Fluorescent nitroheterocycles for identifying hypoxic cells.

Binding of several nitroheterocycles by mammalian cells is a function of the ambient oxygen concentration; anoxic single cells bind up to 10 times as much of these drugs as do aerobic cells. We thus hypothesized that fluorescent nitroheterocyles could be used to quantitate the fraction of hypoxic cells in multicell systems, and this was tested in multicell spheroids using a relatively nontoxic nitrofuran, trans-5-amino-3-[(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole. Binding of trans-5-amino-3-[(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole, as quantified by flow cytometry, was highly responsive to external oxygen concentration. To assess the relevance of the observed fluorescence, fluorescence-activated cell sorting was used to examine the radiosensitivity of cells as a function of their fluorescence intensity. The most fluorescent were indeed the most radioresistant (i.e., contained the least oxygen). Additional results confirm the general feasibility of using fluorescent nitroheterocycles as hypoxic cell probes but also reveal that cellular binding of these agents is not exclusively dependent upon cellular oxygen content.