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Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy
Pei-Lin Hsiung1, Jonathan Hardy1, Shai Friedland2,3, Roy Soetikno2,3, Christine B Du1, Amy P Wu1, Peyman Sahbaie2, James M Crawford4, Anson W Lowe3, Christopher H Contag1 & Thomas D Wang2,3
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-afnity ligands with preferential binding to premalignant tissue. We identied a specic heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with uorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a uorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-lm working distance and 2.5-lm (transverse) and 20-lm (axial) resolution. The uorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specicity. This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer and potentially of other epithelial malignancies. Colorectal cancer develops in a multistep process that arises from genetic alterations and environmental insults1. Most colorectal cancers can be prevented through the detection and removal of premalignant polypoid lesions2. Despite the efcacy of endoscopic screening and polyp removal, colorectal cancer remains the third most common cancer in men and women in the United States3. This high incidence results from noncompliance with screening guidelines and limitations on conventional endoscopic screening, which lacks molecular specicity and reveals only anatomical changes through a macroscopic view of the surface mucosa. Flat or depressed neoplasms are difcult to detect using endoscopic methods4, and patients with chronic inammatory bowel disease are at increased risk of developing malignancy due to undetected dysplastic lesions5. There is therefore a need for improved methods for detecting early changes in high-risk individuals. New approaches that are compatible with conventional endoscopy would allow seamless integration into existing protocols and would be applicable to other cancers of the epithelium, including those of the esophagus, stomach, cervix and bladder, which together result in around 65% of all cancer deaths in the United States3. A number of biomarkers for colorectal cancer have been described that might facilitate surveillance, identication of high-risk populations and earlier detection6. Because of their high specicity, antibodies to these markers have been investigated for cancer detection and drug delivery. However, their in vivo use has been limited by immunogenicity and challenges in development and production7. By contrast, peptides are typically less immunogenic, nontoxic and relatively simple to produce in quantity. Their small size might also allow them to penetrate more easily into diseased mucosa, potentially binding to molecular targets at greater tissue depths. The identication of peptides for mucosal markers would allow topical administration, which maximizes safety and reduces the risk from immunoreactivityimportant considerations for the development of molecular imaging approaches. Peptide libraries displayed on the surface of bacteriophage are highly diverse libraries that can be screened for specic binding properties8. Peptides targeting human colon cancer cell lines have been identied9 and have been used for delivering radioisotopes10 or as imaging agents in animal models11. However, isolated and cultured cells might de-differentiate and lose tissue-specic traits upon prolonged culture12. Cell culture screening protocols might also fail owing to the inaccessibility of relevant receptors in vivo13. Intravenous injection of native phage libraries followed by removal of the organs of interest has identied peptides with tissue-specic homing properties in animal models14. Although ligands for receptors isolated in animals have been useful for understanding their human homologs, species-specic differences might limit their application in humans15. Tissue-homing peptides have also been shown in humans, but the recovery of target tissues after intravenous phage administration is only possible under special circumstances16. Notably, the selection of peptides through intravenous administration is biased toward targeting receptors that are directly accessible through the vasculature, facilitating the discovery of molecules that are more suitable for drug delivery to the endothelium than for binding to mucosal

1Department of Pediatrics, Radiology and Microbiology & Immunology, Stanford University School of Medicine, 318 Campus Dr., Rm. E-150, Stanford, California 94305, USA. 2Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave., Bldg. 100, Palo Alto, California 94304, USA. 3Department of Medicine, Stanford University School of Medicine, 300 Pasteur Dr., Alway Bldg., Rm. M211, Stanford, California 94305, USA. 4Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, P.O. Box 100275, 1600 SW Archer Rd., MSB 649, Gainesville, Florida 32610, USA. Correspondence should be addressed to T.D.W. (thomaswa@umich.edu).

