Evaluation of an Integrin α_vβ₃ and Aminopeptidase N Dual-Receptor Targeting Tracer for Breast Cancer Imaging

Integrin α_vβ₃ and aminopeptidase N (APN, also known as CD13) are two important targets involved in the regulation of angiogenesis, tumor proliferation, invasion, and metastasis. In this study, we developed a heterodimeric tracer consisting of arginine-glycine-aspartic (RGD) and asparagine-glycine-arginine (NGR) peptides targeting α_vβ₃ and CD13, respectively, for PET imaging of breast cancer. The NGR peptide was first modified with N₃-NO^tB₂ and then conjugated to BCN-PEG₄-c(RGDyK) via copper-free click chemistry. The resulting precursor was purified and radiolabeled with gallium-68. Small-animal PET/CT imaging and post-imaging biodistribution studies were performed in mice bearing human breast cancer MCF-7, MDA-MB-231, MDA-MB-468, and MX-1 xenografts and pulmonary metastases models. The expression levels of α_vβ₃ and CD13 in tumors were checked via immunochemical staining. The heterodimeric tracer was successfully synthesized and radiolabeled with gallium-68 at a molar activity of 45-100 MBq/nmol at the end of synthesis. It demonstrated high in vitro and in vivo stability. In static PET/CT imaging studies, the MCF-7 tumor could be clearly visualized and exhibited higher uptake at 30 min post injection of ⁶⁸Ga-NGR-RGD than that of either ⁶⁸Ga-RGD or ⁶⁸Ga-NGR alone. High specificity was shown in blocking studies using Arg-Gly-Asp (RGD) and Asp-Gly-Arg (NGR) peptides. The MCF-7 tumor exhibited the highest uptake of ⁶⁸Ga-NGR-RGD followed by MDA-MB-231, MDA-MB-468, and MX-1 tumors. This was consistent with their expression levels of CD13 and α_vβ₃ as confirmed by western blot and immunohistochemical staining. Metastatic lesions in the lungs were clearly detectable on ⁶⁸Ga-NGR-RGD PET/CT imaging in mouse models of pulmonary metastases. ⁶⁸Ga-NGR-RGD, a CD13 and α_vβ₃ dual-receptor targeting tracer, showed higher binding avidities, targeting efficiency, and longer tumor retention time compared with monomeric ⁶⁸Ga-NGR and ⁶⁸Ga-RGD. Its promising in vivo performance makes it an ideal candidate for future clinical translation.