Primary Study Of Isolation And Identification Of Colon Tumor Specific Phage Peptides

Human colon adenocarcinoma is an ordinary malignant cancer around the world. There are some problems in its diagnosis and therapy. At present, searching for new specific ligands provides hope for the effective diagnosis and therapy of colon cancer.In this study, human colon adenocarcinoma cell line HT-29 is used to screen phage display random peptide libraries(fUSE5/6-mer library; fUSE5/15-mer library).Binding to the whole cells with no directive pressures on the selection scheme has many advantages. These screens may be useful for cells that have complex and frequently changing cell-surface landscapes,such as cancer cells. So intact cells are used to screen two peptide libraries. To select colon cancer cells specific phage display peptides, the procedure of screens are optimized. After three-round selection of"adsorption-elution-amplification", titers eluted from HT-29 increase obviously, which suggests specific peptides binding to the colon tumor cells are enriched. Repeated experiments do not detect the internalized phage-peptides. 50 monoclones of phage peptides selected from two three-round sub-libraries respectively can bind to HT-29 in various extent.To investigate whether these phage peptides are specific ligands of colon cancer, normal selection and negative selection are performed to identify the phage peptides binding to HT-29 but not to normal liver cell line L-02. In addition, further cell binding assay indicates some peptides which can not bind to L-02 can bind to different malignant degree of colon cancer cell lines, such as HT-29, LOVO, SW480.To evaluate colon cancer specificity in tissues, positive phage peptide clones isolated from above are selected to carry out immuonhistochemistry. It shows that colon carcinoma can be stained by phage peptide 15mer19C but corresponding autologous normal tissue can not. 15mer19C are proved to bind none of the 30 colon normal tissues but can recognize all those 17 colon cancer tissues. To ruLe out the possibility that this phage peptide could bind to other normal organs, in vivo experiment are performed to detect its organ distribution in mice. The results demonstrated this peptide does not bind to normal organs.These data suggest that phage display random peptide libraries may be very useful for selecting the intact tumor cells to get colon tumor specific peptides. Peptides got from this way could be designed as novel diagnostic and cell-targeting reagents.