Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults, characterized by diffuse infiltrative lesions, distinct oncogenic alterations, and a suppressive tumor immune microenvironment (TIME). These traits, however, are not fully recapitulated in current syngeneic murine glioma models, thereby limiting the pre-clinical development of novel therapies. Here we developed murine GBM models with patient-specific mutations that shared striking resemblance with human disease. Mosaic analysis with dual-recombinase mediated cassette exchange (MADR) and CRISPR-Cas9 were leveraged to generate autochthonous tumor models with single-copy somatic transgenesis of murine epidermal growth factor receptor variant III (mEGFRvIII) and loss-of-function mutations commonly observed in GBM. Characterization of a model lacking Pten and Cdkn2a (mEGFRvIII-PC) via histopathologic analyses, functional assays, and single-cell RNA sequencing showed these tumors developed as diffuse, infiltrative lesions with ill-defined margins and a necrotic core. The TIME was primarily comprised of microglia with minimal lymphocyte infiltration, recapitulating patient tumors. Furthermore, these tumors displayed intrinsic resistance to immune checkpoint blockade, potentially due to poor myeloid cell antigen presentation and T cell priming. Additionally, mEGFRvIII-PC cells were the most resistant to antigen-specific T cell-induced cytotoxicity and simultaneously promoted T cell exhaustion, while murine interferon-γ (mIFN-γ) stimulation induced a greater response in vitro with enhanced upregulation of PD-L1 relative to MHC-I. In sum, these findings demonstrate the improved fidelity of the mEGFRvIII-PC model to patient tumors and support its use in developing and refining novel therapeutic paradigms for GBM.
