Advanced 3D patient-derived glioblastoma organoid-immune cell co-cultures reveal preserved MHC-I tumor cell surface expression and tumor cell-driven T-cell dysfunction

Abstract Introduction Glioblastoma represents an aggressive, immunologically cold primary brain tumor largely unresponsive to conventional immunotherapies. Dissecting patient-specific immunosuppressive cues is limited by current preclinical models. Understanding the functional cross-talk between tumor and immune cells across distinct microenvironmental niches is crucial for advancing personalized immunotherapy. Methods We assessed the immunogenic features of glioblastoma cells and cross-talk with T-cells using bulk and single-cell RNA-seq, multicolor flow cytometry, immunohistochemistry, and functional assays in patient tumors, patient-derived organoids (PDOs), orthotopic xenografts and cell cultures. We developed an advanced 3D co-culture system combining PDOs with human peripheral blood T-cells isolated from glioblastoma patients or healthy donor blood to functionally investigate T-cell behavior in a patient-specific context. T-cell subsets integrated and non-integrated into the PDOs were examined for T-cell activation, exhaustion, and T regulatory cell development in response to tumor-derived signals and hypoxia. Results Single-cell RNA-seq-based crosstalk predicted direct ligand-receptor and soluble axes between glioblastoma and T-cells. Glioblastoma cells in patient tumors and diverse patient-derived models showed cell surface MHC class I expression across diverse genetic backgrounds and microenvironmental niches, modulable by interferon gamma. Subsets of CD4 + and CD8 + T-cells integrated into 3D PDOs deprived of myeloid component. PDO-integrated T-cells were enriched for T regulatory cells (Treg), and exhibited a heterogeneous spectrum of activation and dysfunction states, with differential expression of PD-1, LAG-3, TIM-3, CD45RO, Granzyme B, TCF1, and TOX. T-cells subjected to soluble factors were largely unaffected and retained capacity to reintegrate PDOs at sequential co-cultures. Hypoxia influenced T-cell phenotype, but did not prevent activation or infiltration within the early stages of co-culture. Conclusion Our work provides insights into tumor-intrinsic cues of immune evasion in glioblastoma and introduces a scalable patient-relevant ex vivo platform for functional studies. Surface MHC-I was detectable in all assessed glioblastoma models; and was retained in contact with T-cells, indicating that MHC-I loss is not their predominant immune-escape mechanism. Immunosuppression occurred in direct contact with glioblastoma cells, involving both TOX-dependent exhaustion and TOX-independent dysfunction. Advanced co-cultures of glioblastoma PDOs may serve as a powerful tool for functional studies ex vivo, predicting clinically-relevant responses to immunotherapeutic treatments in the future.