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

Abstract

<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).<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_Thy1-scFv Thy1 binding of MB_Thy1-scFv to Thy1-expressing cells was evaluated under flow shear stress conditions in flow-chamber experiments. MB_{scFv-scrambled} and MB_Non-targeted were used as negative controls. All microbubble types were tested in both orthotopic human PDAC xenografts and transgenic PDAC mice <i>in vivo</i><b>Results:</b> Thy1-scFv had a <i>K</i>_D 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_Thy1-scFv 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_Thy1-scFv (3.0 ± 0.81 MB/cell) to Thy1-expressing cells than MB_{scFv-scrambled} (0.57 ± 0.53) and MB_Non-targeted (0.43 ± 0.53). <i>In vivo</i> ultrasound molecular imaging using MB_Thy1-scFv 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.<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>.