PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDA) is a deadly disease with a five-year survival rate of 12%. Oncogenic KRAS (Kras*) is a near-universal driver of PDA, and its activity is required for the formation of the PDA precursor lesions, pancreatic intraepithelial neoplasia (PanIN). PanIN formation is accompanied by fibrosis and immune cell infiltration that is maintained and evolves throughout disease progression. Cancer-associated fibroblasts (CAFs) have known roles in modulating the function of immune and cancer cells via intercellular interactions. Displaying high degrees of heterogeneity, CAFs have tumor-promoting and tumor-restraining functions that are thought to be mediated by distinct fibroblast populations. Data from other labs suggest that differential origins of CAFs play a role in programming this heterogeneity. Notably, mesothelial cells, a protective monolayer of cells that lines internal organs, are a potential, understudied source of CAFs in PDA. Preliminary experiments described in this proposal demonstrate that in orthotopic mouse models of PDA, mesothelial cells give rise to CAFs. Moreover, our group found that a potential population of WT1+ mesothelial-derived CAFs comprise a transcriptionally distinct population of fibroblasts expressing high levels of cytokines with known immunomodulatory roles in PDA. Consistent with this observation, depletion of WT1+ cells reduced expression of these cytokines in orthotopic tumors that coincided with a reduction in tumor weight. Interestingly, an increase in immunosuppressive macrophage infiltration was observed upon WT1+ cell depletion, suggesting that this reduction in tumor weight can be augmented by macrophage-targeting therapeutics. Compatible with the hypothesis that origin dictates CAF function, the overall objective of this study is to characterize sources of CAF heterogeneity, identifying novel approaches to selectively target tumor-promoting properties of CAFs in PDA. The central hypothesis is that mesothelial cells give rise to functionally distinct, immunomodulatory CAFs in PDA. This hypothesis will be investigated through the following two Aims: (Aim 1) Determine the contribution of WT1+ mesothelial cells to the fibroblast compartment in the context of PDA, and (Aim 2) Evaluate the functional role of WT1+ cells at different stages of PDA pathogenesis. To complete this investigation, a combination of in vivo models and in vitro assays, including macrophage polarization and migration assays, will be used to determine the properties of mesothelial-derived CAFs and their functional roles in the PDA tumor microenvironment. The data accumulated by this study will further the field's understanding of how cell origin dictates fibroblast function in PDA and pave the way for development of novel fibroblast-targeting therapeutics.