Received 20 September 2007; accepted 14 November 2007; published online 16 March 2008; corrected online 21 March 2008 (details online); doi:10.1038/nm1692

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2008 Nature Publishing Group http://www.nature.com/naturemedicine

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Figure 1 Binding assays for clone bearing peptide VRPMPLQ. (a) Relative binding to HT-29 (transformed) cells over Hs738.st/int (nontransformed) cells compared with binding of other candidate clones eluted from the third round of selection, normalized to wild-type insertless M13 phage (binding 1). Error bars show s.e.m. for four experiments. (b,c) Confocal images of binding to freshly excised colon adenoma (b) and normal mucosa (c). (d,e) Histology of colon adenoma (d) and normal mucosa (e) stained with H&E. Scale bars, 50 mm.

surfaces, the object of endoscopy. Successful studies using phage display illustrate a key feature of this technique: it can be highly selective for the tissues to which the library is exposed. Thus, we reasoned that if peptides binding to premalignant human tissues are desired, these tissues should be used in the selection process. We used an M13 phage library to identify peptides that specically bind dysplastic colonic mucosa. We rst removed nonspecic peptides by clearing with nonmalignant intestinal epithelial cells followed by selection with fresh human colonic biopsies. A peptide that binds colonic dysplasia was identied, synthesized and conjugated with uorescein for in vivo testing in a pilot study involving patients undergoing routine colonoscopy. We observed uorescence signals from topically administered peptide using a exible-bered confocal microscope (Cellvizio-GI, Mauna Kea Technologies)17 that can pass through the accessory channel of standard colonoscopes, allowing high-speed image acquisition and assessment of local peptide binding characteristics. Fluorescence images and videos of bound topically administered peptide collected in vivo showed that our selected peptide bound more strongly to dysplastic colonocytes than to the adjacent normal mucosa in the same subject. RESULTS Isolation of phage with specic binding to colonic dysplasia Two rounds of incubation with nonmalignant human intestinal cells reduced the original titer of the phage library by 97% to 4.25 109 plaque forming units (p.f.u.). Subsequent panning using fresh human colonic tissue further reduced the library to 1.9 105 clones. After two rounds of selection, 7.8 104 p.f.u. were retained, indicating that around 41% of the phage remained unbound. Histology indicated that tissues in the second round of selection were hyperplastic, inamed and focal adenoma, so this round of selection was considered a clearing round and we retained the unbound phage. After the third round of selection, about 6,000 p.f.u. remained. Histology revealed that the polyps used in this round were tubular adenomas, so we retained the bound phage for further characterization. In vitro assays Of the 40 phage clones we tested, one clone displaying a peptide with sequence VRPMPLQ showed 23 times as much uorescence

intensity as control M13 antibody alone. Binding studies indicated that the binding of this peptide to HT-29 human adenocarcinomaderived cells was around 20-fold greater than its binding to Hs738.st/ int nonmalignant human intestinal cells (Fig. 1a). We saw increased binding compared to ten other randomly selected clones also eluted from the third round of selection. Assays performed using this clone on fresh biopsies of tubular adenoma from human colon polyps yielded threefold greater uorescence intensity from abnormal colonocytes lining dysplastic crypts than from adjacent normal mucosa (Fig. 1b,c). Although we identied a number of peptides using this procedure, the evaluation of selected peptides in clinical studies necessitated choosing one initial peptide. As our goal was to identify a peptide reagent that could target dysplasia in a clinically relevant setting, we synthesized and conjugated the peptide VRPMPLQ with uorescein for a pilot human study conducted during screening colonoscopy.

Figure 2 In vivo confocal uorescence images of peptide binding. (a) Binding to dysplastic colon polyp. (b) Binding to adjacent normal mucosa. (c,d) Histology of dysplastic colon polyp (c) and normal mucosa (d) stained with H&E. Scale bars, 20 mm.

