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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

Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of (a) QRH*-Cy5.5 peptide (red) and (b) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). (c) PEH*-Cy5.5 (red) binding is minimal. (d–f) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). (g) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P=0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P=0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 (P=0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. (h) Western blot shows EGFR expression levels. (i) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μm and higher. Nonsignficant difference was found at 0 μm. We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P-values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μm. Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.
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fig2: Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of (a) QRH*-Cy5.5 peptide (red) and (b) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). (c) PEH*-Cy5.5 (red) binding is minimal. (d–f) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). (g) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P=0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P=0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 (P=0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. (h) Western blot shows EGFR expression levels. (i) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μm and higher. Nonsignficant difference was found at 0 μm. We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P-values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μm. Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.

Mentions: We validated specific binding of QRH*-Cy5.5 to EGFR on siRNA knockdown in HT29 cells. On confocal microscopy, QRH*-Cy5.5- (red), Figure 2a, and AF488-labeled anti-EGFR (green), Figure 2b, bind strongly to the surface (arrows) of control HT29 cells, transfected with siCL nontargeting siRNA. PEH*-Cy5.5 shows minimal binding, Figure 2c. Reduced fluorescence intensity was observed for HT29 knockdown cells, transfected with siEGFR-targeting siRNA, Figure 2d, e. PEH*-Cy5.5 shows minimal binding, Figure 2f. QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, Figure 2g. Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR antibody were significantly greater than those for PEH*-Cy5.5. EGFR expression is shown on the western blot, Figure 2h.


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)

Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of (a) QRH*-Cy5.5 peptide (red) and (b) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). (c) PEH*-Cy5.5 (red) binding is minimal. (d–f) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). (g) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P=0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P=0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 (P=0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. (h) Western blot shows EGFR expression levels. (i) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μm and higher. Nonsignficant difference was found at 0 μm. We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P-values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μm. Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.
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fig2: Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of (a) QRH*-Cy5.5 peptide (red) and (b) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). (c) PEH*-Cy5.5 (red) binding is minimal. (d–f) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). (g) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P=0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P=0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 (P=0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. (h) Western blot shows EGFR expression levels. (i) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μm and higher. Nonsignficant difference was found at 0 μm. We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P-values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μm. Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.
Mentions: We validated specific binding of QRH*-Cy5.5 to EGFR on siRNA knockdown in HT29 cells. On confocal microscopy, QRH*-Cy5.5- (red), Figure 2a, and AF488-labeled anti-EGFR (green), Figure 2b, bind strongly to the surface (arrows) of control HT29 cells, transfected with siCL nontargeting siRNA. PEH*-Cy5.5 shows minimal binding, Figure 2c. Reduced fluorescence intensity was observed for HT29 knockdown cells, transfected with siEGFR-targeting siRNA, Figure 2d, e. PEH*-Cy5.5 shows minimal binding, Figure 2f. QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, Figure 2g. Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR antibody were significantly greater than those for PEH*-Cy5.5. EGFR expression is shown on the western blot, Figure 2h.

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