Effect Of Hepatocellular Carcinoma Conditioned Medium On Phenotype And Glucose Metabolism Of Adipose-derived Mesenchymal Stem Cells And Its Mechanism

Aims:Primary liver cancer is one of the most common malignancies in the world,with extremely high morbidity and mortality.Although great progress has been made in the prevention,monitoring,diagnosis,treatment and multidisciplinary cooperation of liver cancer in recent years,the growth rate and long-term survival rate are still not optimistic,and the diagnosis and treatment of liver cancer are facing with enormous challenges.As one of the important components of tumor microenvironment(TME),mesenchymal stem cells(MSCs)can constantly ‘remodel’ the tumor niche in response to signals from tumor cells,thus further promote tumor growth and metastasis,and modify the responsiveness to various treatments.Meanwhile,because of the characteristics of ‘tumor homing’ and ‘immune-privileged status’,MSCs have recently emerged as attractive targets or tools for anticancer approaches.But it is worth noticing that MSCs play an important role in tumor progression;concomitantly,MSCs also undergo profound changes in the TME.Therefore,safety,targeting efficiency and redistribution also restrict the further advancement of MSCs-based anti-tumor therapy.However,few studies have focused on the regulation of MSC fate in TME,which will limit the progress of MSC-based anti-tumour therapy.Herein,we investigated the effects of conditioned medium from human hepatocellular carcinoma cells on the phenotype and glucose metabolism of hAT-MSCs and its mechanism.Methods:Adipose tissue specimens from liposuction were obtained,and hAD-MSCs were isolated and identified.The chemotactic ability of hAD-MSCs to liver cancer environment was determined by Transwell experiment.Then hAT-MSCs were exposed to conditioned medium from Hep3 B,Huh7 and HCCLM3 cells for 4-8 weeks in vitro.Western Blot were used to detect the expression of α-SMA.And the alterations of cytoskeleton structure were determined using immunofluorescent assay.Then CCK-8 assay,Ed U assay,Transwell assay,and flow cytometry were used to detect the change of cell phenotype,as cell proliferation,migration,invasion,cell cycle,and apoptosis.And the expression of mitochondrial apoptosis pathway and cell cycle related proteins was detected by Western Blot.Next,the alterations of glucose metabolism patterns of hAD-MSCs were detected by lactate assay kit,pyruvate assay kit,ATP assay kit,glucose uptake assay kit and mitochondrial membrane potential kits,and glycolysis-related proteins and gene expression were detected by Western Blot and RT-qPCR.Next,cells were subjected to withdrawal from HCC-CM for 2-4 weeks,and alterations in phenotype and glucose metabolism were reevaluated in hAT-MSCs.In terms of the molecular mechanism,the intracellular ROS levels of hAD-MSCs after induction,reversion and blocking by NAC were detected by flow cytometry,and the expression levels of HIF-1α,MAPK(ERK,p38,JNK)and AKT were detected by Western Blot.Results:The identification results showed that the cells isolated from adipose tissue expressed MSCs’ markers,and could transdifferentiate into adipocytes,osteoblasts and chondrocytes.In vitro chemotactic experiments also confirmed that hAD-MSCs possessed the ability of tumor homing.After 4 to 8 weeks of induction with hepatocellular carcinoma conditioned medium,the expression of α-SMA of hAD-MSCs were increased,and the cell morphology were changed to stellate shaped.The results of CCK-8 assay and Ed U assay showed that hAD-MSCs proliferation was significantly inhibited.The results of flow cytometry showed that cell cycle(G2/M)arrested and apoptosis were significantly enhanced.Detection of related proteins via Western Blot showed that cell cycle regulatory proteins were generally down-regulated and mitochondrial apoptotic pathway were activated.However,the results of Transwell experiment suggested that the migration and invasion ability of hAD-MSCs was significantly enhanced.The results of glucose metabolism related assay kit showed that the relative glucose uptake of hAD-MSCs were significantly increased after induced by hepatocellular carcinoma conditioned medium,meanwhile the relative production of lactate,pyruvate and ATP were also increased,but the mitochondrial membrane potential was decreased significantly.The results of Western Blot were consistent with RT-qPCR results,showed that most of glycolytic-related proteins and corresponding m RNA expression levels were significantly up-regulated.However,interestingly,when hAT-MSCs were subjected to withdrawal from HCC-CM,hAT-MSCs still retained the same characteristic of TA-MSCs,but the alterations in phenotype and glucose metabolism could be reversed.In terms of the molecular mechanism,flow cytometry results showed that the intracellular ROS levels of hAD-MSCs induced by hepatocellular carcinoma conditioned medium were significantly increased,and this effect could be reversed after disengaging from liver cancer microenvironment.At the same time,the expression levels of HIF-1α protein of ‘educated’ hAD-MSCs were significantly up-regulated.Under the condition that the total protein was basically consistent,the expression levels of phosphorylated ERK were decreased,phosphorylated p38 and JNK were increased,and phosphorylated AKT remained unchanged.When ‘educated’ hAD-MSCs were disengaged from liver cancer microenvironment or treated by NAC,the expression levels of HIF-1α,phosphorylated ERK,p38 and JNK could basically return to normal control levels.Conclusions:hAD-MSCs possessed the ability of tumor homing,and after induced by hepatocellular carcinoma conditioned medium,they could be transformed into TA-MSCs.At the same time,the proliferation of ‘educated’ hAD-MSCs was inhibited,the cell cycle(G2/M)arrested and the mitochondrial apoptotic pathway was activated.In terms of metabolic patterns,‘educated’ hAD-MSCs have impaired mitochondrial function and increased glycolytic capacity.However,the effects of long-term HCC-CM treatment on phenotype and glucose metabolism in hAT-MSCs are modest and largely reversible after withdrawal.This is most likely caused by the accumulation of intracellular ROS in hAD-MSCs due to environmental stress,which further activated downstream HIF-1α and MAPK signaling pathways.