Detection Of Pancreatic Cancer In Vivo With MUC4 Targeted Peptide ZP-16

Objectives:This study was carried out to identify a peptide ZP-16 that selectively binds to mucin4?MUC4?and to use the peptide for the detection of MUC4 overexpressing pancreatic cancer in vivo.Methods:1.The synthesis and characterization of peptide fluorescent probe ZP-16-Cy5:finished by Shanghai Biotech Company.2.Immunofluorescence cytochemistry experiments of ZP-16-Cy5:each tumor cell line was tested for its MUC4 expression level by Western Blot,and immunofluorescence experiments were carried out with anti-MUC4fluorescent antibodies?1G8-FITC?and ZP-16-Cy5 respectively.The fluorescence intensity of each cell line was observed by laser confocal microscopy,and the fluorescence intensity of BxPC-3 and U87 cells combined with 1G8-FITC and ZP-16-Cy5 was measured by flow cytometry.3.Characterization of the binding of ZP-16-Cy5 to BxPC-3cells:BxPC-3 cells with high expression of muc4 were selected for competitive inhibition experiments of ZP-16-Cy5 and ZP-16 and co-localization experiments of ZP-16-Cy5 and 1G8-FITC;simultaneously,laser confocal microscopy and flow cytometry was used to monitor the dose-effect relationship and time-effect relationship between ZP-16-Cy5 and BxPC-3.4.Immunofluorescence histochemistry experiments of ZP-16-Cy5:normal pancreatic tissue,pancreatic cancer tissue and low-grade fibromyxoid sarcoma tissue were taken for HE staining and immunohistochemistry?IHC?experiments,then ZP-16-Cy5 and 1G8-FITC immunofluorescence chemistry and colocalization experiments were taken.5.Detection of tumors with high MUC4 expression by ZP-16-Cy5 in vivo:BxPC-3 and U87 tumor-bearing nude mice model was prepared,until the tumor is about 1cm long,inject ZP-16-Cy5?4.0nmol/20gwt?,by tail vein and observe the imaging of implanted tumors by small animal live imager.Results:1.Characterization of ZP-16-Cy5:the molecular weight is 2558.1 and the purity is 99.89%.2.The binding of ZP-16-Cy5 and different tumor cells:Western Blot showed that the cells with high to low MUC4 expression were:BxPC-3,CFPAC-1,Capan-1,PANC-1,HPAF-?,PC-12,U87;the binding of ZP-16-Cy5 and 1G8-FITC to each cell line was similar,and the results were consistent with those of Western Blot.Flow cytometry confirmed that the average fluorescence intensity of BxPC-3 binding to 1G8-FITC was 418%higher than that of U87?p<0.01?;the average fluorescence intensity of BxPC-3 binding to ZP-16-Cy5 was 189%higher than that of U87?p<0.01?.3.The characterization of binding of ZP-16-Cy5 and BxPC-3:ZP-16competitively inhibits the binding of ZP-16-Cy5 to BxPC-3,and ZP-16-Cy5and 1G8-FITC have good overlap on BxPC-3,indicating that ZP-16-Cy5 is specific to the cell binding site and haveadifferent binding epitope from1G8-FITC.The combination of ZP-16-Cy5 and BxPC-3 accords with the first-order reaction kinetics:the binding speed is fast[k=0.105min^-1(t_1/2=6.9min)],and the affinity is high?K_d=17.3nmol/L?.4.The binding of ZP-16-Cy5 and human pancreatic cancer tissues:IHC showed that MUC4 express in pancreatic cancer and low-grade fibromyxoid sarcoma,and no MUC4 express in normal pancreatic tissue;The results of immunofluorescence histochemistry of ZP-16-Cy5 and 1G8-FITC were similar to those of IHC:pancreatic cancer showed strong fluorescence,low-grade fibromyxoid sarcoma had relatively strong fluorescence,and normal pancreatic tissue showed no fluorescence;co-localization showed the fluorescence distribution and intensity of ZP-16-Cy5 and 1G8-FITC were similar,and the degree of coincidence was higher after merge.5.Detection of tumors with high MUC4 expression by ZP-16-Cy5 in vivo:BxPC-3 tumor-bearing mice were injected with ZP-16-Cy5?4nmol/20gwt?by vein,and the tumor showed fluorescence after 9 min,then gradually increased.After reaching the peak in 55 minutes,it gradually weakened and disappeared at 180 min,and there was a clear fluorescence signal aggregation process.Frozen sections of implanted tumor tissues conformed to the pathological features of pancreatic cancer,and fluorescent signals were observed under LSCM.There was a certain fluorescence signal in the tumor of U87 tumor-bearing mice after injection of ZP-16-Cy5?4nmol/20gwt?in the tail vein for 5min?120min,but there was no obvious fluorescence signal aggregation process.Conclusion:1.ZP-16 can bind to tumor cells expressing MUC4,its specificity was similar to anti-MUC4 fluorescent antibody?1G8-FITC?.2.ZP-16 is specific for the cell binding site and have a different binding epitope from1G8-FITC.3.The combination of ZP-16 and BxPC-3 accords with the first-order reaction kinetics:the binding speed is fast[k=0.105min^-1(t_1/2=6.9min)],and the affinity is high?K_d=17.3nmol/L?.4.ZP-16 can bind to clinical tumor tissue expressing MUC4 and has good overlap with 1G8-FITC.5.ZP-16 can detect pancreatic tumors with high MUC4 expression in vivo,and it is expected to be used as a specific ligand for the diagnosis and treatment of tumors.