ABSTRACT
Sensitive activity stains for enzymes selectively expressed in human cancers offer valuable tools for imaging with wide applications in experimental, diagnostic, and therapeutic settings. The scant expression of the antioxidant enzyme NQO1 in normal tissues and its great abundance in malignant counterparts due to the increased redox stress and hypoxia is one such example. Previously, we described a potent nontoxic probe that remains nonfluorescent but releases an intense fluorogenic compound after intracellular cleavage by NQO1 catalysis. This infrared probe with a 644 nm emission has excellent tissue penetrating ability and low background absorption. Described here are methods (fluorescence microscopy, flow cytometry, and in vivo animal imaging) to rapidly image NQO1 activity in hypoxic and non-hypoxic cancer cells and tumors developed in live mouse xenograft models. The specificity of the dye for NQO1 in all three procedures was verified, and the methods should be useful for both in vitro and in vivo studies.
