Immunocompetent murine glioblastoma stem-like cell models exhibiting distinct phenotypes.

Abstract

Background: Glioblastoma (GBM) treatment is hindered by a dearth of representative mouse GBM preclinical models in immunocompetent mice. Here, we characterized 5 murine GBM stem-like cell (mGSC) models derived from lentivirus-induced tumors in transgenic mice that are driven by the activation of the Nf1-Ras signaling pathway and inactivation of Tp53.

Methods: MGSC lines (005, RIG, NF53, C1, and C3) were cultured as spheres in serum-free stem cell media. Whole exome sequencing (WES) was employed to quantify single nucleotide polymorphisms (SNPs). Stem cell properties were characterized by stemness in vitro and tumorigenicity after intracerebral implantation in C57BL/6 mice. Tumor phenotypes and the immune microenvironment were characterized by immunohistochemistry, flow cytometry, and RNA sequencing.

Results: WES revealed a large variation in coding sequence SNPs across mGSC lines (~20-fold), likely influenced by the mixed backgrounds of the parental mice. MGSCs exhibited variable clonogenic sphere formation and CD133 expression levels. In vivo, they consistently initiated lethal malignant gliomas, with median survival ranging from 29 to 82 days, and showed strong CD44 expression and variable invasiveness. The tumor microenvironment featured an abundance of CD68+ macrophages and uniform high PD-L1+ myeloid cells, while T-cell infiltration varied among the models, with low mutation burden C1 and C3 exhibiting fewer tumor-infiltrating T cells.

Conclusions: Upon orthotopic implantation in immunocompetent mice, mGSCs generate tumors characteristic of human GBM. Despite similar strategies to generate these mGSCs, they exhibited a range of phenotypes and immune profiles in mGSC-derived orthotopic tumors. These mGSCs provide new preclinical GBM models for developing GBM immunotherapies.