Dissecting Oncolytic Virus Anti-Virus Vs Anti-Tumor Immunity in Glioma: Insights from a Patient Derived Organoid Model

Abstract Introduction. Intratumoral injection of the oncolytic herpes simplex virus (oHSV) CAN-3110 remodels the immunosuppressive microenvironment of recurrent glioblastoma. The immune infiltration triggered by CAN-3110 treatment is correlated with prolonged survival, especially in HSV1-seropositive individuals (Ling et al., 2023). However, it is still unclear if the immune response induced by oHSV therapy is mainly anti-virus or anti-tumor. We aim to dissect this distinction using ex vivo patient-derived glioma organoid (pGBO)-immune co-culture models to study the impact of oHSV treatment on anti-tumor immunity. Methods. pGBOs were generated from surgically resected glioma specimens and characterized by highly multiplexed cyclic immunofluorescence (CycIF), via (up to date) 29 markers. Viral infection kinetics were assessed in these pGBOs using the GFP-expressing oHSV, rQNestin34.5v.1. Following pGBM characterization, we established a co-culture model of several pGBOs clones with rQNestin34.5v.1 and peripheral blood mononuclear cells (PBMCs) from either healthy donors or the pGBO patients. This system was used to assess tumor-immune interaction characteristics and kinetics under viral and non-viral conditions. Results. Histological and immunofluorescent analyses showed that pGBOs retain tissue microstructures, such as vascular architecture - characterized by endothelial walls containing erythrocytes - for several weeks in culture, even after freezing and thawing the models. The cellular diversity in pGBOs mirrors the heterogeneity found in in vivo, comprising neural stem-like, glial, and residual innate immune cell populations. These distinct cell types exhibited metabolic activity, epithelial-to-mesenchymal transition (EMT), and proliferation. Over a three-month culture period, the architecture and phenotype changed to more aggressively glioma, with increased glioma cell expansion and loss of vasculature. Notably, the underlying cellular heterogeneity remained throughout the process. Co-culture of pGBOs with allogeneic PBMCs triggered infiltration of CD3+, CD8+, and CD4+ lymphocytes under viral and non-viral conditions. Staining for cleaved caspase-3 and granzyme B showed active tumor cell killing by granzyme+ CD8 T cells, which appears unrestricted to infected glioma cells. Further, CD20+ lymphocytes were detected in both infected and uninfected pGBOs. Conclusion. We characterized pGBOs as a physiologically valid ex vivo model, observing both vasculature and cellular composition as found in in situ gliomas. Immune-mediated tumor cell killing was detected, both with and without virus treatment in PBMC x pGBO co-culture, using imaging and molecular readouts. This model enables investigation of how oncolytic HSV infection modulates immune response and provides a tractable system to dissect anti-tumor vs. anti-virus immune responses. Citation Format: C. Zoe Linke, Ethan Chen, Jennifer Gantchev, Aanchalika Chauhan, Christopher Jannotta, Keith L. Ligon, Andres Santos, Nathalie Agar, E Antonio Chiocca, Alexander L. Ling. Dissecting oncolytic virus anti-virus vs anti-tumor immunity in glioma: Insights from a patient derived organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4326.