A Colony Formation-Based Assay to Evaluate Natural Killer Cell Antitumor Responses In Vitro.

Natural killer (NK) cells exert a pivotal role in cancer pathology due to their ability to recognize and eliminate virus-infected, stressed, or malignant transformed cells. Although adaptive immunity recognizes cancer cells by detecting tumor-associated antigens, NK cells detect target cells in an antigen-specific independent manner, relying on a complex array of activating and inhibitory receptors. Despite their potential, NK cell-mediated antitumor activity faces challenges such as tumor resistance mechanisms and limited infiltration into tumor tissues. Herein, we describe a colony formation-based assay to evaluate NK cell-mediated tumor cytotoxicity in vitro, providing insights into NK cell responses and tumor cell resistance mechanisms, offering a valuable tool for studying and optimizing NK cell-based immunotherapy for cancer treatment. This method evaluates relative survival of tumor target cells to NK effector cells, and it can be complemented with flow cytometry analyses of NK cell immunophenotype or functional studies of soluble factors in supernatants, such as an Enzyme-Linked ImmunoSorbent Assay (ELISA). This allows the elucidation of molecules and signaling pathways that regulate NK cell anticancer activity.