Study On The Correlation Between The Distribution Of Liver Cancer Stem Cells And The Mechanical Heterogeneity Of Liver Cancer Tissues

Hepatocellular carcinoma(HCC),as the main histological subtype of liver cancer,is a malignant tumor with multiple causes and high morbidity and mortality.Traditional radiotherapy and chemotherapy and liver transplantation are often difficult to eradicate HCC,and it is easy to relapse and metastasize after treatment.Therefore,exploring better prevention and treatment strategies for HCC is still a huge problem that needs to be solved clinically.Studies have shown that liver cancer stem cells(LCSCs)are a subgroup of tumor cells with self-renewal and differentiation potential in liver cancer.They are resistant to both radiotherapy and chemotherapy,and play a key role in the recurrence and metastasis of liver cancer.Its existence may be an important source of difficulty in the treatment of liver cancer.Therefore,targeted therapy strategies for LCSCs are of great significance for preventing the recurrence and metastasis of liver cancer.HCC is typically manifested as uncontrollable nodular hyperplasia,cancer and metastasis after experiencing repeated inflammation and irreversible fibrosis(cirrhosis).Studies have found that changes in the microenvironment of tissue mechanics are the key factor in the development of this type of cirrhotic liver cancer.The deposition of a large number of collagen fibers in the extracellular matrix(ECM)significantly increases the stiffness of the entire liver tissue.The accompanying portal hypertension will lead to abnormal accumulation of high-risk carcinogens in the liver,and eventually induce liver cancer.At the same time,the survival of cancer stem cells is usually limited to this specific tumor microenvironment.Studies have shown that mechanical factors in the microenvironment(matrix stiffness,shear force,etc.)can induce mesenchymal-like cells to acquire tumor initiation or stem cell-like functions to promote cancer development.It can also induce malignant transformation of epithelial cells and promote the expression of their stem cell marker molecules.Therefore,exploring the mechanical microenvironment of liver cancer stem cells in liver cancer tissues and the regulation of mechanical factors on liver cancer stem cells will provide an important theoretical basis for the development of liver cancer stem cell intervention therapies that target the mechanical microenvironment.In this study,the SD rat liver cancer model was induced by combining N-Nitrosodiethylamine(DEN)and N-Nitrosomorpholine(NMOR)to analyze the changes in mechanical properties during liver lesions.Observing the distribution of liver cancer stem cells in liver cancer tissues,and finally demonstrated that the distribution of liver cancer stem cells is related to the mechanical properties of liver cancer tissues.The main research contents and results of this topic are as follows:(1)Construction of liver cancer model and its pathological studyDEN and NMOR were combined to induce hepatitis,liver fibrosis,liver cirrhosis and hepatocellular carcinoma models in SD rats.Used anatomical observation,tissue section and pathological staining to conduct dynamic research on the whole process of liver cancer occurrence and development.The histological and anatomical observations of the hepatitis,liver fibrosis,liver cirrhosis,and hepatocellular carcinoma models obtained were consistent with the pathological characteristics of the models at various stages,indicating that the animal model was successfully constructed.(2)Analysis of mechanical properties of liver cancer tissueAtomic force microscope(AFM)and Sirius red staining were used to perform biophysical and biochemical evaluation and analysis on the fast frozen sections of tissue samples.The results showed that the average Young’s modulus of normal liver tissue,hepatitis tissue,liver fibrosis tissue,early cirrhosis,late cirrhosis tissue,and liver cancer tissue were 0.113±0.06 k Pa,0.19±0.16 k Pa,0.245±0.19 k Pa,0.344±0.23 k Pa,0.79±0.43 k Pa and 1.65±0.87 k Pa respectively.The result indicatied that the matrix stiffness of liver tissue gradually increases with the progress of fibrosis.In liver cancer tissues,the mean Young’s modulus of the tumor core is 0.26±0.16 k Pa,and the mean of the invasion front Young’s modulus is 3.48±3.36 k Pa.In contrast,the Young’s modulus of the adjacent tissue is 1.10±0.83 k Pa,indicating that liver cancer tissue has obvious mechanical heterogeneity.(3)Observation of the distribution of liver cancer stem cells in liver cancer tissuesThree liver cancer stem cell surface markers,Ep CAM,CD44,and CD13,were used to perform immunofluorescence three-color staining on tissue sections of liver fibrosis,cirrhosis and liver cancer tissues.The results showed that a small number of CD44 single-positive cells appeared in liver fibrotic tissues,and they preferd to concentrate in the portal area where collagen is deposited.In early liver cirrhosis tissues,there were a small amount of CD13~+/CD44~+cells,and they preferd to concentrate in the bile duct area.In advanced liver cirrhosis,a small amount of Ep CAM~+/CD44~+/CD13~+cells began to appear,and they prefer to concentrate on the fibrous structure area of the pseudolobule.In liver cancer tissues,there are a large number of Ep CAM~+/CD44~+/CD13~+cells,and they prefer to concentrate on the invasion front area of tumor tissue.A linear regression model was used to curve-fit the ECM stiffness of each area of liver cancer tissue and the corresponding LCSCs infiltration.The correlation curve drawn shows that the distribution of LCSCs is indeed related to the mechanical heterogeneity of liver cancer tissue.To conclude,the mechanical properties of liver tissues gradually changed in the process of carcinogenesis,and eventually developed significant mechanical heterogeneity.The distribution of LCSCs was related to the mechanical heterogeneity of liver cancer tissues.To conclude,the mechanical properties of liver tissues gradually changed in the process of carcinogenesis,and eventually developed significant mechanical heterogeneity.The distribution of LCSCs was related to the mechanical heterogeneity of liver cancer tissues.The results of this study provide an experimental basis for further understanding of the mechanical microenvironment in which LCSCs are located,and provide an important theoretical basis for the subsequent exploration of the regulatory mechanism of the mechanical microenvironment on LCSCs.