Screening For Gastric Cancer Vascular Endothelial Cell Specific Binding Peptides Using Phage Display Peptide Library

[Background]Angiogenesis and vasculature play important roles in the growth and metastasis of solid tumors. Anti-angiogenesis and anti-vascular therapy have become an intensively studies field in cancer research. It was found that tumor vessels have characteristics different from normal vessels. Therefore a great interest has been aroused in finding tumor vascular endothelial cell specific molecules and using them as targeting molecules to combat cancer. However, no molecule has been identified which is specific to gastric cancer vascular endothelial cells. This could be explained by: the difficulty in isolating and culture of gastric cancer micro vessel endothelial cells; the low level expression of the differentiated molecules on gastric cancer vascular endothelial cells, which is difficult to be detected, isolated and purified by common practice. Recent study suggested that co-culture of the cancer cells with vascular endothelial cells could create a similar scenario with the situation in the solid tumor mass. Studying the molecular changes of vascular endothelial cells co-cultured with gastric cancer cells may shed some light onthe gastric cancer vascular endothelial cell specific molecules. Furthermore, the developing phage peptide library panning technique is a kind of efficient selection system and provides a good way to obtain the ligands binding to target molecules with low concentration.[ Objectives ]To establish the co-culture model of gastric cancer cells and endothelial cells, identify the peptides binding specifically to the co-cultured vascular endothelial cells using phage display peptides library and study the affinity of these peptides to gastric cancer vascular endothelial cells.[Methods](1) Co-culture the primary human umbilical vein endothelial cells (HUVECs) with gastric cancer cells SGC7901 in Transwell dishes and study the biological features of the co-cultured cells.(2) Whole-cell subtractive screening with phage display C7C peptide library was performed on the HUVECs co-cultured with gastric cancer cells (Co-HUVECs), and normal gastric epithelium mucosae cells GES and wild type HUVECs were used as controls.(3) After 3-4 rounds of panning, 20 phage colonies picked randomly were sequenced to identify the consensus sequence.(4) The affinity of these 20 phages with Co-HUVECs was examined by enzyme-linked immunosorbent assay (ELISA) to exclude false positive colonies and those binding with equal affinity to Co-HUVEC and controls. According to the sequence analysis and ELISA results, one or several phage colonies were selected to program further identifications.(5) In vitro binding assay and immunocytochemical stain were performed to determine the specificity of the phages to Co-HUVECs, andimmunohistochemical staining was used to examine the binding of the phages to gastric cancer vascular endothelial cells.(6) Peptides displayed on the phages were synthesized and competitive binding assay was performed to observe the competitive inhibition effect of the peptides with their phage counterparts.(7) Immunofluorescence microscopy was used to study the binding of synthesized peptides to Co-HUVECs and vascular endothelial cells in gastric cancer tissue sections.[Results](1) The co-culture model of SGC7901 and HUVECs was successfully established. Co-cultured HUVECs showed accelerated proliferation but no changes in its adhesive ability. Proliferation of the co-culture SGC7901 cells was inhibited, and the cells had weaker adhesiveness. Acidification was found in the microenvironment of the co-culture.(2) After 4 rounds of panning, phages binding to the Co-HUVECs were enriched from 0.4×104pfu(Mp) and 1.5×104pfu(INp) at the first round to 4.352×106pfu(Mp) and 6.873×106pfu(INp) at the end of the fourth round of the panning (with an increase of the 1088 and 458-fold respectively). Binding of these phages to wild type HUVECs and GES cells kept at low level or even decreased.(3) 20 phage colonies were randomly picked and sequenced. 12 sequences were obtained from these 20 colonies. M6, Ml, M9, IN13, IN11 were repeated found in the sequences. Among them, IN11 was the one with most repeats. Certain homology between the peptide sequences displayed by the phages and known proteins was identified by BLAST analysis.(4) ELISA results suggested that M6, M3, M9, IN12, IN11 phages could bind...