A multimodal pipeline for the identification and diagnostic immunoPET validation of hepatocellular carcinoma targets for radiotheranostic development.

Abstract

Background: Identifying tumor selective targets is critical for the development of precision diagnostic and therapeutic agents in oncology. Despite advances in precision oncology elsewhere, there are no FDA-approved hepatocellular carcinoma (HCC) antigen-selective antibody-drug conjugates or radiopharmaceuticals. This study establishes an integrated workflow to identify HCC-enriched plasma membrane targets and assess their suitability for targeted molecular imaging as foundational candidates for future radiopharmaceutical therapy.

Methods: Bulk RNA sequencing (371 tumors), single cell RNA sequencing (34 HCC cases), and a normal liver dataset were analyzed to identify HCC-enriched plasma membrane targets. Candidate molecules were examined on HCC and normal tissue microarrays (TMAs) and further evaluated in liver cancer cell lines by quantitative PCR, Western blot, and flow cytometry. Selected targets were then tested in mouse models of liver cancer using antibody-based positron emission tomography (immunoPET) to assess in vivo target engagement and distribution.

Results: Integrated transcriptomic analysis identified several tumor plasma membrane molecules with strong tumor enrichment, including GPC3, MUC13, TSPAN8, MET, and EGFR. TMAs confirmed prominent membrane expression in HCC with little signal in normal organs. Combinations of four prioritized markers captured up to 88.5% of patient tumors. Antibody-based immunoPET agents directed against prioritized targets demonstrated specific tumor accumulation in vivo, and signal intensity correlated with membrane staining by immunohistochemistry. These results highlight the potential of these markers to serve as diagnostic tools and promising candidate platforms for therapeutic development, demonstrating the utility of this approach to identify novel oncology targets.

Conclusions: This study establishes a multimodal framework integrating transcriptomic predictions and experimental protein validation to identify HCC targets for molecular imaging and radiopharmaceutical development. This translational blueprint successfully advances precision diagnostic and theranostic-ready agents for this disease for HCC, and a similar approach may be useful for identifying and validating targets in other malignancies.