Optimization of Glioblastoma Mouse Orthotopic Xenograft Models for Translational Research.

Glioblastoma is an aggressive primary brain tumor predominantly localized to the cerebral cortex. We developed a panel of patient-derived mouse orthotopic xenografts (PDOX) for preclinical drug studies by implanting cancer stem cells (CSC) cultured from fresh surgical specimens intracranially into 8-wk-old female athymic nude mice. Here we optimize the glioblastoma PDOX model by assessing the effect of implantation location on tumor growth, survival, and histologic characteristics. To trace the distribution of intracranial injections, toluidine blue dye was injected at 4 locations with defined mediolateral, anterioposterior, and dorsoventral coordinates within the cerebral cortex. Glioblastoma CSC from 4 patients and a glioblastoma nonstem-cell line were then implanted by using the same coordinates for evaluation of tumor location, growth rate, and morphologic and histologic features. Dye injections into one of the defined locations resulted in dye dissemination throughout the ventricles, whereas tumor cell implantation at the same location resulted in a much higher percentage of small multifocal ventricular tumors than did the other 3 locations tested. Ventricular tumors were associated with a lower tumor growth rate, as measured by in vivo bioluminescence imaging, and decreased survival in 4 of 5 cell lines. In addition, tissue oxygenation, vasculature, and the expression of astrocytic markers were altered in ventricular tumors compared with nonventricular tumors. Based on this information, we identified an optimal implantation location that avoided the ventricles and favored cortical tumor growth. To assess the effects of stress from oral drug administration, mice that underwent daily gavage were compared with stress-positive and -negative control groups. Oral gavage procedures did not significantly affect the survival of the implanted mice or physiologic measurements of stress. Our findings document the importance of optimization of the implantation site for preclinical mouse models of glioblastoma.

Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Heparanase expression of glioma in human and animal models.

OBJECT: Mammalian heparanase has been shown to function in tumor progression, invasion, and angiogenesis. However, heparanase expression in gliomas has not been well analyzed. To clarify its expression in gliomas, human glioma tissues and glioma animal models were investigated. METHODS: The expression of heparanase mRNA was determined in 33 resected human glioma tissues by semiquantitative real-time polymerase chain reaction. Heparanase expression was verified with a Western blot assay and immunohistochemistry (IHC) staining. Primary neurospheres from human glioblastoma multiforme (GBM) were developed in vitro. Heparanase expression in murine astrocytoma and human primary neurosphere animal models was examined using IHC. RESULTS: The authors found that heparanase mRNA is greatly increased in gliomas including oligodendroglioma (9 samples), anaplastic astrocytoma (11 samples), and GBM (13 samples) as compared with healthy brain mRNA (3 samples). Note, however, that no significant difference was observed among the 3 tumor groups. Increased heparanase expression was also found in tumor tissues on Western blotting. Immunohistochemistry staining demonstrated that heparanase was expressed by neovessel endothelial cells, infiltrated neutrophils, and in some cases, by neoplastic cells. Heparanase-expressing cells, including GBM tumor cells and neovessel endothelial cells, exhibited decreased expression of CD44, a cell adhesion molecule on the cell membrane that is important for regulating tumor invasion. In addition, heparanase-expressing tumor cells showed an elevated density of the cell proliferation marker Ki 67, as compared with its density in non-heparanase-expressing tumor cells, suggesting that heparanase expression is correlated with enhanced tumor proliferation. Two animal glioma models were tested for heparanase expression. Both murine astrocytoma cells (Ast11.9-2) and cultured primary human GBM neurospheres expressed heparanase when grown in animal brain tissue. CONCLUSIONS: Glioma tissues contain increased levels of heparanase. Multiple cell types contribute to the expression of heparanase, including neovessel endothelial cells, tumor cells, and infiltrated neutrophils. Heparanase plays an important role in the control of cell proliferation and invasion. Animal models using Ast11.9-2 and primary neurospheres are suitable for antitumor studies targeting heparanase.