Optical redox ratio identifies metastatic potential-dependent changes in breast cancer cell metabolism

Abstract

The development of prognostic indicators of breast cancer metastatic risk could reduce the number of patients receiving chemotherapy for tumors with low metastatic potential. Recent evidence points to a critical role for cell metabolism in driving breast cancer metastasis. Endogenous fluorescence intensity of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) can provide a label-free method for assessing cell metabolism. We report the optical redox ratio of FAD/(FAD + NADH) of four isogenic triple-negative breast cancer cell lines with varying metastatic potential. Under normoxic conditions, the redox ratio increases with increasing metastatic potential (168FARN>4T07>4T1), indicating a shift to more oxidative metabolism in cells capable of metastasis. Reoxygenation following acute hypoxia increased the redox ratio by 43 ± 9% and 33 ± 4% in the 4T1 and 4T07 cells, respectively; in contrast, the redox ratio decreased 14 ± 7% in the non-metastatic 67NR cell line. These results demonstrate that the optical redox ratio is sensitive to the metabolic adaptability of breast cancer cells with high metastatic potential and could potentially be used to measure dynamic functional changes that are indicative of invasive or metastatic potential.

Keywords: (170.1530) Cell analysis; (170.2520) Fluorescence microscopy; (170.2655) Functional monitoring and imaging.

Fig. 1

Optical redox ratio is sensitive to dynamic changes in oxygen consumption. A. Representative images of NADH, FAD and the calculated optical redox ratio (FAD/FAD + NADH) in response to the addition of mitochondrial inhibitors and uncouplers. All images were acquired from different fields of view within the same cell plate over several minutes after serial addition of each drug indicated above the figure panels. B. Quantification of the dynamic changes in the optical redox ratio in response to serial drug additions. Oligomycin, FCCP, and antimycin A/rotenone were injected at T = 30, 70, and 110 minutes, respectively. C. Normalized oxygen consumption rate measured using the Seahorse XFp analyzer. Oligomycin, FCCP, and antimycin A/rotenone were injected at T = 15, 35, and 55 minutes, respectively. The error bars are a standard deviation of the mean plate value.

Fig. 2

Optical redox ratio of breast cancer cells of different metastatic potential is significantly different at normoxia and after exposure to acute hypoxia. A. Representative redox ratio images for 4T1, 4T07, 168FARN, and 67NR breast cancer cells. The redox ratio was measured at baseline normoxic conditions and 1 hour after exposure to acute hypoxia (60 minutes, 0.5% O₂). B. Quantification of redox ratio images illustrates significant differences in the redox ratio between the different cell lines under normoxic conditions, and within each cell line after exposure to acute hypoxia (except 168FARN). Error bars represent standard deviation of the mean plate value. C. The normalized oxygen consumption rate (calculated as oxygen consumption rate/proton production rate) for all four cell lines and the direction of change after exposure to acute hypoxia are consistent with the optical redox ratio. Asterisks placed above bars indicate statistical significance. *** denotes p < 0.0001 and ** denotes p < 0.01.

Fig. 3

Exposure to acute hypoxia leads to cell-line-dependent changes in redox ratio. Bar plots represent the difference in the mean values of normoxia and post-hypoxia reoxygenation groups. Error bars represent the standard deviation and were calculated as follows:$\sqrt{{\left(s{d}_1\right)}^2+{\left(s{d}_2\right)}^2}$, where sd₁ and sd₂ represent the normoxia and post-hypoxia groups within each cell line.

Fig. 4

Effect of mitochondrial inhibitors and uncouplers on redox ratio. Initial addition of oligomycin inhibits ATP synthase, which decreases mitochondrial respiration, increases NADH that cannot be converted to NAD⁺ and hence decreases the redox ratio. FCCP is a mitochondrial uncoupler that causes protons to leak across the membrane, leading to a loss of proton gradient and hence the ability to generate ATP. Because the cell tries to restore the proton gradient, NADH is consumed, leading to increased redox ratio. Finally, Rotenone and Antimycin inhibit complexes I and III, leading to a shutdown of mitochondrial respiration and a decrease in the redox ratio.