PROJECT SUMMARY With the recent FDA approval of chimeric antigen receptor (CAR) T-cell immunotherapies for B-cell malignancies, CAR T-cell therapies are a promising strategy to cure relapsed and refractory leukemia as well as solid tumors. However, the clinical benefit of CAR-T immunotherapy varies tremendously in many clinical trials and overall patient responses reported in trials of relapsed/refractory leukemia remain unfavorable. Factors that contribute to variable clinical responses may arise from early steps like CAR T- cell manufacturing or administration, CAR T-cell exhaustion and immunological resistance in the leukemic niche, but the key elements leading to variations in CAR T-cell efficacy are not fully understood. The objective of our research is to develop novel engineering systems to probe and analyze both the immunological and biomechanical attributes of CAR T-cells and map the leukemic BM niche for advancing current CAR T-cell immunotherapies. First of all, we aim to reconstruct a novel organotypic leukemic BM immunity niche ex vivo model to dissect the heterogeneity of immunosuppression mechanisms of different B-ALL subtypes and preclinically evaluate and optimize CD19 CAR T-cell immunotherapy efficacy. Secondly, we aim to develop and integrate in situ cellular and molecular immunophenotyping systems at single-cell level and/or in a 3D organotypic setting so as to provide a reliable and accurate screening to characterize the functional status of CAR T-cells. Lastly, we will explore CAR T-cell mechanosensitive mechanisms that regulate CAR T-cell activation and killing process to improve the CAR T-cell efficacy. Based on the new insights from CAR T-cell mechanobiology, we aim to engineer a remote “mechanical switch” and incorporate a “mechanical promoter” to effectively control CAR T-cell activation and cytotoxicity for improved CAR T-cell immunotherapy efficacy and safety. Altogether, we propose an innovative framework to precisely map the spatiotemporal immunological and biomechanical dynamics during CAR T-cell activation and killing, aiming to construct ex vivo leukemic BM niche and mechanical signature of CAR T-cells, ultimately optimize CAR T-cell administration, safety, and efficacy.