Development of Netrin-1-targeted peptide probes derived from the Netrin-1/DCC complex for noninvasive imaging of tumors.

Netrin-1 is a secreted glycoprotein that is overexpressed in non-small cell lung cancer (NSCLC) and breast cancer, making it an emerging biomarker for tumor diagnosis and targeted therapy. However, noninvasive imaging tools for quantitative assessment of Netrin-1 expression remain limited. In this study, leveraging the crystal structure of the Netrin-1/DCC complex, we rationally designed two Netrin-1-targeted peptides YP8 and LE7, through systematic in silico molecular docking, alanine scanning, and virtual amino acid mutation. Their corresponding 68Ga-labeled PET tracers [68Ga]Ga-NOTA-YP8 and [68Ga]Ga-NOTA-LE7, were subsequently developed. Comparative evaluation revealed that [68Ga]Ga-NOTA-LE7 exhibited superior Netrin-1 binding affinity, enhanced in vivo stability, and significantly improved tumor-to-background ratio. PET imaging demonstrated specific and robust uptake of [68Ga]Ga-NOTA-LE7 in Netrin-1-positive A549 and 4T1 tumors, with minimal accumulation in Netrin-1-low MDA-MB-231 tumors. The NIRF probe ICG-LE7 further confirmed specific tumor targeting and enabled fluorescence-guided surgical resection. Collectively, these findings establish [68Ga]Ga-NOTA-LE7 and ICG-LE7 as promising noninvasive diagnostic tools for Netrin-1 expression imaging.