Ionizing radiation and photodynamic therapy lead to multimodal tumor cell death, synergistic cytotoxicity and immune cell invasion in human bladder cancer organoids

Photodynamic therapy (PDT) and radiotherapy using ionizing radiation (IR) are promising options for organ-preserving treatment of bladder cancer (BCa). A combination therapy (IR+PDT) could be beneficial for BCa treatment. For PDT, we used the near-infrared photosensitizer tetrahydroporphyrin-tetratosylate (THPTS) showing high therapeutic efficacy. Treatment responses were analyzed in BCa organoids. Organoids consisting of BCa cells lines, bladder fibroblasts and muscle cells were treated with IR (9 Gy) and/or PDT using THPTS (25, 50 μM; 20 J/cm ). Cytotoxicity was determined by microscopy, cell-based assays and histology. The cell death mode was analyzed by applying specific inhibitors followed by immunofluorescence or qPCR analyses of cell death markers. A matrix-based co-culture model was used to study T cell migration into the environment of treated organoids. PDT and/or IR resulted in concentration-dependent reduction of metabolic activity, organoid diameter and integrity. Higher cytotoxicity of IR+PDT vs. monotherapies was observed after 72 h. Non-malignant organoids showed no cytotoxic effects. While apoptosis, necroptosis and ferroptosis were clearly involved in cell death of T-24 cells, cytotoxicity in RT-112 cells was probably provoked by apoptosis, ferroptosis and pyroptosis. IR+PDT resulted in significant migration of Jurkat cells into ECM-embedded organoids within 3 days after treatment. Treatment with IR+PDT showed tumor-selective cytotoxicity with additive or synergistic effects in BCa organoids. Thereby, IR+PDT led to multimodal cell death depending on the cellular context. Migration of T cells into the organoid environment illustrates the immunogenic potential of IR+PDT. Therefore, it might be a promising approach for organ-preserving BCa treatment.