Selection and identification of a specific binding peptide targeting to blood vessel of hmnan colon cancer

Abstract

Objective To select, identify and analyze a peptide binding specifically to blood vessels of human colon cancer by phage displayed peptide library in vivo. Methods Animal models were established using sub-renal capsular assay （SRCA） in immunosuppressive mice implanted with human colon cancer xenografts. The phage displayed peptide library was injected intravenously into mice. After 4 rounds of selection, 20 clones were picked up randomly and sequenced individually. The homing ability to human colon cancer xenografts and co-cultured human umbilical vein endothelial cells （ Co-HUVECs with human colon cancer Lovo cells） of the positive phage clones were determined by in vivo binding assay and in vitro cell enzyme linked immunosorbent assay （ ELISA ）. The binding ability to Co-HUVECs of peptide-displayed phage clone was identified by immunocytoehemieal stain. Peptide displayed on the phage was syn- thesized and competitive binding assays were performed to observe the competitive inhibition effect of the peptide with their phage counterpart, hnmunofluoreseence microscopy was used to study the binding of synthesized peptides to Co-HUVECs and vascular vessels in colon cancer. Results Two peptide sequences were obtained finally and named CV1 and CV2. In vivo binding assay showed that the homing ability to human colon cancer xenografts of peptides of CV1 or CV2 was higher than that of control organs. In vitro cell ELISA suggested that CV2 phage preferably binded to Co-HUVECs rather than control HUVECs. Then CV2 phage clone was identified further. Immunocytoehemical staining revealed that CV2 phage preferably binded to Co-HUVECs rather than the control. Competition binding assays demonstrated a significant competition between the synthesized peptide CV2 and the phage displaying CV2 while binding to Co-HUVECs or human colon cancer xenografts. Under the immunofluorescence microscopy, fluorescence-labeled CV2 peptide was seen on the membrane and in the perinuclear cytoplasm of Co-HUVECs, and bound to colon cancer xenografts rather than control organs. Conclusion Two phage clones displaying CV1 and CV2 peptide could target to human colon cancer xenografts. The peptide CV2 and its displayed phage were identified binding preferably with Co-HUVECs. And the cyclic nonapeptide （CV2） was binding site of the CV2 phage with Co-HUVECs. Synthesized nonapeptide CV2 had specificity to Co-HUVECs and colon cancer vascular endothelial ceils. The peptide CV2 could be used in target therapy of tumor angiogenesis. Key words: Colon carcinoma; Phage displayed peptide library; Angiogenesis; Peptide