Generation of Tumor Spheroids to Evaluate T Cell and NK Cell Cytotoxicity

Tumor cell spheroids are one of the three-dimensional (3D) culture systems that can be used to study the features of tumor cells in a more physiologic condition. Indeed, this 3D culture system has been validated in preclinical studies to select anticancer drugs. Tumor cell spheroids can be obtained through employing different procedures, and here I describe in detail how to obtain spheroids from tumor cell lines of colorectal carcinoma (CRC). I analyze the procedures employed to evaluate the phenotype and growth of tumor cells in this 3D culture system. Also, interaction with immune cells is considered. Indeed, it is relevant to define whether the antitumor effects exerted by different cytotoxic lymphocyte subsets are similar when tumor cells are used either as cells adherent to a plastic substrate or floating spheroids. To obtain optimal results in this complex system, some parameters must be considered, such as those related to poorly defined experimental variables including biological and biophysical features of tumor cell lines and the quality (purity and time to use after generation) of antitumor effector lymphocyte subsets. Also, I describe in detail the methods to generate these lymphocyte subsets and to characterize their cytotoxic potential and effectiveness in killing tumor cells. Human natural killer cells and T lymphocytes expressing the γδ T cell receptor (in particular Vδ2 T cells) are considered among the cytotoxic lymphocyte populations. Eventually, it should be possible to obtain reliable and feasible results to study the molecular mechanisms involved in recognition and killing of CRC spheroids exerted by effector lymphocytes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Generation of tumor cell spheroids Basic Protocol 2: Evaluation of ATP content of spheroids Basic Protocol 3: Evaluation of antigen expression by cell spheroids Basic Protocol 4: Generation of effector lymphocytes Basic Protocol 5: Coculture of spheroids and lymphocytes Basic Protocol 6: Evaluation of lymphocyte cytotoxicity using crystal violet staining.