Peptide-Based Probe Targeting Angiotensin Type-1 Receptor Identifies Pancreatic Ductal Adenocarcinoma Using Multispectral Optoacoustic Tomography

Figures

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  1. Assessment of AT1R in PDAC and control cells: (a) AT1R expression in various cell lines, including S2VP10L, S2O13Q, SUIT2, and HT-29. Note the absence of the AT1R band in HT-29 cells. β-Actin was used as the loading control. (b–d) Images showing the immunohistochemical localization of AT1R in S2VP10L and SUIT2. (e) Bar graph showing the percentage of positive DAB-stained area in cell lines (Mean ± SD; N = 3), one-way ANOVA, significant difference at P < 0.05.

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  2. Docking analysis and membrane localization of the probe in PDAC cells: (a) schematic showing Angio-1 peptide structure and its conjugation with dye, resulting in Angio-1 probe synthesis. (b) Docking analysis performed with Angio-1 peptide against AT1R receptor (PDB ID: 4YAY) using ClusterPro Version X. (c) AT1R-interacting residues with Angio-1 peptide were resolved using Pymol Viewer. (d) Immunofluorescence localization of membranous AT1R using Angio-1-555 on PDAC (S2VP10L, S2O13Q) and reduced binding with low AT1R-expressing colon adenocarcinoma cells (HT-29 cells). Note the membrane labeling of the Angio-1 555 probe on the cell membrane. The TRITC signal (red) was detected. DAPI stains the nuclei blue.

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  3. In vitro binding of the Angio-1-750 probe with the targeted receptor: (a) Angio-1-750 probe shows maximum absorption at 750 nm. (b) Angio-1-750 probe (10 μM) shows differential binding and detection of S2VP10, S2O13, and SUIT2 cells using NIR imaging. (c) Bar graph showing significant difference in the fluorescence intensity of Angio-1-750 probe-treated S2VP10L, S2O13Q, SUIT2 vs HT-29 cells (Mean ± SD; N = 3; significant difference at P < 0.05).

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  4. Representative axial tomographical slices of S2VP10L and S2O13Q demonstrate the localization of Angio-1-750 signal overlapping the region of the tumor bed in implanted mice. (a) Each slice from 36 to 38 mm demonstrates Angio-1-750 (aqua depicts the signal) localization at 3 h and (d) 24 h for S2VP10L and S2O13Q orthotopic mouse models with an approximate tumor size of 3 mm. Treated mice overlapping the gray scale image at 90 nm (b) MSOT (a.u.) signal at 3 h in S2VP10L and (c) S2O13Q models (Mean ± SD; N = 3). (e) MSOT (a.u.) signal at 24 h in S2VP10L and (f) S2O13Q models.

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  5. Tumor detection and analysis of the Angio-1-750 probe biodistribution using NIR fluorescence. (a) Representative mouse injected with luciferin showed bioluminescence from the tumor in S2VP10L- and S2O13Q-implanted mice. (b) Representative mouse showing biodistribution and clearance of the probe at 3 and 24 h. (c) Ex vivo distribution of the probe in representative organs following 24 h of injection using AMI. (d) Bar graph showing signal intensity (Photon/sec) of the Angio-1-750 probe in ex vivo organs of S2VP10L-implanted mice after 24 h of injection (N = 3). (e) Bar graph showing the signal intensity (photons/sec) of the Angio-1–750 probe in ex vivo organs of S2013Q-implanted mice after 24 h of injection.

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  6. Representative H&E-stained sections depict the architectural details of various organs, including the spleen, liver, and kidney, to assess toxicity following the injection of the Angio-1-750 probe 24 h postadministration. (a, d, g) Spleen sections demonstrating normal architecture of red and white pulp in the treated and control groups. Similarly, (b, e, h) kidney sections of treated mice did not show any microscopic changes in glomeruli and tubules compared to the control. (c, f, i) Liver showed normal architecture detail of hepatocytes as well as the central vein. H&E,10×, Bar: 20 μm.