Investigation of Mitochondrial Metabolic Response to Doxorubicin in Prostate Cancer Cells: An NADH, FAD and Tryptophan FLIM Assay
Prostate cancer (PCa) is one of the leading cancers in men in the USA. Lack of experimental tools that predict therapy response is one of the limitations of current therapeutic regimens. Mitochondrial dysfunctions including defective oxidative phosphorylation (OXPHOS) in cancer inhibit apoptosis by modulating ROS production and cellular signaling. Thus, correction of mitochondrial dysfunction and induction of apoptosis are promising strategies in cancer treatment. We have used Fluorescence Lifetime Imaging Microscopy (FLIM) to quantify mitochondrial metabolic response in PCa cells by tracking auto-fluorescent NAD(P)H, FAD and tryptophan (Trp) lifetimes and their enzyme-bound fractions as markers, before and after treatment with anti-cancer drug doxorubicin. A 3-channel FLIM assay and quantitative analysis of these markers for cellular metabolism show in response to doxorubicin, NAD(P)H mean fluorescence lifetime (τm) and enzyme-bound (a2%) fraction increased, FAD enzyme-bound (a1%) fraction was decreased, NAD(P)H-a2%/FAD-a1% FLIM-based redox ratio and ROS increased, followed by induction of apoptosis. For the first time, a FRET assay in PCa cells shows Trp-quenching due to Trp-NAD(P)H interactions, correlating energy transfer efficiencies (E%) vs NAD(P)H-a2%/FAD-a1% as sensitive parameters in predicting drug response. Applying this FLIM assay as early predictor of drug response would meet one of the important goals in cancer treatment.
Segmented cell analyses to measure redox states of autofluorescent NAD(P)H, FAD & Trp in cancer cells by FLIM
Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio. Furthermore, image data is analyzed in segmented cells thresholded by 2 × 2 pixel Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis in a prostate cancer cell line. Hundreds of data points allow demonstration of heterogeneity in response to intervention, identity of cell responders to treatment, creating thereby different sub-populations. Histograms and bar charts visualize differences between cells, analyzing whole cell versus mitochondrial morphology data, all based on discrete ROIs. This assay method allows to detect subtle differences in cellular and tissue responses, suggesting an advancement over means-based analyses.