Gupta Vanshika, Falciani Francesco, Layman Brady R, Hill Megan L, Rapino Stefania, Dick Jeffrey E
Department of Chemistry, Purdue University, West Lafayette, Indiana 47906, United States.
Department of Chemistry "Giacomo Ciamician", University of Bologna, Via P. Gobetti 85, 40129 Bologna, Italy.
Chem Biomed Imaging. 2025 Mar 10;3(5):310-321. doi: 10.1021/cbmi.4c00105. eCollection 2025 May 26.
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 HO 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 HO electrochemiluminescence reaction on a transparent tin-doped indium oxide electrode. The luminescence of this reaction as a function of [HO] is linear ( = 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--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.
在理解人类疾病方面，二维细胞培养可能并不充分。复杂的三维组织的氧化还原行为对于理解疾病的发生和传播至关重要。不幸的是，对于这种三维模型，几乎没有测量工具能够对活性氧（ROS）如何随时间形成进行定量洞察。在此，我们展示了一个成像平台，用于实时可视化由非癌性人胚肾细胞（HEK - 293）和转移性乳腺癌细胞（MCF - 7和MDA - MB - 231）制成的哺乳动物球体中羟基自由基（HO）的形成。我们利用了鲁米诺和HO在透明的氧化铟锡电极上的电化学发光反应。该反应的发光强度与[HO]的函数关系呈线性（ = 0.98），动态范围在0.5 μM至0.1 mM之间，检测限为2.26 ± 0.58 μM。我们的方法能够在当前技术之前数天观察生长中的球体中的ROS活性，且无需在分析后牺牲样本。最后，我们使用我们的方法证明了通过添加常见的代谢药物鱼藤酮和羰基氰化物 - 三氟甲氧基苯腙，癌性球体中的关键ROS途径如何被上调和下调。我们的结果表明，可以针对单个哺乳动物癌性球体研究瓦伯格效应，并且使用代谢药物能够揭示ROS形成中的特定代谢途径。我们期望这种诊断工具在更接近人体组织的系统中理解人类疾病的实时传播方面有广泛应用。
