Pancreatic duct epithelial cell isolation and cultivation in two-dimensional and three-dimensional culture systems.

Pancreatic ductal adenocarcinoma (PDA) is generally considered to have originated from pancreatic duct epithelial cells (PDEC). The ability to manipulate the growth properties of PDEC is, therefore, critical for understanding the molecular events involved in the initiation of PDA. Here, we describe methods that we have established for the isolation and maintenance of PDEC in two-dimensional and three-dimensional culture systems. The availability of these culture systems should be particularly useful for studying their relationships between specific genetic lesions and the morphogenic changes that accompany pancreatic ductal tumorigenesis.

Oncogenic K-ras drives cell cycle progression and phenotypic conversion of primary pancreatic duct epithelial cells.

We have established a primary pancreatic duct epithelial cell culture (PDEC) system to investigate the relationship between oncogenic activation of K-ras and pancreatic ductal tumorigenesis. We have found that the acute introduction of physiological levels of oncogenic K-ras (K-rasV12) into quiescent PDECs stimulates S-phase entry and induces a pronounced increase in cell size. Both effects are dependent on the functional integrity of the phosphatidylinositol 3'-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. In addition, K-rasV12 promotes the loss of epithelial E-cadherin and the gain of mesenchymal N-cadherin in PDEC. Our observations indicate that the oncogenic activation of K-ras is sufficient to elicit mitogenic and morphogenic responses in pancreatic ductal cells and hence is likely to play an instructive role in the initiation of pancreatic ductal adenocarcinoma.