A New Mouse Model of Diffuse Midline Glioma to Test Targeted Immunotherapies

Abstract

BACKGROUND: Diffuse midline gliomas remain incurable, with consistently poor outcomes in children despite radiotherapy. Immunotherapeutic approaches hold promise, with the integration of the host's immune system fundamental to their design. Here, we describe a new, genetically engineered immunocompetent model that incorporates interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen, which is suitable for further evaluation of the antitumor activity of IL13Rα2-targeted immunotherapeutics in preclinical studies. METHODS: The RCAS-Tv-a delivery system was used to induce gliomagenesis through overexpression of PDGFB and p53 deletion with and without human IL13Rα2 in Nestin-Tva; p53fl/fl mice. Neonatal pups were infected with Cre recombinase and PDGFB+IL13Rα2 or Cre recombinase and PDGFB in forth ventricle or right cortex of the brain to model diffuse midline glioma and pediatric high-grade glioma, respectively. Immunoblotting and flow cytometry was used to confirm target expression. Kaplan-Meier survival curves were established to compare tumor latency in both models. Tumor tissue was analyzed through immunohistochemistry and H&E staining. Cell lines generated from tumor-bearing mice were used for in vitro studies and orthotopic injections. RESULTS: The protein expression of PDGFB and IL13Rα2 was confirmed by flow cytometry and western blot. In both groups, de novo tumors developed without significant difference in median survival between PDGFB and p53 loss (n=25, 40 days) and PDGB, IL13Rα2, and p53 loss (n=33, 38 days, p=0.62). Tumors demonstrated characteristics of high-grade glioma such as infiltration, palisading necrosis, microvascular proliferation, high Ki-67 index, heterogeneous IL13Rα2 expression, and CD11b+ macrophages, along with a low proportion of CD3+ T cells. Orthotopic tumors developed from cell lines retained histopathological characteristics of de novo tumors. Mice orthotopically implanted with cells in the 4th ventricle or right cortex showed a median survival of 42 days and 41 (p=0.56) days, respectively. CONCLUSION: Generation of de novo tumors using the RCAS-Tv-a delivery system was successful, with tumors possessing histopathologic features common to pediatric diffuse gliomas. The development of these models opens the opportunity for preclinical assessment of IL13Rα2-directed immunotherapies with the potential for clinical translation.