Mesenchymal Stem Cells Promotes Chemoresistance Through Activating Autophagy In Intrahepatic Cholangiocarcinoma

Objectives:Intrahepatic cholangiocarcinoma(ICC)is a primary liver cancer that is difficult to treat.Curative resection is the well-established treatment aiming at cure for ICC patients,but the surgical prognosis is still poor due to the high invasiveness of the tumor.Although chemotherapy is an important treatment for patients with unresectable and advanced ICC,the outcomes are usually unsatisfactory due to chemoresistance of the cancer cells.The mechanism of chemoresistance in ICC remains unclear.Mesenchymal stem cells(MSCs)are known to be important components of tumor microenvironment.The current study aimed to investigate the role of MSCs isolated from tumor microenvironment in the chemoresistance of ICC and the underlying mechanism.Materials and Methods:1.MSCs were isolated from the fresh surgical specimens obtained from ICC patients using Fluorescence activated Cell Sorting(FACS).The antibodies specific for SSEA4 were used to identify MSCs.Then,SSEA4 positive cells were collected and herpes virus entry mediator(HVEM)was detected in ICC-MSCs by western blotting and RT-PCR assay.MSCs from normal umbilical cord(UC-MSCs)were used as control.2.HVEM overexpressed adenovirus were constructed and used to transfected MSCs from normal people.HVEM-overexpressed MSCs and UC-MSCs were cocultured with RBE and QBC939,two cholangiocarcinoma cell lines.Meanwhile,the chemotherapeutic drugs 5-Fu and cisplatin were added to the culture system at 48 h.Cell viability was examined using CCK8 assay.3.Bioplex assay was used to detect the cytokines in HVEM-overexpressed MSCs,and the inflammatory factors that may induce high expression of HVEM in MSCs were screened.ICC cells treated with the screened factor were used as the experimental group,while ICC cells treated with blank as the control group.Chemotherapy drugs were added after 48 h of culture.The proliferation and apoptosis of the ICC cells were detected by CCK8 assay and real-time apoptosis detection kit.4.The level of autophagy was detected in IL-6 treated RBE cells by western blot.The proliferation and apoptosis of the ICC cells were detected by CCK8 assay and realtime apoptosis detection kit.5.The role of AMPK/m TOR signaling pathway in the chemoresistance of ICC cells induced by screened inflammatory factors were determined by western blot.6.80 patients with ICC were recruited and their tumor tissues were collected.According to the immunohistochemistry score,patients were divided into two groups:high IL-6 level group and low IL-6 level group.The correlation between IL-6 level and the expression of autophagy-related markers was identified by the IPP analysis.The survival outcomes of ICC patients was evaluated using the Kaplan-Meier survival analysis.Results:1.HVEM was overexpressed in MSCs derived from the ICC microenvironment.2.MSCs with HVEM overexpression promoted the capacity of chemoresistance in cholangiocarcinoma cells.3.MSCs with HVEM overexpression increased chemoresistance in cholangiocarcinoma cells by secreting IL-6.4.IL-6 promoted the chemoresistance of cholangiocarcinoma cells through activation of autophagy.5.IL-6 activated autophagy by upregulating AMPK/m TOR signaling pathway.6.IL-6 expression level was associated with autophagy of ICC and a worse clinical prognosis.Conclusions:Our current study demonstrated that that MSCs in ICC could highly express HVEM and secrete high level of IL-6.IL-6 induced AMPK/m TOR signaling pathwaydependent autophagy supported cholangiocarcinoma cell survival and anti-toxic ability.HVEM level and IL-6 level can provide a new prognostic predictor,and related pathways affecting chemotherapy resistance can provide a potential therapeutic target for ICC.