Epithelial-mesenchymal Transition Promotes The Generation, Invasion And Migration Of Pancreatic Cancer Stem Cells

Objective: To explore the possible association between the epithelial–mesenchymaltransition (EMT) and cancer stem cells in pancreatic cancer.Methods: We used transforming growth factor beta1(TGF-β1) to induce anepithelial–mesenchymal transition in pancreatic cancer cell line PANC-1. Phasecontrast images of pancreatic cancer cells untreated and treated with TGF-β1byinverted microscope. Total RNA was isolated from PANC-1cells treated withTGF-β1and controls separately. Quantitative RT-PCR was done to detect the geneexpression of EMT-associated markers: E-cadherin and Vimentin, and normalizedwith β-actin. Western blot was performed to detect the expression of E-cadherin andVimentin on protein level, and β-actin was used as an internal loading control. Weevaluated the invasion and migration activity of PANC-1cells treated with TGF-β1and controls by Transwell tests. The proportion of pancreatic cancer stem cells wasmeasured by flow cytometry to determine the effect of TGF-β1-induced EMT on thepopulation of cancer stem cells through the surface specific antigen notation(CD44and CD24), and cancer stem cells were sorted into cell culture in the same way. Theexpression of EMT-associated markers was measured by quantitative PCR andWestern blot analysis in CD44~+CD24~+and CD44~–CD24~–cells, and β-actin was usedas an internal loading control. Cell cycle distribution was assessed by flowcytometry. The invasion and migration activity of CD44~+CD24~+and CD44~–CD24~–cells were evaluated by Transwell assay.Results: PANC-1cells showed morphological changes apparently under TGF-β1 treatment: cells arranged in irregular, showed a spindle-type morphology and thenumber of cell–cell contacts was reduced. PANC-1cells exhibited the typicalcharacteristics of epithelial cells: cells arranged in groups and cell–cell contacts weretight. The expression of EMT-associated markers measured by quantitative PCR andWestern blot analysis on mRNA and protein level showed that TGF-β1treatmentreduced the expression of the epithelial marker E-cadherin but increased theexpression of the mesenchymal marker Vimentin. The proportion of pancreaticcancer stem cells measured by flow cytometry to determine the effect ofTGF-β1-induced EMT on the population of cancer stem cells showed that, thenumber of cancer stem cells was significantly increased by30%among48h TGF-β1treated PANC1cells compared with controls. The increase occurred in parallel withthe morphological changes which began12h after treatment, and was maximal after48h. Transwell assay showed that, about the invasion activity, the number ofpenetrated cells among TGF-β1treated PANC-1cells was283±9, and the invasionability was significantly increased by90%,compared with that of controls (146±12);about the migration activity, the number of penetrated cells among TGF-β1treatedPANC-1cells was358±9, and the migration ability was significantly increased by80%,compared with that of controls (195±9). In an isolated cancer stem cell culture,phase contrast images of CD44~+CD24~+and CD44~–CD24~–cells were observed byinverted microscope. The CD44~+CD24~+cells exhibited the characteristics ofmesenchymal cells: cells arranged in irregular and the number of cell–cell contactswas reduced. In contrast, the CD44~–CD24~–cells showed the typical characteristics ofepithelial cells: cells arranged in groups and cell–cell contacts were tight.Quantitative RT-PCR and Western blot showed reduced expression of the epithelialmarker E-cadherin but increased expression of the mesenchymal marker vimentin inCD44~+CD24~+pancreatic cancer cells, compared with CD44~–CD24~–cells. Cell cycle distribution assessed by flow cytometry showed that there was no significantaccumulation of the G1phenotype among the CD44~+CD24~+cells (41.31%)compared with the CD44~–CD24~–cells (40.57%). The invasion and migration abilityof the CD44~+CD24~+cells was significantly increased by70%and60%separatelycompared with that of the CD44~–CD24~–cells.Conclusions: TGF-β1can induce EMT in pancreatic cancer cell line, promote thegeneration of pancreatic cancer stem cells, and increase the invasion and migrationability of pancreatic cancer cells. Pancreatic cancer stem cells have undergone anEMT and exhibited greater invasion and migration activity in vitro thannon-pancreatic cancer stem cells. EMT is very important to the understanding of themechanisms of pancreatic cancer and the molecular targeted therapy for tumors.