Study On Functional Monoclonal Antibody Targeted Human Liver Cancer Stem Cells

Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of cancer mortality in China. The main causes of failure in the treatment of cancer are the development of metastasis, drug resistance and recurrence by the cancer cells. Recent studies show that cancer stem cells (CSCs) play an important role in the tumor proliferation, metastasis, recurrence and drug-resistance. Therefore, the new therapeutic strategy for targeting CSCs will provide a breakthrough to the cancer treatment.In this study, the CSCs in two HCC cell lines with different metastasis potentials were cultured and enriched by serum free suspension medium. Flow cytometry and immunofluorecence were used to analyze twelve candidate CSCs surface markers including PKH26, ESA, CD44, CD133, CD117and so on. The result showed that ESA+cells and CD44+cells in Bel7402-V3cell line were significantly enriched by sphere culture, which might be the liver cancer stem cells (LCSCs). Moreover, only the CD117+cells were enriched in HepG2cell line. Then we applied the Fluorescence-activated cell sorting (FACS) to isolate ESA+, CD44+and CD117+cells from two cancer cell lines for sphere culture. invasion assay, chemosensitivity and tumorigenicity test. The results indicated that ESA+cells possessed the sternness property of self-renewal, invasion, drug-resistance and tumorigenicity. We firstly identified that ESA was the candidate CSCs marker in Bel7402-V3cell lines and CD117was another CSCs marker in HepG2cell line. In conclusion, we successfully established a LCSCs tumorigenic model in vivo, which contributed to study antibodies targeting LCSCs.Base on the establishment of cellular model and tumorigenic model of LCSCs, we studied the biological characteristics and treatment experiment of the anti-LCSCs monoclonal antibody15D2and15B7in vivo and in vitro. The results of two-color immunofluorescence and two-color flow cytometry showed that monoclonal antibody15D2could recognized cells which also were partly co-stained with CSC markers. Then15D2positive cells were isolated by FACS to analyze their CSC property. The result showed that15D2+cells possessed a higher abilities of self-renewal, invasion, drug-resistance and tumorigenicity than15D2-cells. It was suggested that15D2mAb could specifically recognize LCSCs. In vitro functional experiments showed that monoclonal antibody15D2can inhibit the proliferation, migration and invasion of LCSCs, and the inhibition rate were41.7%,35%and11.9%respectively. Furthermore, we applied the tumor treatment experiments in vivo to test the inhibition rates of implanted tumor growth of high, middle, and low-dose of monoclonal antibody15D2mAb, and15D2mAb coupled with cisplatin, and cisplatin monotherapy. Afer a month withdrawal, the inhibitory rate of treated group among high, middle, and low-dose group were82.6%,71.4%and60%respectively. Moreover, the inhibitory rate of15D2mAb high-dose combining with cisplatin and cisplatin monotherapy were91%and56.7%. However, the increasing growth of xenograft tumors took place in chemotherapy alone group in recent three weeks. The result showed that functional monoclonal antibody15D2targeting HCC-CSC could not only inhibit tumor engraftment, but also prevent the recurrence and chemoresistance. Cancer stem-cell-targeting therapy thus represents a promising potential strategy to reduce mortality and prolong survival time.At the same time, we applied the similar technical approach and method to prove that15B7mAb could recognize and suppress LCSCs. The biological assay demonstrated that15B7+cells processed self-renewal and invasive characterics. In vitro, functional experiments showed that monoclonal antibody15B7can inhibit the proliferation, sphere formation and invasion of CSCs. and the inhibition rate were50.3%,32.4%and10%. The results indicated that15B7was a functional monoclonal antibody against LCSCs.