Abstract The dismal prognosis of late stage, unresectable pancreatic adenocarcinoma (PDAC) patients has prompted the search for novel chemotherapeutic agents. However, PDAC tumors are encapsulated by a remarkable fibrotic extracellular matrix (ECM), termed desmoplasia that prevents efficient intratumoral delivery of therapeutics. Thus, current PDAC therapies such as gemcitabine, Abraxane, erlotinib, and folfirinox must be given at elevated doses resulting in systemic toxicity with only marginal improvement in patient survival. The increased deposition of ECM components, such as hyaluronan (HA), surrounding and internal to the tumor mass serves as the ideal target for reducing desmoplasia and improving drug delivery. PEGPH20 is a pegylated hyaluronidase developed by Halozyme Therapeutics that depletes HA and increases vascular permeability in spontaneous murine PDAC tumors. Data from a Phase 1b trial indicates a clinical benefit of using PEGPH20 with gemcitabine in patients with high levels of PDAC-associated HA. In the present work, we tested a unique combination of PEGPH20 with a Salmonella-based therapeutic, shIDO-ST, which expresses an inhibitory RNA (RNAi) that degrades indoleamine 2,3-dioxygenase (IDO) mRNA. We observed striking regression in 100% of mice (N=4/group) with our unique therapeutic combination leading to elimination of orthotopically transplanted PDAC tumors, whereas combinations of gemcitabine and Abraxane marginally stabilized tumor growth (p<0.001, ANOVA). The conditions of these experiments were 90 μg PEGPH20 administered alone or combined with shIDO-ST (5 million CFU on day 14 and 18 (post-tumor implantation)), shScrambled (Scr)-ST (5 million CFU on day 14 and 18), or Abraxane (120 mg/Kg given i.v. on days 14, 18, and 22) + Gemcitabine (100 mg/Kg given i.p. on days 14, 18, and 22). This remarkable and unexpected result is the first demonstration that PEGPH20 facilitates entry of a bacterium into the intratumoral space. This response was uniquely associated with accumulation of shIDO-ST and neutrophils in the tumor which confirms the importance of the shIDO component, since a control scrambled RNAi expressed from Salmonella did not accumulate nor caused regression of PDAC tumors. Tracking experiments in shIDO-ST + PEGPH20 treated mice revealed that the neutrophils originate from the spleen and later migrate into the tumor. Surprisingly, purified splenic neutrophils from these mice were sufficient to kill PDAC tumor cells ex-vivo in cytotoxicity assays. We also observed significant IDO knockdown in purified neutrophils that corresponded to a greater activation phenotype typical of anti-tumor (N1) polarized neutrophils. Neutrophil killing of tumor targets could be inhibited in a dose-dependent manner using heparin or polysialic acid treatment, which specifically acts to neutralize the apoptosis-inducing activity of histones in the DNA matrixes formed as part of neutrophil extracellular traps (NETs). Collectively, these data support continued development of a combination therapy that overcomes desmoplasia in PDAC with remarkable anti-tumor activity. Supported by R21 CA174306, the Tim Nesvig Foundation, and Halozyme Therapeutics Citation Format: Edwin R. Manuel, Melanie G. Lampa, Joseph Kim, Vincent Chung, Massimo D'Apuzzo, Curtis B. Thompson, Zhongdong Huang, Daniel C. Maneval, Don J. Diamond. Control of PDAC tumor progression by RNA interference targeting indoleamine 2,3-dioxygenase using salmonella as a delivery vehicle facilitated by PEGPH20. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A97.