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

Background Currently, radiotherapy-immunotherapy (RT-IO) combinations provide limited and heterogeneous benefit 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 co-cultures (PATEC) as an ex vivo platform to functionally evaluate RT-IO regimens within an immunocompetent tumor microenvironment. Methods 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, TIGIT) in combinatorial regimens. Tumor cell death, T-cell activation, cytokine secretion and CD8⁺ T-cell checkpoint expression was assessed using multiparametric flow cytometry and multiplex immunoassays. Contact dependence of cytotoxicity was evaluated by comparing tumor monocultures, direct co-cultures and transwell separated co-cultures. Results 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 spatial separation of tumor and immune compartments, whereas RT alone produced similar cytotoxicity in monocultures and co-cultures, indicating a predominantly tumor-intrinsic effect. 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. Conclusions PATEC enables functional dissection of RT-IO combinations in a native effusion-derived tumor-immune microenvironment and shows that 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 a patient-specific setting.