Evaluation of an Integrin αvβ3 and Aminopeptidase N Dual-receptor Targeting Tracer for Breast Cancer Imaging.

Integrin αvβ3 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 αvβ3 and CD13, respectively, for PET imaging of breast cancer. NGR peptide was first modified with N3-NOtB2 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 αvβ3 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, MCF-7 tumor could be clearly visualized and exhibited higher uptake at 30 min post injection (p.i.) of 68Ga-NGR-RGD than that of either 68Ga-RGD or 68Ga-NGR alone. High specificity was shown in blocking studies using RGD and NGR peptides. 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 αvβ3 as confirmed by western blot and immunohistochemical staining. Metastatic lesions in lung were clearly detectable on 68Ga-NGR-RGD PET/CT imaging in mouse models of pulmonary metastases. 68Ga-NGR-RGD, a CD13 and αvβ3 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.