A Multimodal Framework to Assess Patient-Derived Colon Tumor Organoid-Immune Cell Co-cultures.

T cells play a key role in cancer immunotherapy, and understanding their interactions with tumors is essential for developing novel immunotherapies. This protocol describes the step-by-step workflow for establishing colorectal cancer patient-derived organoids (PDOs) from our biobank and developing autologous PDO-T cell co-culture systems. Using Annexin V NIR and cleaved caspase-3 staining, we standardized flow cytometry protocols for surface and intracellular staining to assess T cell reactivity and cytotoxicity, as well as tumor organoid killing. In addition to flow cytometry-based analysis, the protocol includes methodological approaches for functional assays, such as ELISpot assays to quantify granzyme B and perforin secretion, and live-cell imaging to monitor T cell-mediated tumor killing over time via Annexin V-based apoptosis detection. This workflow further details the setup of co-culture conditions, including the incorporation of target inhibitors and immune checkpoint inhibitors in both monotherapy and combination treatment settings. Guidelines are provided for selecting and applying key immunological and tumor-associated markers, including CD137 for T cell reactivity, CD107a for T cell degranulation, caspase-3 for tumor apoptosis, and Ki67 for tumor proliferation. Collectively, this paper provides a comprehensive and reproducible framework for colorectal cancer organoid establishment, autologous T cell culture and expansion, co-culture setup, therapeutic perturbations, and downstream analyses using flow cytometry, ELISpot, confocal microscopy, and live-cell imaging.