Molecular Oncology and Angiogenesis Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy.
Curr Protoc. 2022 Feb;2(2):e366. doi: 10.1002/cpz1.366.
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.
肿瘤细胞球状体是可用于研究肿瘤细胞在更生理条件下特征的三维（3D）培养系统之一。事实上，这种 3D 培养系统已在临床前研究中得到验证，可用于筛选抗癌药物。肿瘤细胞球状体可通过采用不同的程序获得，在这里我详细描述了如何从结直肠癌（CRC）的肿瘤细胞系中获得球状体。我分析了用于评估该 3D 培养系统中肿瘤细胞表型和生长的程序。还考虑了与免疫细胞的相互作用。事实上，当使用贴壁于塑料基质的细胞或悬浮球状体的肿瘤细胞时，不同细胞毒性淋巴细胞亚群发挥的抗肿瘤作用是否相似，这一点很重要。为了在这个复杂的系统中获得最佳结果，必须考虑一些参数，例如与实验变量（包括肿瘤细胞系的生物学和生物物理特征以及抗肿瘤效应性淋巴细胞亚群的质量（纯度和生成后使用时间））相关的参数。此外，我还详细描述了生成这些淋巴细胞亚群的方法，并描述了其杀伤肿瘤细胞的细胞毒性潜力和有效性。自然杀伤细胞和表达 γδ T 细胞受体的 T 淋巴细胞（特别是 Vδ2 T 细胞）被认为是细胞毒性淋巴细胞群之一。最终，应该有可能获得可靠且可行的结果，以研究效应淋巴细胞识别和杀伤 CRC 球状体所涉及的分子机制。©2022 年 Wiley 期刊 LLC。基础方案 1：生成肿瘤细胞球状体基础方案 2：评估球状体中的 ATP 含量基础方案 3：评估细胞球状体的抗原表达基础方案 4：生成效应淋巴细胞基础方案 5：球状体和淋巴细胞共培养基础方案 6：使用结晶紫染色评估淋巴细胞的细胞毒性。
