IN-DEPTH CHARACTERIZATION OF THE SECRETOME OF PANCREATIC CANCER STEM CELLS BY iTRAQ-BASED SHOTGUN PROTEOMICS AND IDENTIFICATION OF POTENTIAL MARKERS FOR EARLY DIAGNOSIS OF PANCREATIC CANCER.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer-related death in the world. The prognosis for patients affected by this type of cancer is poor, with the 5-year survival rate after diagnosis being less than 5%. One major reason for this high mortality is the lack of effective diagnostic and prognostic markers. Emerging research has demonstrated that PDAC tumours contain a small sub-population of cancer stem cells (CSCs), which are characterized by self-renewal, anchorage independent growth, long term proliferative capacity, and chemotherapy resistance. These observations render imperative the identification of the specific biological features of CSCs, in order to improve PDAC diagnosis and prognosis. The present research PhD project aim was focused on the identification of markers for early diagnosis of PDAC through the analysis of cancer stem cells. In this investigation, CSCs were obtained ,by using a selective medium, from five out of nine PDAC cell lines; after the formation of spheres which represent the first evidence of staminal traits, CSCs have been characterized for the expression profile of different markers by using FACS and Western blot analysis. CSCs showed increased expression of the stem cell markers EpCAM and CD44v6, decreased expression of epithelial marker E-cadherin, and higher resistance to five anti-cancer drugs. Finally, PDAC CSCs injected into nude mice developed a larger subcutaneous tumour mass and showed a higher metastatic activity compared to parental cells. The results obtained demonstrated the ability to isolate CSCs from different PDAC cell lines and that these cells are differentially resistant to various anticancer agents. This variability render them a model of great importance to deeply understand pancreatic adenocarcinoma biology, to discover new biomarker and to screen new therapeutic compounds. Since it is known that cells secrete proteins for cell-cell communication and that the specificity of secreted proteins can direct cells to distinct microenvironment, it has been analyzed the difference in protein secretion between CSCs and parental cells. It has been performed an iTRAQ-based mass spectrometry proteomic analysis of Panc1 and Panc1 CSCs secretomes identifying a total of 112 secreted proteins, of which 43 were found to have higher abundance in the secretome of Panc1 CSCs as compared to parental cells. Ingenuity Pathway Analysis (IPA) analyses of Panc1 CSCs secretome showed a predominance of proteins involved in glycolysis and gluconeogenesis, pentose phosphate pathway, signalling of glioma invasiveness, of myc-mediated apoptosis, and of ERK5, and remodelling of epithelial adherent junctions. To discover novel potential markers for PDAC diagnosis, it has been analysed the presence of the three proteins that were over-secreted from Panc1 CSCs, i.e. ceruloplasmin, galectin-3, and myristoylated alanine-rich C-kinase substrate (MARCKS) in 100 pancreatic cancer patient and 20 control sera. It has been found that ceruloplasmin and MARCKS levels, were significantly higher in PDAC patient sera compare to healthy controls. In particular, ceruloplasmin was more abundant in patients at an early stage of PDAC. Furthermore, the combination of ceruloplasmin and CA19-9, the current standard serum tumour marker for PDAC, showed an improved area under the receiver operating characteristic curve compared to CA19-9 alone and ceruloplasmin levels were higher than controls in more than 50% of patients negative for CA19-9. This finding suggested that ceruloplasmin might prove to be an important complementary biomarker for CA19-9 in PDAC diagnosis. The study of CSCs and the analyses of secreted molecules turned out to be an approach with a strong potential to improve PDAC biology knowledge and to identify new potential early markers.