Abstract
BackgroundCancer 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.MethodsTM40D 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.ResultsCells 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.ConclusionSuspension culture combined with anticancer regimens provides an effective means of isolating, culturing and purifying BrCSCs.
Highlights
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
Enrichment of the subpopulation of putative breast cancer stem cells (BrCSCs) in TM40D cells under suspension conditions TM40D murine breast cancer cells could be directly adapted from serum-containing medium to serum-free medium
The percentage of CD44+CD24-cells had a marked increase at passages 2 to 10, and it maintained a relatively stable proportion (Fig. 2)
Summary
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. 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. In recent years there has been an increasing focus on the cancer stem cell (CSC) theory. More and more CSCs in solid tumors have been prospectively identified based on expression of cell surface markers [1,2,3,4,5] or Hoechst 33342 dye efflux [6,7,8,9]. More primitive, undifferentiated CSCs ( called true CSCs) remain hidden among them
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