title: [Treatment of liver cancer in vitro and in mice by monoclonal antibody targeting epithelial specific antigen-positive liver cancer stem cells in combination with cisplatin].
abstract: OBJECTIVE
  To investigate the biological characteristics of monoclonal antibodies against human liver cancer stem cells and its therapeutic effect in combination with cisplatin in the treatment of hepatocellular carcinoma.
  
  
  METHODS
  Cell culture in serum-free medium and PKH26 staining were used to determine the existence of cancer stem cells in human liver Bel7402-V3 cell line. The co-expression of antigen recognized by monoclonal antibody (McAb) 15D2 and epithelial specific antigen (ESA) and PKH26-positive cells in the Bel7402-V3 cells were detected by immunofluorescence assay. Serum-free suspension culture was used to detect the self-renewal ability of 15D2-positive Bel7402-V3 cells sorted by flow cytometry and the effect of 15D2 on the self-renewal ability of Bel7402-V3 cells. The effect of 15D2 on cisplatin resistance in the cells was examined by CCK8 method. The inhibitory effect of 15D2 combined with cisplatin on the transplanted tumor growth in mice was also observed.
  
  
  RESULTS
  Single PKH26-positive cells were observed in the Bel7402-V3 cell spheroids cultured for 11 days. Immunofluorescence assay showed that the 15D2-recognized antigen could be conjugated with PKH26 and ESA and co-localized on Bel7402-V3 cells. The spheroid formation rate of 15D2-positive cells in serum-free medium was significantly higher than that of 15D2-negative cells [(30.4±3.4)% vs. (8.8±1.8)%, P<0.01]. The cisplatin resistance of 15D2-positive cells was obviously higher than that of 15D2-negative cells (IC50: 1.014 μmol/L vs. 0.365 μmol/L). McAb 15D2 significantly suppressed the spheroid formation of Bel7402-V3 cells, with an inhibition rate of 37.5%. McAb 15D2 also notably inhibited the cisplatin resistance of Bel7302-V3 cells. The IC50 was 0.211 μg/ml in the 15D2 group and 0.325 μg/ml in the control group. The mouse experiment showed that the tumor growth rates of 50 mg/kg, 25 mg/kg and 12.5 mg/kg 15D2-treatment groups were 82.6%, 71.4% and 60.0%, respectively; that of the 50 mg/kg 15D2 + cisplatin group was 91.0%, and that of the cisplatin monotherapy was 56.7%.
  
  
  CONCLUSIONS
  McAb 15D2 is a functional monoclonal antibody targeting liver cancer stem cells, which could be a potential monoclonal antibody drug for the stem cell-targeted therapy of liver cancer.
authors.affiliation: Experimental Center of Medical Sciences of Guangxi Medical University, Nanning 530021, China.
authors.forename: Y Y
authors.initials: YY
authors.lastname: He
authors.affiliation: State Key Laboratory of Molecular Oncology, National Cancer Center Cancer Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China.
authors.forename: L
authors.initials: L
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Molecular Oncology, National Cancer Center Cancer Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Rong
authors.affiliation: State Key Laboratory of Molecular Oncology, National Cancer Center Cancer Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China.
authors.forename: L X
authors.initials: LX
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Molecular Oncology, National Cancer Center Cancer Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China.
authors.forename: L C
authors.initials: LC
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Molecular Oncology, National Cancer Center Cancer Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China.
authors.forename: Z H
authors.initials: ZH
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Molecular Oncology, National Cancer Center Cancer Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China.
authors.forename: Y L
authors.initials: YL
authors.lastname: Ran
authors.affiliation: Department of Gynecologic Oncology, the Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China.
authors.forename: L
authors.initials: L
authors.lastname: Li
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000628:therapy*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000627:therapeutic use*; D000071858:Epithelial Cell Adhesion Molecule / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008113:Liver Neoplasms / Q000378:metabolism / Q000628:therapy*; D051379:Mice; D014411:Neoplastic Stem Cells / Q000737:chemistry* / Q000276:immunology; D009930:Organic Chemicals; D018874:Spheroids, Cellular / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D000071858:Epithelial Cell Adhesion Molecule; D009930:Organic Chemicals; C070080:PKH 26; D002945:Cisplatin