Tetraspanin-8 as a Tumor-Selective Immuno-PET Target in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) ranks among the leading causes of cancer-related mortality worldwide. Although HCC-selective diagnostics and treatments remain limited for late-stage disease, tumor-targeted radiopharmaceuticals offer a promising strategy to address this unmet need. Here, we identify tetraspanin-8 (TSPAN8) as a novel tumor-selective immuno-PET imaging target in HCC. Methods: TSPAN8 expression was analyzed in 4 HCC cell lines (Huh7, Hep3B, SNU182, and SNU449) and 1 hepatoblastoma cell line (HepG2) using Western blotting and flow cytometry. The binding kinetics of a monoclonal antibody (α-hTSPAN8) against human TSPAN8 were evaluated using enzyme-linked immunosorbent assay and biolayer interferometry. Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-mediated gene editing was used to generate TSPAN8-knockout (TSPAN8^-) variants in Huh7 and Hep3B cells, validated through flow cytometry, immunofluorescence, and immunohistochemistry of xenograft tissues. Subcutaneous TSPAN8⁺ and TSPAN8^- xenografts were established in athymic NU/NU mice. For imaging studies, α-hTSPAN8 was conjugated with deferoxamine (DFO) and radiolabeled with ⁸⁹Zr, a positron emitter. Tumor-specific uptake of [⁸⁹Zr]Zr-DFO-α-hTSPAN8 was evaluated by in vivo PET/CT imaging at 48, 72, and 144 h after injection, followed by an ex vivo biodistribution analysis. Results: Huh7 and Hep3B cells exhibited high membrane TSPAN8 expression. The α-hTSPAN8 antibody demonstrated nanomolar affinity and specific binding to TSPAN8⁺ cells. We synthesized both DFO-α-hTSPAN8 and [⁸⁹Zr]Zr-DFO-α-hTSPAN8 to greater than 98% purity and greater than 99% labeling efficiency, respectively, with the radioconjugate exhibiting excellent stability in human serum at 37 °C. In vivo PET/CT imaging and biodistribution studies showed significant and selective tracer accumulation in TSPAN8⁺ tumors, with negligible uptake in TSPAN8^- controls. Conclusion: Our findings establish TSPAN8 as a promising target for radiopharmaceutical development for the treatment of HCC.