Project Summary Metastasis is the leading cause of cancer-related deaths. But despite much effort in elucidating the molecular mechanisms underlying metastasis, there is still much that remains unknown and unexplored. In many solid tumors, metastasis is closely associated with an epithelial-to-mesenchymal transition (EMT), a form of cellular plasticity by which stationary epithelial cells acquire the motile, invasive phenotypes reminiscent of fibroblasts. And while EMT results in many tumor cell-intrinsic changes, such changes are also known to alter the tumor microenvironment (TME) in ways that facilitate tumor cell invasion into the bloodstream and ultimately metastasis. Thus, to define the molecular changes that occur during EMT in vivo, our lab performed whole transcriptomic analysis on sorted epithelial and mesenchymal tumor cells that were harvested from a well- established genetic mouse model of spontaneous pancreatic ductal adenocarcinoma (PDA). Among the genes that were most significantly upregulated in mesenchymal cells was Serping1, which encodes the hepatocyte- derived plasma protease inhibitor C1-inhibitor (C1-INH). Intriguingly, our preliminary results demonstrate a possible role for tumor-derived C1-INH in the growth and vascularization of mesenchymal PDA tumors. Furthermore, recent studies have shown that complement factor C1Q, whose dissociation from the complement cascade depends on C1-INH, promotes angiogenesis in the context of wound healing, as well as melanoma progression. Based on our preliminary data and these newer studies, we hypothesize that C1-INH is upregulated in PDA cells during EMT to promote angiogenesis and tumor progression in a C1Q- dependent manner. In this proposal, we provide details of two aims to test this hypothesis. Briefly, in Aim 1, we will more deeply investigate the role of tumor-derived C1-INH in angiogenesis by performing genetic gain and loss of function studies in murine PDA cells and using standard in vitro and in vivo angiogenesis assays to determine C1-INH’s effects on endothelial cell proliferation, migration, and differentiation, as well as the functional integrity of intratumoral blood vessels. We will also dissect the molecular mechanism of C1-INH in PDA progression by investigating its functional dependencies on C1Q. Since C1-INH was originally found to be enriched in the mesenchymal state, we will also investigate its potential role in promoting metastasis. Thus, in Aim 2, we will determine whether tumor-derived C1-INH is necessary for metastasis by employing an orthotopic transplantation model and monitoring different stages of the metastatic cascade, from tumor cell dissemination to metastatic colonization. The completed aims of this proposal – which...