Isolation and culture of primary human pancreatic stellate cells that reflect the context of their tissue of origin.

BACKGROUND: Pancreatic stellate cells (PSCs) play a critical role in pancreatic ductal adenocarcinoma (PDAC). Activated PSCs are the main source of fibrosis in chronic pancreatitis and of desmoplasia in PDAC. The majority of studies on PSC are based on in vitro experiments relying on immortalized cell lines derived from diseased human pancreas or from animal models. These PSCs are usually activated and may not represent the biological context of their tissue of origin. PURPOSE: (1) To isolate and culture primary human PSC from different disease contexts with minimal impact on their state of activation. (2) To perform a comparative analysis of phenotypes of PSC derived from different contexts. METHODS: PSCs were isolated from normal pancreas, chronic pancreatitis, and PDAC using a hybrid method of digestion and outgrowth. To minimize activation by serum compounds, cells were cultured in a low-serum environment (2.5 % fetal bovine serum (FBS)). Expression patterns of commonly used markers for PSC phenotype and activity were compared between primary PSC lines derived from different contexts and correlated to expression in their original tissues. RESULTS: Isolation was successful from 14 of 17 tissues (82 %). Isolated PSC displayed stable viability and phenotype in low-serum environment. Expression profiles of isolated PSC and matched original tissues were closely correlated. PDAC-derived PSC tended to have a higher status of activation if compared to PSC derived from non-cancerous tissues. CONCLUSIONS: Primary human PSCs isolated from different contexts and cultured in a low-serum environment maintain a phenotype that reflects the stromal activity present in their tissue of origin.

Epithelial to Stromal Re-Distribution of Primary Cilia during Pancreatic Carcinogenesis.

BACKGROUND: The Hedgehog (HH) pathway is a mediator in pancreatic ductal adenocarcinoma (PDAC). Surprisingly, previous studies suggested that primary cilia (PC), the essential organelles for HH signal transduction, were lost in PDAC. The aim of this study was to determine the presence of PC in human normal pancreas, chronic pancreatitis, and during carcinogenesis to PDAC with focus on both epithelia and stroma. METHODS: PC were analyzed in paraffin sections from normal pancreas, chronic pancreatitis, intraductal papillary-mucinous neoplasia, and PDAC, as well as in primary human pancreatic stellate cells (PSC) and pancreatic cancer cell lines by double immunofluorescence staining for acetylated α-tubuline and γ-tubuline. Co-staining for the HH receptors PTCH1, PTCH2 and SMO was also performed. RESULTS: PC are gradually lost during pancreatic carcinogenesis in the epithelium: the fraction of cells with PC gradually and significantly decreased from 32% in ducts of normal pancreas, to 21% in ducts of chronic pancreatitis, to 18% in PanIN1a, 6% in PanIN2, 3% in PanIN3 and to 1.2% in invasive PDAC. However, this loss of PC in the neoplastic epithelium is accompanied by a gain of PC in the surrounding stroma. The fraction of stromal cells with PC significantly increased from 13% around normal ducts to about 30% around PanIN and PDAC. HH-receptors were detected in tumor stroma but not in epithelial cells. PC are also present in PSC and pancreatic cancer cell lines. CONCLUSION: PC are not lost during pancreatic carcinogenesis but re-distributed from the epithelium to the stroma. This redistribution may explain the re-direction of HH signaling towards the stroma during pancreatic carcinogenesis.