Effect Of Endothelial Cells Conditioned Medium On Cancer Stem Cell Phenotype Of Liver Cancer Cells And Migration Of Liver Cancer Stem Cells

Cancer is one of the most serious diseases threatening human health, where lung cancer, stomach cancer and liver cancer are top 3 causes to cancer relate fatality. Dispite high motility of liver cancer, its morbidity is steadily rising, thus treatments, prevention and the related mechanism exploration of cancers have been being a significant scientific issue for tumor research. The main treatments of HCC are surgical resection,radiotherapy, anti-cancer chemical drugs and liver transplantation, but all those means not sufficient to effectively inhibit recurrence and metastasis, making treatments of liver cancer still a challenge to clinical scientists.Current evidences suggest that the most malignant tumors are derived from Cancer stem cells(CSCs), which are small amount of cells with self-renewal capability and pluripotency, and are essential for tumor initiation, developing and differentiation, and are also the root of poor prognosis, recurrence and metastasisEndothelial cells(ECs) are important parts of vessel wall and tumor micro-environment. Previous researches demonstrated that the interaction between tumor cells and ECs is essential to regulate CSCs phenotype and cancer recurrence and metastasis. Two currently unclear questions about the vessels-rich liver are proposed in this study: 1) Whether tumor microenvironment with ECs participation promotes CSCs phenotype of liver cancer cells during the recurrence and development? 2) Does migration of Liver cancer stem cells(LCSCs) influenced by this microenvironment?The purposes of this paper is to study change of cancer stem cell phenotype of liver cancer cells and migration behavior of cancer stem cells in endothelial micro-environment.Main contents and results of this research are listed as follows:①The effects of EC-CM on cancer stem cell phenotype of MHCC97HHuman umbilical vein endothelial cells and human liver cancer cells MHCC97 H are cultured respectively. EC-CM are collected and Co-cultured with MHCC97 H.Co-culturing, Chemotherapy sensitivity assays, Colony forming assay, Flow cytometry analysis, Matrigel invasion assay, Transwell migration assay, and Sphere formation assay are applied to study the effect of EC-CM on cancer stem cell phenotype of MHCC97 H. MHCC97 H cultured in EC-CM demonstrated a significantly higher survival rate after 5-FU(p<0.05) or Cis(p<0.05) treatment. After treatment of EC-CM,MHCC97 H proliferated significantly faster and induced bigger and greater numbers oftumor colonies(p<0.01) than control medium. The numbers of invasion cell(p<0.05)and migration cell(p<0.05) of MHCC97 H are significantly increased after treated by EC-CM. Upon serial passaging, EC-CM treatment increased sphere forming capability(p<0.05) of MHCC97 H. EC-CM also increased the expression of CD133 on MHCC97H(p<0.01). According to the experimental results above, EC-CM could promote cancer stem cell phenotype of MHCC97 H.②The separation and identification of liver cancer stem cellsIn vitro separation of liver cancer stem cell from tumor sphere are carried out by MHCC97 H serum-free suspension culturing and passaging for every 5-6 days. The third generation of tumor sphere-forming cells are used to detect drug sensitivity, clone forming ability and liver cancer stem cell marker expression(CD133, CD44,Oct3/4).Compared with the MHCC97 H, tumor sphere-forming cells have stronger cancer stem cell phenotype and drug resistance to different concentrations of anticancer drugs cisplatin and 5 fluorouracil(p<0.05). Tumor sphere-forming cells have stronger ability of clone forming(p<0.05), and sphere-forming cells expressed higher levels cancer stem cell marker of CD133, Oct3/4 when Comparing sphere-forming cells with MHCC97 H. These results indicate that the sphere-forming cells separated from MHCC97 H is a different subpopulation of LCSCs with CSCs like properties.③Effect of EC-CM on migration behavior of LCSCsMigration change of LCSCs treated with EC-CM is measured by using transwell system. The results indicate that EC-CM can significantly enhance the migration capacity of LCSCs(p<0.05). Immunoflourescent staining further demonstrates that EC-CM reduced the F-actin expression of LCSCs. Results of AFM suggest that young’s modulus of LCSCs reduced after EC-CM treatment. Those results show that reduction of stiffness by lowering the F-actin expression may be responsible to improve eamoeboid movement of LCSCs and cell migration.Taking together, endothelial micro-environment are able to promote liver cancer stem cell phenotype of MHCC97 H cells, enhance the migration capability of liver cancer stem cells via decreasing cell stiffness by remodeling cytoskeleton. This research may provide theoretical and references basis of liver cancer cell and micro-environment targeted therapy.