A 3D multi-compartment assembloid 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. Standard in vitro cytotoxicity assays measure immune cell killing in obstacle-free, two-dimensional (2D) microenvironments, which preclude the study of 3D immune cell-extracellular matrix (ECM) interactions. Here, we use a 3D multi-compartment assembloid for the combined study of immune cell stromal invasion and matrix migration, followed by invasion of the solid tumor and subsequent cytotoxicity. We compare this 3D cytotoxicity assay to the benchmark 2D cytotoxicity assay using both unmodified immune cells and chimeric antigen receptor (CAR) T cells. This assay is amenable to a range of imaging techniques, allowing for the direct observation and quantification of each stage of infiltration in various immune and oncological contexts. We highlight the value of the 3D infiltration/cytotoxicity assay as an important tool for the mechanistic study of immune cell interactions with the tumor microenvironment.