Data from Thy1-Targeted Microbubbles for Ultrasound Molecular Imaging of Pancreatic Ductal Adenocarcinoma

Abstract

<div>Abstract<p><b>Purpose:</b> To engineer a dual human and murine Thy1-binding single-chain-antibody ligand (Thy1-scFv) for contrast microbubble–enhanced ultrasound molecular imaging of pancreatic ductal adenocarcinoma (PDAC).</p><p><b>Experimental Design:</b> Thy1-scFv were engineered using yeast-surface-display techniques. Binding to soluble human and murine Thy1 and to Thy1-expressing cells was assessed by flow cytometry. Thy1-scFv was then attached to gas-filled microbubbles to create MB<sub>Thy1-scFv</sub>. Thy1 binding of MB<sub>Thy1-scFv</sub> to Thy1-expressing cells was evaluated under flow shear stress conditions in flow-chamber experiments. MB<sub>scFv-scrambled</sub> and MB<sub>Non-targeted</sub> were used as negative controls. All microbubble types were tested in both orthotopic human PDAC xenografts and transgenic PDAC mice <i>in vivo</i>.</p><p><b>Results:</b> Thy1-scFv had a <i>K</i><sub>D</sub> of 3.4 ± 0.36 nmol/L for human and 9.2 ± 1.7 nmol/L for murine Thy1 and showed binding to both soluble and cellularly expressed Thy1. MB<sub>Thy1-scFv</sub> was attached to Thy1 with high affinity compared with negative control microbubbles (<i>P</i> < 0.01) as assessed by flow cytometry. Similarly, flow-chamber studies showed significantly (<i>P</i> < 0.01) higher binding of MB<sub>Thy1-scFv</sub> (3.0 ± 0.81 MB/cell) to Thy1-expressing cells than MB<sub>scFv-scrambled</sub> (0.57 ± 0.53) and MB<sub>Non-targeted</sub> (0.43 ± 0.53). <i>In vivo</i> ultrasound molecular imaging using MB<sub>Thy1-scFv</sub> demonstrated significantly higher signal (<i>P</i> < 0.01) in both orthotopic (5.32 ± 1.59 a.u.) and transgenic PDAC (5.68 ± 2.5 a.u.) mice compared with chronic pancreatitis (0.84 ± 0.6 a.u.) and normal pancreas (0.67 ± 0.71 a.u.). <i>Ex vivo</i> immunofluorescence confirmed significantly (<i>P</i> < 0.01) increased Thy1 expression in PDAC compared with chronic pancreatitis and normal pancreas tissue.</p><p><b>Conclusions:</b> A dual human and murine Thy1-binding scFv was designed to generate contrast microbubbles to allow PDAC detection with ultrasound. <i>Clin Cancer Res; 24(7); 1574–85. ©2018 AACR</i>.</p></div>