IMMU-36. iHOTT: A Human Tumor–Immune Organoid Model for Interrogating Immunotherapy Response in Glioblastoma

Abstract Glioblastoma is a highly aggressive brain tumor with a dismal prognosis, in part due to its ability to evade immune detection through an immunosuppressive microenvironment. While immune checkpoint inhibitors have shown promise in other cancers, glioblastoma remains largely resistant to immunotherapy. There is a critical need for physiologically relevant, patient-derived human models to investigate mechanisms of immune resistance and response. Here, we developed and validated iHOTT, a human tumor–immune organoid transplantation system, in which patient glioblastoma cells and matched circulating immune cells (PBMCs) are co-cultured within mature human cortical organoids. This model was evaluated using single-cell RNA sequencing, cytokine profiling, immunofluorescence, and T cell receptor (TCR) sequencing, and was benchmarked against pembrolizumab-treated and untreated patient tumor samples. iHOTT preserved diverse tumor and immune compartments, supported cytokine production in the presence of both tumor and immune cells, and demonstrated robust immune activation at both the transcriptional and protein levels. Upon pembrolizumab treatment, the model mirrored multiple aspects of patient immune responses, including expansion of T cells, B cells, and ILCs. Cell–cell interaction analyses showed increased signaling involving unconventional T cell subsets such as MAIT and γδ T cells, which were mirrored between patient samples and iHOTT. Furthermore, we noted upregulation of immunologically relevant pathways, including complement, CD226, and CD70 in both iHOTT and patients. TCR sequencing revealed increased clonal diversity driven by CD4 T cells in treated iHOTT samples, consistent with patient tumor profiles. Clonotype clustering revealed largely private, patient-specific responses in both iHOTT and patient datasets, reflecting the inter-tumoral heterogeneity that may underlie inconsistent responses to PD-1 blockade in glioblastoma. This autologous human organoid platform provides a scalable, multimodal system to model patient-specific tumor–immune dynamics, dissect immunotherapy mechanisms, and support biomarker discovery and therapeutic development in glioblastoma.