Glypican 3-Targeted Fluorescent Imaging for Intraoperative Tumor Boundary Identification in Hepatocellular Carcinoma

Abstract Background Intraoperative tumor boundary identification is essential for complete resection of hepatocellular carcinoma (HCC). Glypican-3 (GPC3) is a tumor-associated membrane antigen that is highly expressed in most HCCs but barely in livers. This study evaluated the feasibility of GPC3 as a tumor-specific imaging target and developed a GPC3-targeted fluorescent probe for intraoperative tumor boundary identification. Patients and Methods In the clinical study, intraoperative tumor identification using indocyanine green (ICG) fluorescence was evaluated in 38 patients with HCC, and clinicopathological factors were compared between identifiable and nonidentifiable groups. The tumor specificity of GPC3 was validated by immunohistochemistry of resected specimens. In the experimental study, a GPC3-targeted probe (GPC3-IRDye800) was developed and administered to human HCC cell-line-derived mouse models. Antigen specificity and tumor boundary identifiability were assessed in vivo and ex vivo. Fluorescence-guided surgery (FGS) was performed to examine the feasibility of complete resection. Results ICG enabled tumor identification in 30 patients (78.9%). Cirrhosis was significantly associated with detection failure. GPC3 expression was observed in 35 tumors (92.1%) and was higher in the tumors than in the peritumoral livers ( P < 0.0001). GPC3-IRDye800 demonstrated antigen-dependent uptake, with higher fluorescence intensity and tumor-to-background ratios than controls ( P < 0.0001). At 72 h post-administration in both normal- and fibrotic-liver mouse models, GPC3-IRDye800 provided higher fluorescence intensity at the tumor boundary than ICG. FGS using this probe allowed for complete tumor resection without margin exposure. Conclusions GPC3-IRDye800 enables reliable tumor boundary visualization, regardless of background liver condition, and may contribute to complete tumor resection.