Processing human colon cancer specimens for in vitro cytotoxicity assays

Colorectal cancer (CRC) research demands reliable experimental models to enhance translational potential. Immortalized cancer cell lines, although commonly employed, exhibit limitations such as phenotypic divergence from primary tumors, which underscores the need for more representative models. This method chapter presents a protocol for collecting and processing primary CRC specimens for in vitro assays to assess the cytotoxic potential of antitumor agents, with a focus on adoptive cellular therapies. The protocol emphasizes the importance of immediate processing to minimize ex vivo alterations and includes guidelines for cryopreservation, thawing, enzymatic digestion, and mechanical disruption, which were optimized for increased cell yield and viability. An optional step of immune cell depletion is included to avoid indirect effects of endogenous leukocytes, with the option to retain this fraction for further analysis. Finally, the steps for flow cytometry-based evaluation of tumor cell apoptosis by assessment of Caspase 3/7 staining are detailed. The implementation of this standardized protocol using patient-derived specimens offers a superior alternative to immortalized cell lines for assessing therapeutic efficacy, increasing the probability of translation of preclinical research findings, and bolstering the development of innovative therapeutic strategies for CRC.