Project summary Cell-based immunotherapy such as chimeric antigen receptor (CAR)-T and CAR-natural killer (NK) cells have shown tremendous successes in hematomalignancies. However, it has had limited positive outcomes in the treatment of solid tumors. For effective killing of tumor cells, immune cells must be physically engaged with the tumor cells and form an immunological synapse. In solid tumors, a main challenge is for the immune cells to infiltrate to the tumor core and to physically surround each tumor cells. Here, we hypothesize that mechanical stresses within the target tumor critically regulate the immune cell infiltration into the tumor core, as well as their ability to form immunological synapse with tumor cells. As such, mechanical stresses can be utilized to improve immune cells’ ability to kill tumor cells. To test this hypothesis, we propose the following specific aims. Aim 1: We will determine roles of mechanical forces in regulating infiltration of natural killer cells into the tumor spheroid core. Aim 2: We will determine roles of mechanical compression in potentiating tumor spheroids for effective immune killing. We will use a 3D microfluidic compression device developed under parent grant to control mechanical stresses within the tumor spheroids. Natural killer cells and breast tumor spheroids embedded within a collagen matrix will be used to recreate the 3D tumor microenvironment. The proposed project extends the current mechano-immune regulation assay from a 2D to 3D platform. The delivery of this research project will be a set of mechanical conditions, extracellular matrix architecture, compression rate and frequencies for effective tumor killing. These results can be used to guide future CAR- NK cell based immunotherapies for the treatment of solid tumors. An equally important component of this project is to provide a training opportunity for a first year URM graduate student to set a solid foundation for a PhD thesis program, and also to enable her to reach a personal goal of supporting the educational advancement of underrepresented communities.