Purpose: Molecular imaging using positron emission tomography (PET) radiotracers targeted to tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The previous in vitro results demonstrated that the GX1 peptide, identified by phage display technology, is a tumor vasculature endothelium-specific ligand. In this study, we evaluated a ⁶⁴Cu-labeled GX1 peptide as a potential radiotracer for microPET imaging of tumor vasculature in a U87MG tumor xenografted mouse model.
Methods: Macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA)-conjugated GX1 peptide was synthesized and radiolabeled with ⁶⁴Cu (t(1/2) = 12.7 h) in ammonium acetate buffer. The ⁶⁴Cu-labeled GX1 peptide was then subjected to in vitro tumor cell uptake study, small animal PET and direct tissue sampling biodistribution studies in a U87MG tumor xenografted mouse model.
Results: The in vitro experiment demonstrated that ⁶⁴Cu-DOTA-GX1 is stable in PBS with more than 91% of ⁶⁴Cu-DOTA-GX1 peptide remaining intact after 24 h of incubation. Cellular uptake and retention studies revealed (64)Cu-DOTA-GX1 binds to U87MG glioma cells and has good tumor cell retention. For small animal PET imaging studies, the U87MG tumors were all clearly visible with high contrast to contralateral background at all measured time points after injection of ⁶⁴Cu-DOTA-GX1 while high accumulation in liver and kidneys were also observed at early time points. The U87MG tumor uptake was determined to be the highest (7.97 ± 0.75%ID/g) at 24 h pi. The blocking experiment was achieved by co-injection of ⁶⁴Cu-DOTA-GX1 with non-radiolabeled GX1 peptide (20 mg/kg) at 24 h pi, suggesting ⁶⁴Cu-DOTA-GX1 is a target-specific tracer. Furthermore, the biodistribution results were consistent with the quantification of microPET imaging, demonstrating the highest ratio (16.09 ± 1.21) of tumor/muscle uptake of ⁶⁴Cu-DOTA-GX1 at 24 h pi for non-blocking group and significant decreased ratio (6.57 ± 0.58) for blocking group. Finally, metabolic studies suggested that ⁶⁴Cu-DOTA-GX1 is stable in mouse blood and urine in vivo at early time point while the metal transchelation may also occur in mouse liver and kidneys.
Conclusion: Our studies demonstrate that ⁶⁴Cu-DOTA-GX1 is a promising radiotracer for imaging tumor vasculature.