Matrix Stiffness Modulates Hepatocellular Carcinoma Invasion And Metastasis And Its Molecular Mechanism

Hepatocellular carcinoma (HCC) is one of the most frequentmalignant tumors characterizedby insidious onset, high incidence and mortality rates, and poor prognosis. Although successful curative hepatectomy has significantly improved survival in last two decades, the prognosisof HCC is still dismaldue to high aggresisiveness and metastasis. Therefore, to further clarify the mechanism of metastasis and recurrence and discover effective ways to inhibit these pathological process becomes the key point to further improve the survival of HCC.Increasing evidences suggest that tumor microenvironment involves in the regulation of HCC invasion and metastasis. Extracellular matrix (ECM), asan important component of cell surrounding environment,not only maintains solid cell/tissue structure, but also provides key biochemical and physical signals to regulation of normal cell functions. The excessive deposition and conformational change of ECM proteins result in an increase of extracellular matrix stiffness, and these mechanical forces influenc thebiological behavior changes of cells by distorying the cellular surface force balance.A lot of studiesshow that increasing matrix stiffness may involve in pathological progress of some diseases such as HCC, gastric cancer, breast carcinoma, oophoroma, glioma,etc. and seriously affect prognosis of these tumors. The recent studies also demonstrates that matrix stiffness can regulate proliferation, migration and invasion of tumor cells. Generally, matrix stiffness could induce integrin congestion to transduce mechanic signals into cells, affect cell growth and the secretion of cytokines.On the other hand, matrix stiffness alsomodulates cell migration through cystoskeleton remodeling. The effectsof matrix stiffness on regulating breast and ovarian cancer have been partly elucidated. However, little is known about its role in HCC. Actually, matrix stiffness is important for HCC development,80% of HCC patients emerge on a background of advanced fibrosis or liver cirrhosis, and these patients with advanced cirrhosis have lower median survival time. At present, liver stiffness measurement has become a strong predictor of HCC development and progress in clinic. Undoubtedly, study on matrix stiffness modulating invasion and metastasis of HCC willbe helpful to better understand of HCC pathogenesis.In the present study, wefirst determine the effects of matrix stiffness on the invasion and metastasis of HCC using the established rat HCC models with different liver stiffness backgrounds, andthen investigate the related mechanism of matrix stiffness modulating HCC invasion and metastasis (VEGF expression and EMT ocurrence) basedon an in vitro cell culture system of mechanically tunable COL1-coated polyacrylamide gel. From perspective of physical microenvironment, this study will provide new experinmental and theoretical evidences for HCC development and therapy.Parti Establishment of rat HCC models with different liver stiffness backgrounds and an in vitro cell culture system of mechanically tunable COL1-coated polyacrylamide gel, and analysis of the characteristics of invasion and metastasis in rat HCC models.Objective To establish rat HCC models with different liver stiffness backgrounds and an in vitro cell culture system to analyze the effects of matrix stiffness on HCC invasion and metastasis., as well as the characteristics changes of invasion and metastasis in rat HCC models.Methods Buffalo rat models with different liver stiffness backgrounds were first induced by long term intraperitoneal injection with different dose of carbon tetrachloride (CC14), and then they were treated by orthotopic implantation of a piece of subcutaneous HCC tissue derived from McA-RH7777 cells.Twenty-five days later, they growed into rat HCC models with different liver matrix stiffness backgrounds.Pathological features of rat HCC modelsincludingmatrix stiffness, histological morphology, liver function, proliferation, lung metastasis as well as invasion/metastasis associated genes, etc were evaluated respectively. An in vitro cell culture system of mechanically tunable COL 1-coated polyacrylamide gel was established to simulate the variation of tissue stiffness, and the coated collagen 1 provide the biological matrix interface. Cell morphology, motility and invasion/ metastasis associated genes expression of HCC cells were detected using this culture system.Results HCC growth(tumor size and wet weight of tumor) and malignant characteristics were all remarkably increased in parallel with liver matrix stiffness increasing. Cell proliferation index Ki-67, HCC invasion/metastasis asssociated genes (MMP9, MMP2, CD44, SPP1), Integrinβ1, AFP were all upregulated significantly in HCC tissues with higher liver matrix stiffness background, as compared with those of the control. Expression of COL1 and LOX indicating the level and cross-linking of collagen in HCC were also significantly upregulated. Meanwhile, levels of serum ALT, AST, ALP and y-GT in HCC rats with higher liver matrix stiffness background were increased, level of serum albumin was decreased, indicating poor liver function, and high expressions of Integrinβ1 and FAK were also found.In vitro cell culture system, with increasing