Noninvasive in vivo discrimination between mitochondrial ROS and global ROS production in solid tumors using EPR spectroscopy.

Because the precise site of ROS production plays a key role in cellular redox signaling and its (patho)physiological consequences, it is crucial to develop tools that enable site-specific detection of ROS in complex systems, including in vivo. Here, we propose the use of Electron Paramagnetic Resonance (EPR) and dual nitroxide sensors composed of mitoTEMPO and carbamoyl-proxyl (3CP) to probe ROS production in the mitochondrial and intracellular/extracellular compartments, respectively. For the proof-of-concept, the decay rates of the nitroxides were measured in 4T1 breast tumor models, both in vitro and in vivo, using 9 GHz and 1 GHz spectrometers, respectively. To modulate the level of ROS either in the cytosol or in the mitochondria, cells and mice were treated with either the glutathione synthesis inhibitor l-Buthionine Sulfoximine (L-BSO) or Antimycin A, an inhibitor of the complex III of the mitochondrial electron transport chain, or their appropriate controls. In mice, an increase in relative decay rate was observed for 3CP, but not for mitoTEMPO, 1 and 2 days after starting L-BSO treatment, while the opposite result was obtained after Antimycin A treatment. These observations were consistent with results obtained on cells in vitro. Ex-vivo analyses of tumors, with or without ferricyanide that converts hydroxylamines back to nitroxides, revealed non-significant changes in the total amount of nitroxide + hydroxylamine, suggesting that the blood wash-out did not play a role in the decay of the nitroxide signal. In addition, the use of genetically engineered 4T1 cells that overexpress the mitochondrial isoform superoxide dismutase 2 (SOD2) allowed the assessment of the contribution of superoxide production to EPR signal decay. Overall, this study identifies a new protocol to noninvasively discriminate the site of ROS production in vivo.