Abstract 5221: Immune-organoid cocultures to investigate the role of herpesvirus serology in oncolytic viral therapy for glioblastoma

Abstract Background: Complex relationships between treatment and intratumoral immunity are challenging to study in glioblastoma patients owing to the dynamic nature of these relationships and the difficulty in obtaining serial tissues from brain tumors. The importance of these processes necessitates the development of robust model systems that enable researchers to study immune dynamics in controlled and clinically relevant ways. To this end, we have generated a panel of patient derived organoids from glioblastoma patients with paired PBMCs and known clinical features. We are leveraging these models to more deeply study our recently published finding that patients with pre-existing seropositivity to herpesvirus 1 (HSV1) survive significantly longer than seronegative patients when treated with an engineered oncolytic HSV1 agent. Methods: Resected tumor tissues were freshly obtained and cultured to establish glioblastoma organoids alongside paired PBMC and plasma samples. To assess differential immune activation between HSV1 seropositive and seronegative models, PBMCs were expanded for all antigen-experienced immune cells using IL-21, IL-15, and IL-7 and cocultured with their paired organoid in the presence or absence of oncolytic HSV1 infection. Cocultures were observed for spatial dynamics and viral infectivity under time lapse microscopy over 72 hours, followed by flow cytometric analysis at endpoint. Results: Viable organoid cultures were successfully generated for 8 patients over a 7 month period, with paired PBMCs/plasma being available for 5 of these patients, of which two were HSV1 seropositive. Optimization experiments confirmed that expanded T cells and organoids could be cocultured for 72 hours, enabling interrogation of tumor-immune interaction dynamics. Initial results from our cocultures revealed serology-dependent T cell activation in the context of HSV1 infection, with effector CD4 T cells increasing with virus infection in seropositive but not seronegative coculture models. Conclusion: Our results demonstrate that we can rapidly generate glioblastoma organoids with a high success rate. By coculturing these models with paired patient PBMCs, we were able to study dynamic tumor-virus-immune interactions with a high degree of spatiotemporal resolution. We believe this approach holds significant promise for enabling novel insights into the dynamic impact of immunotherapy on the tumor-immune microenvironment-potentially providing critical insights into why these therapies often show limited efficacy in glioblastoma and how they can be improved. Citation Format: Ethan Chen, Alexander L. Ling, Michael C. Prabhu, Connor C. Bossi, Keith L. Ligon, E Antonio Chiocca. Immune-organoid cocultures to investigate the role of herpesvirus serology in oncolytic viral therapy for glioblastoma [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 5221.