In Vivo Fluorescence-Based Endoscopic Detection of Colon Dysplasia in the Mouse Using a Novel Peptide Probe

Abstract

Colorectal cancer (CRC) is a major cause of cancer-related deaths in much of the world. Most CRCs arise from pre-malignant (dysplastic) lesions, such as adenomatous polyps, and current endoscopic screening approaches with white light do not detect all dysplastic lesions. Thus, new strategies to identify such lesions, including non-polypoid lesions, are needed. We aim to identify and validate novel peptides that specifically target dysplastic colonic epithelium in vivo. We used phage display to identify a novel peptide that binds to dysplastic colonic mucosa in vivo in a genetically engineered mouse model of colo-rectal tumorigenesis, based on somatic Apc (adenomatous polyposis coli) gene inactivation. Binding was confirmed using confocal microscopy on biopsied adenomas and excised adenomas incubated with peptide ex vivo. Studies of mice where a mutant Kras allele was somatically activated in the colon to generate hyperplastic epithelium were also performed for comparison. Several rounds of in vivo T7 library biopanning isolated a peptide, QPIHPNNM. The fluorescent-labeled peptide bound to dysplastic lesions on endoscopic analysis. Quantitative assessment revealed the fluorescent-labeled peptide (target/background: 2.17±0.61) binds ∼2-fold greater to the colonic adenomas when compared to the control peptide (target/background: 1.14±0.15), p<0.01. The peptide did not bind to the non-dysplastic (hyperplastic) epithelium of the Kras mice. This work is first to image fluorescence-labeled peptide binding in vivo that is specific towards colonic dysplasia on wide-area surveillance. This finding highlights an innovative strategy for targeted detection to localize pre-malignant lesions that can be generalized to the epithelium of hollow organs.

Molecular Fluorescence Endoscopy Targeting Vascular Endothelial Growth Factor A for Improved Colorectal Polyp Detection

“…Therefore, confocal laser endomicroscopy is not suitable for screening purposes, as our technique is, but rather a tool for lesion characterization. Second, other research groups have described topical spraying of tracer products to improve detection of dysplastic regions, but this is most attractive for relatively small areas and therefore not practical in the colon (28)(29)(30). An approach using intravenously injected tracers can circumvent these issues.…”

Targeted detection of murine colonic dysplasia in vivo with flexible multispectral scanning fiber endoscopy

“…Specific binding of the candidate peptides to primary dysplastic colonic epithelial cells were validated on flow cytometry ex vivo and with small animal endoscopy in vivo using FITClabeled peptides using a nonscanning white light endoscope. 19 …”

“…19 If found, the adenomas were rinsed of debris, including stool and mucous, by delivering tap water through a 3 Fr instrument channel. Then the fluorescent-labeled peptides were delivered topically at a concentration of 100 μM in 1 × PBS in a volume of 1 mL to the distal 4 cm of the colon and rectum.…”

“…With topical application to the mucosa, we have previously demonstrated that peptides derived from phage display technology can preferentially bind to diseased mucosa in the esophagus 18 and colon. 19 Current wide-field imaging methods being developed as an adjunct to conventional endoscopy for the early detection of colorectal cancer include narrow band imaging (NBI), chromoendoscopy, and autofluorescence imaging. NBI relies on enhanced contrast from vasculature by enhancing hemoglobin absorption, allowing diseased tissue to appear darker in color than surrounding normal mucosa.…”

Screening and Identification of a Phage Display Derived Peptide That Specifically Binds to the CD44 Protein Region Encoded by Variable Exons

“…First, peptides can penetrate and accumulate within the lesion with rapid binding kinetics. 20 Second, peptides exhibit minimal immunogenicity and rapid clearance, which are characteristics that are associated with low toxicity. 21 Finally, because of their simple structure, peptides are easily synthesized and conjugated with drugs or contrast agents.…”