Isolation And Characterization Of Side Population From Breast Cancer Cell Line MCF-7

It is generally accepted that transformation of normal cell to tumor cell is amultistep process caused by accumulation of mutations and epigeneticchanges.Recently,a new model was proposed mammary carcinogenesis is driven bytumor stem cells derived from mutated adult stem or progenitor cells since adultstem cells are slowly dividing, long-lived cells with a high proliferative capacitywho are able to accumulate the multiple mutations that occur during carcinogenesis.Another presumption is that cancer stem cells might be derived from progenitorcells that have acquired the ability to self-renew as a result of oncogenic mutations.Some studies have been reported that stem-like cells are isolated from brain tumorsand breast cancer. But more attempts are needed.Since SP phenotype is consideredas a wide marker of stem cell, we try to isolate SP in MCF-7 cells. Then thestem-like properties are proved by experimental systems, based on tumor formingcapacity in vivo and in vitro. In both cases disregulation of self-renewal in stemcells is a key event which drives the process of tumorigenesis and aberrantdifferentiation in tumorigenesis. It was found that the Wnt pathways are involved inregulation of self-renewal of normal breast stem cells. So we still detected theexpression of several signaling proteins to find if Wnt pathway plays an importantrole in SP cells isolated from MCF-7.1 Isolation of SP from MCF-7 cellsMETHODS: The MCF-7 cells from log-phase cultures were trypsinized andresuspended at 106 cells/ml in complete RPMI1640. Hoechst33342 was added to afinal concentration of 5μg/ml in the presence of verapamil or not and cells wereincubated for 90 minutes in a 37°C water bath. The MCF-7/Adr,MCF-7 and the SPcells from MCF-7 cultured 2 weeks were analyzed by FACS. In order to observethe SP cells directly ,another flask of attached MCF-7 cells was examined byfluorescence microscoped after being stained with Hoechst33342 and CFSE.Bcrp1expression has been proved to be the molecular determinant of the SP phenotype,sowe detected and compared the expression of bcrp mRNA by RT-PCR between SPand MP, MCF-7/Adr and MCF-7 cells.RESULTS:SP is contained in MCF-7 for about 4%.The SP percentage inMCF-7/Adr is higher than in MCF-7.As we speculated, the expression of bcrp ishigher when the percentage of SP is higher.The SP cells can be separated into SPand MP after being cultured 2 weeks .There is no difference between thepercentages of SP in the parental cells and the descendant cells.CONCLUSION:SP was identified in MCF-7,MCF-7/Adr cell lines. Thepercentage of SP in MCF-7/Adr is higher. The expression of Bcrp1 conferred the SPphenotype. The ability of SP cells developing into SP and MP means that SP is inthe higher hierarchy.2 The property of SP cellsMETHODS:SP and MP cells were placed in the incubator to observe theattaching efficiency at 6h,12h,18h by the microscope. SP and MP cells were fixedin cold 70% ethanol for 18 hours. Then cells were treated with ribonuclease andPIbefore analysed for cell cycle by FACS.For in vitro clonal analysis, SP and MPcells were plated at clonal density in culture dishes. After 1 to 2 weeks, the disheswere fixed with methanol and stained with Gimsa. The percentage of cells thatinitiated a clone was presented as cloning efficiency. For in vivo self-renewalanalysis , various numbers of cells, either SP or MP ,were injected in 200ul ofPBS/Matrigel (1:1) subcutaneously in the region of the upper left mammary fat padsinto 24 female NOD/SCID mice. All animals were terminated when the diameter ofany tumor gets 3cm Tumors harvested were fixed in formalin and paraffin sectionswere made for HE staining.RESULTS:The attaching efficiency of MP cells are higher than MP at 6h and12h.They are equal at 18h. The percentage of G0/G1 in SP is the highest among SP,MP and unsorted cells.The cloning efficiency of SP cells is higher than that of MPwhen culture was ended on the 16th day. All the SP cells gave rise to tumor .Only 2tumors were observed in mice injected with MP cells. Tumor latency was alsoreduced with increased numbers of side population cells injected.CONCLUSION:SP cells are more nonadherent,quiensent,clonogenetic andtumorigenic. Only 103 SP cells can give rise to tumor. So SP really have some stemcell properties.3 The role of Wnt signaling pathway in SP cellsMETHODS:SP and MP cells spread on slides for 10hours and fixed inpolyoxymethylene. The expression of β -catenin was examined byimmunocytochemistry(SABC).Total RNA was isolated and used in RT-PCRanalysis of Wnt1 and cyclinD1.RESULTS:As expected, β-catenin was accumulated in cytoplasm of SP cellswhile absent in MP cells. Wnt1 mRNA was detected neither in SP nor in MPcells.The MP of MCF7 cells expressed higher levels of cyclinD1 mRNA than theSP cells.CONCLUSION:The accumulation of β-catenin implys the wnt pathway isactivated in SP.But it is not Wnt1 that leads to the downstream activation.And thetarget gene cyclinD1 may not play important role in stem cells as in progenitorcells.The research of breast cancer stem cells signals the beginning of a new era ofbreast cancer research.To our knowledge, this is the first time to use the SP analysistechnician by FACS in China. It's also the first time to isolate SP in MCF-7 in ourcountry and the second time in the world. Patrawala's result is consistent withours'.But we first analyzed the character of SP such as cell cycle, Wnt signalingpathway et al. proving the stem-like property of SP cells in breast cancer cells.Ourwork provides some evidence and methods for breast cancer stem cells research andsome cues for the role of Wnt pathway in selfrenewal of breast cancer stem cells.The efforts to elucidate molecular differences in normal and cancer stem cells areexpected to produce promising approach to targeted causal therapy of malignanttumors.