A Novel Peptide Multimer for Enhanced Imaging and Multivalent Detection of Hepatocellular Carcinoma.

The global incidence of hepatocellular carcinoma (HCC) is rising steadily, yet early detection methods remain a major clinical challenge due to genetic heterogeneity and variable target expression in these tumors. Conventional imaging approaches rely on non-specific or single targets, and often fail to achieve sufficient sensitivity or specificity, in particular, for early stage disease. A peptide multimer capable of simultaneously targeting 3 early HCC targets, including GPC3, CD44, and EpCAM, using a single biochemical construct was demonstrated. The multimer was labeled with either Cy5.5 for fluorescence imaging or Gd-DOTA for MRI to enable dual-modality detection. In vitro analysis using patient-derived HCC cell lines and organoids, and demonstrated significantly enhanced binding kinetics and affinity, including a 2.6-fold increase in fluorescence intensity and a 2.18-fold faster association rate compared with individual monomers. In vivo MRI in orthotopic patient-derived xenograft (PDX) models, both with and without cirrhosis, showed a peak tumor-to-background ratio of 3.05 at 0.5 hours post-injection and rapid renal clearance by about 4 hours. Ex vivo immunofluorescence of human liver specimens yielded 87% sensitivity and 80% specificity for distinguishing HCC from cirrhosis. These findings highlight multimer potential as a clinically translatable platform to improve early HCC diagnosis via enhanced molecular imaging.