Study On The Activation Of Pancreatic Intraepithelial Neoplasia (PanINs) Stellate Cells And The Mechanism Of Promoting Malignant Transformation

Background:Pancreatic cancer is one of the most lethal malignancies that human being could ever have.Early diagnosis,disease prevention and treatment at its early stage is critical to the prognosis of patients,thus,studies on precancerous lesions and"inflammation-cancer" transition are regarded as hope for future direction.Desmoplasia,as one of the most important components of the microenvironment in pancreatic cancer,can promote the progression of tumor.However,the characteristics and mechanisms of its involvement in pancreatic precancerous lesions are largely unknown.Methods:We observed the desmoplasia and activation of pancreatic stellate cells(PSCs)in human PanIN lesions with Masson trichrome staining and immunostaining for a-smooth muscle actin(a-SMA),respectively;Based on the semi-quantitative analysis on the pathological slides with different staining techniques with Image J software,correlationship between the desmoplasia and activation of PSCs with grading of PanIN lesions were analyzed;Expression pattern of common profibrogenic growth factors and their receptors were analyzed with immunohistochemistry staining(IHC)in human PanIN tissue sections;Genetic mouse model of PanINs was built with mutant-type KRAS gene under the control of pancreatic tissue specific Pdxl gene promoter;With this murine model,we analyze the desmoplasia,activation of PSCs and expression of various profibrogenic factors and their receptors in mouse PanINs with Massson trichrome staining or IHCs;With in vitro co-culture system of human pancreatic ductal cell lines HPNE with primarily cultured PSCs,we verified the promotion of PSCs activation by HPNE cells;By utilizing specific inhibitors for different signaling pathways we analyzed the mechanisms of the promotive effect on PSCs activation by HPNE cells.Resutls:Evident desmoplasia and activation of PSCs could be observed in human PanINs,and were positively correlated with PanINs gradings;Results from IHC in tissue sections have shown that various profibrogenic factors,including TGF-β1,PDGF-BB and Sonic hedgehog,were expressed in epithelial compartment in PanINs,while their receptors could be expressed by activated PSCs in the stroma;Expression of mutant-tye KRAS gene in pancreas under the control of tissue specific Pdx-1 promotor could lead to a full disease spectrum of PanINs in mice,and desmoplasia,activation of PSCs and expression of various profibrogenic factors could be observed in mouse PanINs as well;HPNE with expression of mutant-type KRAS gene could promote the activation of PSCs in vitro,which was mediated by multiple signaling pathways including TGF-β,PDGF and Hedgehog.Conclusions:Desmoplaisa and activation of PSCs could be observed in PanINs.Diseased pancreatic epithelium could promote the activation of PSCs via TGF-β,PDGF and Hedgehog pathways.Background:Activated pancreatic stellate cells(PSCs),as one of the most important components in the microenvironment of pancreatic cancer,could promote the growth,invasion,metastases of pancreatic cancer and played a role in inducing immunological evasion and resistance of chemo-and radiotherapy.However,its significance in the progression of PanINs was largely unknown.Our studies showed that there were activated PSCs around the pancreatic intraepithelial neoplasms(PanINs).Whether the activated PSCs would promote the progression of the precancerous lesion in pancreas and its mechanism were unclear.Methods:Cells counting,CCK-8 assay were carried out to evaluate the effect of co-culture with PSCs on the growht of HPNE cells,with or without KRAS gene mutation;EdU assay were carried out to assess the proliferation of HPNE cells with or without co-culture of activated PSCs;Flow cytometry was used to examine the regulation of HPNE cell cycle and apoptosis by PSCs;Quantitative RT-PCR,ELISA,WB and ICC/IF were utilized to study the expression of IL-6 and its receptors IL-6R and gp130 in PSCs,HPNEs and pancreatic cell lines;Immunohistochemistry were carried out to evaluate the expression of ligand and receptor of IL-6 signaling in PanINs tissue;In co-culture system of PSCs and HPNEs,specific siRNA of gp130 were utilized to explore potential role of IL-6 in pro-proliferative ability of PSCs on HPNE cells;Western blot were also utilized to evaluate the activation of different signaling pathways in HPNEs after co-cultured with PSCs;Specific pathway inhibitors for Stat3 was used in co-culture system of HPNEs and PSCs to assess the roles of Stat3 pathways in mediating the proliferative activity of PSC on HPNE;Expression profiling and analysis on differentially expressed genes with GSEA were carried out to explore the possible molecular mechanism in HPNE under co-culture with PSCs.Results:Cell counting and CCK-8 assay showed that both co-culture with PSCs or conditioned media of PSCs could promote the growth of HPNE-e6e7 and HPNE-Kr;EdU and flow cytometry revealed that the growth promotive effect were related to the increased proliferative rate in HPNE cells,as well as regulated cell cycle,but not in relationship with apoptotic status of HPNE cells;Primarily culture PSCs expressed high level of IL-6,while HPNE cells and various pancreatic cancer cell lines expressed IL-6R and gp130;Immunohistochemistry showed that the ligand and receptors of IL-6 signaling pathways were positive in stromal cells and epithelium in PanINs,respectively;siRNA for gp130 could inhibit the proliferative role of PSCs on HPNE-Kr cells;Western blot showed that multiple intracellular signaling pathways were activated in HPNE cells after co-culture with PSCs;Blocking Stat3 pathways with specific inhibitor could significantly inhibit the proliferation of HPNE-Kr cells,and could block the proliferative effect of co-cultured PSCs on HPNE-Kr cells;Results from expression profiling showed that a significant change in gene expression was induced after co-culture with PSCs,and GO analysis on differentially expressed genes resulted in enrichments of various gene sets.Conclusions:Activated PSCs could promote the proliferative activity of HPNE cells,which was possibly mediated by IL-6/Stat3 signaling pathway.Co-culture with activated PSCs could results in moderate chage in gene expression of HPNE-Kr cells.