title: Suspension culture combined with chemotherapeutic agents for sorting of breast cancer stem cells.
abstract: BACKGROUND
  Cancer stem cell (CSC) hypothesis has not been well demonstrated by the lack of the most convincing evidence concerning a single cell capable of giving rise to a tumor. The scarcity in quantity and improper approaches for isolation and purification of CSCs have become the major obstacles for great development in CSCs. Here we adopted suspension culture combined with anticancer regimens as a strategy for screening breast cancer stem cells (BrCSCs). BrCSCs could survive and be highly enriched in non-adherent suspension culture while chemotherapeutic agents could destroy most rapidly dividing cancer cells and spare relatively quiescent BrCSCs.
  
  
  METHODS
  TM40D murine breast cancer cells were cultured in serum-free medium. The expression of CD44+CD24- was measured by flow cytometry. Cells of passage 10 were treated in combination with anticancer agents pacilitaxel and epirubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The rate of apoptosis was examined by flow cytometry with Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining method. Selected cells in different amounts were injected subcutaneously into BALB/C mice to observe tumor formation.
  
  
  RESULTS
  Cells of passage 10 in suspension culture had the highest percentage of CD44+CD24- (about 77 percent). A single tumor cell in 0.35 PPC could generate tumors in 3 of 20 BALB/C mice.
  
  
  CONCLUSIONS
  Suspension culture combined with anticancer regimens provides an effective means of isolating, culturing and purifying BrCSCs.
authors.affiliation: Department of General Surgery, First Affiliated Hospital, Nanjing Medical University, Nanjing, China. ligengquan@163.com
authors.forename: Hai-zhi
authors.initials: HZ
authors.lastname: Li
authors.forename: Tong-bo
authors.initials: TB
authors.lastname: Yi
authors.forename: Zheng-yan
authors.initials: ZY
authors.lastname: Wu
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051927:CD24 Antigen / Q000096:biosynthesis; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D015251:Epirubicin / Q000008:administration & dosage; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D008325:Mammary Neoplasms, Experimental / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000473:pathology*; D017239:Paclitaxel / Q000008:administration & dosage; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D051927:CD24 Antigen; C476399:Cd44 protein, mouse; D016895:Culture Media, Serum-Free; D018960:Hyaluronan Receptors; D015251:Epirubicin; D017239:Paclitaxel