Heterogeneous Matrix Stiffness Regulates The Cancer Stem-like Cell Phenotype In Hepatocellular Carcinoma

BackgroundPrimary liver cancer(PLC)is one of the cancers with high morbidity and mortality in the world,and hepatocellular carcinoma(HCC)accounts for about 80% of all cases.HCC usually occurs on the basis of chronic liver disease,which mainly caused by hepatitis virus infection and alcohol abuse.Effective treatments for early HCC include liver transplantation,ablation,and partial hepatectomy,which are potential cures for HCC.But even then,there is still a certain probability of recurrence after surgery.In addition,due to the lack of obvious early symptoms and specific diagnostic markers of HCC,most HCC patients have progressed to middle and late stages,and the prognosis is worse.Due to the special drug resistance of HCC,the therapeutic effect of chemotherapy drugs has been unsatisfactory.The tyrosine kinase inhibitor sorafenib is the first approved first-line therapy for advanced HCC,but some patients still develop resistance to the drug.HCC is prone to relapse and has strong drug resistance.In order to achieve better therapeutic effect,it is urgent to deeply understand the process of cancer occurrence and development in HCC patients,so as to develop new targeted therapeutic drugs.Common therapeutic resistance and relapse are usually caused by the inherent heterogeneity within cancers,and a common model to explain this heterogeneity is cancer stem cells(CSCs)model.CSCs constitute only a small fraction of the cancer cell composition and are defined as cells with unlimited self-renewal ability,setting a differentiation hierarchy in cancers,resulting in a range of different cell types.However,this differentiation is not unidirectional.Terminally differentiated cancer cells can obtain CSCs characteristics through interaction with tumor microenvironment(TME).In the past,TME was regarded as a bystander in the process of cancer occurrence and development.In recent years,with the in-depth understanding of TME,it has been found that it has a complex structure and plays an important role,and can even be used as a resource library for the development of anti-cancer drugs.As an important component of TME,extracellular matrix(ECM)not only acts as a scaffold for cell growth,but also plays a role in tissue repairing and homeostasis maintenance.In solid tumors,ECM structure undergoes dramatic changes,the most obvious of which is abnormal production and crosslinking of matrix proteins,resulting in changes in tumor stiffness.Stiffness,as the most easily recognized mechanical feature in solid tumors,can reflect the rigidity of tissues.As is known to all,tumor tissues are generally stiffer than normal tissues.Stiffness can regulate tumor cell proliferation,invasion,metabolism and other functions.Therefore,the aim of this study is to explore the heterogeneity of matrix stiffness within HCC tissues,the regulatory effect of matrix stiffness on stemness expression of cancer cells and related transduction pathways,and to further verify whether inhibiting the function of proteins related to mechanical transduction pathways can block the regulatory effect of stiffness on cells,so as to provide new ideas for the development of cancer therapeutic drugs in the future.MethodsThree parts of each sample were randomly selected for sampling,labeled as group A,B and C.The tissue elastic modulus was measured by atomic force microscopy(AFM)after frozen section.Then polyacrylamide(PA)hydrogel was prepared according to the measured elastic modulus and HCC cells were cultured on the surface to simulate mechanical forces imposed by matrix stiffness in vivo.Real-time quantitative PCR,Western blotting,immunofluorescence staining and flow cytometry were used to detect the expression levels of stemness-related markers in cells under different interventions of stiffness.Cell proliferation and drug resistance were also detected.In order to explore the mechanical signaling pathways involved in stiffness transduction,functional inhibition was performed on the pathway-related proteins,integrin and yes-associated protein(YAP),and the expression levels of stemnessrelated markers in HCC cells were detected again.Next,HCC cells mixed with polysaccharide hydrogel with adjustable stiffness were used for subcutaneous tumor formation in nude mice,and YAP inhibitor was used for intervention.According to the elastic modulus of hydrogel,nude mice were divided into soft,medium,stiff and stiff+ VP groups,and the formation of subcutaneous tumor in each group was observed.In addition,immunofluorescence and immunohistochemical staining were performed on tumor tissues to verify the results of cell experiments in vitro.Finally,real-time quantitative PCR,cell scratch,transwell and immunofluorescence staining were used to explore the role of stiffness in the process of fibroblast activation induced by cancer cells.Results(1)There is intra-tumoral heterogeneity in matrix stiffness of HCC tissues.The elastic modulus values of groups A,B and C were 1051.61 ± 434.27 Pa,4540.35 ±2666.98 Pa,9307.37 ± 4989.91 Pa,P < 0.001.The results of Sirius red staining showed significant differences in collagen fiber deposition and crosslinking in different parts of the tumor.The higher the stiffness,the more collagen fiber deposition and the more disordered the arrangement.