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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy remaining incurable to date. The tumor cells and the relevant oncogenic signaling have been extensively investigated, still, the exact molecular and cellular pathology of the disease is not well known due to its fibrous stroma. The tumor microenvironment of PDAC is characterized by a dense desmoplastic stroma consisting of fibroblast, pancreatic stellate cells (PSC), immune cells and the extracellular matrix components. PSCs and tumor associated macrophages (TAM) are the most abundant and important cells in the tumor microenvironment of PDAC and their cellular communication play a significant role in PDAC progression and immune escape. The molecular mechanisms of PSCs activation and the role of their secreted proteins in macrophage polarization as well as the possibility of defining biomarkers from the secretome of pancreatic cancer cells were studied in this thesis. The secretome of tumor necrosis factor-alpha (TNF-α) activated PSCs were analyzed and validated using different techniques. Activation of PSCs resulted in regulating the secretome profile of PSCs and increased ROS production. The study also demonstrated that activated PSCs secrete numerous proteins which are associated with the cancer cell proliferation and immune cells activation. Co-culturing PDAC cells with the secretome of activated PSCs resulted in a significant increase of cells proliferation. Moreover, the secretome of PDAC cells and activated PSCs promoted the expression of HLA-DR on monocytes and induced the polarization of macrophages into the immunosuppressive M2 phenotype. The M2 polarization was confirmed by their ability to express CD163 and CD206 surface markers, higher arginase activity and interestingly, their conditioned medium increased Panc-1 cell proliferation. Additionally, interleukin-6 (IL-6) differentially regulated the expression of M1 and M2 surface markers pre- and post-polarization. Moreover, the secreted molecules by pancreatic cancer cells, sera of PDAC patients, chronic pancreatitis (CP) and sera from healthy donors were studied for the establishment of a set of defined biomarkers. The study indicated that eight markers which were similarly regulated in the PDAC cell secretome and serum of PDAC patients could increase the accuracy of PDAC diagnosis up to 10%. Abstract 8 Taken together, these results will provide insight into the complex and dynamic microenvironment of pancreatic cancer and show the critical role of tumor and stromal cell’s secretome in PDAC development and cellular communications in the tumor microenvironment.