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Generation of Tumor Spheroids to Evaluate T Cell and NK Cell Cytotoxicity
1Molecular Oncology and Angiogenesis Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy.
Abstract:
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.
Insights
This study details methods for generating colorectal cancer (CRC) spheroids and evaluating immune cell interactions. These 3D models improve preclinical anticancer drug screening and understanding of tumor cell recognition by effector lymphocytes.
Area of Science:
- Oncology
- Immunology
- Cell Biology
Background:
- Three-dimensional (3D) tumor cell spheroids offer a more physiologically relevant model than traditional 2D cultures for studying cancer.
- These 3D models are crucial for preclinical anticancer drug selection and understanding tumor cell behavior.
- Investigating immune cell interactions with tumor spheroids is vital for assessing antitumor efficacy.
Purpose of the Study:
- To provide detailed protocols for generating colorectal carcinoma (CRC) spheroids.
- To analyze methods for evaluating CRC spheroid phenotype, growth, and immune cell interactions.
- To describe the generation and characterization of effector lymphocytes for cytotoxicity assays.
Main Methods:
- Generation of tumor cell spheroids from CRC cell lines.
- Evaluation of spheroid characteristics including ATP content and antigen expression.
- Isolation, generation, and characterization of effector lymphocytes (e.g., natural killer cells, γδ T cells).
- Coculture of spheroids with effector lymphocytes and assessment of lymphocyte-mediated cytotoxicity.
Main Results:
- Established protocols for generating and characterizing CRC spheroids.
- Detailed methods for preparing and assessing effector lymphocyte populations.
- Demonstrated feasibility of evaluating effector lymphocyte recognition and killing of CRC spheroids.
- Provided a framework for studying molecular mechanisms of immune cell-mediated tumor killing.
Conclusions:
- Tumor cell spheroids represent a valuable 3D model for preclinical cancer research.
- Standardized methods are essential for reliable assessment of immune cell interactions with spheroids.
- This work facilitates the study of effector lymphocyte functions against colorectal cancer in a 3D context.
