| Background:Lung cancer, one of the most common malignant tumor and the leading cause of cancer death in male and female, leads to much more death than the sum of breast, colon, prostate, and pancreatic cancer. Lung adenocarcinomas, the most common type of NSCLC, usually begin in tissues that lie near the outer part of the lungs, and they may be present for a long time before causing symptoms and being diagnosed. Distant metastasis is the most common reason for failure to treat advanced NSCLC, is also the key factor to determine the prognosis of patients. The pathogenesis and the mechanism of metastasis of lung cancer is still not clear, effective treatment to metastasis of lung cancer is still lacking, so it is important for studying the pathogenesis of lung cancer's genesis and process and indentify effective treatments.There are two main ways to study the metastasis of lung cancer, one is establishing high metastastic potential cell line by animal model in vivo, the other is establishing artificial basement membrane which can imitate the process of metastasis in vitro. They both had limitations,which delayed the process of studying metastasis of lung cancer to a large extent. A549 and SPC-A1, the most widely used lung adenocarcinoma, were established from surgical section on earlier stage, which makes it limitationly to study the metastasis of lung cancer. The widely used lung cancer cell line with high metastastic potential is usually subcloned from the lung cancer cell line by limited dilution method and then sorted in vivo by experimental animal models. The characteristics of cells will change with the differences of growing environment in vitro. So if we established a lung cancer cell line with metastastic potential by primary c?Lture and passage, we co?Ld study the mechanisms of lung cancer's genesis and process. In the meantime, we can sort cells with higher metastastic potential to find the genes involved in metastasis and treatment against metastasis by this model. Therefore, establishing lung cancer cell line with metastastic potential by primary cell culture and passage is of magnificent to study the genesis and process of lung cancer.Malignant pleural or peritoneal effusion is one of the complications of advanced cancer, it also indicates that there are distant metastasis. We are going to establish a lung adenocarcinoma cell line with metastastic potential from malignant peritoneal effusion, and reduce the influences to cell line's biological behaviors by experimental factors in vitro, provide a good experimental model to study the the mechanisms of lung cancer's metastasis and identify tumor therapeutic targets.In the basic research on lung cancer metastasis, tumor stem cell (cancer stem cells, CSCs) theory provides a new research direction to study the treatment of lung cancer metastasis, and has become a research hot spot. Exploring the biological mechanisms of tumor metastasis from the perspective of tumor stem cell theory will provide theory of targeting treatment for lung cancer metastasis. Epithelial-mesenchymal transition(EMT) refers to cells' transition from epithelial phenotype to mesenchymal phenotype, this change not only plays an important role in the process of embryonic development in a variety of animals, at the same time, a lot of research res?Lts show that EMT is the key step in tumor distant metastasis. So, to explore the existence of the phenomenon of EMT in tumor cells, in order to further reveal the nature of the tumorigenesis and development opens a new angle of view, also has provided new mentality of the molec?Lar basis for anti-tumor therapy.Objectives:1. To establish a human lung adenocarcinoma cell line M-LAC with metastastic potential by culturing cells primarily from malignant peritoneal effusion from a patient with adenocarcinoma of lung;2. To study the basic biological characteristics of human lung adenocarcinoma cell line with metastastic potential;3. To study the biological characteristics on tumor stem cells and EMT of human lung adenocarcinoma cell line M-LAC with metastastic potential preliminarily.Methods:1. To optimize a series of conditions that is suitable to culture primarily human lung adenocarcinoma cell line M-LAC with metastastic potential by optimizing a sepa ??n method and a purification method using different adherence and collagenase digestion ways;2. To identify the biological characteristics of the human lung adenocarcinoma cell line with