96-Well Live-Cell Assays for Immune Cell Killing of 3D Tumour Spheroids

Immunotherapies such as checkpoint inhibitors, CARTs and immune-targeting Abs have great promise for cancer treatment. Translational cell-based assays are required to optimise these approaches.

Here we describe image-based, immune cell-killing assays of 3D tumour spheroids, geared for assessing the efficacy of novel immune-modulators. Human tumour cell lines expressing RFP were used to form spheroids in 96-well ULA plates. Immune cells were then added and activated to kill. Spheroid viability was assessed over time (up to 10 days) by measuring the loss of RFP fluorescence using Incucyte^® live-cell analysis. 

This method is exemplified with a range of immune cell types (PBMCs, T-cells, NK-cells) and activators, including anti-CD3 & IL-2. In an ADCC format, Herceptin induced a concentration-dependent specific killing of Her-2 expressing tumours. Higher concentrations of Herceptin were required in 3D vs 2D ADCC assays. These data demonstrate how immune-cell killing and ADCC assays can be extended from traditional 2D mono-cultures to 3D spheroid assays, providing the potential for greater translational relevance. These assays will be highly valuable in the search for novel immune-modulators.