Pancreatic ductal adenocarcinoma (PDAC) is now the 3rd leading cause of cancer-related death in the US and remains a deadly disease for US veterans. Although pancreatic cancer cells show susceptibility to standard chemotherapeutic agents, most patients eventually develop resistance, leading to poor survival. Myeloid cells have been reported to mediate chemotherapy resistance, however the mechanisms by which this occurs specifically in humans have yet to be elucidated. Such studies are challenging due to fresh biospecimen acquisition, patient heterogeneity, and a diverse tumor microenvironment. I propose to address these obstacles by exploiting opportunities to obtain specimens during endoscopic diagnostic biopsy (treatment-naïve) and surgical resection (post-treatment). In this proposal, I will develop a novel pipeline to generate a comprehensive dataset of single-cell sequenced human PDAC tumors longitudinally, before and after treatment, using pancreatic cancer patients at the Ann Arbor VA Healthcare System. I will also use a patient-derived co-culture system in vitro to parse out the myeloid-mediated mechanisms of chemoresistance in PDAC. The overarching hypothesis is that chemotherapy alters the tumor microenvironment in pancreatic cancer through reprogramming of the local and systemic immune system, and if better understood, can be exploited to uncover mechanisms of therapy resistance. The overall goal will be to uncover the role of myeloid cells in tumor aggressiveness and identify putative therapeutic targets to overcome chemo-refractory disease. In Aim 1, I will define immune signatures of PDAC response to chemotherapy in patient tumors and peripheral blood with the goal of correlating signatures to disease outcomes. Aim 1 will utilize single-cell RNA sequencing, multiplex immunostaining, and mass cytometry on longitudinal matched PDAC patient biospecimens, allowing for an individualized patient-specific comparison of treatment-naïve and post-treatment states. In Aim 2, I will use a patent-derived co-culture system of PDAC tumor organoids and peripheral myeloid cells to dissect the crosstalk responsible for myeloid-mediated therapy resistance. Here I will test the role of candidate ligand-receptors pairs between myeloid cells and tumor epithelial cells in tumor aggression and resistance. This proposal supports the development of an invaluable single-cell sequencing dataset of matched longitudinal pre- and post-treatment tumor tissue in PDAC patients, as well as an in vitro platform for mechanistic studies, altogether allowing for a comprehensive study of the tumor microenvironment and providing new immunotherapeutic targets in this deadly disease. Furthermore, this proposal will allow for the development of a robust pancreatic cancer biospecimen program at the Ann Arbor VA using samples from veterans that have previously been untapped for translational research purposes, with the goal of eventually building a precision medicine platfor...