Comprehensive Multiparametric Assay Of Hypoxia And Reactive Oxygen Species In Live Cells Using Different Instrumentation Platforms

Hypoxia is a pathological condition in cells and tissue arising from oxygen deprivation. Cancer-associated hypoxia has been linked to drug treatment resistance, increased cell proliferation and metastatic potential, which overall contributes to poor patient prognosis. Many of the cellular responses to hypoxia are now known to be mediated by the production of reactive oxygen species at mitochondrial complex III. The importance of the maintenance of cellular oxygen supply requires further investigation into the role of ROS as hypoxia signaling molecules. Despite sustained interest in the association between hypoxia, ROS production and disease, methods for conveniently and reliably identifying and quantifying hypoxic cells as well as redox status in patients have remained elusive. We designed and synthesized a series of non-fluorescent or weakly fluorescent aromatic compounds that become fluorescent once activated by hypoxic conditions. The probes were validated using a variety of in vitro cell-based hypoxia model systems involving exposure to hypoxia-inducing compounds (CoCl 2 , desferrioxamine or dimethyloxalylglycine) or by incubation in an anoxic environment (95% N 2 , 5% CO 2 ). In the in vitro cell-based hypoxia model systems, all tested probes exhibited strong fluorescent cytoplasmic signal, readily detectable by different instrumental platforms. For the real-time profiling of both hypoxic and redox cellular status, these self-immolative hypoxia probes were combined with various ROS detection probes and stained samples were analyzed using conventional fluorescence microscopy or flow cytometry. The described assay workflow facilitates rapid, reproducible, specific and relatively simple functional and quantitative detection of cellular oxygen status in a real-time format, and can readily be implemented on widely available instrumentation. The assay may be applicable to the screening of putative modulators of hypoxia or ROS production. Profiling of ROS is also possible when specific inhibitors of ROS are implied. The outlined fluorescence detection method offers an attractive approach for studying hypoxia and ROS pathways in a noninvasive manner, providing the ability for spatial and temporal resolution of cellular signaling events. The described multiparameter assay should find use in diverse applications relating to detection, quantification and localization of hypoxic cells in vivo and in vitro, as tools for the development of predictive assays or possibly as biosensors for early treatment monitoring and validation of potential endogenous hypoxia biomarkers and signaling molecules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2060. doi:10.1158/1538-7445.AM2011-2060