode (1991). "Measurement of the blood-brain barrier permeability

Tourdias, T., S. Rodrigo, et al. (2008). "Pulsed arterial spin labeling applications in brain

Tropine, A., G. Vucurevic, et al. (2004). "Contribution of diffusion tensor imaging to delineation of gliomas and glioblastomas." J Magn Reson Imaging 20(6): 905-12. Vander Elst, L., A. Roch, et al. (2002). "Dy-DTPA derivatives as relaxation agents for very

Warach, S., D. Chien, et al. (1992). "Fast magnetic resonance diffusion-weighted imaging of

Warmuth, C., M. Gunther, et al. (2003). "Quantification of blood flow in brain tumors:

Witwer, B. P., R. Moftakhar, et al. (2002). "Diffusion-tensor imaging of white matter tracts in

Wolf, R. L., J. Wang, et al. (2005). "Grading of CNS neoplasms using continuous arterial spin labeled perfusion MR imaging at 3 Tesla." J Magn Reson Imaging 22(4): 475-82.

tumors: practical review." J Neuroradiol 35(2): 79-89.

relaxivities." Magn Reson Med 47(6): 1121-30.

acute human stroke." Neurology 42(9): 1717-23.

enhanced MR imaging." Radiology 228(2): 523-32.

patients with cerebral neoplasm." J Neurosurg 97(3): 568-75.

bundles using diffusion tensor imaging." Int J Med Robot 1(3): 80-6.

Radiology 255(2): 622-8.

Radiology 217(2): 331-45.

Reson Med 17(2): 357-67.

42(1): 288-292.

9(1): 53-60.

7.

antiangiogenic chemotherapy by using MR perfusion imaging--pilot study."

Presence of a Time-Dependent Field Gradient." The Journal of Chemical Physics

planar technique in the evaluation of cellularity in gliomas." J Magn Reson Imaging

diffusion-weighted echoplanar pulse sequences." AJR Am J Roentgenol 162(3): 671-

and leakage space using dynamic MR imaging. 1. Fundamental concepts." Magn

high field MRI: the beneficial effect of slow water exchange on the transverse

comparison of arterial spin labeling and dynamic susceptibility-weighted contrast-

There is a strong need to obtain precise surrogate biomarkers to improve the accuracy of diagnosis for gliomas, and to effectively evaluate therapeutic response. Often pre-clinical models of disease are used to develop diagnostic procedures and assess the effectiveness of a potential therapy. For gliomas, there are a variety of rodent models that have been investigated by numerous investigators over the past few decades, ranging from intracranial rodent glioma cell implantation models, intracranial human glioma xenografts, orthotopic implantation of human glioma stem cells, multipotent human glioblastoma stem-like neurosphere lines, transgenic mouse models, to viral-induced progenitor or stem cell derived glioma models. Tumor grades in these models vary from low to high grade tumors, with many of the high-grade glioma models sharing some of the characteristics of human grade IV glioblastoma multiforme (GBM). Many of the characteristic features of gliomas can be assessed diagnostically with *in vivo* imaging techniques such as magnetic resonance imaging (MRI). MRI has the capability of obtaining morphological/anatomical, functional, biophysical, molecular and metabolic information of a disease at various pathological stages of development. Tumor characteristics often associated with aggressive gliomas include an invasive growth pattern, angiogenesis, necrosis, hypoxia, edema, and alterations in major metabolic pathways. Morphological features, such as tumor size and position, infiltrative growth, hemorrhaging, necrotic lesions, edema, mass effect, heterogeneity, and cyst formation, can be followed using standard contrast-enhanced T1-weighted MR imaging or non-contrast T2-weighted imaging.

Although conventional MRI provides us with some indication about the nature of the lesion or tumor, it has limited sensitivity and specificity in determining histological type and grade, delineating margins and differentiating edema, as well as effectively evaluating therapeutic effects or side-effects. Incorporating some advanced MR techniques, such as MR angiography (MRA), perfusion-weighted MR imaging (PWI), diffusion-weighted MR imaging (DWI), MR spectroscopy (MRS), and molecular MRI (mMRI), may help to overcome some of those limitations. Angiogenesis associated with major blood vessels can be assessed using MR angiography, whereas perfusion-weighted imaging can be used to monitor angiogenesis associated with capillary vessels in tumors. Biophysical parameters such as water diffusion, as measured by diffusion-weighted imaging, have also provided

