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Glioblastoma (GBM) tumors represents diverse genomic epigenomic, and transcriptional landscapes, with significant intratumoral heterogeneity that challenges standard of care treatments involving radiation (RT) and the DNA-alkylating agent temozolomide (TMZ). In this study, we employed targeted proteomics to assess the response of a genomically-diverse panel of GBM patient-derived cancer stem cells (CSCs) to astrocytic differentiation, growth factor withdrawal and traditional high fetal bovine serum culture. Our findings revealed a complex crosstalk and co-activation of key oncogenic signaling in CSCs and diverse patterns of response to these external stimuli. Using RNA sequencing and DNA methylation, we observed common adaptations in response to astrocytic differentiation of CSCs across genomically distinct models, including BMP-Smad pathway activation, reduced cholesterol biosynthesis, and upregulation of extracellular matrix components. Notably, we observed that these differentiated CSC progenies retained a subset of stemness genes and the activation of cell survival pathways. We also examined the impact of differentiation state and genomic background on GBM cell sensitivity and transcriptional response to TMZ and RT. Differentiation of CSCs increased resistance to TMZ but not to RT. While transcriptional responses to these treatments were predominantly regulated by p53 in wild-type p53 GBM cells, its transcriptional activity was modulated by the differentiation status and treatment modality. Both mutant and wild-type p53 models exhibited significant activation of a DNA-damage associated interferon response in CSCs and differentiated cells, suggesting this pathway may play a wider role in GBM response to TMZ and RT. Our integrative analysis of the impact of GBM cell developmental states, in the context of genomic and molecular diversity of patient-derived models, provides valuable insights for pre-clinical studies aimed at optimizing treatment strategies.

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.