P18.23.A AUTOLOGOUS ORGANOTYPIC MULTICELLULAR SPHEROIDS (OMS) AS A PRECLINICAL PLATFORM FOR STUDYING PERSONALIZED GLIOBLASTOMA IMMUNOTHERAPY

Abstract BACKGROUND Immunotherapies for glioblastoma (GBM), including oncolytic viruses, have shown encouraging responses in a small subset of patients, but overall clinical efficacy remains limited. A major hurdle is GBM’s inter- and intra-tumoral heterogeneity, particularly within its immunosuppressive tumor microenvironment (TME), which plays a role in therapy resistance. While multiple ex vivo models exist to address this, most fail to preserve the patient-specific TME cell populations and spatial architecture of the parental tissue - limiting their utility in predicting responses to TME-directed treatments. In this study, we evaluate an advanced patient-derived and autologous ex vivo model, organotypic multicellular spheroids (OMS), which retains unique TMEs and could serve as a platform for personalized therapy selection. We compare the impact of different medium formulations on the preservation of TME integrity within an ex vivo setting. Ultimately, our goal is to determine the optimal culturing conditions under which OMS can serve as a reliable tool to functionally assess and stratify patients for immunomodulatory therapies within a preclinical setting. MATERIAL AND METHODS Freshly resected GBMs are processed to generate OMS under distinct medium formulations: serum-free, FBS, and autologous serum. We analyze key parameters, including time to spheroid formation, viability and growth. TME integrity is assessed by IHC for multiple immune and cell type markers and compared against matched parental tumor tissue. Further TME validation will occur through analysis of secreted cytokines, metabolites and bulk RNAseq. RESULTS The majority of OMS generated within autologous conditions are formed and available for therapy testing within two weeks of the initial resection, with other conditions taking additional weeks to form. The autologously generated OMS also demonstrated robust preservation of parental tissue histology and multicellularity - revealing immune cell infiltrates and (remnant) vascular structures preserved after two weeks in culture. Various cell populations, such as those positively marked by Ki-67, nestin, CD163, CD68, CD31, or CD3, mirrored naturally occurring distributions and patterns found within matched parental tissues. CONCLUSION The use of autologous serum in the generation of OMS offers a faster and higher rate of spheroid generation whilst still preserving key TME cell populations to a higher degree than other conditions. The functional relevance of OMS will be assessed via transcriptomic analysis to further validate the preservation of TME components and by assessment of intertumoral responses to oncolytic viral therapies. Altogether, this indicates that autologously generated OMS retain the parental tissue’s TME better, laying the foundation for a potentially more representative platform to access immunotherapies on a personal level.