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We calculated the uorescence intensity between adenoma and normal mucosa using mean colonocyte uorescence signal values, and this yielded an optimum sensitivity of 81% and specicity of 82% for a signal threshold of 6.1, with P o 0.01 using a two-sided Student t-test (Fig. 4). We saw no preferential binding for three adenomas, and it was necessary to rinse with water for several seconds before imaging to remove excess peptide, which might contribute to nonspecic uorescence (Supplementary Table 1 online). In addition, imaging performed after B10 min (owing to difculty in positioning the probe or subject peristalsis) yielded substantially decreased uorescence signal. We observed photobleaching during the imaging procedure on rare occasions when the probe was held stationary on the tissue surface for more than 5 s. We saw no toxicity associated with peptide administration in any subject (Supplementary Table 2 online). DISCUSSION New endoscopic imaging techniques such as optical coherence tomography (OCT)18, autouorescence imaging (AFI)19,20, and narrowband imaging (NBI)21 have been investigated for the detection of dysplasia in the gastrointestinal tract. Fluorescent imaging in combination with 5-aminolevulinic acid (5-ALA)22, as well as uorescently labeled monoclonal antibodies, have also been investigated23,24. It has been reported that endoscopic confocal microscopy with systemically administered uorescein has advantages over locally applied contrast agents such as acriavine25. However, these nonspecic dyes provide anatomical and not functional data and are complementary to approaches that target molecular markers of disease. The combination of targeted peptide probes and visualization using confocal microendoscopy offers a new method for facilitating in vivo optical imaging for early cancer detection. We performed peptide selection using freshly excised tissue, which mimics the colonic environment as closely as is experimentally feasible, to identify peptides that bind to mucosal surface markers of colonic dysplasia. Pilot in vivo human studies showed that at least one of these peptides, VRPMPLQ, shows selective binding to dysplastic tissue over normal mucosa. Imaging performed using a exible-bered confocal microscope showed up to 50 times

Figure 3 In vivo confocal uorescence 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 mm.

In vivo imaging In vivo imaging of dysplastic colon treated with our peptide showed uorescence that correlated with areas containing dysplastic colonocytes (Fig. 2a and Supplementary Fig. 1 online). By contrast, normal mucosa sprayed with peptide showed decreased uorescence associated with residual peptide collecting in regions between crypts (Fig. 2b). Histology conrmed the presence of both dysplastic and normal crypts in the biopsy specimens (Fig. 2c,d). All peptide uorescence signals were greater than background autouorescence (Supplementary Fig. 2 online). Peptide also collected in lumens of normal crypts and appeared as round spots of increased uorescence in the center of a low-uorescence colonocyte layer. The contrast between adenomatous and normal colonocytes was 17.9 4.2 (s.e.m.), with an average signal-to-noise ratio (SNR) of 9.3 0.9 (s.e.m.), for n 18 polyps diagnosed as either tubular adenoma or tubulovillous adenoma. The contrast ranged from 0.9 17.2 (s.d.), indicating variable binding, to 52.3 6.8 (s.d.), indicating substantial peptide binding. Imaging of a border between a dysplastic polyp and normal mucosa provided a reference point that controlled for variables such as incubation time and probe contact pressure, which contributes to imaging depth (Fig. 3 and Supplementary Video 1 online). The contrast between polyp and normal colonocytes calculated at this border region was 22.6 5.7 (s.d.) with an SNR of 17.0 2.5 (s.d.). In contrast to dysplastic polyps, the uorescence associated with hyperplastic polyps (n 5) was predominantly associated with residual peptide collecting in regions between crypts and in crypt lumens. Hyperplastic crypts often had slit-like or stellate-shaped lumens. Fluorescence from an additional four endoscopically polypoid lesions was similar to that of normal mucosa. These specimens were subsequently diagnosed as containing no signicant abnormality upon pathological examination. Imaging using a scrambled control peptide (QLMRPPV) showed no enhanced binding for either dysplastic (n 5) or hyperplastic (n 2) polyps relative to normal mucosa. We subsequently conrmed this result using ex vivo frozen sections (Supplementary Fig. 3 online). Imaging of dysplastic polyps was similar to that of hyperplastic polyps for scrambled control peptide.

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Figure 4 Receiver operating characteristic (ROC) for peptide binding to adenoma versus normal colonocytes. Fluorescein-conjugated peptide showed preferential binding to dysplastic colonocytes relative to adjacent normal cells with 81% sensitivity and 82% specicity.

