A new genetically engineered transplant model of glioma recapitulates key phenotypes of low- and high-grade gliomas

Abstract

Immune competent animal models are essential in preclinical glioma research. The ability to investigate tumor development with key tumor microenvironment components such as immune infiltration, stromal cells and extracellular matrix allows for the investigation of these complex tumors. However, the current range of syngeneic models of glioma possess intrinsic limitations which must be acknowledged when designing a preclinical study. To address this gap, we developed genetically engineered mouse cell line models (GEM-CLeMs) by introducing common glioma driver mutations into immortalized astrocytes. A high-grade glioma model was generated by combining Pten knockdown with RAS V12 overexpression, while a low-grade glioma model was produced through p53 knockdown with mutant IDH1R132H overexpression. The RAS/Pten GEM-CLeM tumors grew rapidly in vivo, displayed necrosis, multinucleated pleomorphisms, abundant vascularization, and showed strong enrichment for extracellular matrix remodelling and mesenchymal genes, features closely aligned with human glioblastoma. Importantly, the RAS/Pten GEM-CLeM tumors showed similar survival trends and immune infiltration patterns as a corresponding KrasG12D/PtencKO GEMM, but could be grown to larger sizes, facilitating better stromal and immune analyses. In contrast, the IDH1R132H/p53 GEM-CLeM formed slow-growing tumors with distinctive immune infiltration and vascular patterns, consistent with low-grade glioma phenotypes. Compared with the commonly used cell line GL261, the GEM-CLeM tumors had higher levels of stromal integration and immune suppression, making them a more faithful model of the glioma tumor microenvironment. This system enables rapid generation of transplantable glioma models with defined driver mutations in a low-mutational background, offering a flexible platform for dissecting glioma biology and evaluating immunotherapies. Importance of the studyWe have generated a set of customizable, modular Genetically Engineered Mouse Cell Line Models (GEM-CLeMs) of "high grade" and "low grade" glioma. They reliably form tumors when transplanted intracranially into immune-competent C57BL/6 mice, and they are cost- and time-effective at capturing the important characteristics of glioma, both mutant IDH1 low grade glioma and high grade glioblastoma. Critically these characteristics include the myeloid-rich immune suppressive tumor immune microenvironment, a key weakness of existing murine glioma cell lines like GL261. These GEM-CLeM models can be used in multiple ways. The cells are amenable to further manipulation, so therapeutic targets and drug mechanism of action can be assessed. The activity of candidate genes in tumour formation and phenotype can be determined. Most importantly the models can be used to develop effective immunotherapies, including strategies to target macrophage and myeloid cell immune suppression. Key pointsO_LICombinations of driver mutations were engineered into an immortalised mouse astrocyte. C_LIO_LIEngineered cells formed tumours on intracranial transplant into immune competent mice. C_LIO_LITumors had key histological and immune suppressive features of human glioma. C_LI