Screening and identification of a CD44v6 specific peptide using improved phage display for gastric cancer targeting

Abstract

Background: Peptide probes can be applied for biomarker targeting to improve the diagnostic accuracy. Cluster of differentiation 44 (CD44) is up-regulated in gastric cancer (GC). Among all the variants of CD44, CD44v6 is reported the most promising biomarker for GC. The purpose of this study was generating and identification a peptide ligand specific to CD44v6.

Methods: A 12-mer phage peptide library was screened on CD44v overexpressed HEK-293 cells with an improved subtractive method. Five candidate sequences emerged. Candidate phages were selected using enzyme-linked immunosorbent assay and competitive inhibition assays. Then the sequence (designated ELT) was chosen for further study. Its binding affinity and specificity were verified on recombinant protein, GC cells, GC tissues and xenograft models based on BALB/c-nu/nu mice using dissociation constant calculation, immunofluorescence, immunohistochemistry and in vivo imaging separately.

Results: The dissociation constant of ELT with recombinant protein was 611.2 nM. ELT stained CD44v overexpressed HEK-293 but not the cell expressing wild-type CD44s. On GC cell lines, ELT co-stained with anti-CD44v6 antibody. ELT binding on tumor tissues significantly increased compared with that of paracancer tissues, also showed a linear positive correlation with CD44v6 expression. ELT specifically accumulated in tumor and eliminated in short time in vivo.

Conclusions: ELT can target GC in vitro and in vivo via CD44v6, indicating its potential to serve as a probe for GC targeting diagnosis and therapy.

Keywords: CD44v6; Peptide probe; gastric cancer (GC); in vivo imaging; phage display.

Figure 1

Screening and identification of candidate phage clones. (A) Recovery rate of each panning round; (B) binding of candidate phage clones (presented as OD value at 450 nm) to HEK-293 cells transfected with CD44v recombinant plasmid (striped column) and empty plasmid (white column). Data were presented as mean ± SD. For each data point, measurements were made three times. OD, optical density.

Figure 2

Binding of the candidate peptides to the recombinant CD44v protein. (A) Competitive inhibition of candidate phages binding to CD44v protein by corresponding displayed peptide; (B) binding curve of ELT peptide at different concentrations to CD44v protein; (C) Scatchard analysis of ELT binding data with CD44v protein; (D) binding curve of ELT peptide at different concentrations to BSA protein; (E) Scatchard analysis of ELT binding data with BSA protein. A scrambled peptide served as the control. Data were presented as mean ± SD. For each data point, measurements were made three times.

Figure 3

Binding of ELT peptide to GC cells. (A) GC cell lines MKN-28, MKN-45 and SGC-7901 were stained with fluorescence dual-labeling technique. Green staining presented FITC-labeled peptide probe binding, while red staining showed CD44v6 expression that was detected by immunofluorescence; (B) binding of rhodamine B labeled peptide probes to CD44v3–v10 recombinant plasmid and empty plasmid. GFP served as a report gene. Cell nuclei were stained with DAPI. Original magnification, ×400. GC, gastric cancer.

Figure 4

Immunohistochemical staining of ELT and its correlation with CD44v6 expression in gastric tissues (A) Biotin labeled ELT peptide staining and immunohistochemistry detection of CD44v6 on serial sections of GC and adjacent normal tissues from the same patient. (a) ELT stained GC; (b) ELT stained adjacent normal tissue; (c) immunohistochemistry detection of CD44v6 on GC; (d) immunohistochemistry detection of CD44v6 on adjacent normal tissue; (B) statistical analysis of HSCORE of GC tissues (striped box) and pericarcinous tissues (white box) targeted with ELT peptide and anti-CD44v6 antibody. Single asterisk (*) denotes significant difference; (C) regression line generated using Pearson correlation analysis presents correlation. A linear positive correlation (r=0.596 P<0.05) was observed between ELT binding and CD44v6 expression. GC, gastric cancer; HSCORE, immunohistochemistry score.

Figure 5

In vivo fluorescence imaging of FITC-ELT probe (A) Serial in vivo fluorescence imaging of FITC labeled ELT and scrambled peptides in nude mice with subcutaneous xenograft tumor at different post-injection time points; (B) fluorescent intensity values of tumor at different time after peptide probe injection. (C) in vivo fluorescence imaging of excised mice organs (1, tumor; 2, liver; 3, stomach; 4, heart; 5, kidney; 6, lung); (D) fluorescent intensity values of excised mice organs. Data were presented as mean ± SE. For each data point, measurements were made five times.