An ex vivo assay to assess CAR T cell cytotoxicity in viable tumor tissues using a two-step staining protocol.

Chimeric Antigen Receptor (CAR) T cell therapy has shown remarkable efficacy in hematologic malignancies, yet its success in solid tumors remains limited due to the complexity of the tumor microenvironment (TME) and limited penetration. To address this, we developed a versatile two-step staining assay to assess CAR T cell-mediated cytotoxicity both in vitro and ex vivo on viable tumor tissues. The method combines detection of caspase-3/7 activity with phenotypic characterization of the tumor cells, offering a reliable readout of CAR T cell efficacy. The assay is compatible with live tissue slices and tumor cell lines, and can be customized for various tumor types and antigens. This model provides a robust tool for preclinical screening of CAR T cell function in physiologically relevant contexts.