Cathepsin L‐Activatable Fluorescent Probe for Real‐Time Monitoring of Drug‐Resistant Breast Cancer Cells

Abstract Precise identification of drug‐resistant tumor cells remains a critical challenge for optimizing therapeutic strategies and improving clinical outcomes. Herein, we present FR‐DCO, a cathepsin L (CTSL)‐activatable fluorescent probe, engineered for targeted imaging of doxorubicin‐resistant breast cancer cells (MCF‐7/ADR). Capitalizing on the overexpression of CTSL in chemoresistant malignancies, FR‐DCO integrates a CTSL‐specific dipeptide substrate (Phe‐Arg, FR) with a dicyanoisophorone (DCO) fluorophore, enabling fluorescence activation via a single‐step enzymatic cleavage. Upon hydrolysis, FR‐DCO transitions from an intramolecular charge transfer‐quenched state to an activated DCO fluorophore, achieving a 12.8‐fold enhancement in quantum yield. In vitro studies demonstrated both caveolin‐ and clathrin‐mediated endocytosis in MCF‐7/ADR cells, yielding 2.3‐fold higher fluorescence intensity than its nonresistant counterparts (MCF‐7 and 4T1). In vivo imaging in murine models revealed rapid and specific activation within drug‐resistant tumors, with intratumoral fluorescence intensity doubling that of nonresistant lesions within 20 minutes post‐injection. Notably, dual‐tumor xenograft models confirmed precise discrimination of MCF‐7/ADR tumors from 4T1 tumors, highlighting the spatial resolution of the probe. This work not only provides a molecular tool for spatiotemporal resistance monitoring, but also establishes a modular design paradigm for enzyme‐responsive probes targeting tumor microenvironment biomarkers, advancing precision oncology strategies.