Assessment of Rodent Glioma Models Using Magnetic Resonance Imaging Techniques 253

potential new therapies. This review will provide examples of commonly used and new experimental animal models for gliomas, which make up a large portion of primary brain tumors. The majority of models involve intracerebral implantation of rodent (rat or mouse) or human glioma cells into synergetic rats or mice, or immunocompromised rodents (e.g. nude or athymic rats or mice). There are also a limited number of transgenic mouse models for gliomas. One approach to better simulate a human tumor is to obtain human glioma neurospheres from patients during tumor resection, and then culture the cells prior to intracerebral implantation into immunocompromised rodents. Another recent approach is to implant non-replicating viruses that can stimulate neuronal stem cells to turn into glioma cells which develop into diffuse tumors similar to those found in high-grade or malignant

Glioma cells (rat, mouse or human origin) are injected into the cerebral cortex of rats or mice (synergetic if cells are transplanted into the same species and strain that they were obtained from, or immune-compromised rats or mice if human cells are used) using a stereotaxic device for precise implantation into a brain region. As tumors grow over a period of 1-2 months, this model is considered a short-term model. Different cell lines varying in their degree of malignancy, such as rat C6, 9L/LacZ, F98 and RG2 cells, mouse GL261 cells and human U87 cells, provide a range of gliomas from moderately aggressive to GBM-like. Many of these models have some characteristics associated with human gliomas, such as aggressive tumor growth, angiogenesis, and tumor necrosis (in a few models), however the diffuse nature of high-grade gliomas, glioblastoma multiforme (GBM), is not well represented. In many instances the intracerebrally-implanted rodent tumors have defined tumor boundaries, which do not represent the infiltrative nature of GBMs well. A comprehensive review that discusses the advantages and disadvantages of rat brain tumor models, most of them involving intracerebral implantation of rat glioma cells, is discussed in

The rat C6 cell line produces diffusively invasive astrocytomas (Barth, 1998; Barth and Kaur, 2009), which have been found to be similar to human glioma cells regarding the expression of genes mainly involved in tumor progression (Sibenaller *et al*., 2005). C6 gliomas were induced in an outbred Wistar rat strain repeatedly injected with methylnitrosourea (MNU), which makes it non-syngeneic in inbred strains, and increases its potential to evoke an alloimmune response (Barth and Kaur, 2009). As a result of some genetic similarities to human gliomas, the C6 model has been widely used as a GBM model for a number of years (Grobben *et al*., 2002; Barth and Kaur, 2009). The 9L/LacZ-derived tumors are aggressive and infiltrative, and are angiogenic (Plate *et al*., 1993), which are some of the characteristics associated with human GBM (Weizsaecker *et al*., 1981). Although the aggressive 9L/LacZ gliomas are highly invasive (Szatmori *et al*., 2006) and have extensive neovascularization (Plate *et al*., 1993), due to their pronounced immunogenicity (Barth, 1998) and the fact that they are classified as gliosarcomas (Sibenaller *et al*., 2005), makes these cells a poor choice for glioma studies. F98 gliomas are classified as anaplastic malignant tumors, which have an infiltrative pattern of growth, and also have attributes associated with human GBM (Barth, 1998; Barth and Kaur, 2009). The aggressive and invasive nature (Barth, 1998; Barth and Kaur, 2009) of RG2 tumors (Groothuis *et al*., 1983), as well as the highly tumorigenic human glioblastoma U87 MG cell line *(Martens et al*., 2006; Cheng *et al*., 1996), both mimics human

gliomas called, glioblastoma multiforme (or GBM).

**3.1 Intracerebral cell implantation models** 

a paper by Barth and Kaur (2009).

information regarding alterations in tissue structure associated with glioma tumors. An extension of the diffusion-weighted imaging technique is diffusion tensor imaging (DTI), which can provide information on white matter neuronal fiber tractography. MR spectroscopy can be used to assess alterations in tumor metabolites associated with glucose, bioenergetics, amino acid or lipid metabolism, for example. Many of these advanced MR techniques are used in a clinical setting. More recently, molecular MRI (mMRI) which incorporates a MRI contrast agent as a signaling molecule and an affinity component that targets specific tumor markers associated with tumor growth, angiogenesis, cell invasion, inflammation, or apoptosis, can be used to characterize *in vivo* molecular events associated with gliomas.

This review will focus on various glioma models that have been studied *in vivo*, with particular emphasis on MR image and/or spectroscopy evaluation of these models, and the use of MR image criteria (morphological, biophysical, molecular and metabolic) to evaluate therapeutic treatments. The aims are to: (1) provide an overview on current rodent glioma models being studied; (2) provide an overview regarding currently used MR methods, including advanced MR techniques (e.g. MRA, DWI, PWI, MRS and mMRI), relevant to glioma research; and (3) summarize studies that have used MR methods to evaluate therapeutic response in pre-clinical models for gliomas.
