Limits...
EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging.

Zhou J, Joshi BP, Duan X, Pant A, Qiu Z, Kuick R, Owens SR, Wang TD - Clin Transl Gastroenterol (2015)

Bottom Line: No downstream signaling was observed.We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively.On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT

Objectives: Colorectal cancer initially lies dormant as dysplasia, a premalignant state that provides an opportunity for early cancer detection. Dysplasia can be flat in morphology, focal in size, and patchy in distribution, and thus it appears "invisible" on conventional wide-field endoscopy.

Aims: We aim to develop and validate a peptide that is specific for epidermal growth factor receptor (EGFR), a cell surface target that is overexpressed in colonic adenomas and is readily accessible for imaging.

Methods: We expressed and purified the extracellular domain of EGFR for use with phage display to identify a peptide QRHKPRE that binds to domain 2 of this target. A near-infrared fluorescence endoscope was used to perform in vivo imaging to validate specific peptide binding to spontaneous colonic adenomas in a mouse model with topical administration. We also validated specific peptide binding to human colonic adenomas on immunohistochemistry and immunofluorescence.

Results: After labeling with Cy5.5, we validated specific peptide binding to EGFR on knockdown and competition studies. Peptide binding to cells occurred within 2.46 min and had an affinity of 50 nm. No downstream signaling was observed. We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively. On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

Conclusions: We have selected and validated a peptide that can be used as a specific contrast agent to identify colonic adenomas that overexpress EGFR in vivo on fluorescence endoscopy.

No MeSH data available.


Related in: MedlinePlus

Peptide specific for EGFR. (a) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). (b) Scrambled peptide PEHKRRQ (control). (c) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). (d) Fluorescence spectra of Cy5.5-labeled peptides with λex=671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4816258&req=5

fig1: Peptide specific for EGFR. (a) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). (b) Scrambled peptide PEHKRRQ (control). (c) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). (d) Fluorescence spectra of Cy5.5-labeled peptides with λex=671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.

Mentions: After four rounds of biopanning with phage display, 10 sequences showed enrichment. We evaluated the binding of each candidate on a structural model,38, 39 and found a minimum energy of Et=−504.1 for docking of QRHKPRE labeled with Cy5.5 to EGFR (1IVO) (http://www.rcsb.org/pdb). We synthesized this sequence (black) and attached a Cy5.5 (red) fluorophore via a GGGSK linker (blue) on the C terminus, hereafter QRH*-Cy5.5, to prevent steric hindrance, Figure 1a. Cy5.5 was chosen for its high-quantum yield and photostability.40 We used this model to develop a scrambled sequence PEHKRRQ for control, hereafter PEH*-Cy5.5, Figure 1b, and found Et=−493.1. In the model, QRH*-Cy5.5 binds to amino acids 230–310 of EGFR (domain 2), Figure 1c. The fluorescence spectra of QRH*-Cy5.5 and PEH*-Cy5.5 at 10 μm concentration in PBS with λex=671 nm excitation revealed a peak emission at 710 nm, Figure 1d. We purified the Cy5.5-labeled peptides to >97% on high-performance liquid chromatography, and measured an experimental mass-to-charge (m/z) ratio on mass spectrometry of 1900.05 for both QRH*-Cy5.5 and PEH*-Cy5.5 that agreed with expected values, Supplementary Figure S2A, B.


EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging.

Zhou J, Joshi BP, Duan X, Pant A, Qiu Z, Kuick R, Owens SR, Wang TD - Clin Transl Gastroenterol (2015)

Peptide specific for EGFR. (a) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). (b) Scrambled peptide PEHKRRQ (control). (c) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). (d) Fluorescence spectra of Cy5.5-labeled peptides with λex=671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816258&req=5

fig1: Peptide specific for EGFR. (a) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). (b) Scrambled peptide PEHKRRQ (control). (c) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). (d) Fluorescence spectra of Cy5.5-labeled peptides with λex=671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.
Mentions: After four rounds of biopanning with phage display, 10 sequences showed enrichment. We evaluated the binding of each candidate on a structural model,38, 39 and found a minimum energy of Et=−504.1 for docking of QRHKPRE labeled with Cy5.5 to EGFR (1IVO) (http://www.rcsb.org/pdb). We synthesized this sequence (black) and attached a Cy5.5 (red) fluorophore via a GGGSK linker (blue) on the C terminus, hereafter QRH*-Cy5.5, to prevent steric hindrance, Figure 1a. Cy5.5 was chosen for its high-quantum yield and photostability.40 We used this model to develop a scrambled sequence PEHKRRQ for control, hereafter PEH*-Cy5.5, Figure 1b, and found Et=−493.1. In the model, QRH*-Cy5.5 binds to amino acids 230–310 of EGFR (domain 2), Figure 1c. The fluorescence spectra of QRH*-Cy5.5 and PEH*-Cy5.5 at 10 μm concentration in PBS with λex=671 nm excitation revealed a peak emission at 710 nm, Figure 1d. We purified the Cy5.5-labeled peptides to >97% on high-performance liquid chromatography, and measured an experimental mass-to-charge (m/z) ratio on mass spectrometry of 1900.05 for both QRH*-Cy5.5 and PEH*-Cy5.5 that agreed with expected values, Supplementary Figure S2A, B.

Bottom Line: No downstream signaling was observed.We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively.On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT

Objectives: Colorectal cancer initially lies dormant as dysplasia, a premalignant state that provides an opportunity for early cancer detection. Dysplasia can be flat in morphology, focal in size, and patchy in distribution, and thus it appears "invisible" on conventional wide-field endoscopy.

Aims: We aim to develop and validate a peptide that is specific for epidermal growth factor receptor (EGFR), a cell surface target that is overexpressed in colonic adenomas and is readily accessible for imaging.

Methods: We expressed and purified the extracellular domain of EGFR for use with phage display to identify a peptide QRHKPRE that binds to domain 2 of this target. A near-infrared fluorescence endoscope was used to perform in vivo imaging to validate specific peptide binding to spontaneous colonic adenomas in a mouse model with topical administration. We also validated specific peptide binding to human colonic adenomas on immunohistochemistry and immunofluorescence.

Results: After labeling with Cy5.5, we validated specific peptide binding to EGFR on knockdown and competition studies. Peptide binding to cells occurred within 2.46 min and had an affinity of 50 nm. No downstream signaling was observed. We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively. On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

Conclusions: We have selected and validated a peptide that can be used as a specific contrast agent to identify colonic adenomas that overexpress EGFR in vivo on fluorescence endoscopy.

No MeSH data available.


Related in: MedlinePlus