CD105/CD133 As Cell Surface Markers For Screening And Identification Of Liver Cancer Stem Cell

Background The incidence and mortality of liver cancer was ranked second among the malignant tumors in China. The death caused by liver cancer in China accounts for 53% of the total death in the world. At present, surgical resection or liver transplantation for treating primary hepatocellular carcinoma(HCC) is only limited to patients with early HCC. However, there are 60 to 70% cases with metastatic recurrence even after radical resection in primary HCC after five years. Moreover, radiotherapy and chemotherapy is limited for treating the primary HCC. So, the overall 5-year survival rate of HCC patients is only about 5% in the present time. It failed to significantly improve the prognosis of HCC over the years based on classical theory of HCC. Therefore, there needs novel theory, such as cancer stem cell theory for understanding the specific mechanism of HCC development, and then finding earlier detection and prognosis and more effective treatment for HCC.There is common viewpoint in tumor classical theory: most of the tumor cells are derived from mature tissue cells. The biological characteristics and behavior of tumor cells in tumor tissues are almost consistent. However, the theory of cancer stem cells proposes that tumor cells are derived from tissue stem cells with heterogeneity of tumor cells in the same tumor tissue. The key description of cancer stem cells theory is: the tumor tissue is composed of heterogeneous tumor cell populations, of which only a small number of cells can self-renew, with unlimited proliferation and differentiation potential. These cells acts as tumor-initiating cells and maintain continued tumor growth. On the contrary, most of the remaining tumor cells keep only restrictedly proliferation and differentiation. These small numbers of cells are called cancer stem cells, due to their feature similar to a normal physiological state of the body stem cells or embryonic stem cells. Cancer stem cells play a key role in tumorigenesis, progression, metastasis and recurrence. That may be the key target for the cancer treatment.Cancer stem cell theory has indeed been verified in leukemia, breast, glioma, colon, melanoma and other tumors. In recent years, both the animal model studies and clinical research confirmed the presence of primary liver cancer stem cells. However, there is still no definitive marker, accurately detecting human liver cancer stem cells. In order to understand clearly the nature of liver cancer stem cells, the first task is to purify and identify the liver cancer stem cells from human HCC tissues and HCC cell lines.Objective The objective of this research is to compare the expression of CD105 and CD133 in membrane of human HCC cell line SMMC-7721, different biological characters among the subpopulations in vitro, and different tumorigenicity in vivo; And to explore preliminarily the characteristics of CD105+CD133+ subpopulation, which may be the cancer stem cells in HCC.Methods SMMC-7721 cell line was cultured in DMEM containing 10% FBS. Flow cytometer(FCM)was used to detect CD105, CD133 expression in SMMC-7721 cell in vitro. Four cell sub-populations CD105 + CD133 +, CD105-CD133 +, CD105 + CD133-, CD105-CD133- were sorted by flow cytometry. Cell proliferation of these four sub-populations was detected by CCK-8 assay. Sphere formation ability of these four sub-populations was examined by soft agar test. Invasion ability of these four sub-populations was examined by transwell cell invasion test. Bmil gene expression of these four sub-populations was detected by RT-PCR. In vivo tumorigenicity assay was also performed on these four sub-populations using nude mice.Result The proportions of four sub-populations CD105+CD133+、CD105-CD133+、CD105+CD133-、CD105-CD133- were 1.61%, 0.01%, 97.88% and 0.50% sorted by flow cytometers, respectively. CD133+ subsets cells grow more quickly than those of CD105+ and unsorted cells. The numbers of colonies formed by CD133+ subsets cells were more than those of CD105+ and unsorted cells. Furthermore, the metastatic capacity of CD105+ subset cells observed by Transwell assay was higher than that of the CD133+ subsets cells and unsorted cells. Semi-quantitative RT-PCR optical density ratio(Bmil/β-actin m RNA) results suggest that the Bmil expression of two sub-groups, CD105+CD133+, CD105-CD133+ were significantly elevated statistically significantly(P<0.01), compared with unsorted groups, while the Bmil expression of two sub-groups, CD133-CD105+、 CD105-CD133- were decreased statistically significantly(P<0.01), compared with unsorted group. In vivo tumor experiments showed CD105+CD133+ needed a shorter time and fewer cells, compared with unsorted group and CD105-CD133- groups was statistically significant(P<0.05). Conclusion: Our study found that human live cancer cell line SMMC-7721 can be sorted into four sub-groups CD105+ CD133+, CD105-CD133+, CD105+ CD133- and CD105-CD133- successful by flow cytometer. It was found that CD105+CD133+ sub-group showed high proliferative potential, high capacity for self-renewal by proliferation assay, clone forming assay, in vitro invasion assay and RT-PCR semi-quantitative optical density ratio(Bmil/ β-actin m RNA). In vivo tumor experiment proved CD105+CD133+ sub-group showed a higher degree of tumorigenicity. Therefore, CD133+CD105+ subset cells are a subpopulation with stem properties in SMMC-7721 cells. CD133+CD105+ subgroup may represent a higher degree of malignancy of the HCC.