The Quasimesenchymal Pancreatic Ductal Epithelial Cell Line PANC-1-A Useful Model to Study Clonal Heterogeneity and EMT Subtype Shifting.

Abstract

Simple SummaryMalignant tumors often escape therapy due to clonal propagation of a subfraction of drug-resistant cancer cells. The underlying phenomenon of intratumoral heterogeneity is driven by epithelial–mesenchymal plasticity (EMP) involving the developmental programs, epithelial–mesenchymal transition (EMT), in which epithelial cells are converted to invasive mesenchymal cells, and the reverse process, mesenchymal–epithelial transition (MET), which allows for metastatic outgrowth at distant sites. For therapeutic targeting of EMP, a better understanding of this process is required; however, cellular models with which to study EMP in pancreatic ductal adenocarcinoma (PDAC) are scarce. Using single-cell clonal analysis, we have found the PDAC cell line, PANC-1, to consist of cells with different E/M phenotypes and functional attributes. Parental PANC-1 cultures could be induced in vitro to shift towards either a more mesenchymal or a more epithelial phenotype, and this bidirectional shift was controlled by the small GTPases RAC1 and RAC1b, together identifying PANC-1 cells as a useful model with which to study EMP.Intratumoral heterogeneity (ITH) is an intrinsic feature of malignant tumors that eventually allows a subfraction of resistant cancer cells to clonally evolve and cause therapy failure or relapse. ITH, cellular plasticity and tumor progression are driven by epithelial–mesenchymal transition (EMT) and the reverse process, MET. During these developmental programs, epithelial (E) cells are successively converted to invasive mesenchymal (M) cells, or back to E cells, by passing through a series of intermediate E/M states, a phenomenon termed E–M plasticity (EMP). The induction of MET has clinical potential as it can block the initial EMT stages that favor tumor cell dissemination, while its inhibition can curb metastatic outgrowth at distant sites. In pancreatic ductal adenocarcinoma (PDAC), cellular models with which to study EMP or MET induction are scarce. Here, we have generated single cell-derived clonal cultures of the quasimesenchymal PDAC-derived cell line, PANC-1, and found that these differ strongly with respect to cell morphology and EMT marker expression, allowing for their tentative classification as E, E/M or M. Interestingly, the different EMT phenotypes were found to segregate with differences in tumorigenic potential in vitro, as measured by colony forming and invasive activities, and in circadian clock function. Moreover, the individual clones the phenotypes of which remained stable upon prolonged culture also responded differently to treatment with transforming growth factor (TGF)β1 in regard to regulation of growth and individual TGFβ target genes, and to culture conditions that favour ductal-to-endocrine transdifferentiation as a more direct measure for cellular plasticity. Of note, stimulation with TGFβ1 induced a shift in parental PANC-1 cultures towards a more extreme M and invasive phenotype, while exposing the cells to a combination of the proinflammatory cytokines IFNγ, IL1β and TNFα (IIT) elicited a shift towards a more E and less invasive phenotype resembling a MET-like process. Finally, we show that the actions of TGFβ1 and IIT both converge on regulating the ratio of the small GTPase RAC1 and its splice isoform, RAC1b. Our data provide strong evidence for dynamic EMT–MET transitions and qualify this cell line as a useful model with which to study EMP.