PROJECT SUMMARY Therapeutic resistance is a major contributor to high lethality in pancreatic ductal adenocarcinoma (PDAC). A prominent feature of PDAC is desmoplasia, the formation of fibrous tissue that not only plays a critical role in reducing drug perfusion but also in limiting anti-tumor immune cell infiltration and function. The fibrous tissue is primarily composed of the extracellular matrix (ECM) components hyaluronan (HA) and collagen (CN). Previous methods to target these major ECM components have caused severe systemic side effects in patients and, thus, finding a safe, effective approach to disrupt the PDAC ECM and improve drug delivery remains a critical unmet need. Our long-term goal is to develop tumor-specific, microbial-based agents that express functional ECM- degrading enzymes. This novel strategy will remediate tumor desmoplasia, minimize systemic toxicity, and maximize the penetration and efficacy of therapeutics against primary PDAC tumors, as well as distal metastases. The objective of this proposal is to determine the utility of attenuated Salmonella typhimurium (ST)-based agents, engineered to express the ECM-degrading enzymes hyaluronidase (ST-HAse) and collagenase (ST-CNase), in triggering collapse of dense tumor stroma and in enhancing therapeutic efficacy in clinically-relevant models of PDAC. The rationale underlying this proposal is that successful completion of these studies will identify a feasible, tumor-targeting approach to ameliorate desmoplasia in PDAC, which will enable anticancer agents to achieve their greatest therapeutic effects. Our central hypothesis is that degrading both HA and CN in PDAC using tumor-specific ST vectors will induce the greatest stromal collapse, ultimately leading to enhanced penetration and efficacy of therapeutic treatment. This central hypothesis will be tested in relevant models of PDAC by pursuing three specific aims: (1) Determine the effect of dual ST-HAse/CNase treatment on the antitumor efficacy of standard-of-care chemotherapy; (2) Determine the impact of dual ST-HAse/CNase treatment on efficacy of immune checkpoint blockade therapy; and (3) Develop and characterize recombinant STs expressing HAse and CNase under tumor-inducible promoters. The use of tumor-colonizing ST and tumor-inducible bacterial promoters to express ECM-degrading enzymes is an innovative strategy to limit the effects of stromal degradation to tumor tissues. Furthermore, simultaneously degrading HA and CN will result in greater tumor permeability than targeting either component alone. The results of this work will have a significant impact for PDAC patients, because it is predicted to yield an agent(s) that can, in the short-term, be optimized for manufacturing and Investigational New Drug (IND)-enabling studies and, in the long-term, become a first-in-class, microbial-based agent used to significantly improve drug permeability of desmoplastic primary and metastatic tumors that are inaccessible to conve...