The Study Of Ovarian Cancer Stem Cells Enhancing Ovarian Cancer Lymphangiogenesis And Lymph Node Metastasis

Ovarian cancer is the fifth leading cause of cancer deaths and has the highest mortalityrate among gynecologic cancers. Whereas standard cytoreductive surgery andchemotherapy followed by taxane/platinum,70%of patients will relapse within two yearswith progressive disease. Novel therapeutic approaches are urgently needed to improveprognosis. Lymphangiogenesis, the formation of the lymphatic, play a crucial role in thespread of ovarian cancer. However, the role of lymphangiogenesis in the growth anddissemination of ovarian cancer has not been clearly established.The theory of cancer stem cells (CSCs) has been developed during the last decade.CSCs have been the focal spot of the cancer research field, and have been considered as theorigin of tumorigenesis, metastasis and recurrence. CSCs, successfully isolated andidentified from ovarian cancer, are thought to have the ability to initiate tumour, a highcapacity of self-renewal, and the propensity to differentiate into actively proliferatingtumour cells. CSCs are often associated with elevated expression of the stem cell surfacemarker. In order to investigate the ovarian cancer lymphangiogenesis and lymph nodemetastasis, it is important that studing the interaction between CSCs and lymhaicendothelial cells. SDF-1/CXCR4has been known to have broad activities in different kindsof tumours. Previous studies on CXCR4have been shown that CXCR4are prognosticmarkers in ovarian cancer, and anti-CXCR4may have anti-tumour activity such asinhibition of invasion, migration, and angiogenic potential. Recent studies indicated that thesignaling of SDF-1/CXCR4was participated in lymphangiogenesis.It is known that circulating CD34~+and VEGFR3~+lymphatic endothelial progenitorcells (LEPCs) involved in the lymangiogenesis. And recent studies found that LEPCs weresignificantly increased in patients with small cell lung cancer patients and LEPCs werecorrelated with tumor lymphangiogensis. However the role of LEPCs in thelymphangiogensis has not been clearly established.In the current study, using flow cytometry, we isolated and characterized ovarian CSCs by expression antibody against CD133in anchorage-independent spheres from humanovarian cancer cell line A2780. We investigated proliferation and immigration oflymphatic endothelial cells while lymphatic endothelial cells cultured with ovarian cancerCD133~+cells by transwell chamber. The supernatants were detected by cytokines chips.VEGF-C and SDF-1production in the supernatants were measured with ELISA. Weobserved the effects of SDF-1down-regulation and up-regulation in the supernatantsolution of transwell chamber on immigration of lymph endothelial cells. Then weinvestigated the expression of CXCR4in CD133~+cells by WB, observed CD133~+cellsinvasion co-cultured with lymphatic endothelial cells by transwell assay, and studiedexpression of VEGF in CD133~+cells by WB. At the same time we studied levels ofcirculating LEPCs and lymph node metastasis in epithelial ovarian cancer patients, and alsotested plasma levels of VEGF-C and SDF-1to find out their possible relationships withLEPCs in patients with epithelial ovarian cancer.The main results and conclusions are as follows:1、The experiments demonstrate that only a minority of A2780cells were stainedpositive for CD133, a cell surface marker of CSCs. These CD133~+cells were capable ofself-renewal, multipotent differentiation and tumor initiation.2、Lymphatic endothelial cells proliferation and immigration enhanced when lymphaticendothelial cells were cultured with ovarian CSCs. Cytokines chip and ELISA resultsshowed higher level of SDF-1in their cell supernatants. Up-regulation of SDF-1promotedmigration of lymphatic endothelial cells and down-regulation of SDF-1inhibited migrationof lymphatic endothelial cells. We found that higher level of CXCR4expression in ovarianCSCs. Invasion and VEGF expression of ovarian CSCs enhanced when ovarian CSCscultured with lymphatic endothelial cells.3、We found that the level of circulating lymphatic endothelial progenitor cell wassignificantly correlated with lymph node metastasis and FIGO stage in patients with ovariancancer. There was also a statistically significant correlation between lymphatic endothelialprogenitor cells and SDF-1.In summary, CD133positive cells may represent ovarian cancer stem cells in ovariancancer cell line A2780. Lymphatic endothelial cells proliferation and immigration enhanced,and ovarian CSCs invasion enhanced when ovarian CSCs were cultured with lymphatic endothelial cells. SDF-1/CXCR4axis may be one of the mechanisms responsible for thehigh migration of lymphatic endothelial cells and high invasive capacity of ovarian CSCs.High level of circulating lymphatic endothelial progenitor cells was significantly correlatedwith lymph node metastasis in patients with ovarian cancer.