A nitroreductase-triggered NIR fluorescent probe for selective visualization in orthotopic breast cancer

Nitroreductase (NTR) plays a crucial role in the hypoxic metabolism of breast tumors and serves as an important indicator of tumor aggressiveness and therapeutic response. However, visualization of NTR activity remains challenging due to the limited photophysical properties of existing probes, particularly their short emission wavelengths and small Stokes shifts. Here, we report a near-infrared (NIR) activatable fluorescent probe, DHM-NO₂, which we constructed by coupling the large-Stokes-shift fluorophore DHM-OH with a nitrobenzyl recognition unit. DHM-NO₂ exhibited negligible background fluorescence; however, it could undergo NTR-mediated reduction that triggered self-immolative cleavage and the release of DHM-OH, which emitted fluorescence at 880 nm upon 590 nm excitation. The probe had a detection limit of 25.6 pg/mL, excellent selectivity, and could be rapidly activated. DHM-NO₂ could differentiate MCF-7 breast cancer cells from normal MCF-10 A cells, resist interference from ROS/RNS, and respond to pharmacological modulation of reductive metabolism. In an orthotopic breast cancer model, DHM-NO₂ could rapidly produce tumor-localized fluorescence with a markedly increased tumor-to-normal ratio. Inhibitor, hypoxia-enhancing, and oxygenation treatments further confirmed its NTR-dependent activation. Collectively, these results show that DHM-NO₂ is a sensitive NIR-I probe that can monitor NTR activity in breast cancer and is highly compatible with conventional fluorescence instrumentation.