Preclinical Evaluation of [68Ga]Ga-CS03 as a Novel GPC3-Targeted Probe for Imaging GPC3 in Hepatocellular Carcinoma Models

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Due to its high specificity and peak expression in hepatocellular carcinoma (HCC), Glypican-3 (GPC3) is a heavily researched target. While current GPC3-targeted therapies demonstrate remarkable efficacy, traditional GPC3 assessment methods are limited by time-dependent and spatial heterogeneity, invasiveness, and prolonged waiting periods, highlighting an urgent need for new GPC3 evaluation strategies. In this study, first, we selected MHCC97H as a GPC3-high expressing HCC cell line (named GPC3high MHCC97H) and constructed a GPC3-low expressing MHCC97H cell line (named GPC3low MHCC97H) using lentiviral transfection technology. Second, a novel radiopharmaceutical precursor, CS03, was synthesized. Molecular docking analysis revealed that CS03 exhibits the potential to bind to multiple amino acid residues of GPC3, indicating its promise as a targeting molecule for GPC3. Therefore, we labeled it with Ga-68 to synthesize [68Ga]Ga-CS03. The radiolabeling yield of [68Ga]Ga-CS03 exceeded 80%. After purification, the radiochemical purity of [68Ga]Ga-CS03 was greater than 98%, with a specific activity of (3.00 ± 0.22) × 106 MBq/mmol. [68Ga]Ga-CS03 exhibited excellent in vitro stability, rapid blood clearance (T1/2α = 0.93 ± 0.49 min and T1/2β = 17.22 ± 1.42 min), and good in vitro affinity (36.30 ± 5.17 nM). Micro-PET/CT imaging of HCC models revealed significant [68Ga]Ga-CS03 radiotracer accumulation in GPC3high MHCC97H tumors 0.5 h postinjection. In contrast, minimal or no significant accumulation was detected in GPC3low MHCC97H tumors and blocking group tumors, indicating the excellent in vivo affinity and specificity of [68Ga]Ga-CS03. [68Ga]Ga-CS03 showed rapid tumor clearance, with a significant decrease in tumor radiotracer distribution by 1 h, a finding corroborated by subsequent biodistribution studies. Furthermore, micro-PET/CT images and biodistribution results indicated that [68Ga]Ga-CS03 was cleared by the kidneys, which explains its rapid blood clearance. Finally, we collected tumor tissues from the animal models after PET imaging and performed hematoxylin and eosin (H&E) staining and GPC3 immunofluorescence (IF) staining. We found that the fluorescence intensity of IF staining was positively correlated with the radiotracer distribution in the tumor sites on PET images, which further validated the specificity of [68Ga]Ga-CS03 for GPC3 binding. In conclusion, we developed [68Ga]Ga-CS03, a novel GPC3-targeted PET radiotracer. Extensive in vitro and in vivo evaluations demonstrated its favorable stability, high affinity, and specificity. These promising preclinical findings highlight its potential for the noninvasive assessment of GPC3 expression in HCC.

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