Metabolic Profiling of Live Cancer Tissues Using NAD(P)H Fluorescence Lifetime Imaging

Blacker, Thomas S.; Sewell, Michael D E; Szabadkai, György; Duchen, Michael R.

doi:10.1007/978-1-4939-9027-6_19

Cited by 10 publications

(9 citation statements)

References 93 publications

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“…12,30,[32][33][34][35][36][37] These assays are of particular interest in measuring responses to treatment in various cancer pathologies, cancer being a metabolically heterogeneous pathology, being able to generate energy by oxidative phosphorylation (OXPHOS) and (often preferentially) by glycolysis. 9,[38][39][40][41] A third coenzyme, nicotinamide adenine dinucleotide (phosphate) [NAD(P)H], the phosphorylated form of NADH, cannot spectrally be differentiated from NADH, so this paper follows convention to describe the mixed lifetime/intensity signal as NAD(P)H. A heightened interest in expanding the application and simplification of this FLIM assay arises from its potential to test suitability and earliest response to drug treatment in cancer therapies. Chemotherapy response in animal models and patients typically take days or weeks; this in vitro assay has been shown to predict results in hours, 35 potentially helpful to devise more individualized treatment modalities for patients.…”

Section: Introductionmentioning

confidence: 99%

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

2020

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Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)-essential coenzymes in cellular respiration-have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

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Section: Introductionmentioning

confidence: 99%

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

2020

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“…Importantly, FLIM is compatible with confocal or multiphoton laser scanning microscopy as well as wide-field illumination. To obtain more details from each methodology, readers may refer to the following excellent publications [138][139][140][141].…”

Section: Analysis Of Nadh Autofluorescence By Flimmentioning

confidence: 99%

Analyzing Olfactory Neuron Precursors Non-Invasively Isolated through NADH FLIM as a Potential Tool to Study Oxidative Stress in Alzheimer’s Disease

Gómez-Virgilio

Luarte

Ponce

et al. 2021

IJMS

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Among all the proposed pathogenic mechanisms to understand the etiology of Alzheimer’s disease (AD), increased oxidative stress seems to be a robust and early disease feature where many of those hypotheses converge. However, despite the significant lines of evidence accumulated, an effective diagnosis and treatment of AD are not yet available. This limitation might be partially explained by the use of cellular and animal models that recapitulate partial aspects of the disease and do not account for the particular biology of patients. As such, cultures of patient-derived cells of peripheral origin may provide a convenient solution for this problem. Peripheral cells of neuronal lineage such as olfactory neuronal precursors (ONPs) can be easily cultured through non-invasive isolation, reproducing AD-related oxidative stress. Interestingly, the autofluorescence of key metabolic cofactors such as reduced nicotinamide adenine dinucleotide (NADH) can be highly correlated with the oxidative state and antioxidant capacity of cells in a non-destructive and label-free manner. In particular, imaging NADH through fluorescence lifetime imaging microscopy (FLIM) has greatly improved the sensitivity in detecting oxidative shifts with minimal intervention to cell physiology. Here, we discuss the translational potential of analyzing patient-derived ONPs non-invasively isolated through NADH FLIM to reveal AD-related oxidative stress. We believe this approach may potentially accelerate the discovery of effective antioxidant therapies and contribute to early diagnosis and personalized monitoring of this devastating disease.

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“…Fluorescence lifetime imaging was extended to determine the concentration of NADH. 36 The assessment of mitochondrial activity through NADH autofluorescence by live cell microscopy gives a range of outputs reflecting the activity of ETC as well as substrate supply which is conceptually and practically appealing. 37…”

Section: Can Nad+ and Nadh Be Responsible For Mas?mentioning

confidence: 99%

Potential biomarkers in septic shock besides lactate

Yang

Zhang

2020

Exp Biol Med (Maywood)

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Septic shock can be defined as sepsis with persisting hypotension and is required for vasopressors after initial unsuccessful fluid resuscitation. Elevated lactate is a biomarker of tissue perfusion and oxygenation and a useful prognostic tool for resuscitation in septic shock, as it is a byproduct of anaerobic glycolysis due to inadequate oxygen delivery and tissue hypoxia. Early and serial systematic lactate measurement will prompt physician more rapid intervention and lactate normalization, which is associated with better outcome. However, lactate formation during septic shock is neither entirely related to tissue hypoxia, nor reversible by increasing oxygen delivery. Meanwhile, lactate can be oxidized via tricarboxylic acid cycle after being transferred into mitochondria via lactate shuttle, which indicates elevated lactate can be used rather than only accumulation. Glycolysis and elevated lactate can be initiated by hypoxia, but persistent hyperlactatemia may not only represent persistent hypoxia. Some other potential biomarkers have been reviewed in the article including intermediates of tricarboxylic acid cycle, malate-aspartate shuttle, the nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD+/NADH) ratio, NAD+, NADH, malate, and malate dehydrogenase from the point of view of energy metabolism. Among them, malate dehydrogenase participates in both malate-aspartate shuttle and tricarboxylic acid cycle, and it can also indirectly reflex the NAD+/NADH ratio. It is reasonable to hypothesize that the combination of lactate and malate dehydrogenase will be more comprehensive to reflex hypoxia in septic shock. Impact statement Elevated lactate has been commonly considered as a biomarker and a useful prognostic tool for resuscitation in septic shock, facilitating physician more rapid intervention and treatment. However, it can be initiated by hypoxia, but persistent hyperlactatemia may not represent persistent hypoxia only. In the article, it is the first time to review potential biomarkers in septic shock from the point of view of energy metabolism including intermediates of TCA cycle, MAS, the NAD+/NADH ratio, NAD+, NADH, malate, and MDH. And the combination of lactate and MDH is also proposed in septic shock for the first time, as MDH in cytoplasm and mitochondria participates in both MAS and TCA cycle for ATP generation. Its feasibility in clinic has been analyzed at the end, although related research is still limited. It is reasonable the combination of lactate and MDH will be more comprehensive to reflex hypoxia in septic shock.

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Metabolic Profiling of Live Cancer Tissues Using NAD(P)H Fluorescence Lifetime Imaging

Cited by 10 publications

References 93 publications

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

Analyzing Olfactory Neuron Precursors Non-Invasively Isolated through NADH FLIM as a Potential Tool to Study Oxidative Stress in Alzheimer’s Disease

Potential biomarkers in septic shock besides lactate

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