Screening And Identification Of Novel Specific Polypeptide Markers Of Breast Cancer Stem Cells

Background and Objective:Breast cancer is the malignant tumor with the highest incidence in women.The incidence of breast cancer has been increasing year by year in the past 20 years.Although the early diagnosis and comprehensive treatment of breast cancer have made great progress,some patients still suffer local recurrence or distant metastasis.With the developing of tumorigenesis mechanism and tumor stem cells theory,solid tumor stem cells have attracted more and more attention.Breast cancer stem cells are a group of pluripotent cells with self-renewal and highly tumorigenic nature,which was closely related to the occurrence,development,metastasis,recurrence and resistance to treatment of breast cancer.In the treatment of breast cancer,only the targeted elimination of breast cancer stem cells is the key to the complete cure of breast cancer.Therefore,it is of great significance for the treatment of breast cancer to study the phenotypic characteristics and biological characteristics of breast cancer stem cells and establish treatment strategies for breast cancer stem cells.Phage random peptide library and its screening technology are a kind of technology for finding high specific protein molecules,which have been widely used in the researches of tumor targeted therapy and tumor diagnostic markers.We cultured and enriched the stem cells in breast cancer cell line MDA-MB-231 and MCF7 stem cells by "serum-free suspension" method,screened the positive phage that could specifically combined with breast cancer stem cells.We identified the positive phage by ELISA and extracted the positive phage DNA.Then we isolated the DNA pellet and sent them to biotechnology companies.According to the sequencing results,polypeptides were synthesized and labeled with FITC.Finally,the specificity of polypetides to breast cancer stem cells was identified in vivo and vitro.MethodsWe chose the human breast cancer cell line MDA-MB-231,the human breast cancer cell line MCF-7 and the human mammary gland cell line hs578bst for use in the present study,and these cell lines were preserved in our laboratory.The bacteriophage random 12-peptide library kit(Ph.The D?12 TM phage display peptide library)was purchased from New England BioLabs,Inc.,Ipswich,MA,USA,and 100 ?l,1.5×1013 pfu/ml,complexity 2.7×109 transformation was stored in TBS containing 50%glycerol solution at-20?.The following antibodies were used:FCM antibody;FITC anti-human CD44 antibody(purchased from BioLegend,Inc.,San Diego,CA,USA;cat.no.338804);PE anti-human CD24 antibody(purchased from BioLegend,Inc.,San Diego,CA,USA;cat.no.311106);and Alexa Fluor647 anti-human CD326 antibody(purchased from BioLegend,Inc.;cat.no.324212).Stem cells were enriched in serum-free medium supplemented with EGF,bFGF,and B27 growth factors,and the microspheres were observed microscopically.Centrifugation was conducted to change the medium every 2-3 days.After 1 week of serum-free culture,the medium was changed to medium with 10%FBS for one passage to remove any dead cells.The cells were cultured alternately with serum and serum-free culture medium to maximize breast cancer stem cells.Then,we chose the the CD44+/CD24-/low and ALDH+as the marker of breast cancer stem cells,and the CD44+/CD24+/low cell group was sorted using a flow cytometer and cultured for 15 days.The microspheres were observed under a microscope.A phage random peptide library was amplified and screened by culturing with breast cancer cells and breast cancer stem cells.We identified the positive phage by ELISA and extracted the positive phage DNA.We isolated the DNA pellet and sent them to biotechnology companies for sequencing with primers 96 g? 5'-HOCCC TCA TAG TTA GCG TAA CG-3'.We synthesized polypeptides according to the sequencing results,and then labeled them with FITC.The breast cancer cells and breast cancer stem cells were incubated together with the polypeptides labeled with FITC.Then,we observed the distribution of the FITC-tagged polypeptides in different cell lines and captured images.We injected the breast cancer stem cells into nude mice to stimulate the growth of breast carcinoma.Then,we injected the polypeptides labeled with FITC into the nude mice.The tumor tissue and the control tissue(of the liver)were observed under a microscope.We chose the nude BALB/c mice(6-8 weeks)to establish animal model after we acquired the approval of Etiics Committee of Shandong Cancer Hospital and Institute.Firstly,we centrifuged MDA-MB-231 and MCF7 stem cells,adjusted the concentration of breast stem cells to 5×105/ml,then implanted them subcutineously under the right breast pad or intravenously into the tail vein of nude BALB/c mice.Secondly,we measured the tumor size and divided nude mice into different groups.Lastly,we injected the polypeptide into the vein of nude mice and dissected them to observe the polypeptide distribution in liver tissue.Results:The pretreated primary breast cancer cells were cultured overnight to adhere to the surface.Elongated flat cells were observed under a microscope.After 30 days of "serum and serum-free" culture,the cells were bright,round and suspended in the culture medium under the microscope.The cells form balloon shapes and are able to grow in complete medium.We analyzed the breast cancer stem cells to sort the CD44+/CD24-/low cell group by flow cytometry,and we found that the proportion of induced MDA-MB-231 and MCF7 breast cancer stem cells was 77.7 and 82.2%,and while we chose the ALDH+as the marker of breast cancer stem cells,the proportion of induced MD A-MB-231 and MCF7 breast cancer stem cells was 75.8%and 58.7%.The phage peptide library was subjected to three rounds of "affinity-elutriation-amplification" affinity screening.After three rounds of elutriation,the phage recovery rate increased and the recovery rate stabilized in the third round,indicating that the phage can specifically bind to human breast cancer cell lines.The third round of screening enriched-200 times more than the first round of screening,and the titer was monitored for each screening product.A total of 10 clones were randomly selected from the final screening products and sequenced.In total,10 positive clones were obtained through MDA-MB-231 and MCF7 screening,and eight and six sequencing results were obtained,respectively.A total of two of the eight 12-peptides sequences obtained from the MDA-MB-231 cells differed by only one amino acid.Given the complexity of the cell surface,this screening result is reliable and credible.Further studies on the two polypeptides,A3 for MCF7 stem cells and B8 for MDA-MB-231 stem cesss,are needed.A total of six distinct polypeptide sequences with no similar or identical sequences were obtained in the 10 sequences from the MCF7 cell screening.According to the sequencing results,the peptides were synthesized and labeled with FITC.The peptides were identified to be specific for breast cancer stem cells;the cells were observed and imaged under a fluorescence microscope after co-incubation in vitro.Polypeptide B8(sequenced twice)was able to specifically bind to the MDA-MD-231 stem cells without binding to the original MDA-MD-231 cells,whereas,the negative control polypeptide B11 did not bind to the MDA-MD-231 stem cells or to the original MDA-MB-231 cells.Polypeptide A3(sequenced once)was able to specifically bind to the MCF7 stem cells without binding to the original MCF7 cells,whereas,the negative control peptide A13 did not bind to the MCF7 stem cell or to the original MCF7 cells.The tumor tissues and the control tissues(of the livers of the nude mice)were observed under a microscope.It was observed that the polypeptide B8 and A3 labeled with FITC were rich in the tumor tissue and poor in the control liver tissue.Conclusions:Specific phage polypeptides that can specifically bind to breast cancer stem cells were successfully screened through stem cell enrichment and phage display technology,which may lay the foundation for targeted therapy and further study of breast cancer stem cells.