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inhibitory molecule27, or alterations in tight junction structure in dysplastic tissue28, might allow peptides to bind to previously inaccessible sites. Therefore, reagents discovered through intact tissue panning might have the advantage of selecting for targets that are not differentially expressed, but are clinically relevant because of accessibility. The molecular target of the VRPMPLQ peptide is unknown and the sequence does not show full homology to ligands for known receptors. This peptide does have partial homology (6 of 7 amino acids, V/LRPMPLQ) to the laminin-G domain of contactin-associated protein 1 (Caspr-1), which is present in human intestine and might be involved in contactin-mediated cell signaling and in tumor metastasis and invasion29,30. Given the short amino acid sequences of the candidate peptides, however, homology is of limited predictive value. Several peptides, each targeted towards a different molecular marker, might be required to optimize sensitivity and specicity for detecting dysplasia. Additional biopsy-screened phage are therefore being characterized. The use of a targeted contrast agent for screening would ideally involve an instrument capable of wide-area surveillance used in combination with microscopic high-resolution detection. Currently available prototype uorescence endoscopes measure bulk tissue uorescence, are low in resolution, and tend to wash out contrast from peptide binding owing to considerable autouorescence background. This study used the real-time microstructural information provided by confocal microendoscopy to validate peptide binding, which provides a powerful adjunct to macroscopic imaging. Future wide-area imaging using targeted peptide reagents could be performed by modifying current prototype uorescence endoscopic systems to excite and collect longer wavelengths. The identication of dysplasiatargeting peptides might substantially enhance efforts to detect premalignant lesions in patients with inammatory bowel diseases or Barretts esophagus, conditions for which the in vivo assessment of risk is currently challenging. This methodology could also be developed for detecting other epithelial cancers, assessing disease margins during interventional procedures, monitoring biomarkers for the progression and recurrence of disease, and facilitating targeted drug delivery. METHODS more uorescence due to peptide binding to dysplastic polyps than to normal mucosa. In vivo imaging was performed at a 50-mm working distance, enabling us to image through the surface layer and visualize peptide binding to crypt colonocytes as conrmed by histopathology. Although this peptide currently does not improve on the reported performance of endoscopic confocal microscopy using intravenously administered uorescein25, intravenously administered agents are less practical for clinical use as a screening tool. Peptides, as well as other targeted molecular probes, have the potential to provide quantitative in vivo measures and molecular subtyping of dysplasia for directed therapy with minimal subjective interpretation. They might also serve to mark tumor margins and thereby improve tissue removal. The range in contrast values between individuals in our study indicates that variations in mucus consistency and bowel cleanliness, as well as biological variations between subjects, might affect the performance of this approach. The process of applying and washing specic probes is expected to improve as the technique is used in more subjects. Dysplasia-modied mucins might also be viable targets for peptide reagents. A number of membrane-bound and secreted mucins are differentially expressed in colon adenocarcinomas, and these might serve as targets, given the presence of mucin in our intact tissue-panning specimens26. In addition, the absence of a particular
Panning with excised tissue. We selected peptides that preferentially bound to dysplasia through several rounds of clearing with cell lines and normal tissue and panning with dysplastic polyps (Fig. 5). The rst round of panning was performed using a cleared library from which nonspecically bound phage was removed using Hs738.st/int cells (Supplementary Methods online). We performed clearing with biopsies of normal mucosa before each selection step to reduce nonspecically binding phage before panning with intact polyps from the same individual. We chose this procedure to bias selection towards markers that were present only on dysplastic mucosa rather than those that were overexpressed, with the intention of optimizing uorescence signal-to-background during endoscopic imaging. All polyps used for panning were subsequently stained using hematoxylin and eosin (H&E) and read by two pathologists independently. Cell-binding assays. Of the roughly 6,000 phage eluted from adenomas in the third round of selection, we randomly selected 40 to be tested using HT-29 cells. We performed imaging on four pools of ten phage clones, and one pool was split into ten individual clones for further testing. After antibody incubation with M13 monoclonal antibody conjugated with FITC (Fitzgerald Industries), cells were imaged with a uorescence confocal microscope (Leica TCS SP2). We used increased uorescence intensity as an indicator of phage binding to select the most promising phage clone for further assays. We amplied and assessed this most promising phage clone plus ten additional randomly selected clones from the third round of panning. We determined preferential binding to HT-29 cells over Hs738.st/int cells by measuring the titer of bound phage relative to wild-type insertless phage used as a control.

