Center for Hematology and Regenerative Medicine (HERM), Karolinska Institutet, ANA Futura, Huddinge, Sweden.
Department of Oncology-Pathology, Karolinska Institutet, Karolinska vägen, Solna, Sweden; Department of Molecular Biology, Princeton University, Princeton, NJ, United States.
Methods Cell Biol. 2023;178:43-61. doi: 10.1016/bs.mcb.2022.07.011. Epub 2022 Sep 13.
Understanding the anti-tumor activity of immune cells and testing cancer immunotherapies requires conditions that are as life-like as possible. The tumor microenvironment (TME) describes a complex sum of cellular and acellular actors that influence both immune cells and tumor cells as well as their interplay. Yet in development phases of new immunotherapies, the screening of drugs and adoptive cell products benefits from reproducible and controlled conditions. Two-dimensional (2D) cell cultures cannot simultaneously meet these two challenges therefore lacking considerably predictive power owing to their artificial nature. Various 3D tumor models have therefore been implemented to mimic the architecture and intrinsic heterogeneity of a microtumor. This protocol provides an easy-to-follow, time-efficient, material-limited method for live cell killing and infiltration of single tumor spheroids. It uses multicellular tumor spheroids grown scaffold-free and allows co-culture with immune cells. This protocol is optimized for natural killer (NK) cell functionality assays. However, it can be transferred to other immune cells, in particular cytotoxic T cells. This assay can be analysed using life cell imaging (here with the IncuCyte S3 system) and/or flow cytometry.
理解免疫细胞的抗肿瘤活性并测试癌症免疫疗法需要尽可能逼真的条件。肿瘤微环境（TME）描述了一个复杂的细胞和无细胞因子的总和，这些因子影响免疫细胞和肿瘤细胞及其相互作用。然而，在新免疫疗法的开发阶段，药物筛选和过继细胞产品受益于可重复和可控的条件。二维（2D）细胞培养不能同时满足这两个挑战，因此由于其人为性质而缺乏相当大的预测能力。因此，已经实施了各种 3D 肿瘤模型来模拟微肿瘤的结构和内在异质性。本协议提供了一种简单易行、耗时短、材料有限的方法，用于活细胞杀伤和单个肿瘤球体的浸润。它使用无支架生长的多细胞肿瘤球体，并允许与免疫细胞共培养。该方案针对自然杀伤（NK）细胞功能测定进行了优化。然而，它可以转移到其他免疫细胞，特别是细胞毒性 T 细胞。该测定可以使用活细胞成像（这里使用 IncuCyte S3 系统）和/或流式细胞术进行分析。
