Overexpression Of Podoplanin Correlates With Poor Prognosis And Promotes Invasion In Cholangiocarcinoma

Backgrounds and Objects:Cholangiocarcinoma(CCA)is classified into intrahepatic cholangiocarcinoma(ICC),perihilar cholangiocarcinoma(pCCA)and distal cholangiocarcinoma(dCCA)based on its anatomical location.In recent years,the morbidity and mortality of cholangiocarcinoma appears to be increasing globally.Due to the characteristics of occult onset,high degree of malignancy,rapid development and early invasiveness and metastasis,more than half of the cholangiocarcinoma patients are diagnosed with advanced stage.Surgical resection is considered the best option for cholangiocarcinoma treatment,but radical resection of the 5-year survival rate is only 20%to 40%.Therefore,It is important to find more effective molecular targets for the cholangiocarcinoma treatmentCholangiocarcinoma has a highly desmoplastic nature and is surrounded by a large number of stroma that contain many activated myofibroblasts known as cancer-associated fibroblasts(CAFs).CAFs could impact on tumors biological behavior.Podoplanin,a 38-kDa type Ⅰ transmembrane glycoprotein,is a specific marker for lymphatic endothelial cells.Recent studies also indicated that podoplanin can be expressed in several malignant tumors including cancer cells,CAFs and correlated with the prognosis.But its mechanism is unclear.Several studies reported that podoplanin expression in cells can enhance their migration and invasion ability.Some studies showed podoplanin could mediate the epithelial mesenchymal transition(EMT)and cause tumor invasion and metastasis.Other studies have also suggested that podoplanin can lead the tumor migration and invasion by changing the activities of Rho family small GTPases.There is a little research about the expression of podoplanin in cholangiocarcinoma.Studying the molecular mechanism of podoplanin in cholangiocarcinoma is conducive to finding new biomarkers and potential therapeutic targets.Materials and Methods1.66 paired ICC and 42 paired pCCA paraffin-embedded tissues were obtained from the Department of Pathology,Affiliated Drum Tower Hospital of Nanjing University Medical School.All patient samples included tumor and para-tumor tissues.Expression of podoplanin on paraffin clinical samples were investigated by IHC.Correlations between podoplanin expression in tumor cells and CAFs and clinical features were analyzed.2.CAFs were isolated from cholangiocarcinoma tumor xenografts and characterized by the double immunofluorescence and flow cytometry.CAFs were transfected with adenovirus packaging the podoplanin gene(Ad-podoplanin)or not(Ad-vector).Impact of podoplanin overexpression on cell proliferation and migration was evaluated by the CCK-8 cell proliferation assay and transwell migration assay.Western blotting was used to detect the expression of ERM,Cofilin and MLC-2.Tube formation assay was performed to analyze the affection of Ad-podoplanin CAFs on the HDLECs’ tube formation ability.Trans well invasion assay was performed to analyze the effect of Ad-podoplanin CAFs on the QBC939 cells.Xenotransplantation of CCA cells in nude mice was performed to evaluate the effect of Ad-podoplanin CAFs on the cholangiocarcinoma tumorigenesis(This experiment included three groups:QBC939 cells、QBC939 cells and Ad-vector CAFs,QBC939 cells and Ad-podoplanin CAFs).3.The invasion and metastatic ability of podoplanin overexpressed QBC939 was evaluated by transwell invasion assay and liver metastatic model in mice.Two human cholangiocarcinoma cell lines,QBC939 and HCCC9810,were transfected with the Ad-podoplanin.Western blotting was used to detect the expression of EMT related markers and the activation of Rho family small GTPases(Racl,Cdc42,and RhoA)in the podoplanin overexpressed cholangiocarcinoma cell lines.Results1.42 pCCA patients’ IHC results indicated that the ratio of podoplanin-positive CAFs to podoplanin-negative CAFs in the para-tumor tissue was 8/42,and all of these podoplanin-positive CAFs were stained weakly or moderately.In comparison,the ratio of podoplanin-positive CAFs in the tumor tissue was 20/42,and stained strongly.Compared to the podoplanin-negative CAFs group,the podoplanin-positive CAFs group was significantly related to lymph node metastasis(P=0.014)and TNM staging(P＝0.002);The ratio of podoplanin-positive CAFs was 29/66 in ICC tissue and 13/66 in para-tumor tissue.Podoplanin-positive CAFs stained weakly in 9 of these para-tumor tissues.The podoplanin-positive CAFs group was related to the neural invasion(P=0.011)and lymph node metastasis(P=0.045);Kaplan-Meier survival analysis showed that the overall survival rate in the podoplanin-negative CAFs group was significantly higher than in the podoplanin-positive CAFs group.(log rank test,P=0.018(ICC)and P=0.042(pCCA)).Therefore,podoplanin expression in CAFs was an important prognostic marker for the cholangiocarcinoma patients.There wasn’t significant difference between podoplanin expression in tumor cells with patients clinical features and overall survival time in pCCA tissues.However,the expression of podoplanin in tumor cells was correlated with TNM stage(P<0.01)and lymph node metastasis(P=0.02)in ICC tissues.Kaplan-Meier survival analysis also showed that podoplanin expression in ICC was correlated with the reduction of overall survival time(log rank test,P=0.037).Together,our data revealed podoplanin expression in tumor cells appears to be a predictor of survival rate,tumor invasion and metastasis for cholangiocarcinoma patients.2.Isolated CAFs was characterized through immunofluorescence staining and flow cytometry with antibodies to a-SMA,vimentin and FSP1.Podoplanin overexpression in CAFs could enhance its migration ability,but didn’t significantly affect the proliferation and the tube-formation ability of HDLECs.Additionally,Western Blot results indicated that ERM,MLC-2 and Cofilin were phosphorylated in podoplanin overexpressed CAFs.Compared to the QBC939 cells,QBC939 cells were cultured with the Ad-podoplanin CAFs’ and Ad-vector CAFs’ conditioned medium showed stronger invasion ability,but there was no significant differences between the two CAFs groups.In nude mice model,QBC939 cells with Ad-podoplanin CAFs injected group resulted a significant increase of tumor volume.3.The overexpression of podoplanin in QBC939 cells can enhance its invasiveness.It can also affect the liver metastasis,but there is no significant differences with control group.IHC results indicated that there was a significant correlation between podoplanin expression and N-cadherin(P=0.039),S100A4(P=0.0029)expression in ICC tissues.Podoplanin overexpression in QBC939 cells can increase its stromal phenotype.Moreover,overexpression of podoplanin can induce the EMT-like changes in HCCC9810 cells.The expression of activated Cdc42 protein in the Rho GTPase family was significantly increased in podoplanin overexpressed QBC939 and HCCC9810 cells.ConclusionsPodoplanin expression can be detected in tumor cells and CAFs in cholangiocarcinoma tissues.There are positive correlations between podoplanin expression in tumor cells and CAFs with TNM stage and lymph node metastasis.Podoplanin overexpression in cholangiocarcinoma cell lines can promote tumor invasion by inducing EMT-like changes and activating Cdc42 protein.Meanwhile,podoplanin expression in CAFs also significantly enhance its migration ability.Therefore,podoplanin has an important role in cholangiocarcinoma progression and may be expected to become a potential target for anti-cancer therapy and important biomarker to evaluate prognosis.