Thy1-Targeted Microbubbles for Ultrasound Molecular Imaging of Pancreatic Ductal Adenocarcinoma
Lotfi Abou-Elkacem, Huaijun Wang, Sayan M Chowdhury, Richard H. Kimura, Sunitha V Bachawal, Sanjiv S Gambhir, Lu Tian, Juergen K. WillmannClinical Cancer Research2018
Purpose: To engineer a dual human and murine Thy1-binding single-chain-antibody ligand (Thy1-scFv) for contrast microbub- ble–enhanced ultrasound molecular imaging of pancreatic ductal adenocarcinoma (PDAC). Experimental Design:Thy1-scFvwere 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 micro- bubbles to create MBThy1-scFv. Thy1 binding of MBThy1-scFv to Thy1-expressing cells was evaluated under flow shear stress conditions in flow-chamber experiments. MBscFv-scrambled and MBNon-targeted were used as negative controls. All microbubble types were tested in both orthotopic human PDAC xenografts and transgenic PDAC mice in vivo. Results: Thy1-scFv had a KD 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. MBThy1-scFv was attachedtoThy1withhighaffinity compared with negative controlmicrobubbles (P < 0.01) as assessed by flow cytometry. Similarly, flow-chamber studies showed significantly (P < 0.01) higher binding of MBThy1-scFv (3.0 ? 0.81 MB/cell) to Thy1- expressing cells than MBscFv-scrambled (0.57 ? 0.53) andMBNon- targeted (0.43 ? 0.53). In vivo ultrasound molecular imaging using MBThy1-scFv demonstrated significantly higher signal (P < 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.). Ex vivo immunofluorescence confirmed sig- nificantly (P < 0.01) increased Thy1 expression in PDAC com- pared with chronic pancreatitis and normal pancreas tissue. Conclusions: A dual human and murine Thy1-binding scFv was designed to generate contrast microbubbles to allow PDAC detection with ultrasound.