A tractable microscopy- and flow cytometry-based method to measure natural killer cell-mediated killing and infiltration of tumor spheroids.

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.