PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy characterized by a fibrotic and immunosuppressive tumor microenvironment. Innovative approaches to overcome the lack of an effective immune response in PDAC are desperately needed. Although the concept of therapeutic infection, using bacteria as immunotherapy, has been around for over a century, recent advances in our understanding of bacterial interaction with immune cells and molecular pathways with relevance in cancer suggest new opportunities to harness natural bacterial immunomodulatory properties in PDAC. We have developed preliminary data demonstrating that injection of Group A Streptococcus (GAS) activates anti-tumor immunity resulting in profound tumor regression in murine pancreatic cancer. We have identified an extracellular protein, Scl1, on GAS, which selectively binds oncofetal fibronectin (cFn) in the pancreatic tumor microenvironment, suggesting the potential for bacterial localization to pancreatic tumors. Importantly, cFn is expressed in pancreatic tumors, but not healthy tissues, therefore Scl1/cFN interaction may be harnessed as a mechanism to limit off-target effects of PDAC treatment following direct injection into tumors. Additionally, we have discovered that Scl1 on GAS blocks cancer promoting neutrophil extracellular traps (NETs). Since NETs are responsible for promotion of tumor growth, metastatic disease and immunosuppression in PDAC, this has tremendous implications for utilizing GAS-Scl1 to alter cancer pathogenesis and anti-tumor immunity. Tumor growth and survival after intra-tumoral injection of 3 unique strains of GAS will be assessed in murine orthotopic, metastatic and transgenic PDAC. The formation and structure of GAS microcolonies within tumors will be imaged by two-photon microscopy. Circulating and intra-tumoral NETs after GAS injection will be measured and a robust immune profile generated to identify immunomodulatory effects. The proposed grant will decipher novel mechanisms driving the capability of GAS-Scl1 to stimulate anti-tumor immunity and elucidate the therapeutic potential of non-infectious strategies harnessing GAS-Scl1 in a future treatment for PDAC .