Pancreatic cancer stem cells characterization and secretome analysis

Pancreatic ductal adenocarcinoma (PDAC) is generally asymptomatic until the late stage of the disease and it often metastasizes. Cellular dissemination leading to metastasis occurs prior to the formation of an identifiable primary tumour and this event strictly correlates with the presence of cancer stem cells (CSC). These observations render imperative the identification of the specific biological features of CSC, in order to improve PDAC diagnosis and prognosis. We obtained in vitro CSC from different pancreatic ductal adenocarcinoma (PDAC) cell lines, named parental cell lines (P), using a selective medium. After the formation of spheres, which represent the first evidence of the staminal traits, cells have been characterized for the expression profile of different markers, e.g. CD44v6, Ep-CAM, E-caderin via FACS or Western Blot (WB) analyses. Furthermore, since CSC have been shown to represent the most threatening and resistant portion of the tumour, we tested their aggressiveness in mice subcutaneously injected with Panc1 P or CSC, at different cell concentrations. At the higher cell concentration (1*106 cells/mouse), mice injected with CSC displayed a significant higher tumour volume, as well as the tumour cellular morphology appeared to be very different, by histochemical analysis, compared to the mice injected with P cells. We are now performing other analyses, in particular immunohistochemistry on the tumour tissues, WB and real-time PCR on the tumour cells, to evaluate differences in morphology and marker expression between the two cell types. Moreover, to test the capacity of CSC to metastasize, we injected Panc1 P or CSC in the spleen, which was then excised after 5 minutes, with the advantage to follow metastasis formation, without the presence of a primary tumour. The invasive capacity of CSC is still under evaluation, through the monitoring of metastasis formation by MRI. Since it is known that cells secrete proteins for cell-cell communication and that the specificity of the secreted proteins can direct cells to distinct environments, we analysed the difference in protein secretion between Panc1 P and CSC by iTRAQ, an innovative protein quantification technique. The results showed that 71 proteins were secreted by CSC with an average fold change higher than 1.5 (p<0.001) relative to P cells, and 9 proteins were secreted only by CSC. We are validating these results with other techniques, mainly by WB on the secreted proteins in the culture medium, and by ELISA, using patient serum. The study of CSC and the analyses of secreted molecules is an approach with the strong potential to improve PDAC biology knowledge and to identify new potential early diagnostic markers.