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Figure 5 Schematic of peptide reagent development procedure. (1) An M13 phage library was cleared with nonmalignant epithelial cells and freshly excised normal human colonic mucosa. (2,3) Panning was then performed using freshly resected human colon polyps. (4,5) Peptides displayed by phage that bound to dysplastic tissue were characterized, synthesized and conjugated to uorescein for use as imaging reagents during endoscopy.

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Ex vivo imaging of phage binding. In addition to cell binding assays, the 40 clones above were also assessed in pools and then as individual clones for binding to intact biopsy specimens. Fresh biopsies were rinsed in fresh DMEM, then incubated for 15 min at room temperature with 5 109 p.f.u. phage in 300 ml DMEM. Biopsies were then rinsed twice with PBS, blocked using 1% BSA for 5 min, rinsed again with PBS, and incubated with 300 ml of 1:1,000 mouse M13 monoclonal antibody conjugated with FITC for 1 h at room temperature. After antibody incubation, biopsies were rinsed twice with PBS and imaging was performed using a commercial confocal microscope (Bio-Rad MRC-1024) at magnications of 1060. All biopsies were subsequently formalin-xed, H&E-stained, and read by staff pathologists at the Palo Alto VA Hospital. Human study. IRB approval was granted by Stanford University Medical Center and the VA Palo Alto Health Care Systems. We recruited individuals undergoing routine screening colonoscopy and obtained informed consent from all individuals. Polyps that were identied endoscopically during routine colonoscopy were washed with water for B5 s to remove excess mucus. Approximately 36 ml of peptide at a concentration of 100 mM was then administered topically to 12 cm2 of the surface of the colon using a standard endoscopic spray catheter. Excess peptide was removed by gently rinsing the region with water. Imaging was performed within B5 min of peptide administration using the Cellvizio-GI confocal uorescence imaging system (Mauna Kea Technologies). The bered confocal microscope was passed through the instrument channel of a standard colonoscope (Olympus CFQ160). Imaging of the polyp and adjacent endoscopically normal-appearing mucosa was performed before and after peptide administration. After imaging, the polyp was removed according to standard protocol, submitted for routine histology, and analyzed by staff pathologists at the Palo Alto VA Hospital. Image and statistical analysis. For each polyp and normal region imaged in vivo, we selected ve representative images according to the following criteria: minimum motion artifact; probe dwell time of less than 2 s; lack of stool or excess mucus preventing contact with the mucosa; and recognizable crypt structure in the images. We calculated uorescence signal intensity by taking the mean of a pixel array corresponding to a 25 25 mm square region within the colonocyte layer for three sites within each image. The signal noise was calculated by taking the standard deviation (s.d.) of the values in this pixel array. The uorescence contrast was obtained from the ratio of the mean polyp uorescence intensity to the mean normal uorescence intensity over all sites within each subject. The SNR was obtained from the ratio of the mean of the polyp signal to signal noise for each site within each subject. Average uorescence signal contrast and SNR attributed to the peptide were then calculated by averaging the values obtained for each subject. Standard errors of the mean (s.e.m.) were reported based on n 18 polyps. The receiver operating characteristic (ROC) for adenoma versus normal was calculated using mean uorescence signal values for each image site using n 270 values for adenoma and n 270 values for normal.
Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS The authors acknowledge funding support from the US National Institutes of Health, including U54 CA105296 (NCI), K08 DK067618 (NIDDK) and P30 DK56339 (DDC Pilot Award), from the Doris Duke Charitable Foundation, from the Stanford School of Medicine Deans Fellowship (P.-L.H.) and from the Cynthia Fry Gunn Research Fund. P.-L.H. is supported by the Canary Foundation/American Cancer Society Early Detection Postdoctoral Fellowship. We thank J. Kosek for support with histopathological interpretation and Mauna Kea Technologies for technical support and use of their clinical Cellvizio-GI imaging system. AUTHOR CONTRIBUTIONS P.-L.H. conducted most of the experiments, with help from A.P.W. and J.H. (library clearing and cell line screening), C.B.D., P.S. and T.D.W. (clinical coordination), and T.D.W., S.F. and R.S. (in vivo studies). P.-L.H., J.H. and T.D.W. conducted the data analysis. P.-L.H., J.H., J.M.C., A.W.L., C.H.C. and T.D.W. were responsible for the concepts and writing of the manuscript. R.S., C.H.C. and T.D.W. supervised the project.
Published online at http://www.nature.com/naturemedicine Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions

