Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: On confocal microscopy, fluorescence from binding of candidate heterodimer with linkers A) E2, B) Hex, C) E3, D) E6, and E) E10 to the surface (arrow) of SKBr3 cells can be seen. F) Quantified results show that the E3 linker provides the highest mean fluorescence intensity. P-values were determined using unpaired t-test. Measurements are an average of 10 randomly chosen cells from 4 images collected independently. G) Western blot shows EGFR and ErbB2 expression in SKBr3 and QhTERT cells.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Confocal Microscopy, Fluorescence, Binding Assay, Western Blot, Expressing

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: On representative confocal microscopy images of human esophageal specimens ex vivo, QRH*-KSP*-E3-Cy5.5 (red) shows minimal staining to A) squamous (SQ) and B) Barrett’s esophagus (BE), and increased intensity with C) high-grade dysplasia (HGD) and D) esophageal adenocarcinoma (EAC). Similar results were found with AF568-labeled anti-EGFR antibody (yellow) and for AF488-labeled anti-ErbB2 antibody (green). Merged images show co-localization of peptide and antibody binding.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Confocal Microscopy, Ex Vivo, Staining, Labeling, Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: In siRNA knockdown experiments, QRH*-KSP*-E3-Cy5.5 (red) shows significantly greater binding to the surface (arrows) of A) siCL (control) SkBr3 cells compared with that for B) siEGFR (knockdown) cells. Similar results were found for C) siCL and D) siErbB2 (knockdown) cells. E) Quantified results show significantly greater intensity for siCL versus siEGFR and siCL versus siErbB2, P=3.6×10−4 and P=7.8×10−3, respectively, by unpaired t-test. The mean value was calculated from 5 cells chosen randomly from 3 images collected independently. F) Western blot shows EGFR and ErbB2 expression in control and knockdown cells. G) The apparent dissociation constant (binding affinity) for QRH*-KSP*-E3-Cy5.5 was found to be kd = 23 versus 98 and 54 nM for QRH*-Cy5.5 and KSP*-Cy5.5. H) The apparent association time constant for QRH*-KSP*-E3-Cy5.5 was found to be k = 0.22 min−1 (4.5 min) versus 0.21 min−1 (4.8 min) and 0.35 min−1 (2.9 min) for QRH*-Cy5.5 and KSP*-Cy5.5. Results for each measurement are representative of 3 independent experiments.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Binding Assay, Western Blot, Expressing

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: We evaluated the effect of the QRH*-KSP*-E3-Cy5.5 on downstream cell signaling after binding to SKBr3 cells. On Western blot, we observed no change in phosphorylation of EGFR (p-EGFR), ErbB2 (p-ErbB2) or of downstream AKT (p-AKT) and ERK (p-ERK) with incubation of heterodimer at 1, 5, and 20 μM. By comparison, the addition of EGF, an endogenous ligand for EGFR, showed increased expression of p-AKT and p-ERK. The addition of 100 nM of lapatinib, a tyrosine kinase inhibitor known to interrupt EGFR/ErbB2 signaling in solid tumors, showed reduced expression of p-EGFR, p-ErbB2 and p-AKT. Cells treated with 1% DMSO and untreated cells showed no suppression of EGFR and ErbB2 mediated signaling. β-tubulin is used as loading control.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Binding Assay, Western Blot, Incubation, Expressing

Journal: Journal of medicinal chemistry

Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

doi: 10.1021/acs.jmedchem.8b00405

Figure Lengend Snippet: A) On confocal microscopy, serial sections of HGD in human esophageal specimens are shown following staining with QRH*-KSP*-E3-Cy5.5 (red), anti-EGFR antibody labeled with AF568 (yellow) and anti-ErbB2 antibody labeled with AF488 (green). Fluorescence intensities were quantified from the mean of a set of 3 boxes with dimensions of 20×20 μm2 placed over random crypts. Co-localization of binding can be appreciated on the merged image. B) High-magnification images are shown from dashed boxes. On the merged image, Pearson’s correlation coefficient of ρ = 0.60 and 0.75 was measured for EGFR and ErbB2, respectively. C) From n = 31, 8, 23, and 12 specimens of SQ, BE, HGD, and EAC, respectively, we found significantly greater mean fluorescence intensity from HGD and EAC compared with that for BE and SQ with QRH*-KSP*-E3-Cy5.5, the P-value for difference are calculated by Tukey’s multiple comparisons. A similar result was found for anti-EGFR-AF568 and anti-ErbB2-AF488. D) ROC curve shows 88% sensitivity, 87% specificity and 0.95 AUC with QRH*-KSP*-E3-Cy5.5; 74% sensitivity, 69% specificity, and 0.79 AUC with QRH*-Cy5.5, and 85% sensitivity, 79% specificity, and 0.91 AUC with KSP*-Cy5.5.

Article Snippet: Anti-EGFR antibody (#2232S), anti-HER2 antibody (#2165), anti-phospho-EGFR sampler kit (#9922s), anti-phospho-HER2/ErbB2 (Tyr1248) antibody (#2247), anti-AKT (#4691P), anti-ERK1/2 (#4695P), anti-phospho-AKT (pS473; #4060P), anti-phospho-ERK1/2 (#4370P) were used per manufacturer’s instructions (Cell Signaling, Inc).

Techniques: Confocal Microscopy, Staining, Labeling, Fluorescence, Binding Assay