Modl-49. new murine glioblastoma stem-like cell models

Abstract BACKGROUND There is a dearth of mouse glioblastoma (GBM) preclinical models in immunocompetent mice for the development of immunotherapeutic agents. In this study, we characterized five murine GBM stem-like cell (GSC) models derived from lentivirus induced tumors in transgenic mice that are driven by the activation of the Nf1-Ras MAP kinase signalling pathway due to HRasV12 or NF1 shRNA, and inactivation of Tp53. METHODS Murine GSC lines, 005, NF53, RIG, C1, and C3, were cultured as spheres in serum-free stem cell media. Whole exome sequencing (WES) was conducted to quantify single nucleotide polymorphisms (SNPs) using the C57BL/6J genome as reference. Stem cell properties were characterized by in vitro assays and tumorigenicity was examined by intracerebral implantation into C57BL/6 mice. Brain tissues were processed for histological analysis, such as immunohistochemistry, or for FACS to examine markers of stemness and tumor-infiltrating immune cells. RESULTS WES identified a large variation in coding sequence SNPs, likely due to the mixed backgrounds of the parental mice, with 005 having the highest number of SNPs, and C1 and C3 the lowest. All GSC lines were capable of clonogenic sphere formation, indicative of self-renewal, and showed varying levels of cell surface CD133. In vivo, all the GSCs efficiently established tumors that were histologically consistent with malignant glioma with strong CD44 expression. Median survival varied, ranging from 27 days (005) to 82 days (C1), upon 20,000-cell implantation. CD68+ macrophages were consistently dominant in the tumor microenvironment, while C1 and C3 exhibited fewer CD4+ and CD8+ T cells. CONCLUSIONS Despite the similar molecular strategy used to generate these GBMs, we observed a range of tumor phenotypes and immune microenvironmental profiles. These models can serve as valuable tools for studying GBM biology and exploring immunotherapeutic strategies. Keywords: Glioblastoma, glioblastoma stem-like cell (GSC), genetically engineered mouse models, Tumor microenvironment.