Identification Of A Subpopulation Of Liver Cancer Stem Cells Mediating The Tumor Metastasis

Liver cancer is one of the most common malignant tumor in our country ,and it's mortality is the highst in the world.though the clinic diagnosis and treatment have made great improvement in last half century,the long term curative effect remain still unsatisfied .,the most important reason is tumor metastasis and relapse .the 5-year recurrent rate of liver cancer is as high as 61.5% after radical resection.so study on the recurrence and metastasis has brought about extensive attention in our country and become a emphasis in tumor prevention at present. The metastasis of liver cancer is a multi-step process including falling of tumor cells from the primary tumor, degradation of the base membrane,invasion into the circulation,anchoring on the target organ , proliferation and growth into metastasis tumor. Through gene microarray on primary tumor and accordance metastasis of liver cancer ,we find similar gene profile between the two samples. It's suggest that the tumor cells formed metastasis have the ability to copy the heterogeneity of cells in primary tumor.In the study on malignant tumors ,a rare cell population defined as cancer stem cells(CSCs) in the primary and metastasis has been found which has the ability of unlimited selfrenew and tumorformation . moreover in the research on breast cancer and pancreatic cancer we found that the CSC could induce tumor metastasis. Research on CSC in liver cancer is just in a development stage. And the liver cancer stem cells are believed to origin from liver stem cells or liver progenitor cells at present . recently , by the cell surface marker CD133,some researchers separated a subpopulation possessed the character of cancer stem cells from liver cancer tissue and cell lines. It's found a new proof of the presence of CSCs in liver cancer. These CD133+ tumor cells not only possess extensive capability of proliferation and clonformation in vitro,but appear advantages of tumorformation in immunodeficiency mice. And some stem cell-related genes such as catenin,oct-3/4.bmi,notch-1 could be found highly expressed in these cell proliferation .cultured in proper condition in vitro,these cells could differentiate into endothelia cells and skeletal muscle cells or myocardial cells.Whether these CD133+ tumor cells mediate liver cancer metastasis? And whether all the CD133+ cancer stem cells could induce metastasis? Our research lay emphasis on the role of CD133+ cancer stem cells in liver cancer metastasis. Through immuno-histological analysis of human HCC samples, we found the increased expression of CD133 correlated with HCC metastasis. Although CD133+ tumor cells displayed an increased proliferation rate, they were not advantageous in cell invasion in vitro. We then identified CD44+/high as a marker to isolate a high invasive population in HCC cells in vitro. CD44+ tumor cells were found more in metastatic HCC samples and enriched in the tumor boarder. We then combined CD133+ and CD44+/high to define a putative metastatic population in HCC cells. CD133+CD44+ tumor cells were found more abundant in metastatic HCC samples, compared with those in non-metastatic HCC samples. These cells were present in both primary tumor bulk and their derived tumor thrombus. Moreover only CD133+CD44high HCC cells could generate intrahepatic and lung metastasis in animal models. Down-regulating CD133 or CD44 led to inhibition of the liver cancer growth or invasion, respectively. Taken together, we here identified a subpopulation of liver cancer stem cells, characterized by the CD133+CD44+/high phenotype, which mediated the tumor metastasis and these cells might be a new target for treatment of HCC metastasis. This study is civided into 3 parts.Part one,the role CD133+ tumor cells in liver cancer. First ,by IHC we detected a rare population of CD133+ cells in liver cancer samples,and their proportions are corelatrd with tumor metastasis. then we separated CD133+ cells from HCC cell lines by flow cytometry. The CD133+ cancer cells appared stem cell characters in comparing their capability of cloneformation and drug-resistant. by RNAi,we found its functional role of CD133+ cells in HCC tumor formation. Whereas we do not find its invasion advantage through transwell in vitro. So we make a conclution that CD133+ tumor cells might be the cancer stem cells in HCC, but do not contribute to cancer cells invasion.Part two,CD44 play a important role in HCC metastasis. Also by IHC ,we found the expression of CD44 in HCC tissue mostly at the edge of invasion ,and had relations with tumor embolism and intrahepatic metastais. By clone formation and charmber invasion assay, we detected that CD44+ cancer cells had marked advantage of invasion but not proliferation. So we conclude that the expression of CD44 took advantage of tumor invasion and metasitasis.Part three ,CD133+CD44+ tumor cells mediate HCC invasion and metastasis. We could detect CD133+CD44+ tumor cells in primary tumor and metastasis with immunofluorescence or flow cytometry, and the percentage of these cells have relations with tumor embolism and tumor stage. Moreover only CD133+CD44+ tumor cells separated from HCC cell lines could mediate intrahepatic or lung metastasis in nude mice. Here we prove CD133+CD44+ cancer cells play an important role in HCC metastasis. Conclusion: in HCC, CD133 may be the cells surface marker of CSCs, and help tumor formation, proliferation and apoptosis-resistant. While CD44 can increase the invasive ability of tumor cells. It's necessary for the cancer cells to combine these two molecules to mediate intrahepatic and lung metastasis in HCC.