Isolation and characterization of breast cancer stem cell-like phenotype by Oct4 promoter-mediated activity

Cancer stem cells (CSCs) are a small subset of cancer cells responsible for self-renewal activity, drug resistance, and tumor recurrence. CSCs have been derived from diverse tumors and cell lines. The expression of stemness markers has been identified in CSCs. Oct4 is a well-established transcription factor expressed in stem cells and CSCs. In this study, we isolated and characterized breast CSC-like cells from murine MC4-L2 cells by Oct4 promoter-mediated activity. The MC4-L2 cells were electroporated by a plasmid expressing puromycin resistance (Puro^R ) gene from the Oct4 promoter and then selected by puromycin. The isolated cells were named as the MC4-L2^puro cells and characterized for CSCs properties. Immunostaining indicated CD44^high and CD24^high phenotype for the MC4-L2 and MC4-L2^puro cells. The enhanced expression of stem cell markers was detected in the puromycin-selected cells compared with the parental cells. Moreover, the isolated cells only grew up in sphere-formed shape in low attachment plates. Serial dilution transplantation in syngeneic mouse models showed increased tumorigenicity of the MC4-L2^puro cells, as they induced new tumors when injected into the mammary fat pad as few as 10⁴ cells. In conclusion, we designed a novel genetic construct, which allows the isolation of Oct4-positive cells in a cancer population by a simple selection step in a puromycin-containing medium. Transfection of this construct into the MC4-L2 cells resulted in growing a subpopulation of cells having tumor-initiating cell characteristics. To the best of our knowledge, this is the first report on the isolation of CSC-like cells from the mouse breast cancer MC4-L2 cells.