1. Vogelstein, B. et al. Genetic alterations during colorectal-tumor development. N. Engl. J. Med. 319, 525532 (1988). 2. Winawer, S.J. et al. Prevention of colorectal cancer by colonoscopic polypectomy. The National Polyp Study Workgroup. N. Engl. J. Med. 329, 19771981 (1993). 3. American Cancer Society. Cancer Facts & Figures 2007 (American Cancer Society, Atlanta, 2006). 4. Kudo, S. et al. Colonoscopic diagnosis and management of nonpolypoid early colorectal cancer. World J. Surg. 24, 10811090 (2000). 5. Mayer, R., Wong, W.D., Rothenberger, D.A., Goldberg, S.M. & Madoff, R.D. Colorectal cancer in inammatory bowel disease: a continuing problem. Dis. Colon Rectum 42, 343347 (1999). 6. Garcea, G. et al. Molecular biomarkers of colorectal carcinogenesis and their role in surveillance and early intervention. Eur. J. Cancer 39, 10411052 (2003). 7. Goldsmith, S.J. Receptor imaging: competitive or complementary to antibody imaging? Semin. Nucl. Med. 27, 8593 (1997). 8. Scott, J.K. & Smith, G.P. Searching for peptide ligands with an epitope library. Science 249, 386390 (1990). 9. Kelly, K.A. & Jones, D.A. Isolation of a colon tumor specic binding peptide using phage display selection. Neoplasia 5, 437444 (2003). 10. Abraham, J.M. et al. Novel decapeptides that bind avidly and deliver radioisotope to colon cancer cells. PLoS ONE 2, e964 (2007). 11. Kelly, K., Alencar, H., Funovics, M., Mahmood, U. & Weissleder, R. Detection of invasive colon cancer using a novel, targeted, library-derived uorescent peptide. Cancer Res. 64, 62476251 (2004). 12. Zangani, D. et al. Multiple differentiation pathways of rat mammary stromal cells in vitro: acquisition of a broblast, adipocyte or endothelial phenotype is dependent on hormonal and extracellular matrix stimulation. Differentiation 64, 91101 (1999). 13. Walters, R.W. et al. Basolateral localization of ber receptors limits adenovirus infection from the apical surface of airway epithelia. J. Biol. Chem. 274, 1021910226 (1999). 14. Pasqualini, R. & Ruoslahti, E. Organ targeting in vivo using phage display peptide libraries. Nature 380, 364366 (1996). 15. Carson-Walter, E.B. et al. Cell surface tumor endothelial markers are conserved in mice and humans. Cancer Res. 61, 66496655 (2001). 16. Arap, W. et al. Steps toward mapping the human vasculature by phage display. Nat. Med. 8, 121127 (2002). 17. Wang, T.D. et al. Functional imaging of colonic mucosa with a bered confocal microscope for real-time in vivo pathology. Clin. Gastroenterol. Hepatol. 5, 13001305 (2007). 18. Fujimoto, J.G. Optical coherence tomography for ultrahigh resolution in vivo imaging. Nat. Biotechnol. 21, 13611367 (2003). 19. Wang, T.D. et al. Fluorescence endoscopic imaging of human colonic adenomas. Gastroenterology 111, 11821191 (1996). 20. Haringsma, J. et al. Autouorescence endoscopy: feasibility of detection of GI neoplasms unapparent to white light endoscopy with an evolving technology. Gastrointest. Endosc. 53, 642650 (2001). 21. Machida, H. et al. Narrow-band imaging in the diagnosis of colorectal mucosal lesions: a pilot study. Endoscopy 36, 10941098 (2004). 22. Messmann, H., Knuchel, R., Baumler, W., Holstege, A. & Scholmerich, J. Endoscopic uorescence detection of dysplasia in patients with Barretts esophagus, ulcerative colitis, or adenomatous polyps after 5-aminolevulinic acid-induced protoporphyrin IX sensitization. Gastrointest. Endosc. 49, 97101 (1999). 23. Folli, S. et al. Immunophotodiagnosis of colon carcinomas in patients injected with uoresceinated chimeric antibodies against carcinoembryonic antigen. Proc. Natl. Acad. Sci. USA 89, 79737977 (1992). 24. Keller, R., Winde, G., Terpe, H.J., Foerster, E.C. & Domschke, W. Fluorescence endoscopy using a uorescein-labeled monoclonal antibody against carcinoembryonic antigen in patients with colorectal carcinoma and adenoma. Endoscopy 34, 801807 (2002). 25. Kiesslich, R. et al. Confocal laser endoscopy for diagnosing intraepithelial neoplasias and colorectal cancer in vivo. Gastroenterology 127, 706713 (2004). 26. Ho, S.B. et al. Heterogeneity of mucin gene expression in normal and neoplastic tissues. Cancer Res. 53, 641651 (1993). 27. Moore, A., Medarova, Z., Potthast, A. & Dai, G. In vivo targeting of underglycosylated MUC-1 tumor antigen using a multimodal imaging probe. Cancer Res. 64, 18211827 (2004). 28. Soler, A.P. et al. Increased tight junctional permeability is associated with the development of colon cancer. Carcinogenesis 20, 14251431 (1999). 29. Peles, E. et al. Identication of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein-protein interactions. EMBO J. 16, 978988 (1997). 30. Su, J.L. et al. Knockdown of contactin-1 expression suppresses invasion and metastasis of lung adenocarcinoma. Cancer Res. 66, 25532561 (2006).

