Isolation, Culture And Identification Of Murine Breast Cancer Stem Cells

In recent 5 years,noteworthy achivements have been achieved in the field of solid tumor stem cells.More and more cancer stem cells(CSCs) have been identified in solid tumors of the breast,brain,skin,prostate, colon,liver and many others.Cancer stem cell theory has led to a number of considerable advances in our understanding of the origin of tumor cells and new concept for tumor therapy.However,further research on molecular mechanisms of CSCs has been hindered owing to the scarcity in quantity and the instinct feature of continuous differentiation.CSCs identified from several solid tumors,based on cell surface antigens sorted by fluorescence-activated cell sorting(FACS)or Hoechst 33342 dye efflux, are only a small subpopulation of tumorigenic cells that most probably represents a heterogeneous mixture of CSCs and their differentiated progeny.More primitive,undifferentiated CSCs(also called true CSCs) remain hidden among them.Research facilities available at the moment are not adequate to support the cancer stem cell theory with convincing experimental evidence.In this study,we explored the possibility of exploiting the feature of drug resistance in cancer cells for screening CSCs on the basis of serum-free suspension culture for isolation and in vitro propagation of tumorigenic cancer cells.1,Isolation and in vitro propagation of tumorigenic murine breast cancer cells TM40D from suspension cultureOBJECTIVE:To enrich tumorigenic TM40D cells under serum-free culture conditions.METHODS:TM40D cells were direct cultured in serum-free medium (SFM).CD44~+CD24~- and CD44~+CD24~+ subsets were sorted by FACS. Half of the sorted CD44~+/CD24~- subset were replated at 1000 cells/ml in SFM,whereas the remaining half were grown under serum-supplemented medium.The same culture procedure was followed in the sorted CD44~+/CD24~+ subpopulation.The expression of CD44~+/CD24~- in three group(the sorted CD44~+CD24~- group,the sorted CD44~+CD24~+ group and TM40D suspension culture group)was determined by flow cytometry at passages 2,4,6,8,10,12 and 14.alpha-smooth muscle actin(α-SMA)and cytokeratin(CK)18 were detected by immunocytochemical staining in these three group.The rate of apoptosis induced by serum withdrawal was analyzed by flow cytometry using an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide(PI)double staining method.RESULTS:TM40D cells could be directly adapted in SFM.After 4 days in suspension culture,about 78.5%cells underwent apoptosis.Floating tumorspheres could be observed in serum-free culture after 5 days.The CD44~+/CD24~- subset of TM40D accounted for 1.2%in serum medium. Under suspension culture,the percentage of CD44~+/CD24~- cells had a marked increase,which had a maximum percentage at passage 10(85.17% in the sorted CD44~+CD24~- group and 77%in TM40D suspension culture group),and then maintained a stable ratio.TM40D cells expressedα-SMA and CK 18 in adherent state,whereas they failed to express these two antigens under suspension culture.CONCLUSION:Putative breast cancer stem cells(BrCSCs)subpopulation, CD44~+CD24~- subset,could be enriched in suspension culture.Under such culture conditions,CD44~+CD24~- cells could maintain undifferentiated state. An in vitro suspension culture growth pattern is further proposed to interpret the proliferation of BrCSCs.2,Suspension culture combined with anticancer regimens for screening BrCSCsOBJECTIVE:To determine optimal concerntration of chemotherapeutic regimens for sorting BrCSCs based on suspension culture.METHODS:Cells of passage 10 were treated in combination with anticancer agents pacilitaxel and epirubicin(PE chemotherapy protocol) at different peak plasma concentrations for 24 hours,and then maintained under suspension culture.At the 7 days of cultivation,the rate of apoptosis was examined by flow cytometry with Annexin-V FITC/PI double staining method.RESULTS:all cells treated with 1 and 0.5 PPC of TE underwent apoptosis and necrosis.Cells treated with 0.25 and 0.1 PPC of TE survived, proliferated,and formed tumorspheres.The vast majority of cells treated with 0.35 PPC of TE(～98%)suffered death.CONCLUSION:0.35 PPC of TE were the most desirable research subjects used to tumor xenograft experiment.3,tumorigenicity of BrCSCs in BALB/C miceOBJECTIVE:To observe tumorigenicity of the sorted breast cancer cells in BALB/C mice.METHODS:Cells treated with 0.35 PPC of chemotherapeutic drugs in different amounts were injected subcutaneously into the abdominal wall of the mice with normal or suspressed immune function to observe tumor formation.By contrast,CD44~+CD24~- and CD44~+/CD24~+ cells were chosen as control group.All tumors removed were examined by pathologic sections.RESULTS:A single cell of 0.35 PPC of TE group reconstituted an entire tumor in 3 of 20 mice,one with normal immune function,the other two with suppressed immune function.Regardless of whether mice have normal immune function or not,1000 or 100 CD44~+/CD24~+ cells failed to form tumors.Tumors could be observed in all of seven,five of seven mice with normal immune function at the injection site of 1000 and 100 CD44~+/CD24~- cells,respectively.10 CD44~+/CD24~- cells in all of seven mice with normal immune function failed to form tumors.CONCLUSION: A single breast cancer cell injected into BALB/C mice could generate a tumor.This study suggests that suspension culture combined with anticancer regimens is a feasible approach for screening cancer stem cells.