(2)HCC cells grown on the surface of PA hydrogel with different stiffness showed different cell morphology and cytoskeletal structure,and the higher the stiffness,the more extended the cell morphology,the clearer the cytoskeletal structure,and the larger the cell surface area(P < 0.05).The expression levels of intra-cellular stemness-related markers NANOG and OCT4 increased with the increase of stiffness(P < 0.05),and the expression trend of CD133 on the cell surface was consistent with that.The proliferative ability of HCC cells decreased with the increase of stiffness(P < 0.05).Under different stiffness conditions,sorafenib induced apoptosis of HCC cells also showed differences:the proportion of apoptotic cells decreased with the increase of stiffness.There were no significant differences in the proliferation and pellet-forming ability of HCC cells among all groups after being removed from the environment with different stiffness.(3)There were no significant differences in the expression levels of YAP m RNA and protein in HCC cells grown on PA hydrogel with different stiffness,but the expression levels of its downstream target genes ANKRD and CTGF increased with the increase of stiffness(P < 0.05).The expression of phosphorylated YAP protein decreased with the increase of stiffness,and the results of immunofluorescence staining showed that the accumulation of YAP in the nucleus increased with the increase of stiffness.There were no significant differences in the expression levels of intra-cellular stemness-related markers among the groups treated with integrin inhibitors or YAP inhibitors on the PA hydrogel with different stiffness.Compared with the group without inhibitor intervention,the expression of total YAP protein and phosphorylated YAP protein in HCC cells decreased after YAP inhibitor treatment.There was no significant change in total YAP protein expression after integrin inhibitor treatment,but phosphorylated YAP protein expression increased.(4)The tumor size of nude mice was different among all groups,and the higher the stiffness of polysaccharide hydrogel,the smaller the tumor volume(P < 0.05).Immunohistochemical staining showed that the protein expressions of NANOG and OCT4 were increased with the increase of stiffness(P < 0.05).Tissue immunofluorescence staining showed that the accumulation of YAP protein in the nucleus increased with the increase of stiffness(P < 0.01).After intervention of subcutaneous tumor formation with YAP inhibitors,tumor size did not change significantly in stiff + VP group compared with stiff group,but nuclear aggregation of YAP protein was significantly reduced(P < 0.01).(5)The conditioned medium derived from HCC cells increases the expression level of hepatic stellate cells(HSCs)activation-related markers(P < 0.05),and the expression of proliferation-related protein PCNA is upregulated in cellular immunofluorescence results.Faster healing of scratches was seen in the cell scratch experiment,and cells were seen to cross the compartment membrane more in the transwell experiment.Under the dual effects of conditioned medium of HCC cells and high stiffness,the expression level of HSCs activation-related markers was further increased(P < 0.001),and the secretion of type Ⅰ collagen was promoted(P < 0.01).Conclusions(1)There is intra-tumoral heterogeneity in the matrix stiffness of HCC tissues,which is positively correlated with the amount of collagen fiber deposition and the degree of crosslinking.(2)High stiffness can increase the expression level of stemness-related markers in HCC cells and enhance drug resistance.Cells need constant mechanical stimulation to maintain this function.(3)Stiffness stimulation enters cells through integrin-cytoskeleton-YAP pathway and regulates transcriptional activity of stemness-related markers.(4)HCC cells can activate HSCs in a paracrine way to promote their stronger proliferation and migration.High stiffness can enhance the activation of HCC cells on HSCs,and activated HSCs can produce more matrix proteins to further stiffen the ECM.