Abstract
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Objectives Due to hepatic clearance of the vast majority of contrast agents, molecular imaging detection of hepatic malignancies is challenging. To our advantage, overexpression of CD146 has been associated with aggressiveness and metastatic potential in liver cancer. Our goal is to develop a CD146-targeted probe for high-contrast noninvasive in vivo positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of liver cancer.
Methods In vitro expression levels of CD146 were characterized in two liver cancer cell lines, HEPG2 (+) and Huh7 (-) via western blot, flow cytometry, and immunofluorescent staining. YY146, an anti-CD146 monoclonal antibody, was conjugated to the NIRF dye ZW800-1 and to deferoxamine (Df) for radiolabeling with 89Zr. Sequential PET and NIRF imaging were performed after intravenous injection of 3.7 - 7.4 MBq (400 pmol) of 89Zr-Df-YY146-ZW800 in athymic nude mice bearing HepG2 or Huh7 subcutaneous (s.c.) xenografts. Orthotopic tumors were generated by stereotactic injection of luciferase-transfected HepG2 cells into the left lobe of the liver and their progression was monitored by bioluminescence imaging. Multimodality imaging was carried out in mice with confirmed orthotopic liver tumors using the condition described for s.c. tumors. A terminal imaging time-point was acquired 168 h post-injection (p.i.) of the tracer, after which tissues were collected for ex vivo NIRF imaging, biodistribution, and histological studies.
Results In vitro assays revealed a high and low CD146 expression levels in HepG2 and Huh7 cells, respectively. Flow cytometry showed similar CD146-binding affinity between the native and conjugated YY146 mAb. PET and NIRF imaging unveiled a prominent and persistent uptake of 89Zr-Df-YY146-ZW800 in HepG2 tumors that peaked at 31.7 ± 7.2 %ID/g (n=4), 72 h p.i.. Owing to such marked accumulation, the detection of orthotopic HepG2 tumors was successful, despite the relatively high liver background. CD146-negative Huh7 and CD146-blocked HepG2 tumors exhibited significantly lower 89Zr-Df-YY146-ZW800 accretion (6.1 ± 0.5 and 8.1 ± 1.0 %ID/g at 72 h p.i., respectively; n=4), demonstrating the CD146-specificity of the tracer in vivo. Ex vivo biodistribution and immunofluorescent staining corroborated the accuracy of the imaging data and correlated 89Zr-Df-YY146-ZW800 uptake with in situ CD146 expression.
Conclusions Overall, 89Zr-Df-YY146-ZW800 showed excellent properties as PET/NIRF imaging agent, including high in vivo affinity and specificity for CD146-expressing liver cancer. CD146-targeted molecular imaging using dual-labeled YY146 has great potential for noninvasive detection and image-guided surgical resection of liver malignancies.