Analysis of murine CAR-T cell-mediated cytotoxicity in solid tumor models.

Chimeric Antigen Receptor T-cell (CAR-T) based immunotherapy represents a ground-breaking advancement in the treatment of certain haematological malignancies, such as acute lymphoblastic leukemia (ALL) and multiple myeloma (MM), often leading to remarkable remission rates. Despite these successes, many patients relapse within the first-year post-treatment, highlighting a critical limitation of these therapies. Furthermore, translating the success of CAR-T therapies to solid tumors has been challenging, with limited clinical efficacy, highlighting the need for innovative designs and strategies to achieve durable responses. There is strong scientific interest in enhancing CAR-T antitumor efficacy in solid tumors. Current research efforts are focused primarily on two main strategies: first, the development of enhanced fourth- and fifth-generation CAR-T cells through genetic modification; and second, the optimization of the manufacturing protocols to improve cell quality and functionality. All these novel CAR-T cell designs must be rigorously characterized using standardized and robust protocols. In this review, we describe a protocol for generating murine CAR-T cells and, in detail, the methodology to assess their lytic capacity-both through a real-time impedance-based assay and a conventional endpoint cytotoxicity assay.