Real-Time Visualizationof Endogenous H₂O₂ Production in Mammalian Spheroidsby Electrochemiluminescence

Two-dimensional cell culture may be insufficient when it comes to understanding human disease. The redox behavior of complex, three-dimensional tissue is critical to understanding disease genesis and propagation. Unfortunately, few measurement tools are available for such three-dimensional models to yield quantitative insight into how reactive oxygen species (ROS) form over time. Here, we demonstrate an imaging platform for the real-time visualization of H₂O₂ formation for mammalian spheroids made of noncancerous human embryonic kidney cells (HEK-293) and metastatic breast cancer cells (MCF-7 and MDA-MB-231). We take advantage of the luminol and H₂O₂ electrochemiluminescence reaction on a transparent tin-doped indium oxide electrode. The luminescence of this reaction as a function of [H₂O₂] is linear (<i>R</i>² = 0.98) with a dynamic range between 0.5 μM to 0.1 mM, and limit of detection of 2.26 ± 0.58 μM. Our method allows for the observation of ROS activity in growing spheroids days in advance of current techniques without the need to sacrifice the sample postanalysis. Finally, we use our procedure to demonstrate how key ROS pathways in cancerous spheroids can be up-regulated and downregulated through the addition of common metabolic drugs, rotenone and carbonyl cyanide-<i>p</i>-trifluoromethoxyphenylhydrazone. Our results suggest that the Warburg Effect can be studied for single mammalian cancerous spheroids, and the use of metabolic drugs allows one to implicate specific metabolic pathways in ROS formation. We expect this diagnostic tool to have wide applications in understanding the real-time propagation of human disease in a system more closely related to human tissue.