A 3D in vitro assay to study combined immune cell infiltration and cytotoxicity
Immune cell-mediated killing of cancer cells in a solid tumor is prefaced by a multi-step infiltration cascade of invasion, directed migration, and cytotoxic activities. In particular, immune cells must invade and migrate through a series of different extracellular matrix (ECM) boundaries and domains before reaching and killing their target tumor cells. These infiltration events are a central challenge to the clinical success of CAR T cells against solid tumors. The current standard in vitro cell killing assays measure cell cytotoxicity in an obstacle-free, two-dimensional (2D) microenvironment, which precludes the study of 3D immune cell-ECM interactions. Here, we present a 3D combined infiltration/cytotoxicity assay based on an oil-in-water microtechnology. This assay measures stromal invasion following extravasation, migration through the stromal matrix, and invasion of the solid tumor in addition to cell killing. We compare this 3D cytotoxicity assay to the benchmark 2D assay throu