Identification of liver cancer stem cells-containing cell subgroups based on resistance characteristics and unicellular culture

AIM: To identify liver cancer stem cells-containing cell subgroups based on resistance characteristics and unicellular culture. METHODS: The BEL-7404 cell line was inoculatwcjd ed subcutaneously in nude mice to induce tumor formation, and adriamycin (8 mg/kg) intervention was given. When the tumor diameter was 1.5 cm, tumor tissues were collected for primary culture. The cells were inoculated subcutaneously in nude mice again. After four consecutive generations in nude mice, the tumor cells were named "BEL-7404-ADM-P4". Based on the tumor forming time, the enrichment of liver cancer stem cells was tested. Using unicellular culture, clones from BEL-7404 and BEL-7404-ADM-P4 cells were divided into holoclones, meroclones and pareclones to test the cancer stem cell characteristics (renewal ability, clone formation and sphere formation). Holoclones, meroclones and pareclones were stained with hoechst33342 and observed under a confocal microscope. Based on the renewal ability, clone formation rate and sphere formation rate and hoechst33342 staining properties, cancer stem cells subpopulations were identified. RESULTS: The tumor formation rate was 100%, and tumor formation time was shortened from the first generation to the fourth generation, which suggested the preliminary enrichment of liver cancer stem cells. Holoclones showed the fastest growth and the largest cell volume, followed by meroclones and pareclones. On the 10th day, pareclones started to shrivel and die. The clone formation rate of BEL-7404-ADMP4- H cells was significantly higher than those of BEL-7404 and BEL-7404-ADM-P4-M cells (P < 0.05). Only BEL-7404-H and BEL-7404-ADMP4- H cells could form spheres, and there was no significant difference in sphere formation rate between BEL-7404-H and BEL-7404-ADM-P4-H cells. In BEL-7404 and BEL-7404-ADM-P4 monoclones and holoclones, there were a small number of lowly hoechst33342 stained or non-stained cells, but cells in meroclones and pareclone cells were all stained. The hoechst33342 fluorescence intensity of BEL-7404 holoclones, meroclones and pareclones was stronger than that of BEL- 7404-ADM-P4 clones. CONCLUSION: Based on resistance charac-teristics and unicellular culture, we found that holoclones contain a higher proportion of liver cancer stem cells. ? 2014 Baishideng Publishing Group Co., Limited.