Abstract 955: Single cell sequencing delineates immune features of oncolytic virus therapeutic response in murine high-grade gliomas

Abstract Oncolytic virotherapy (OV) comprises a developing family of cancer treatments that exploit cancer-specific vulnerabilities in viral defense through treatment with therapeutic viruses modified for safety and tumor-selective infection. Beyond direct infection and lysis of cancer cells, OVs are additionally capable of stimulating powerful immune responses that pose an opportunity to redefine the immune system’s behavior in the tumor microenvironment. This may hold particular significance for highly immunosuppressive malignancies, including glioblastoma and other high-grade gliomas (HGG). Our labs developed OV C134, now under evaluation in a phase I clinical trial of recurrent glioblastoma (NCT03657576). While additional refinements to the activity and control of OVs within the tumor microenvironment may lead to superior performance, little is known about the exact features or timing of immune changes undergone in the setting of positive therapeutic response to oncolytic virus treatment. Here, using the widely-adopted C57BL/6 syngeneic high-grade glioma model CT2A modified to express luciferase, we applied bioluminescence imaging to measure tumor progression in vivo and identify therapeutic responders after intracranial treatment with C134, C002 (IL-12-expressing C134), or control (saline). We then performed single cell RNA sequencing of CD45-sorted high-grade glioma immune infiltrate (n=10 mice total; n=3 C002 responder and n=3 C002 nonresponder). Analysis of single cell sequencing data revealed that OV treatment produced diverse changes in gene expression consistent with activation of antigen presentation and antigen-specific immune responses throughout a variety of innate and adaptive immune populations. Further, cell type-specific immune signatures of C002 therapeutic responders as compared to nonresponders were identified, including responder-specific increases in major histocompatibility complex class II in macrophages/monocytes, interferon gamma in CD4+ and CD8+ T cells, tumor necrosis factor-alpha in CD4+ T cells, and a decrease in interferon gamma receptor 1 in T regulatory cells. Collectively, our findings contribute to the knowledge of how OV therapy alters tumor immune microenvironments and reveal prioritized candidates whose further study could advance the understanding of how to achieve positive responses to OV therapy in patients. Citation Format: Jack Hedberg, Alexia Martin, Doyeon Kim, Jaye Navarro, Adithe Rivaldi, Yeaseul Kim, Elizabeth Garfinkle, Katherine Miller, Ravi Dhital, Kevin Cassady, Elaine Mardis. Single cell sequencing delineates immune features of oncolytic virus therapeutic response in murine high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 955.