A novel co-culture assay to assess anti-tumor CD8+ T cell cytotoxicity via luminescence and multicolor flow cytometry

T cell immunotherapies have shown great promise in patients with advanced cancer disease, revolutionizing treatment. T cell cytotoxicity is crucial in its efficacy, therefore developing ex vivo methods testing tumor and T cell interactions is pivotal. Increasing efforts have been made in developing co-culture assays with sophisticated materials and platforms aiming to mimic the tumor microenvironment (TME), but its complexity makes it difficult to develop the ideal model. In this study, we developed a simple co-culture assay, reproducible in any lab, but respecting the multicellular nature of the TME. Our goal is to combine in a single assay well-established techniques such as a luciferase assay for target cell viability analysis, a CD107a degranulation assay, and multicolor flow cytometry for the detection of cytokines and cytotoxicity markers. Cell suspensions of whole spleens and tumors containing splenic or tumor-infiltrating effector T cells of mice bearing Lewis lung carcinoma (LLC) or CT26 colon carcinoma tumors treated with radiation alone or in combination with immunotherapies were used for co-culture. LLC and CT26 cell lines transduced with the firefly luciferase gene were used as target cells. We demonstrated that splenocytes and tumor-infiltrating T cells derived from mice treated with combination therapy were able to kill approximately 50% of target cells after 48 h of co-culture. This effect was tumor cell-specific and dependent on CD8⁺ T cells evidenced by in vitro CD8⁺ T cell depletion. Flow cytometry demonstrated increased expression of CD107a and production of granzyme B, IFNγ, and TNFα by CD8⁺ T cells. Our co-culture assay is therefore suitable as proof of principle for in vivo therapeutic studies testing immunotherapies, and specifically to assess the involvement of cytotoxic CD8⁺ T cells in treatment response in LLC and CT26 tumor models. We also propose this assay as an ex vivo platform for high-throughput screening of immunomodulating agents to be tested in these two murine tumor models. This assay can be adapted to other tumor models after optimizations.