Background and purpose:The scarcity in quantity and continuous differentiation ability severely compromise the isolation and purification of cancer stem cells(CSCs).The existing sorting methods can only enrich CSCs as a heterogeneous population.However,more primitive CSCs closer to the source still have not been identified.Based on the CSC hypothesis,we hereby explored the possibility of isolation,culture,purification and identification of murine breast cancer stem cells in suspension culture combined with anticancer regimens. Methods :TM40D murine breast cancer cells were cultured in serum-free medium.The expression of CD44 + CD24-was measured by flow cytometry.Cells from the passage of TM40D cells with the highest expression of CD44 + CD24-were treated in combination with anticancer agents pacilitaxel and epirubicin at different peak plasma concentrations(PPC) for 24 hours,and then maintained under suspension culture.The rate of apoptosis was examined by flow cytometry with Annexin-V FITC/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).As few as 100 cells in 0.35 PPC could generate tumors in BALB/C mice. Conclusions :TM40D cell line contains breast cancer stem cells.This study suggested that suspension culture combined with anticancer regimens is a feasible approach for screening tumor stem cells.

Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 3'-PO4 end of the substrate and generates 3'-OH,TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10-3 U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.