metastastic potential, M-LAC:cell morphology was observed under light microscope; cell growth curve, cell clonoy formation, and karyotype analysis were measured; Matrigel transwell chamber was used to test the invasion ability of M-LAC; heterotransplantation of M-LAC cells into nude mice in situ was applied to examine oncogenicity of of M-LAC cells and pathological characteristics with HE and immunohistochemistry staining were used to observe tissue morphology of xenograft tumors and their metastatic tumors in nude mice; mycoplasma contamination was tested to verify.3. To identify the biological characteristics on tumor stem cells and EMT of human lung adenocarcinoma cell line M-LAC with metastastic potential preliminarily:(1) CD133 expression in human lung adenocarcinoma M-LAC and A549 control was analyzed by Flow cytometric;(2) The protein expression of Oct4, ABCG2, E-Cadherin, and Vimentin in human lung adenocarcinoma M-LAC and A549 control was analyzed by Western blot;(3) Genes such as CD133, Oct4, ABCG2, VEGF-A, P53, Rb, c-myc, and TNF-ain human lung adenocarcinoma cell line, M-LAC andA549 control were detected by RT-PCR analysis.Results:1. Human lung adenocarcinoma cell line M-LAC with metastastic potienal was successfully established by applying different adherence and collagenase digestion.2. The biological characteristics of human lung adenocarcinoma cell line M-LAC with metastastic potienal:An ultrastructural examination revealed that M-LAC cells grow in a monolayer way, its diverse morphology can be characterized by fusiform, triangular or polygonal. The pop?Lation doubling time of this cell line was about 29.07±0.278 h. Clonal formation rate was about 17.8%. With a large subpop?Lation of aneuploid cells, about 35% was hypotetraploid. Of M-LAC cells,90% expressed SFTPC and Cytokeratin 7,85% E-Cadherin, and 10% Vimentin. The invasion ability of M-LAC was tested higher than that of A549 controll.2×106 cultured cells were all capable of forming tumors in 8 immunodeficient nude mice with 50% lymphatic metastasis rate, and 25% hematogenous lung metastasis rate. The tumor and metastastic lymph node expressed SFTPC(+), Cytokeratin 7(+), and E-Cadherin(+).3. The biological characteristics on tumor stem cells and EMT in human lung adenocarcinoma cell line M-LAC with metastastic potential:The relative expression amount of CD133 in M-LAC and A549 control is respectively 8.095% and 5.0%. The relative expression amount of Oct4 in M-LAC and A549 control is respectively 3.586±0.024 and 2.912±0.013. The relative expression amount of ABCG2 in M-LAC and A549 control is respectively 3.834±0.028 and 1.561±0.016.These res?Lts indicated that the metastastic potential of M-LAC may be associated with its higher proportion of tumor stem cells. The relative expression amount of E-Cadherin in M-LAC and A549 control is respectively 1.236±0.017 and 1.431±0.018. The relative expression amount of vimentin in M-LAC and A549 control is respectively 2.064±0.028 and 1.022±0.027. These res?Lts suggested that M-LAC may be of EMT which may give rise to metastastic potential.4. Genes such as CD133, Oct4, ABCG2, p53, Rb, c-myc, TNF-?, and VEGF-A showed higher expression in M-LAC than those in A549 control.Conclusions:1. Human lung adenocarcinoma cell line M-LAC with metastastic potienal was successfully established from malignant peritoneal effusion from a patient with adenocarcinoma of lung,100% xenograft tumors formed in 8 immunodeficient nude mice with 50% lymphatic metastasis rate and 25% hematogenous lung metastasis rate.2. The population doubling time of M-LAC was about 29.070±0.278 h. Clonal formation rate was about 17.8%. With a large subpopulation of aneuploid cell, about 35% was hypotetraploid.90% M-LAC cells expressed SFTPC and Cytokeratin 7,85% expressed E-Cadherin, and 10% expressed Vimentin. The invasion ability of M-LAC was tested higher than that of A549 controll. These results indicate that Human lung adenocarcinoma M-LAC with metastastic potienal accords with the standard of establishing a cell line.3. Human lung adenocarcinoma cell line M-LAC is of metastastic potential may due to its higher proportion of tumor stem cells, and M-LAC cells are of EMT which may contribute to their metastastic potential.4. Genes like TNF-?,VEGF-A, p53, Rb and c-myc expressed higher in Human lung adenocarcinoma cell line M-LAC with metastastic potiena than those in A549 control. |
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