Colorectal cancer (CRC) develops in a multistep process that arises from genetic alterations and environmental insults1
. Most CRCs can be prevented through detection and removal of pre-malignant polypoid lesions2
. Despite the proven efficacy of endoscopic screening and polyp removal, colorectal cancer remains the third most common cancer in men and women in the United States3
. The high incidence of colon cancer remains, in part, due to noncompliance to screening guidelines, but also to limitations in conventional endoscopic screening which lacks molecular specificity and reveals only gross anatomic changes through a macroscopic view of the surface mucosa. Flat or depressed neoplasms, which have recently been appreciated as another presentation of colorectal cancer4,5
, are difficult to detect using endoscopic methods and are associated with an increased risk of submucosal invasion when compared to polypoid lesions of similar size6
. In addition, patients with a long-standing history of inflammatory bowel disease are at increased risk for developing malignancy due to undetected dysplastic lesions7,8
. These observations support the need for improved methods for detecting early changes in high-risk patients. Novel approaches that are also compatible with conventional endoscopy would allow seamless integration into existing protocols, and offer greater potential for reducing morbidity and mortality. Such an approach could be applicable to other cancers of the epithelium including the esophagus, stomach, cervix, and bladder, which result in ~65% of all deaths by cancer in the U.S.3
A number of biomarkers for CRC have been discovered that may lead to new surveillance methods, identification of high-risk populations, and earlier detection9
. Because of their high specificity, monoclonal antibodies to these markers have been investigated for tumor detection and drug delivery. Their in vivo
use, however, has been limited by immunogenicity and challenges associated with development and production.10
In contrast, peptides are typically less immunogenic, non-toxic, and relatively simple to produce in quantity. The small size of peptides may also allow them to penetrate more easily into diseased mucosa, potentially binding to molecular targets at greater tissue depths. Bacteriophage that display peptides on their surface enables screening of highly diverse libraries for peptides with highly specific binding properties, without prior knowledge of the target.11
Phage display experiments have typically been performed using purified receptor targets or established cell lines in culture, leading to selection of phage for those preselected targets enriched on cell lines. Such peptides have been identified through biopanning with human colon cancer cells and have been used as imaging agents in animal models12,13
. Isolated and cultured cells, however, may de-differentiate and lose tissue-specific traits upon culture14
. In vitro
targeting systems may also fail due to inaccessibility of relevant receptors in vivo15
. Because phage display enables selection through an iterative process of selection and enrichment, the use of primary premalignant human tissues could eliminate any bias imposed by prolonged culture or over expression of receptors present on end-stage cancer.
Peptides with tissue-specific homing properties have been isolated from animal models in vivo
by intravenous injection of native phage libraries and harvesting organs of interest16,17
. Although ligands and receptors isolated in animal models have been useful for understanding their human homologs, species-specific differences in protein expression and ligand-receptor accessibility may limit their application in humans. For example, TEM7, an endothelial marker corresponding to a transmembrane protein expressed in endothelial cells of human colorectal tumor stroma, is not present in tumor blood vessels but in Purkinje cells in mice18
. Tissue-homing peptides have also been demonstrated for human tissues, however, recovery of target tissues after intravenous phage administration can only be achieved under special circumstances19,20
. These approaches have been useful for identifying peptides that bind to abnormal endothelial markers because intravenous phage are confined to the lumen of the blood vessels due to their relatively large size. Selection of peptides through intravenous administration is also biased towards targeting receptors that are directly accessible via the vasculature, and facilitates discovery of molecules more suitable for drug delivery and not for binding to mucosal surfaces that are the object of endoscopic observation. Successful studies using phage display to generate unique probes illustrates a key feature of this method: it is highly selective for the tissues to which the library is exposed. Therefore, if peptides that bind to premalignant human tissues are desired, these tissues should be used in the selection process.
The primary motivation for identifying mucosal markers is to enable topical administration of probes, which maximizes safety and reduces the risk from immunoreactivity–important considerations in the development of molecular imaging approaches. Panning using freshly harvested human tissue has been shown to successfully isolate peptides specifically binding to polarized luminal surfaces of umbilical vein endothelial cells21
. In addition, peptides specific for endothelial markers for dysplasia have been identified in mice22
. Previous studies with non-specific dyes have shown that confocal microendoscopy is capable of visualizing cellular and subcellular features associated with neoplasia23,24
. The use of a flexible fibered confocal microscope capable of high speed image acquisition thus may enable subsurface imaging with subcellular resolution, permitting local binding of topically administered dysplasia-targeting reagents to be assessed.
We used an M13 phage library to identify peptides that specifically bind dysplastic colonic mucosa. Nonspecific peptides were first removed with non-malignant intestinal epithelial cells followed by panning with fresh human colonic biopsies. A peptide that binds colonic dysplasia was identified, synthesized, and conjugated with fluorescein for in vivo
testing in patients undergoing routine colonoscopy. Fluorescence from topically administered peptide was collected using a confocal microendoscope (Cellvizio®-GI, Mauna Kea Technologies)25–28
that can pass through the accessory channel of standard colonoscopes was used to visualize topically administered peptide. Fluorescence images and videos of bound topically administered peptide collected in vivo
showed enhanced binding to dysplastic colonocytes compared to the adjacent normal mucosa in the same patient.