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

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.