substrate stiffness,the spread area of HCC cells,pseudopodia extension were all increased, and the expressions of invasion/metastasis associated genes (MMP9, MMP2, CD44, SPP1) were also upregulated.Conclusions The in vivo rat HCC models and an in vitro cell culture system were successfully establishedfor elucidating the effects of matrix stiffness on HCC metastasis. Tumor growth and malignancy characteristics (invasion/metastasis) were significantly increased in ratHCC models with higher liver stiffness backgrounds. The expression of invasion/metastasis in HCC cells cultured on substrate rigidity were also upregulated, which indicates that matrix stiffness may modulate the invasion and metastasis of HCC.Part2 Increasing matrix stiffness upregulates the expression of VEGF in HCC cells and its related mechanism analysisObjective To investigate the effects of matrix stiffness on VEGF expression in HCC cells and explore its related mechanism.Methods Tumor tissue samples of the rat HCC model with different liver stiffness backgrounds established in parti were collected and constructed into a tissue microarray to analyze the relationship between matrix stiffness and HCC angiogenesis. An in vitro system of mechanically tunable Collagenl (Coll)-coated polyacrylamide gel was used to clarify the regulatory mechanism of VEGF expression in HCC cells.Results Tissue microarray analysis showed that the expression levels of VEGF and CD31 were gradually upregulated in tumor tissues with increasing Coll and lysyl oxidase(LOX) expression, indicating a positive correlation between tumor angiogenesis and matrix rigidity. The expression of VEGF and the phosphorylation levels of PI3K and Akt were all upregulated in HCC cells on high-stiffness gel than on low-stiffness gel. Meanwhile, alteration of intergrin β1 and a 5 expression was found to be the most distinctive, implying that it might mediate the response of HCC cells to different matrix stiffness simulation. Using specific monoclonal antibody to block integrin β1 in HCC cells, the expression of VEGF and the phosphorylation levels of PI3K and Akt at different culture times were suppressed and downregulated respectively in the treatment group as compared with those in the control group. It suggested that the extracellular matrix stiffness stimulation signal was transduced into HCC cells via integrin β1, and this signal activated the PI3K/Akt pathway and upregulated VEGF expression.Conclusions Matrix stiffness modulates VEGF expression in HCC cells via the integrin β1/PI3K/Akt pathway. Matrix stiffness-initiated HCC angiogenesis is a potential target for preventing tumor growth and metastasis.Part3 Increasing matrix stiffness induces EMT ocurrence of HCC cells and its related mechanism explorePurpose To investigate the effects of matrix stiffness on EMT of HCC cells and its related mechanism.Methods EMT associated markers (N-cadherin, vimentin, a-SMA, E-cadherin and β-catenin) of HCC cells cultured on tunable substrate stiffness were detected, then HCC cells were further stimulated with TGF-β1, and EMT associated marker expression of the treated HCC cells were analyzed,meanwhile phosphorylation levels of signaling molecule smad2/3 in HCC cells were alsomeasured at different time points of TGF-β1stimulation. Additionally, FN-coated and LN-coated substrate gellwere used to examine HCC cell morphology and snail gene expression.Results Withincrease of the substrate stiffness, the expressions of E-cadherin and β-cateninwereall downregulated, whereas the expressions of N-cadherin, vimentin, and a-SMA were all upregulated, indicating that increasing substrate stiffness counld inducethe ocurrenece of EMT in HCC cells. After stimulation with TGF-β1, the changes of EMT markers in HCC cells were more obvious. Moreover, with gel stiffness increasing and TGF-β1 stimulation time prolonging, the phosphorylation levels of smad2/3 were all significantly enhanced. In addition, the changes of cell morphology and snail gene expression of HCC cellscultured in COL1, FN, LN-coatedsubstrate showed no apparent difference with substrate stiffness increasing, suggesting that the ocurrence of EMT in HCC cells was mainly induced bysubstrate rigidity rather than the coated matrix proteins.Conclusionslncreasing matrix stiffness could independently induce EMT ocurrence of HCC cells viasmad2/3 signaling pathway.Innovation1. Two novel experinment platforms in vivo and in vitro, such asrat HCC models with different liver stiffness backgroundsand in vitro cell culture system with tunable substrate stiffness, were sucesssfully establishedand used for explainning the effects of matrix stiffness on HCC cells.2. This study firstly reports that matrix stiffness can induce EMT ocurrence of HCC independently and upregulate VEGF expression in HCC cells, furthermore affect HCC invasion and metastasis.Potential merits for clinical application1. This study on matrix stiffness provides experimental data which can better reflect the pathological changes of HCC patients with cirrhosis background, and this physical mechanics parameter can be used to guide the diagnosis and treatment of HCC.2. This study could lay foundations for further research about matrixstiffness modulating biological behaviors of HCC cells and the mechanism in matrix stiffness promoting the invasion and metastasis of HCC.