Effect Of Matrix Stiffness On Cancer Stem Cell Phenotype Of Hepatocellular Carcinoma Cells And Molecular Mechanism

Cancer is a malignant disease that seriously endangers human life.Every year,the mortality and morbidity of cancer is the second highest after cardiovascular and cerebrovascular diseases.Hepatocellular carcinoma(HCC)is one of the most malignant cancers with greater mortality and poor prognosis.In China,the morbidity and mortality of hepatocellular carcinoma are much higher than the world average.Liver cancer stem cells(LCSCs)are a small group of HCC cells.They have the ability of self-renewal and metastasis.They are insensitive to drugs and radiotherapy and play a key role in the metastasis and recurrence of hepatocellular carcinoma.Studies have shown that cancer stem cells(CSCs)have phenotypic plasticity,that is,cancer cells can be transformed into CSCs under certain conditions,and CSCs can also differentiate into cancer cells.However,the specific condition and mechanisms regarding the phenotypic change between CSCs and cancer cells are still remain controversial.Liver cancer stem cell(LCSC)is a rare subpopulation of hepatocellular carcinoma cells,which have strong self-renewal ability and metastasis potential,and insensitive to drugs and radiotherapy.LCSC plays a critical role in the metastasis and recurrence of hepatocellular carcinoma.Studies have shown that CSCs have phenotypic plasticity,that is,under certain conditions,they also display plasticity by reversible transition between stem and non-stem cell states.Previous studies have shown that mechanical factors can influence the differentiation of stem cells.For example,matrix stiffness can induce mesenchymal stem cells(MSCs)to differentiate into different types of cells.CSCs have similar character with stem cell.Researchers have also found that mechanical factors can induce CSCs to differentiate into cancer cells or cancer cells to dedifferentiate into CSCs.For example,fluid shear stress can induce LCSCs to differentiate into normal cancer cells,and fluid shear stress can induce lung cancer cells to acquire cancer stem cell phenotype.These results suggest that mechanical factors can induce phenotypic changes of cancer cells/CSCs.Liver cancer tissue has mechanical heterogeneity.In a single tumor tissue,there is a significant difference in the stiffness of the matrix between the invasive front and the core of the tumor.More importantly,the distribution of CSCs in cancer tissue is not uniform.The increasing matrix stiffness of the liver caused by liver fibrosis or cirrhosis can lead to liver cancer.Moreover,the stiffness of liver cancer tissue is much higher than that of normal liver tissue.Both reasons suggest that the development of liver cancer is a direct result of matrix stiffness.Whether the matrix stiffness can affect the CSCs phenotype of liver cancer cells need to be research.In previous studies,integrin-?1 is known as a transmembrane protein that has been considered as a mechanically sensitive molecule which can regulate cell migration,proliferation,and adhesion.Integrin-?1 has been found to play an important role in the development of cancer in recent studies.Besides,research shows that Integrin-?1 is highly expressed in some CSCs,which may be a potential new marker of CSCs.In summary,this work investigated whether matrix stiffness can effect HCC stemness,and explored the molecular mechanism.The main research contents and results are described as follows:(1)Establishment of different stiffness matrix cell culture modelsIn this work,polyacrylamide hydrogel was used to construct the experimental model.Hydrogels with different stiffness were constructed by adjusting the ratio of Acrylamide and Bis-acrylamide.The stiffness of polyacrylamide hydrogel constructed in this study can be adjusted to mimic the matrix stiffness of normal liver(5.9 kPa)or diseased liver(24.8 kPa,48.1 kPa).At the same time,the stiffness can be adjusted to provide a good growth interface for cells and to provide a new platform for studying the mechanism of liver lesions in vitro(2)Matrix stiffness affects HCC cells stemnessTo investigate whether matrix stiffness can affect the phenotype of hepatocellular carcinoma cell stem cells,we have analyzed various CSCs character which include morphology,cytoskeleton,stem cell-related genes,liver cancer stem cell surface marker molecules,SP cells,cell cycle,sphere ability,clonality,drug resistance,and tumorigenicity.We found that the soft matrix reduced the cell spreading,reduce the cytoskeleton polymerization,increased the expression of stem cell-related genes Oct-4,Sox-2,CXCR4 and the number of CD133 and CD90 positive cells on the surface of liver cancer stem cells,and SPs.Cells had a slower cell cycle,greater sphere ability,and colony forming ability on soft matrix.Increasing the matrix stiffness will enable cells to reduce apoptosis under the treatment of the anticancer drugs Sorafenib and fluorouracil(5-FU).The softer matrix stiffness allows the cells to still have greater colony forming ability under the treatment of the anticancer drugs Sorafenib and 5-FU.Finally,we demonstrated from animal models that liver cancer cells cultured on a softer matrix have stronger tumorigenic ability.The experimental results show that the soft matrix can induce the cancer stem cell phenotype of hepatoma cell line MHCC97 H.(3)The role of Integrin-?1 on the effect of matrix stiffness in HCC cells stemnessIn this study,we found that the expression of Integrin-?1 decreased with the increase of matrix stiffness,suggesting that Integrin-?1 may be associated with the phenotypic changes of CSCs.In this experiment,the expression of Integrin-?1 had successfully knocked down by siRNA.After knocked down the expression of Integrin-?1,the stimulatory effects of soft(5.9 kPa)matrix on the expression of stem cell-related genes and the positive number of surface markers of hepatocellular carcinoma stem cells were inhibited.The results suggest that the softer(5.9 kPa)matrix stiffness may up-regulate the expression of CD90 and CD133,markers of hepatocellular carcinoma stem cells,by increasing the expression of Integrin-?1.Our results suggest that soft(5.9 kPa)matrix can induce the HCC stem cells phenotype,which may be mediated by the increase of Integrin-?1 expression and upregulate the expression of HCC stem cell markers CD90 and CD133.