Isolation and characterization of breast cancer stem cell‐like phenotype by Oct4 promoter‐mediated activity
Zahra Ghanei, Abbas Jamshidizad, Morteza Daliri Joupari, Mehdi Shamsara
Abstract
Abstract 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 10 4 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.