PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is characterized by resistance to therapy and is often diagnosed at a late stage, limiting treatment options. A contributing factor to therapeutic failure is profound desmoplasia and a well-documented hypoxic and immunosuppressive tumor microenvironment (TME). In PDAC, several therapeutic approaches, including chemotherapy and radiation alone or combined with immune checkpoint inhibitors, have shown minimal therapeutic success. Endoscopic ultrasound guided radiofrequency ablation (EUS-RFA) is a promising local ablative, stromal and immunomodulator therapy for PDAC. We have established a comprehensive research program to evaluate therapeutic benefits of EUS-RFA in patients with resectable PDAC. In tandem, we have published a preclinical model to test how RFA treatment alters the TME in the local ablation site or systemically through evaluation of contralateral tumors (abscopal effect). Successful amalgamation of our clinical and murine data will reveal mechanistic understanding of RFA-mediated immune stimulation, immune inhibitory checkpoints, and RFA-immunotherapy combination strategies to improve PDAC survival outcomes. We recently established the safety and feasibility of a minimally invasive, repeatable technique that can be used with systemic chemotherapy: EUS-RFA. Our phase II clinical trial (PANCARDINAL- 1), with 12 enrolled patients, demonstrates the tolerability, safety, and feasibility of repeated EUS-RFA with standard chemotherapy for resectable PDAC. We further found CD40 in patient serum is elevated post EUS- RFA, indicating immune activation and anti-tumor immunity. Using our preclinical model, we have also shown CD73 or PD-L1 inhibition augments RFA-mediated tumor growth reduction. Given these findings, we hypothesize that a multipronged approach that targets immune checkpoint blockade and immunosuppression in combination with RFA will improve future clinical trial design with EUS-RFA and improve PDAC survival outcomes. We propose the following Specific Aims: Aim 1: Evaluate effects of chemotherapy with repeated EUS-RFA on tumor growth, long-term outcomes, and anti-tumor immunity mechanisms in resectable PDAC patients (PANCARDINAL-1 Trial) and Aim 2: Determine impact of repeated RFA treatment in sustaining anti- tumor immunity and improving drug delivery with and without novel combined immunotherapies. Impact and Innovation: This proposal is the first to execute a clinical trial examining EUS-RFA for improving chemotherapy- based treatment of PDAC. Through complementary incorporation of clinically relevant animal models, we will identify novel therapeutic strategies for future studies. This funding will solidify establishment of a PANCARDINAL Network to serve as a pipeline for ongoing bench-to-bedside approaches to establish a new standard of care for the treatment of PDAC.