Selection Of Peptide Specifically Binding To Human Breast Medullary Cancer Cells Bcap-37 By In Vivo Phage Display

Objective:To select the peptide specifically binding to human medullary breast cancer cells Bcap-37 by in vivo phage display and to provide molecular targeting probe for early diagnosis of breast cancer.Methods:The human medullary breast cancer Bcap-37 cells tumor-bearing nude mice model was prepared and three rounds selection by in vivo phage display were performed with Ph.D.-C7C^TM Peptide Library.The distribution of screened phages in tumors and normal tissues was detected by using immunohistochemistry.The affinity of monoclonal phage to Bcap-37cells was identified by using enzyme-linked immunosorbent assay?ELISA?.The positive monoclonal phage DNA was taken and sequenced,and the DNA sequence with high repetition rate was selected to synthesize peptide by using chemical methods.Optical molecular probe was prepared and the toxicity about the peptide and optical molecular probe on Bcap-37 cells were detected by using MTT assay.The specificity of optical molecular probe for Bcap-37 cells was identified by using fluorescent invered microscopy and flow cytometry.At last,the in vivo specificity and targeting ability of optical molecular probe for breast cancer transplantation tumor were tested in tumor-bearing nude mices.Results1.The phages targetting to Bcap-37 cell transplantational tumor were obtained after three rounds in vivo screening and the recovery rate of phages was increased successively with each round of screening.The recovery rate of phages in the third round in vivo screening was 107.2 times than that in the first round.2.Immunohistochemical results show that the phages binding to the tumor tissues were increased successively with the increasing screening rounds in vivo.The number of phages targetting to tumor tissues was obviously more than that binding to normal tissues.The absorbancy?A?value of section scanning image in the tumor tissues was higher than that in the normal tissues,and the difference was statistically significant?P<0.05?.3.ELISA results show that 22 phages?affinity?2?among the 50 randomly selected monoclonal phages were positive.4.The results of DNA sequence analysis showed that there were 4 amino acid sequences with frequency?2.Among them,the sequence?CSPLNTRFC?with the highest repetition rate had no homology with the known amino acid sequence in the database and it was named BCSP1.5.The peptide?BCSP1?,the fluorescein isothiocyanate-labelled peptide?FITC-BCSP1?and the control peptide?FITC-svBCSP1?were synthesized.The purity of the synthesized peptide was identified more than 98%by using mass spectrometry.6.MTT results show that the proliferation and activity of Bcap-37 cells were not effected by the targetting peptide BCSP1,the optical molecular probe?FITC-BCSP1 and FITC-svBCSP1?at different concentrations.7.After incubation of the optical molecular probe?FITC-BCSP1 and FITC-svBCSP1?with Bcap-37 cells,the specific binding of FITC-BCSP1 to Bcap-37 cells could be observed by using fluorescent invered microscopy,while the FITC-svBCSP1 could be not.8.FCM results show that the negative threshold was set as 0.18%,according to the blank control of Bcap-37 cells.The percentage of positive cells in Bcap-37 cells was 94.53±2.26%,which was obviously higher than that in other cells?P<0.05?.The results showed that FITC-BCSP1could be specifically bindind to Bcap-37 cells.9.200?l optical molecular probe?FITC-BCSP1?with a concentration of 280?mol/L was injected into Bcap-37 cells tumor-bearing nude mice via tail vein..2.5 hours later,FITC-BCSP1 peptide could specifically enriched in the tumor tissues and the intensity of fluorescence signal on the transplantational tumor in vivo reached the strongest?gray value78.68?.Strong fluorescence signal?gray value 80.43?could also be detected in the isolated tumor tissue,but almost no fluorescence signal could be seen in normal tissues except the gallbladder.Conclusion:BCSP1 peptide?CSPLNTRFC?specifically targetting to human breast camcer cells Bcap-37 was screened out successfully by using in vivo phage display,and its specificity and targeting ability were preliminarily identified by in vitro and in vivo experiment.It will be helpful to vitro studies on the early diagnosis of breast cancer.