PROJECT SUMMARY The past decade has witnessed a breakthrough in cancer immunotherapy, from checkpoint inhibitors to adoptive T cell therapies, a new pillar in our armament of anti-cancer drugs now exists. However, our current therapies are based on the premise that adaptive immunity alone, mediated by activated T cells, can eliminate tumors. While the generation of anti-tumor T cells is paramount, many additional factors must be considered in an immune response, including access of therapies to solid tumors, the role of innate immunity, and the suppressive tumor microenvironment (TME). Microbial-based cancer therapies have the potential of addressing all of these challenges that can impede the success of immunotherapy. This proposal is based on extensive preclinical and clinical experience using an attenuated (DactA) strain of Listeria monocytogenes as a therapeutic cancer vaccine. In this proposal, we focus on the direct impact of L. monocytogenes (Lm) on shaping the immune phenotype of the TME. We find that injection of Lm intratumorally (IT) results in profound changes in the TME, including the reduction of Tregs which we trace to activation of TLR2. Surprisingly, bacteria injected IV also localize to tumors, but while bacteria in the liver and spleen are eliminated, bacteria injected both IT and IV, persist indefinitely in the tumors, although neither treatment results in reduction of tumor volume. However, mice previously immunized with Lm followed by IT injection, dramatically reduce the tumor burden, which we show requires CD8+ T cells. Based on our preliminary data, we hypothesize that Lm injected either IT or IV localizes and persists in tumor and increases the inflammatory milieu. Upon prior immunization, influx of Lm-specific CD8+ T cells mediate clearance of both the remaining bacteria and substantial shrinkage of the tumor, although the mechanism remains to be determined. Collectively these data reveal the importance of both innate and adaptive immunity in mediating a productive response to the tumors.