Autologous tumor-immune effusion cocultures enable ex vivo functional profiling of radiotherapy-immunotherapy combinations

Currently, radiotherapy-immunotherapy (RT-IO) combinations provide limited and heterogeneous benefits in human solid cancers and are frequently selected empirically, partly because human models that preserve native autologous tumor-immune interactions are lacking. We developed patient-derived autologous tumor-immune effusion cocultures (PATECs) as an ex vivo platform to functionally evaluate RT-IO regimens within an immunocompetent tumor microenvironment. Malignant pleural and peritoneal effusions (n = 29) from patients with metastatic solid cancers were processed for biobanking and primary tumor culture. Expandable tumor cultures were established in six effusions and recombined with matched autologous immune cells to generate PATEC. PATEC were treated with radiotherapy (RT), innate immune agonists (STING, TLR7/8), and immune checkpoint inhibitors (CTLA-4, PD-1, PD-L1, and TIGIT) in combinatorial regimens. Tumor cell death, T-cell activation, cytokine secretion, and CD8⁺ T-cell checkpoint expression were assessed using multiparametric flow cytometry and multiplex immunoassays. Contact dependence of cytotoxicity was evaluated by comparing tumor monocultures, direct cocultures, and transwell-separated cocultures. Across conditions, regimens combining RT with a stimulator of interferon genes (STING) agonist were the most tumoricidal in PATEC, with marked interpatient variability and Bliss-defined synergy in a subset of effusions (3/6). STING agonist-mediated cytotoxicity required immune cells and was attenuated by the spatial separation of the tumor and immune compartments, whereas RT alone produced similar cytotoxicity in monocultures and cocultures at 72 h, suggesting that the observed RT effect in this assay was predominantly tumor-intrinsic. STING-based RT-IO induced early T-cell activation and a type I interferon-rich cytokine milieu, followed by increased expression of multiple inhibitory checkpoints on CD8⁺ T-cells. A composite CD8⁺ checkpoint co-expression score correlated with both overall and contact-dependent tumor cell death. PATEC enables the functional dissection of RT-IO combinations in a native effusion-derived tumor-immune microenvironment. The additional tumor cell killing conferred by STING-based RT-IO depends on immune cells and direct tumor-immune contact and varies between patient samples. These findings support the use of PATEC as a functional ex vivo system for testing therapeutic combinations in patient-specific settings.