Abstract: Objective The objectives of this study were to screen and identify monoclonal antibodies against hepatoma stem cells by screening for hepatoma spheroid cells,and to provide candidate therapeutic monoclonal antibodies for targeting cancer stem cells to treat hepatic cancer.Methods Hepatic cancer stem cells were enriched by serum-free suspension culture.Immunofluorescence,cisplatin resistance assay,Real-time qPCR,subcutaneous tumor formation in nude mice,and other methods were used to screen and identify anti-hepatocarcinoma stem cell monoclonal antibodies.Immunohistochemistry was used to identify the expression of antigen recognized by monoclonal antibody in liver cancer tissues.The antigen was identified by mass spectrometry.Results MHCC97L cells were able to form cell spheres in serum-free suspension culture and were labeled with PKH26 dye.Flow cytometry showed that the expression of CD90⁺ in MHCC97L spheroid cells was 3.4 times higher than that in the parental cells.In the inhibition experiment of serum-free spheroid,6 monoclonal antibodies significantly inhibited MHCC97L cells in serum-free medium,and inhibitory rates were 54.67%,50.33%,45.73%,42.26%,39.11%,and 37.63%,respectively.The results of immunofluorescence showed that monoclonal antibodies 28C10 and CD90 were colocalized in MHCC97L cells.The results of real-time qPCR showed that the expression of Sox-2 and Oct-4 in MHCC97L 28C10⁺ cells was significantly higher than those of MHCC97L 28C10^- cells.Flow cytometry showed that the ratio of 28C10⁺ in MHCC97L cells and its sphere cells were 7.98% and 10.7%,respectively.The ratio of 28C10⁺ cells was increased by 1.34 times.The in vitro globing ability and invasive ability of 28C10⁺ cells obtained by flow cytometry were significantly higher than those of 28C10^- cells.The results of CCK-8 assay showed that 28C10⁺ cells were resistance to cisplatin in 28C10^- cells,which are 1.96 g/ml and 1.16 g/ml,respectively.Tumorigenic assay showed that 28C10⁺ cells were inoculated subcutaneously with 2×10⁴ cells into the nude mice,and tumors were formed in 2 months,with 40% of tumor formation rate.Another nude mouse that did not form a tumor had formed a lung metastasis(1/5).Immunohistochemistry showed that the target antigen positive rate of monoclonal antibody 28C10 in hepatic cancer tissues was about 72.0%(77/107),while it was lowly expressed in adjacent tissues,and the difference was significant.Mass spectrometry showed that the antigen recognized by 28C10 was HSP90α.Conclusion The MHCC97L spheroid cell model is successfully used to identify a monoclonal antibody that specifically recognizes hepatoma stem cells,which provides a foundation for antibody therapy targeting hepatic cancer stem cells.

Key words: Hepatic cancer, Cancer stem cell, Monoclonal antibody, CD90, HSP90α