Fig 1: Binding of FGFR2 peptide to Barrett's neoplasiaOn representative images collected with confocal microscopy of human esophageal specimens ex vivo, SRR*-Cy5.5 (red) shows minimal staining to (A) squamous (SQ) and (B) Barrett's esophagus (BE) and strong binding (arrows) to (C) high-grade dysplasia (HGD) and (D) esophageal adenocarcinoma (EAC). (E-H) Anti-FGFR2 antibody labeled with AF488 (green) was used as a positive control, and shows weak staining to SQ and BE but strong binding (arrows) to HGD and EAC. We quantified the fluorescence intensities from the mean of a set of 3 boxes with dimensions of 30×30 µm2 placed over cells, shown in panels (C) and (G). From n = 28, 33, 22, and 17 specimens of SQ, BE, HGD, and EAC, respectively, we found significantly greater mean fluorescence intensity from HGD and EAC compared with that for BE with (I) SRR*-Cy5.5 and (J) AF488-labeled anti-FGFR2 using an ANOVA model with terms for 4 means on log-transformed data. (K) ROC curve shows 87% sensitivity and 70% specificity for detecting Barrett's neoplasia (HGD and EAC) at a T/B ratio of 3.0. (L-O) Merged images shows co-localization of peptide (red) and antibody (green) binding. We determined a Pearson's correlation coefficient of ? = 0.59, 0.54, 0.52 and 0.59 for SQ, BE, HGD and EAC, respectively. Representative histology (H&E) are shown for (P) SQ, (Q) BE, (R) HGD, and (S) EAC.
Fig 2: Peptide specific for FGFR2Chemical structure is shown for 12 amino acid (aa) peptide sequence (A) SRRPASFRTARE (SRR*) found to be specific for FGFR2, and (B) scrambled peptide SPSRERTFRARA (SPS*) used for control. A Cy5.5 fluorophore (red) is attached via a GGGSK linker (blue) to prevent steric hindrance. (C) SRR*-Cy5.5 was found using a structural model (1EV2) to bind to the extracellular domain (ECD) of FGFR2c (147-366 aa) with Et = -290.43 while SPS*-Cy5.5 resulted in Et = -277.37. (D) Fluorescence spectra of SRR*-Cy5.5 and SPS*-Cy5.5 at 10 µM concentration in PBS with excitation at ?ex = 671 nm shows peak emission at ?em = 710 nm in the NIR spectrum.
Fig 3: FGFR2 peptide does not affect cell signalingWestern blot shows no obvious change in phosphorylation for either FGFR2 (p-FGFR) or downstream AKT (p-AKT) and ERK (p-ERK) with addition of SRR* peptide at 5 and 100 µM to QhTERT cells that express FGFR2b or FGFR2c compared with untreated cells. Addition of FGF1 as positive control to bind FGFR2b and FGFR2c shows phosphorylation activity for FGFR2 (p-FGFR), downstream AKT (p-AKT) and ERK (p-ERK), especially in QhTERT cells expressing FGFR2c.
Fig 4: Validation of specific FGFR2 peptide binding to cellsOn confocal microscopy, we observed strong binding of SRR*-Cy5.5 (red) to surface of QhTERT cells that express (A) FGFR2b and (B) FGFR2c compared with (C) wild type. (D-F) Minimal signal is seen with the scrambled peptide SPS*-Cy5.5. (G-I) Strong binding is seen with anti-FGFR2 antibody labeled with AF488 (green) used as positive control. All experiments were performed in triplicate. (J) Quantified results show significantly higher mean fluorescence intensities for SRR*-Cy5.5 versus SPS*-Cy5.5 (control). We log-transformed and averaged measurements for 3 random cells on each of 3 slides per condition, and fit an ANOVA model with terms for 6 means. (K) Western blot shows protein expression level of FGFR2 for each cell.
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