In vivo Application of BL660-NTR to Detect Tumor Hypoxia Finally, we employed BL660-NTR in vivo to image tumor hypoxia (via NTR activity). Hypoxia is defined as a condition where the demand for oxygen from rapidly dividing cancer cells, outweigh the available supply. This condition emerges as the distance between the cancer cells and nutrient-rich blood vessels become greater as a function of tumor growth. Although it is estimated that ~50% of solid tumors are hypoxic, it is impossible to know whether a given lesion is oxygen-deficient without a reliable readout. This knowledge becomes critical when using luciferase-expressing cells to study cancer biology because hypoxia can strongly influence tumor properties (e.g., drug resistance, tumorigenesis, etc.). Of note, although several NTR probes for BL have been developed previously, 50,55-58 BL660-NTR is the first example capable of deep-tissue detection of hypoxia via NIR BL imaging. NU/J mice (male, ~5-weeks old) were inoculated with A549-Luc2 lung cancer cells (5 × 10 6 ) in both flanks. Testing of BL660-NTR commenced after tumors had grown to a volume of ~300 mm 3 (~4 weeks). First, we performed imaging using BL660-NTR, which was administered systemically via retroorbital injection (Figure and). ROIs were drawn around each tumor and the luminescent count was summed for each animal. By the first scan (5 min), strong BL signals were apparent in all tumors. The BL intensity appears to decrease slightly after 15 mins and levels off until the end of the experiment (75 mins) (Figure ). Next, to distinguish between NTR activation and off-target ester hydrolysis, we employed compound 4 as a control reagent, which only differs in that the nitro trigger is absent (Figure ). After approximately 20 hours, the same animals were treated with compound 4 and imaged (Figure and). This delay was to ensure the probe had fully cleared from the first experiment. Despite dosing at the same concentration, the BL signal in the tumors at all time points were barely discernable from background (Figure ). The total luminescent count after 15 mins for compound 4 was only 4.0 ± 0.7 × 10 3 ; whereas the corresponding readout was 16.6 ± 2.5 × 10 3 for BL660-NTR. This represents a 5.2-fold difference, indicating successful detection of tumor hypoxia (Figure ).