Enrichment and identification of androgen receptor-positive breast cancer stem cells

Abstract

Objective To isolate the mammospheres (MSs) from the androgen receptor positive(AR +) breast cancer cell lines for culture and identification to screen AR + breast cancer stem cell model for further study.Methods The three types of AR + breast cancer cell lines were seeded in culture medium DMEM/F12 supplemented with growth factors.The formation and growth of MSs were observed.Immunofluorescence was used to detect the expression of AR in MCF-7,MDA-MB-231 and T47D cell lines.Flow cytometry (FCM) was used to detect CD44 + CD24-/low cell content in adherent cells and mammospheres.Clonogenic assay was used to detect the self-renew capcity of every single cell of the MSs.Results Breast cancer cell lines in serum-free medium showed suspension growth,and MSs formed during 7-10 days.Immunofluorescence showed MCF-7 and T47D cells expressed AR mainly in the cytoplasm,and MDA-MB-231 cells in both the nucleus and cytoplasm.FCM revealed that CD44+ CD24-/low cell levels in MCF-7,MDA-MB-231 and T47D adherent cells were 50.0％,86.4％ and 3.2％,and those in suspended cells were 87.4％,98.8％ and 38.4％,respectively.Colony formation assay demonstrated that the volume of MSs was gradually increased with time.Conclusion Suspension growth of MDA-MB-231 and MCF-7 cells can be used as an optimal model for the study of AR-positive breast cancer stem cells. Key words: Breast cancer stem cells; Androgen receptor