Mechanism And Significance Of Intrahepatic Cholangiocarcinoma Cells Promote Epithelial-mesenchymal Transition Of Hepatocellular Carcinoma Cells In Combined Hepatocellular Cholangiocarcinoma Metastasis

Background: Hepatocellular carcinoma and intrahepatic cholangiocarcinoma cells are common primary hepatic tumor cells in the liver.There is a form of primary liver carcinoma contains a mixture of hepatocellular carcinoma cells and intrahepatic cholangiocarcinoma cells,namely combined hepatocellular cholangiocarcinoma(CHC),with an incidence varying from 1.0% to 14.3% of primary liver cancer in different studies.As the third common liver cancer,CHC has a dismal prognosis because of metastasis.The data from a long-term prognosis study of liver cancer indicates that CHC has a significantly worse prognosis than hepatocellular carcinoma and intrahepatic cholangiocarcinoma even after curative resection.Although it has been speculated progenitor that CHC may arise from hepatic progenitor cells,epithelial-mesenchymal transition(EMT)is an important contributing factor to the development and progression of liver cancer,including CHC.Given the special histological characteristics of CHC,we speculate that there might be interactions between the hepatocellular carcinoma and intrahepatic cholangiocarcinoma components in CHC,which contribute to the poor prognosis of CHC.Methods: We incubated HCC cells with culture supernatant from ICC cells,and examined possible mechanisms of action through CRISPR-Cas9 and cell experiments.Results: We found that the hepatocellular carcinoma cells grown in the culture supernatant of intrahepatic cholangiocarcinoma cells showed expansive and discrete morphology.In wound healing assays,the culture supernatant of intrahepatic cholangiocarcinoma cells induced rapid wound healing in hepatocellular carcinoma cells(Hep G2 and SNU-449).The results from the transwell assay also showed that culture supernatant from intrahepatic cholangiocarcinoma cells enhanced the invasion ability of Hep G2 and SNU-449 cells.Although CCK8 assays and Ed U-labeling assays showed that the culture supernatant from HCCC-9810 cells did not accelerate the proliferation of hepatocellular carcinoma cells,it enhances the chemoresistance of hepatocellular carcinoma cells.The epithelial marker E-cadherin was found to be downregulated in Hep G2 cells incubated with culture supernatant from intrahepatic cholangiocarcinoma cells.Conversely,mesenchymal-associated proteins Vimentin and Snail were upregulated in Hep G2 and SNU-449 cells treated with culture supernatants from intrahepatic cholangiocarcinoma cells.Laminin subunit gamma 2(LAMC2)was detected in the culture supernatant of intrahepatic cholangiocarcinoma cells but not in that of hepatocellular carcinoma cells.Using established LAMC2 knockout intrahepatic cholangiocarcinoma cells,our results demonstrated that intrahepatic cholangiocarcinoma cells promoted the EMT of hepatocellular carcinoma cells through secreting LAMC2.Conclusions: Our results have revealed that intrahepatic cholangiocarcinoma cells promote epithelial-mesenchymal transition of hepatocellular carcinoma cells by secreting LAMC2,which may provide new insight into developing effective treatments for CHC.