Mitochondrial Redox Imaging for Cancer Diagnostic and Therapeutic Studies

Abstract

Mitochondrial redox states provide important information about energy-linked biological processes and signaling events in tissues for various disease phenotypes including cancer. The redox scanning method developed at the Chance laboratory about 30 years ago has allowed 3D high-resolution (~ 50 × 50 × 10 μm3) imaging of mitochondrial redox state in tissue on the basis of the fluorescence of NADH (reduced nicotinamide adenine dinucleotide) and Fp (oxidized flavoproteins including flavin adenine dinucleotide, i.e., FAD). In this review, we illustrate its basic principles, recent technical developments, and biomedical applications to cancer diagnostic and therapeutic studies in small animal models. Recently developed calibration procedures for the redox imaging using reference standards allow quantification of nominal NADH and Fp concentrations, and the concentration-based redox ratios, e.g., Fp/(Fp+NADH) and NADH/(Fp+NADH) in tissues. This calibration facilitates the comparison of redox imaging results acquired for different metabolic states at different times and/or with different instrumental settings. A redox imager using a CCD detector has been developed to acquire 3D images faster and with a higher in-plane resolution down to 10 μm. Ex vivo imaging and in vivo imaging of tissue mitochondrial redox status have been demonstrated with the CCD imager. Applications of tissue redox imaging in small animal cancer models include metabolic imaging of glioma and myc-induced mouse mammary tumors, predicting the metastatic potentials of human melanoma and breast cancer mouse xenografts, differentiating precancerous and normal tissues, and monitoring the tumor treatment response to photodynamic therapy. Possible future directions for the development of redox imaging are also discussed.

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

“…Their levels reflect the varying balance of glycolysis and oxidative phosphorylation, depending on environmental signals (availability of oxygen, and/or lactate/pyruvate, the presence of inhibitors or uncouplers, etc) and genetic makeup of cells (Warburg effect) [43,44]. We emphasise that in this work, we interfere with the activity of the mitochondria by using FCCP at such high levels that they, eventually, become inactive leading to cell death (see cell viability data in Table 1), due to increased production of reactive oxygen species (ROS) and other effects [45,46]. A purely chemical effect of NADH/NADPH oxidation, which decreases NADH/NADPH fluorescence, as quantified in Fig.…”

Breast Cancer Redox Heterogeneity Detectable with Chemical Exchange Saturation Transfer (CEST) MRI

“…The overall CEST contrast of this reaction is also expected to correlate with NADPH and NADPH/NADP + . Thus, both NAD- and NADP-coupled reactions can contribute to the CEST contrast, while the redox signals imaged by the optical redox scanning are mainly from the bound forms of NADH and Fp in the mitochondria [39]. However, the mitochondrial NADH may be coupled to NADPH in the cytosol via NADH transhydrogenase [40].…”

Quantitative mitochondrial redox imaging of breast cancer metastatic potential

“…3 In this study, we employed the mitochondrial redox scanning technique to distinguish between aggressive and indolent human breast tumor mouse xenografts. Redox scanning developed by Chance and collaborators [10][11][12][13] utilizes a cryogenic nicotinamide adenine dinucleotide ͑NADH͒/flavoproteins fluorescence imaging instrument to image the mitochondrial redox state in biological samples snap-frozen under liquid nitrogen. The light emissions originate from the two major classes of intrinsic fluorophores in tissue, NADH and oxidized flavoproteins ͑Fp͒ containing flavin adenine dinucleotide ͑FAD͒, whose fluorescence intensities are enhanced 10-fold at liquid nitrogen temperature.…”