| Hepatocellular carcinoma (HCC) is one of the most common tumor in our country, which is a serious threat to the health and life of the people. More and more research shows that the main reason of failure and high mortality in the treatment of liver cancer are the development of metastasis, recurrence and drug resistance by the liver cancer stem cells, and the liver cancer stem cells (LCSCs) play an important role in tumor metastasis, recurrence and drug resistance. Therefore, the new therapeutic strategy of targeting liver cancer stem cell is expected to improve the efficacy and reduce mortality. This study details and results are summarized as follows:1 Identification of liver cancer stem cellsObjective:To explore the existence of liver cancer stem cells in human liver cancer cell lines Bel7402-V13 and MHCC97-L.Methods:Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cell in human hepatic carcinoma cell lines Bel7402-V13 and MHCC97-L with different metastasis capability. Flow cytometry and immunofluorecence were used to analyze the CSCs surface markers.Results:Bel7402-V13 and MHCC97-L cells could be formed into sphere by serum free culture, and spheroid forming rate was 55% and 22%, respectively. The single PKH26 positive cell was observed in spheroids when Bel7402-V13 and MHCC97-L cells had been cultured for 11 days. The flow cytometry result showed that the ESA+ and CD90+ phenotype of the cells were respectively enriched in Bel7402-V13 and MHCC97-L sphere cells, they were enriched 3.4 and 3.0 times, respectively.Conclusion:Liver cancer stem cells were presented in human liver cancer cell lines Bel7402-V13 and MHCC97-L, and their stem cell markers were ESA and CD90.2 Screening and identification of monoclonal antibodies against human liver cabcer stem cellsObjective:To investigate the biological characteristics of monoclonal antibodies against human liver cancer stem cells and the functions in vivo and in vitro.Methods:The proportion of cells recognized by McAb 21 A3 in Bel7402-V13 and MHCC97-L parent and sphere cells was detected by flow cytometry. Two colour flow cytometry and immunofluorescence were used to detect the expression of stem cell markers and antigen recognized by McAb 21A3 in the two cell lines.21A3 positive cells were isolated by FACS in Bel7402-V13 sphere cells, and then serum-free suspension culture, Transwell assay, CCK8 assay and in mice vivo induced tumor assay were use to analyze the ability of self-renewal, invasion, drug resistance and tumorigenicity in mice. The effects of McAb 21 A3 on self-renewal, invading ability and drug-resistance of Bel7402-V13 and MHCC97-L sphere cells were detected by the the same experimental methods. Meanwhile, the inhibitory effect of monoclonal antibody 21 A3 combined with cisplatin on the growth of Bel7402-V13 transplanted tumor in nude mice.Results:The result of study found that the expression of McAb 21 A3 has increased 4 times in sphere cells compared with Bel7402-V13 parent cells, and showed that the cells identified by 21 A3 have the characteristics of CSCs. The flow cytometry and immunofluorescence assay showed that 21 A3 could recognize cells which were stained by FITC-conjugated anti-ESA antibody, which indicated that McAb 21 A3 could be used to identify LCSCs. Meanwhile, 21 A3 positive cells were isolated by FACS to analyze their CSC characteristics. The 21A3+ cells also showed the higher abilities of self-renewal, invasion, drug-resistance and tumorigenicity than 21 A3- cells and parental cells, It was suggested that McAb 21 A3 is an anti-LCSCs monoclonal. In vitro functional experiments showed that McAb 21 A3 could significantly inhibit the abilities of self-renewal and invasion of LCSCs, and the inhibition rate were 23.9% and 50.8% respectively. Further, McAb 21A3 also notably inhibited the cisplatin resistance of Bel7302-V1.3 cells. The IC50 was 0.334μg/mL in 21A3 group, and 0.9μg/mL in control group. we applied the tumor treatment of nude mouse experiments in vivo to test the inhibition rates of implanted tumor growth. After withdrawal, the results showed that McAb 21 A3 could inhibit the growth of gastric cancer xenograft, the inhibitory rate of treated group among high and low-dose group were 71.5% and 54.4% respectively. The cisplatin monotherapy-does inhibition rate was 83.5%. However,21 A3 combined with cisplatin treatment showed excellent result with 97% of inhibition rate which is not only inhibited growth of xenograft, but also reduced recurrence and drug resistance of tumo. After six months, the survival curve showed that McAb 21 A3 combined with chemotherapy group of the mice survival status was better than other groups.Study on the in vitro function of McAb 21 A3 in the cell line MHCC97-L of hepatocellular carcinoma by the same routes and methods. The flow cytometry and immunofluorescence assay showed that 21A3+ cells could be enriched by serum free culture, and McAb 21 A3 could be co-stained with the liver cancer stem cell marker CD90, which showed that McAb 21 A3 could identified liver cancer stem cells. In vitro functional study suggested that McAb 21 A3 significantly inhibited the ability of sphere formation, invasion and drug resistance of liver cancer stem cells, indicated that McAb 21 A3 is one of functional monoclonal antibodies targeting liver cancer stem cells.Conclusion:McAb 21A3 is one of functional monoclonal antibodies targeting liver cancer stem cells, which provided a new idea and method for targeting LCSCs for the treatment of liver cancer, and then expected to reduce the mortality and prolonged the survival period. |
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