A modified method for isolation of bladder cancer stem cells from a MB49 murine cell line

BACKGROUND The vaccine was efficiently effective against bladder cancer in earlier studies. However, a part of the mouse bladder tumour regrew due to regression after a period of time as the cancer stem cells could not be eliminated. In this study, we showed a modified method for the isolation of MB49 bladder cancer stem cells (MCSCs). METHODS Through a comparison of different serum-free culture mediums (SFM), MCSCs were isolated by a combination of the limited dilution method and the optimal SFM method. The characterizations of MCSCs were verified by the fluorescence activated cell sorting, the quantitative polymerase chain reaction, the western blotting, the cell proliferation assay, the soft agar assay, the transwell assay, the resistance to chemotherapy assay and the tumor xenograft formation assay. RESULTS The optimal SFM contained a RPMI1640+ epidermal growth factor (20 ng/ml), a basic fibroblast growth factor (20 ng/ml), a leukemia inhibitory factor (20 ng/ml), a B-27 serum-free supplement (20 μl/ml), and a bovine serum albumin (4 μg/ml). MCSCs possessed the high expression of cancer stem cell markers (CD133, CD44, OCT4, NANOG, and ABCG2) and the ability of differentiation. In functional comparisons, MCSCs had higher proliferative abilities, lower susceptibility to chemotherapy, greater migration in vitro, and stronger tumorigenic abilities in vivo. CONCLUSION MCSCs displayed specific cancer stem cells properties. Our study showed MCSCs were isolated successfully with a modified method using a combination of limited dilution and SFM methods.