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

• Published in: Journal of cellular physiology 2020-11, Vol.235 (11), p.7840-7848
• Main Authors: Ghanei, Zahra, Jamshidizad, Abbas, Joupari, Morteza Daliri, Shamsara, Mehdi
• Format: Article
• Language: English
• Subjects: Animal models; Animals; Breast cancer; Breast Neoplasms - pathology; cancer stem cell; Cell Line, Tumor; Cell self-renewal; Dilution; Drug resistance; Electroporation - methods; Female; Flow Cytometry - methods; Markers; MC4‐L2 cells; Mice; mouse; Neoplastic Stem Cells - pathology; Oct-4 protein; Oct4; Octamer Transcription Factor-3 - metabolism; Phenotype; Phenotypes; Promoter Regions, Genetic; Puromycin; Stem cell transplantation; Stem cells; Syngeneic grafts; Transfection; Transplantation; Tumorigenicity; Tumors
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.29437

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 (PuroR) gene from the Oct4 promoter and then selected by puromycin. The isolated cells were named as the MC4‐L2puro cells and characterized for CSCs properties. Immunostaining indicated CD44high and CD24high phenotype for the MC4‐L2 and MC4‐L2puro 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‐L2puro 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. We designed a novel genetic construct, which can be used to isolate Oct4‐positive cells from a cancer cell population using a simple puromycin selection step in the cell culture medium. By this method, there is no need for any additional sorting instrument for the purification of Oct4‐positive cells from a cell mixture. Transfection of this construct into the mouse‐derived mammary gland MC4‐L2 cells resulted in growing a puromycin‐resistant subpopulation showing tumor‐initiating cell characteristics. To the best of our knowledge, this is the first report on the isolation of cancer stem cell‐like cells from the mouse breast cancer MC4‐L2 cells.