Project Summary Chimeric antigen receptor (CAR) T cells represent an exciting technology for targeting drug-resistant cancers. These cells have been developed to effectively target hematological malignancies, however have yet to find clinical success against solid tumors, which account for the majority of cancer mortality. Solid tumors have myriad immunosuppressive mechanisms that prevent effective CAR T cell therapies, and new methods are needed to both elucidate these mechanisms as well as direct the engineering of these cells towards targeting solid tumors. This proposal will take an integrated bioinformatic approach to identifying adjuvant targets to enhance CAR T cell therapy at the site of a solid tumor or early metastatic site. Stromal and immune cells will be sequenced at the single cell level at these sites and compared to efficacy of CAR T cell therapy. These sequencing results will guide live cell imaging experiments, in which key transcription factors and effector proteins will be dynamically imaged in CAR T cells in culture as they recognize and target antigen-producing cells. Computational integration of these disparate datasets will result in adjuvant targets that will be built into the CAR design. These new constructs will be validated both in vitro and in vivo and will provide the basis for more advanced clinically available CAR T cell therapies.