| Backgrounds and aims: Cancer is an important cause of death. The annual number of cancer deaths worldwide continues to increase, and the degree of malignancy is also rising. Hepatocellular carcinoma(HCC) ranks second in cancer mortality ranking of the world, which has a high metastasis rate, poor prognosis, and is so difficult to treat. Tumor invasion and metastasis are the main reasons for relapse and poor prognosis. Movement and migration abnormalities are the characteristics of malignant cells. There is a certain degree of abnormal in cytoskeleton of tumor cells in the development and expression of adhesion molecules, which cause tumor cell migration and the unusual ability to move. Tumor cell migration is not limited to a single cell. Most invasive solid tumours display predominantly collective invasion, which is commonly observed in many tumor types, and malignant function is decided by the behavior of groups. 2D(two-dimensional) cell culture has been widely used in biological research, but the cells grown on 2D culture systerm does not accurately simulate the state of cells in vivo. Compared to 2D cell culture, 3D(three-dimensional) cell culture can simulate the environment of the cell in vivo, through the use of recycled extracellular matrix(such as Matrigel, collagen I, etc.). 3D cell culture can provide closer to the body's living conditions of the microenvironment. It's more conducive for tumor cells to invasion and migration into the surrounding tissue. HAb18G/CD147 is a plasma membrane-bound glycoprotein that functions as an adhesion molecule, belonging to the immunoglobulin family of adhesion molecules. It induces hepatocellular carcinoma and surrounding stromal fibroblasts secrete matrix metalloproteinases, and degradede the extracellular matrix surrounding tumor tissue cells, promote the invasion and metastasis of hepatocellular carcinoma. Our laboratory previously identified HAb18G/CD147 may be involved in ollective hepatocellular carcinoma cell movement. Although the concept of collective cell movement has been proposed for a long time, previous studies have focused on collective cell movement which are involved in the normal development process, including gastrulation, tubular form, angiogenesis in zebrafish germ layersi, etc. The mechanism of collective cancercell invasion and movement is still not very clear. Therefore, defining the specific movement phenotype of hepatocellular carcinoma cell movement can contribute to the research of molecular mechanism of HAb18G/CD147 regulating incollective cancer cell movement.Method: We tested the migration and invision abilities of three HCC cell lines by scratching and transwell experiments.Using western blot, we tested the key molecules expression related to migration and invision in 2D and 3D culture conditions.We used Collagen I to establish a 3D culture model. Three HCC cell lines were seeded on collagen I and cultured for several days. After paraffin-embedded sections, HE staining, evaluation model was built. Based on established 3D model of cultured hepatocellular carcinoma cell movement, immunofluorescent staining was used to preliminarily study the expression and distribution of the critical molecular motion of hepatocellular carcinoma cell movement, such as HAb18G/CD147, E-cadherin, adhesion molecules, Vinculin, etc. Clinical samples of HCC were selected for HE staining and immunohistochemical staining to study the expression and distribution of key molecules in hepatocellular carcinoma cell movement.Results: Three HCC cell lines had a strong ability of migration and invasion. Western blot results illustrated that the key molecules related to migration and invision expressed more highly in 3D culture condition than in 2D. After paraffin-embedded and HE staining of HCC cells cultured in 3D model, we found that three HCC cells were able to grow and invade in Collagen I and moved by groups. Immunofluorescence staining and immunohistochemical staining showed that HAb18G/CD147 was highly expressed in HCC cells and mainly distributed in the cell membrane. E-cadherin was highly expressed in HCC cells and mainly distributed in the cancer cell-cell connections.Paxillin was highly expressed in HCC cells,promoting the formation of focal adhesion. Vinculin exhibited polarity distribution which mainly at the leading edge of the group, providing a strong adhesion to move forword for HCC cell movement.FAK was expressed in HCC cells and normal tissue. But activated FAK, also named p-FAK, was only expressed in HCC cells. Rac1 mainly distributed in the leading edge of the group to ensure the continued protrusion.Conclusion: We successfully established 3D HCC cell culture model. Using 3D culture model for immunofluorescence staining, and using clinical specimens of hepatocellular carcinoma for immunohistochemical staining, we analyzed the key molecule expression and distribution of hepatocellular carcinoma cell movement. Finally,we preliminarily defined the henotype of hepatocellular carcinoma cell movement... |
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