Study On The Relationship Between Macrophages And The Epithelial-Mesenchymal Transition Of Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is one of the most prevalent human malignant tumors worldwide and especially in Asia and South Africa. Surgical intervention is the most common form of therapy of it, but the prognosis of HCC remains poor because of tumor invasiveness, frequent intrahepatic spread, and extrahepatic metastasis.The vast majority of previous studies focused solely on malignant cells themselves, regarding tumors just as masses of autonomous cells and aiming at identifying the molecular and genetic changes associated with this malignant transformation. Recent research on tumor-host interactions collectively reveals that microenvironments co-evolve during tumor initiation and progression. As an important part of tumor microenvironment, immune microenvironment plays a critical role in carcinogenesis, metastatic invasion and dissemination. HCC is usually secondary to inflammation conditions due to chronic hepatitis and cirrhosis resulting from either HBV/HCV infection or non viral-related causes such as alcohol or obesity.Metastasis is a complex process and various factors are involved in each step of metastasis. Recent studies suggest that numerous genes and proteins play vital roles in metastasis of cancer cells, and epithelial-mesenchymal transition (EMT) which describes that epithelial cells can convert into mesenchymal cells has been shown to be critical for the development of the invasiveness and metastatic potential of human cancers. During the transition process, epithelial cells reduce intercellular adhesion and acquire fibroblastoid propertis including down-regulation of E-Cadherin which is epithelial marker, up-regulation of N-Cadherin which is mesenchymal marker, and Snail which is the EMT regulator. EMT can help the cancer cells enter the surrounding tissues by increased invasive properties and finally establish a suitable microenvironment for its progression and metastasis. Tumor associated macrophage (TAM) is a special subset of tumor immune cells which is located in the tumor tissues and plays a critical role in tumor immune microenvironment. TAM can secret some cytokines and growth factors which involved in promoting tumor development, stimulate the growth and metastasis of tumor cells, induced tumor angiogenesis and lymphangiogenesis, immune suppression process. In our previous study we found that high peritumoral macrophage colony-stimulating factor (M-CSF) and macrophage density, especially at the tumor edge, predicted high risk of recurrence and metastasis in HCC patients who had received curative live resections. It is reported that dense CD68+macrophage infiltration at the tumor border line related to poor survival in melanoma, and in some other reports (bladder, esophageal, and breast cancer), the same phenomenon was observed. These phenomena suggest that in the area of the tumor edge, macrophages can increase the ability of invasion and metastasis of tumor cells through the secretion of certain cytokines, and promote their recurrence and metastasis.In this study, by establishing a macrophage-HCC cell lines co-culture system, we can perform a similar microenvironment of tumor edge in vitro. This model can assist us in exploring whether the macrophage promote the procedure of EMT. In order to observe the phenomenon in vivo, double staining immunohistochemistry and tissue micro-array was used to localize the biomarker of macrophage and EMT in specimens obtained from HCC patients simultaneously. Cell signal microarray was applied to discover the cytokines during the interaction between paritumoral macrophage and EMT.These experiments are divided into3parts, as following,1. Research on the biological characteristics of macrophages effected human HCC cell lines with different metastatic potential;2. Study on the role of macrophages in the epithelial-mesenchymal transition of HCC cells.3. Study on the effect and mechanism of macrophages secreted IL-8in the epithelial-mesenchymal transition of HCC cells. Part Ⅰ. Research on the Biological Characteristics of Macrophages Effected Human HCC Cell Lines with Different Metastatic PotentialObjective:To study the biological characteristics of macrophages effected human HCC cell lines with different metastatic potential, and explore the correlation of macrophages and the biological changes of characteristics in human hepatoma cell line.Method:Raise the THP-1human leukemia monocyte cell line, and make it differentiate into macrophages through PMA stimulation; Raise the high metastatic potential hepatocellular carcinoma cell lines:MHCC-97H, MHCC-97L and the low metastatic potential of hepatocellular carcinoma cell lines:Hep-G2, Huh-7, in six-well plates and co-cultured with macrophages throught the transwell system. A wound-healing assay was used to evaluated the ability of cell migration. Cell invasion assays were performed using24-well transwells precoated with Matrigel.Results:The human HCC cell lines which were co-cultured with macrophages were significantly changed in morphology and express enhanced wound closured rates and the ability of invasiveness.Conclusion:Macrophages may enhance the migration of invasive ability of the human HCC cell line by certain cytokines.Part Ⅱ. Study on the role of macrophages in the epithelial-mesenchymal transition of HCC cellsObjective:To study the role of macrophages in the epithelial-mesenchymal transition of human HCC cells and explore the potential relationship between them and the cytokines which probably have some effect on the certain process.Method:Randomly choose30HCC samples from the patients who have received curative resection in2009, and double staining immunohistochemistry was applied to observe the relationship between macrophages and the HCC cell which undergoing EMT in the tissues. The levels of E-Cadherin, N-Cadherin and Snail in HCC cell lines co-cultured with macrophages were detected by WesternBlot and immunofluorescence. Antibody micro-array was used to explore the cytokines which probably take part in the certain course.Result:The double staining immunohistochemistry showed that in the macrophages infiltrated area, the expression of N-Cadherin and Snail were increased. WesternBlot and immunofluorescence showed that HCC cell lines with high metastatic potential have low levels of E-Cadherin and high levels of N-Cadherin and Snail. By contrast, HCC cell lines with low metastatic potential have high levels of E-Cadherin and low levels of N-Cadherin and Snail. After co-cultured with macrophages, the HCC cell lines can obtain enhanced expression of N-Cadherin and Snail. The result of antibody micro-array revealed that IL-6and IL-8may be the major cytokines in the certain process.Conclusion:Macrophages can involve in the epithelial-mesenchymal transition of HCC cells through IL-6and IL-8.Part Ⅲ. Study on the effect and mechanism of macrophages secreted IL-8in the epithelial-mesenchymal transition of HCC cellsObjective:To investigate the mechanism of epithelial-mesenchymal transition in HCC cells, the expression the IL-8and CD68in human HCC tissues and the effect of macrophages secreted IL-8in the co-culture system was explored.Method:Tissue micro-array and immunohistochemistry were used to analyze the correlation among IL-6, IL-8and CD68. The level of IL-8in Hep-G2, macrophage and co-cultured system culture supernatants were measured by ELISA. Immunofluorescence was applied to explore the different expression of E-Cadherin, N-Cadherin and Snail in Hep-G2between the control group and exogenous IL-8group. A wound-healing assay was used to evaluate the ability of cell migration and cell invasion assays were performed using24-well transwells precoated with Matrigel in Hep-G2treated with exogenous IL-8. WesternBlot was applied to explore the cell signal pathway caused by exogenous IL-8.Result:IL-6in human HCC tissues had no specific expression, while IL-8and CD68were significant correlated. The levels of IL-8in culture supernatant in macrophage and the co-cultured system were extremely higher than that in Hep-G2(P<0.001). The exogenous IL-8can enhance the wound closure rates of Hep-G2and the promote the invasion of it. The process of epithial-mesenchymal transition can be started by the JAK2/STAT3pathway by activation of Snail.Conclusion:Macrophage secreted IL-8can start the process of EMT in HCC cells.