Pancreatic cancer stem cell characterization and study of the microenvironment impact on their biological features.

It has been reported that cancer stem cells (CSCs) are responsible for tumor initiation, metastasis, chemoresistance, and relapse. Furthermore, the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) is rich of extracellular matrix (ECM), which supports tumor growth and chemotherapy resistance. The aim of this thesis was to obtain and characterize CSCs derived from PDAC established cell lines. PDAC CSCs were generally more resistant to the action of five anti-cancer drugs than parental cell lines and were characterized by an increased expression of the stem cell markers EpCAM and CD44v6, and a decreased expression of the epithelial state marker E-cadherin. Furthermore, PDAC CSCs were more tumorigenic and possessed a higher metastatic activity than parental cells when injected into nude mice. When cultured on the top of several matrices, CSCs and the parental cells acquired a different morphology. In particular, only CSCs developed tube-like structures in the presence of Matrigel, and showed an increased expression of the endothelial cell markers CD34, CD31, and CD144, and of the pro-angiogenic factors IGFBP1 and eNOS. Furthermore, PDAC CSCs demonstrated a higher angiogenic profile with respect to the parental cells, as demonstrated by the secretion of several pro-angiogenic factors. When injected into nude mice, PDAC CSCs gave rise to tumors with a more intense vascular network with vessels with larger caliper than the tumors generated by parental cells. Additionally, when directly co-cultured with microenvironment bone marrow-mesenchymal stem cells (BM-MSCs), PDAC cells showed a decreased expression of the stem cell markers EpCAM and CD24. Taken together these results demonstrate that CSCs derived from PDAC cell lines possess all the characteristics of the clinically relevant tumor, rendering them a model to deeply understand PDAC biology. Three-dimensional cell culture models are of crucial relevance to study the role of the microenvironment on tumor biology and of the capability of PDAC CSCs for the sprouting of new vessels at the initial phases of tumor development. Finally, BM-MSCs may play a role in the regulation of PDAC cellular differentiation.