Summary This is a competitive renewal of the originally funded R01 that was focused on dissecting Annexin A2 mediated mechanisms for the invasion and metastasis of pancreatic ductal adenocarcinoma (PDAC). PDAC is one of the major causes of death from malignant diseases. The poor prognosis of PDAC is attributed to early onset of metastasis and lack of effective treatments for PDACs. The tumor microenvironment (TME) of PDAC is believed to hold a key for overcoming the challenge of treating PDAC. Previously, we showed that tyrosine phosphorylation of Annexin A2 is essential for PDAC invasion and metastasis. We further discovered that Annexin A2 controls the secretion of Semaphorin 3D (Sema3D) and the autocrine effect of Sema3D on PDAC invasion and metastasis through binding to its receptor, Plexin D1/Neuropilin-1 (PlxnD1/NrP1). Interestingly, Sema3D, PlxnD1, and NrP1 all belong to the nerve axon guidance protein family, which is among the most frequently altered gene families in PDACs. The neuronal component of the TME in PDACs cannot be negligible as perineural invasion (PNI) is an important pathological characteristic in many malignancies, particularly PDAC. Semaphorins and plexins have also been implicated to regulate immune functions by controlling differentiation and trafficking of macrophages. Accumulated evidence has suggested that macrophages promote the development of the acinar to ductal metaplasia (ADM), which is thought to be a mechanism underlying the initiation of PDACs. Therefore, we propose to test the hypothesis that the interaction between the tumor cells and nerves plays an essential role in promoting ADM, growth, invasion, and metastasis of PDAC. First, this new project will dissect a paracrine Sema3D-PlxnD1 signaling pathway that mediates the PNI and subsequent metastasis of PDAC. Second, the project will study the nerve-derived Sema3D in controlling PDAC development through the PlxnD1 receptor on tumor cells in a mutant Kras- dependent manner. It will examine whether the Warburg effect induced by the Sema3D/PlxnD1-mutant Kras- ARF-GTP signaling cascade will affect the function of macrophages in vitro and whether tumor associated macrophages (TAM) will be reprogrammed in vivo when Sema3D is tissue-specifically knocked out from sensory nerves. Third, this project will investigate the role of Sema3D and the genetic alterations of the axon guidance pathway in vivo in pro-cancerous inflammatory response and PDAC development in both mouse models of PDAC and human PDAC specimens. The relationship between the expression of axon guidance molecules in pancreatic premalignant and malignant epithelia, in intra-pancreatic nerves, and in immune cells and the quantity and distribution of macrophages and other immune cells will be assessed. 1