Immunotherapies such as checkpoint inhibitors, CAR-Ts and immune-targeting Abs have great promise for cancer treatment. Tumour spheroid models potentially offer more translational biological insight than 2D cell models and more relevant analysis of cancer immunotherapy agents in vitro. Here we describe novel image-based, immune cell-killing assays of 3D tumour spheroids based on IncuCyte live-cell analysis.

Single spheroids were formed from human tumour cell lines expressing RFP in 96-well ULA plates. Immune cells were then added and spheroid viability was assessed over time (up to 10 days) by measuring the loss of RFP fluorescence. This method is exemplified with a range of immune cell activators, stimulating T cell (anti-CD3 and IL-2) or natural killer cell (IL-12 & IL-2) PBMC sub-populations. As expected the magnitude and rate of cytotoxicity was effector-to-target cell ratio dependent. In an antibody-dependent cell-mediated cytotoxicity (ADCC) format, Herceptin induced a concentration-dependent specific killing of Her-2 expressing tumour cells (SKOV-3). Higher concentrations of Herceptin were required in 3D vs 2D ADCC assays.

These data demonstrate the capability to kinetically visualise and quantify 3D immune cell killing and ADCC assays, and illustrates how these assays can be extended from traditional 2D cultures to 3D. These assays will be highly valuable in the search for novel immuno-modulators.