119 - Molecular Profiling of Hepatocellular Carcinoma with Contrast-ultrasound Using a Novel Antibody-conjugated Microbubble via Light-activated Site-specific Chemistry

Purpose: To develop and evaluate a microbubble-based contrast agent using light-activated site-specific conjugation (LASIC) for targeted molecular ultrasound imaging of glypican-3 (GPC3) positive hepatocellular carcinoma (HCC).

Materials and Methods: Microbubbles were conjugated with anti-GPC3 antibody using oYo-Link DBCO chemistry, which enables site-specific labeling of the antibody heavy chain with up to two DBCO groups per antibody by employing a LASIC-based approach that ensures site-specific conjugation without compromising antigen affinity. GPC3 expression levels in HCC cell lines (SNU423, SNU449, and HepG2) were confirmed by Western blotting. Binding specificity was validated by pre-blocking with free anti-GPC3 antibodies in the HCC cell lines. Fluorescence microscopy and quantitative image analysis were used to assess cell-binding efficiency. For in vivo analysis, conjugated microbubbles were intravenously injected into mice bearing orthotopic HepG2 xenografts, and contrast-enhanced ultrasound (CEUS) imaging was performed at multiple time points post-injection (0 sec, 30 sec, 10 min) to evaluate tumor signal enhancement and persistence.

Results: LASIC-based chemistry enabled stable and homogeneous antibody attachment to microbubbles. In vitro assays showed significantly increased binding of targeted microbubbles to GPC3-positive cells compared to antigen blocked control groups (85% vs 2%; p=0.001). In vivo CEUS imaging revealed enhanced tumor-specific signal within 30 seconds post-injection, which persisted up to 10 minutes, confirming specific and sustained tumor targeting. Compared to the targeted group, the control group showed a noticeable decrease in signal intensity beginning at 30 seconds post-injection.

Conclusion: Conjugation of anti-GPC3 microbubbles enable efficient, specific, and durable binding to GPC3-expressing HCC in vitro and in vivo. This approach allows for real-time, image-guided molecular profiling using CEUS, paving the way for precision imaging and potential theranostic integration in liver cancer management.