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Corrigendum: Protein kinase CK2 links extracellular growth factor signaling with the control of p27Kip1 stability in the heart
Ludger Hauck, Christoph Harms, Junfeng An, Jens Rohne, Karen Gertz, Rainer Dietz, Matthias Endres & Rdiger von Harsdorf Nat. Med. 14, 315324 (2008); published online 2 March 2008; corrected after print 21 March 2008 In the version of this article initally published, one author, Junfeng An, was missing from the author list and the Author Contributions section. The errors have been corrected in the HTML and PDF versions of the article. 2008 Nature Publishing Group http://www.nature.com/naturemedicine

Corrigendum: Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability
Christopher A Jones, Nyall R London, Haoyu Chen, Kye Won Park, Dominique Sauvaget, Rebecca A Stockton, Joshua D Wythe, Wonhee Suh, Frederic Larrieu-Lahargue, Yoh-suke Mukouyama, Per Lindblom, Pankaj Seth, Antonio Frias, Naoyuki Nishiya, Mark H Ginsberg, Holger Gerhardt, Kang Zhang & Dean Y Li Nat. Med. 14, 448453 (2008); published online 16 March 2008; corrected after print 18 April 2008 In the version of this article initially published, the affiliation of Rebecca A. Stockton was incorrect. Her correct affiliation is affiliation 5: the Department of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, California 92093-0726, USA. The error has been corrected in the HTML and PDF versions of the article.

Erratum: Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy
Pei-Lin Hsiung, Jonathan Hardy, Shai Friedland, Roy Soetikno, Christine B Du, Amy P Wu, Peyman Sahbaie, James M Crawford, Anson W Lowe, Christopher H Contag & Thomas D Wang Nat. Med.14, 454458 (2008); published online 16 March 2008; corrected 21 March 2008 In the version of this article initially published online, the name of the first author, Pei-Lin Hsiung, was misspelled as Pei-Lei Hsiung. The error has been corrected in all versions of the article.

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