Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy

Abstract

A combination of targeted probes and new imaging technologies provides a powerful set of tools with the potential to improve the early detection of cancer. To develop a probe for detecting colon cancer, we screened phage display peptide libraries against fresh human colonic adenomas for high-affinity ligands with preferential binding to premalignant tissue. We identified a specific heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with fluorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a fluorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-microm working distance and 2.5-microm (transverse) and 20-microm (axial) resolution. The fluorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specificity. This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer and potentially of other epithelial malignancies.

Figure 1

Schematic of peptide reagent development procedure. An M13 phage library was first cleared with non-malignant epithelial cells and freshly excised normal human colonic mucosa. Panning was then performed using freshly resected human colon polyps. Peptides displayed by phage that bound to dysplastic tissue were characterized, synthesized, and conjugated to fluorescein for use as imaging reagents during endoscopy.

Figure 1

Schematic of peptide reagent development procedure. An M13 phage library was first cleared with non-malignant epithelial cells and freshly excised normal human colonic mucosa. Panning was then performed using freshly resected human colon polyps. Peptides displayed by phage that bound to dysplastic tissue were characterized, synthesized, and conjugated to fluorescein for use as imaging reagents during endoscopy.

Figure 2

Cell binding assay comparing clone ‘VRPMPLQ’ against other candidate clones eluted from Round 3 of selection. Data shows relative binding to HT29 (transformed) cells over HS738 (non-transformed) cells, normalized to wild-type insertless M13 phage (binding = 1). Error bars reflect standard error of the mean (s.e.m.) for n = 4 experiments.

Figure 3

Confocal images of phage binding to (a) freshly excised colon adenoma, (b) normal mucosa. Specimens were incubated with 5×10⁹ pfu phage bearing peptide ‘VRPMPLQ.’ Presence of phage is detected through FITC-conjugated M13 monoclonal antibody. (c, d): Histology, H&E, 4x. Scale bars: 50 μm.

Figure 4

In vivo confocal fluorescence images of peptide binding to (a) dysplastic colon polyp, (b) adjacent normal mucosa. (c, d): Histology, H&E, 20x. Scale bars: 20 μm.

Figure 5

In vivo confocal fluorescence images of the border between colonic adenoma and normal mucosa showing peptide binding to dysplastic colonocytes: (a) Endoscopic view. (b) Border. (c) Dysplastic crypt. (d) Adjacent mucosa. Scale bars: 20 μm.