Characterization And Radioimmunotargeting Of CD133-positive HepG2Cancer Stem Cells

Objective: To identify the stem cell characteristics of hepatocellularcarcinoma CD133positive (CD133+)-HepG2cells, and to researchthe biological distribution and in vivo tumor-radioimmunotargeting ofhuman hepatic carcinoma HepG2cells in nude mice model through the131I labeled CD133monoclonal antibody.Part1Sorting and confirmation of CD133+-HepG2liver cancer stemcellsMethods：Magnetic-activated cell sorting (MACS) method was usedto isolated CD133+and CD133-cells from human hepatic carcinomaHepG2cells. Flow cytometry was used to detect the expression of CD133before and after cells isolation. The stem cell properties of sorted CD133+cells were validated by sphere-forming assay, plate colony formation assayin vitro and xenograft experiments in vivo. The xenografts were separatedfor histological analysis using H&E staining to observe the histology andmorphology of tumor tissues.Results：After sorting by Magnetic-activated cell sorting (MACS) method, CD133+and CD133-cells were successfully obtained fromHepG2HCC cell line.The sorted CD133+cells showed a high purity of(93.58±3.74)%, as compared with the purity of (3.69±1.52)%before cellssorting. Tumor-sphere formation assay showed that as time passed CD133+cells could form spheres and gradually increasing tumorspheres wereobserved in CD133﹢cells cultured in serum-free medium containing growfactors, whereas CD133﹣cells failed to stay alive and showed aberrant cellshapes under the same cultural conditions. The results of plate colonyformation assay showed that CD133+cells possess higher colony-formingability than CD133-cells(**P﹤0.01, vs CD133﹣cells). CD133+cellsformed more and larger colonies than their CD133-counterparts.Xenografttumorigenicity assay showed that as few as1,000CD133+cells weresufficient to form subcutaneous xenografts in1of4inoculated BALB/cmice5weeks after inoculation, and10,000CD133+cells formed grafts in4of4inoculated BALB/c mice. However, no tumors wereb observed ininoculation of10,000CD133-cells.Hematoxylin and eosin (H&E) staininganalysis showed a highly cellular mass below the CD133+cell injectionsite.Conclusion：These results reveal that the sorted CD133+-HepG2cellsshowed a higher tumor sphere formation ability, clonality andtumorigenesis capacity compared with CD133--HepG2cells.CD133+-HepG2cells could be considered as CSC-like subsets of liver cancer cells.Part2Biological distribution and tumor-radioimmunotargeting of131I-labeled CD133monoclonal antibody in HepG2tumor-bearing miceMethods:131I-labeled anti-CD133antibody was prepared usingchloramine T method.Labeling efficiency,radiochemical purity and stabilityin human serum of131I-labeled anti-CD133antibody were measured bypaper chromatography, respectively. Cell Binding Assay was used toestimate cell binding property and immunocompetence. Five-week-oldBALB/c were subcutaneously injected with HepG2cells. When the tumorswere8to10mm in the diameter, tumor-bearing mice were intravenouslyinjected with7.4MBq of131I-labeled anti-CD133antibody and thepercentage activity of injection dose per gram of tissue (％ID/g) wasdetermined in major organs at2,12,24, and48h after injection. Using thesame isotype IgG as a control, in vivo biodistribution and tumor tonon-tumor tissue (T/NT) ratio were analyzed at24h after injection of131I-labeled anti-CD133antibody.Results: The labeling efficiency and radiochemical purity of131I-labeled anti-CD133antibody were (86.95±1.16)%and98.07%,respectively. After48h incubation with serum, the radiochemical purity of131I-labeled anti-CD133antibody was (89.63±0.64)%. The relative cellbinding ratios of131I-labeled anti-CD133antibody to CD133+cellsincreased with an increase in the cell number and reached a plateau when the cell density was higher than2×106cells/mL.The highest relative cellbinding ratio was (69.3±0.69)%and in contrast,negligible binding of131I-labeled anti-CD133antibody was observed in CD133-cells（P＜0.01).The uptake rate (％ID/g value) of131I-labeled anti-CD133antibody in thetumors of HepG2tumor-bearing mice at12,24and48h was (1.09±0.08)%,(0.80±0.10)%and (0.60±0.10)%, which were relatively higher than those innon-tumor tissues (except for blood and lung). The131I-labeled anti-CD133antibody showed a modest but significantly higher accumulation in theHepG2xenograft compared to the control IgG at24h after injection.Conclusion: In the present study,we successfully obtained131I-labeledanti-CD133antibody which was stable and had good immunocompetenee．The radioactivity in the tumor was significantly higher for CD133antibodycompared to the control IgG in HepG2tumor-bearing mice,which couldreflect the expression of CD133.These results suggest that the noninvasivein vivo targeted therapy for CD133+liver cancer stem cells may be possiblewith radiolabeled antibodies against CD133, and CD133can become a newtarget for liver cancer treatment.