Abstract
Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells—the “optical redox ratio” (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.
Highlights
Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues
Since the first publication by Chance et al.,[4] the optical redox ratio” (ORR) has been used in a broad range of applications spanning from cancer detection and diagnosis to predicting drug response to monitoring cellular function and stem cell differentiation.[5,6,7,8,9]
In order to test the robustness of the ORR for measuring cell metabolism, a number of studies have attempted to validate the correlation between ORR and the oxidation–reduction ratio of NADþ∕NADH.[18,19]
Summary
Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. We use TPEF to measure ORR in living MDAMB-231 breast cancer cells under normal culture conditions and in MCF-7 breast cancer cells to dynamically measure metabolic changes in response to a series of mitochondrial inhibitors and uncouplers [oligomycin, carbonyl cyanide-4-phenylhydrazone (FCCP) and rotenone/antimycin A].
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