TMOD-40. Genetic drivers shape tumor phenotype and composition of the tumor microenvironment in syngeneic mouse models of glioblastoma

Abstract INTRODUCTION Glioblastoma (GBM) poses a great challenge for therapeutics development due to its heterogeneity and the lack of immunocompetent models that faithfully recapitulate human GBM. We sought to generate and characterize syngeneic mouse models for major GBM subtypes. METHODS Postnatal electroporation using the PiggyBac transposon/transposase for overexpression (OE) and CRISPR/Cas9 for knockout (KO) was used to generate models with common GBM alterations including EGFRvIII-OE/Cdkn2a-KO/Pten-KO, Nf1-KO/Pten-KO/p53-KO, and RasV12-OE/Cdk4-OE/p53-KO. Model characterization was done by HE staining, immunofluorescence staining, RNA sequencing and flow cytometry. RESULTS All models displayed histopathological and molecular features of GBM such as pseudopalisiding necrosis, microvascular proliferation, and cellular atypia, as well as Gfap and Nestin expression. On the transcriptomic level, Nf1/Pten/p53 tumors showed enrichment for the human mesenchymal signature, while RasV12/Cdk4/p53 and EGFRvIII/Cdkn2a/Pten tumors resembled the classical and proneural signatures. Each tumor was comprised of four cellular states, i.e., astrocytic, mesenchymal, OPC-like, and NPC-like, suggestive of intratumoral heterogeneity. On examining the microenvironment, EGFRvIII/Cdkn2a/Pten tumors had a higher relative abundance of T cells (48.6% vs 6.3%, p=0.0084, 48.6% vs 8.8%, p=0.0376,), NK cells, (1.2% vs 0.4%, p=0.1425, 1.2% vs 0.19%, p=0.0315) and dendritic cells (7.5% vs 0.4%, p=0.0272) than the Nf1/Pten/p53 and RasV12/Cdk4/p53 models. RasV12/Cdk4/p53 tumors had greater infiltration of monocyte-derived macrophages than Nf1/Pten/p53 (35.9% vs 4.18%, p=0.0012) and EGFRvIII/Cdkn2a/Pten (35.9% vs 10.4%, p=0.0620) tumors as well as an elevated CD4+/CD8+ T cell ratio. In the Nf1/Pten/p53 model, microglia were the most abundant cells within the CD45+ compartment, surpassing levels observed in EGFRvIII/Cdkn2a/Pten (60.2% vs 4.6%, p=0.0548) and RasV12/Cdk4/p53 (60.2%vs7.5%, p=0.0564) tumors. CONCLUSION We developed an array of syngeneic mouse models of GBM that recapitulate human inter- and intra- tumoral heterogeneity. Future studies will define the functional states of immune cells in the microenvironment and evaluate the impact of immune cell composition on the response to immunotherapies.