GPC-3 targeted hydrophilic peptides for PET imaging of hepatocellular carcinoma

Objectives: Glypican-3 (GPC-3) is a cell surface protein which is overexpressed on the membrane of hepatocellular carcinoma (HCC) cells. Noninvasive imaging of GPC-3 expression in vivo is of particular interest for both GPC-3 targeted HCC diagnosis and treatment. In this study, we radiolabeled GPC-3 targeted peptides (GTPs) with ⁶⁴Cu, and the resulting PET tracers were subsequently subject to biological evaluations in animal models. A highly hydrophilic linker was incorporated into the GTP sequence to construct L-GTP with an aim of reducing the tracer uptake in mouse liver.

Methods: The immunofluorescence assay was used to identify the expression of GPC-3 and determine the binding of GTP in tumor cells. Both GTP and L-GTP were conjugated with NOTA chelator and radiolabeled with ⁶⁴Cu in ammonium acetate buffer. The potency of the hydrophilic linker was investigated by comparing the biodistribution of ⁶⁴Cu-NOTA-L-GTP vs. ⁶⁴Cu-NOTA-GTP in normal mice. The HCC targeting efficacy of ⁶⁴Cu-labeled GTPs was also studied by PET imaging in HepG2 tumor mouse model.

Results: The immunofluorescence assay showed that GPC-3 is highly overexpressed on the membrane of HepG2 cells, and HepG2 cell uptake of L-GTP is strongly associated with the GPC-3 expression. ⁶⁴Cu labeling was achieved in 85% decay-corrected yield with radiochemical purity of >98%. The specific activity of ⁶⁴Cu-labeled GTPs was estimated to be ~40 MBq/nmol. ⁶⁴Cu-NOTA-L-GTP demonstrated high hydrophilicity as determined by octanol-water partition coefficient measurements. PET imaging of ⁶⁴Cu-NOTA-L-GTP at 1 h post intravenous injection in normal mice showed radioactivity was predominately accumulated in kidneys (8.74±1.31%ID/g) and bladder. Significantly lower liver uptake of ⁶⁴Cu-NOTA-L-GTP (2.16±0.62%ID/g) was observed as compared to that of ⁶⁴Cu-NOTA-GTP, suggesting that the hydrophilic linker greatly facilitates the tracer excretion via the renal system. PET study showed ⁶⁴Cu-NOTA-L-GTP has preferential tumor uptake in HepG2 tumor xenografts with limited radioactivity uptake in mouse liver and intestine. The biodistribution results were consistent with the quantitative analysis of PET imaging.

Conclusion: New ⁶⁴Cu-labeled GTPs have been successfully developed for PET imaging of GPC-3 expression in HCC. The incorporation of a hydrophilic linker into the GTP significantly reduces the trace uptake in liver and improves the overall imaging efficacy of radiotracer in HCC. Convenient preparation, good GPC-3 specificity in mouse tumor xenografts, and favorable excretion profile of ⁶⁴Cu-NOTA-L-GTP warrant further translational studies. Research Support: