PROJECT SUMMARY Chimeric antigen receptor (CAR)-T cell therapy has emerged as a powerful new class of living medicines for cancer therapy, especially for patients with resistant or refractory blood cancers. However, application of CAR- T cell therapy to solid cancers has had little success, due to challenges of heterogenous antigen expression, limited tumor infiltration and CAR-T cell dysfunction in the immunosuppressive tumor microenvironment. Combinatorial treatment approaches, such as integration of radiotherapy to CAR-T cells, may provide an effective strategy to propagate antitumor immunity and overcome obstacles posed by solid tumors. In a preliminary study using CAR-T cells co-expressing somatostatin receptor 2 (SSTR2) for the targeted delivery of an immunomodulatory dose of 177Lu-DOTATATE, we noted rapid tumor localization of 177Lu-DOTATATE and subsequent regression of large established tumors. Furthermore, our preliminary data show an increase in serum IFN-γ and perforin following treatment with 177Lu-DOTATATE, suggesting a radiation-induced effect on T-cell immunity. Our objective in this R21 proposal is to explore the potential of target radiotherapy (TRT), delivered systemically through SSTR2-expressing CAR-T cells and an FDA-approved theranostic endoradiotherapy (177Lu-DOTATATE, Lutathera), to enhance CAR-T cell therapy. Our Specific Aims are to: 1) Investigate the impact of image-guided TRT on antitumor immune responses. We hypothesize that an optimized TRT dose can inflame tumors, rendering them more susceptible to T-cell cytotoxicity and thereby facilitating tumor eradication. Guided by CAR-T cell imaging and dosimetry estimation, we will conduct a dose response study to identify the optimal radiation dose for this innovative combination therapy. Additionally, utilizing a heterogenous tumor model, we will explore the potential of immunomodulatory TRT to promote tumor killing through TRAIL- and Fas-mediated mechanisms, partially independent of CAR-T cells. This approach holds the promise of addressing some mechanisms of tumor resistance, such as antigen escape. 2) Elucidate the underlying mechanism for the synergistic combination of TRT and CAR-T cell therapy. Using the syngeneic mouse model with an intact immune system and tumor microenvironment, we aim to validate and elucidate key phenotypic behaviors and molecular pathways driving the synergy between immunomodulatory low-dose TRT and CAR-T cell therapy. Our experimental approach will encompass techniques such as flow cytometry, immunohistochemistry, and single cell multi-omics analysis. The proposed work offers a new strategy for combining endoradiotherapy with CAR-T cell therapy that will be applicable to systemic disease with widespread metastases. Furthermore, it will add mechanistic insights in understanding the immunomodulatory effects of radiation.