Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy (original) (raw)
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Biosensors
Isocitrate dehydrogenase 1 (IDH1) mutations in gliomas, fibrosarcoma, and other cancers leads to a novel metabolite, D-2-hydroxyglutarate, which is proposed to cause tumorigenesis. The production of this metabolite also causes vulnerabilities in cellular metabolism, such as lowering NADPH levels. To exploit this vulnerability, we treated glioma and fibrosarcoma cells that harbor an IDH1 mutation with an inhibitor of nicotinamide adenine dinucleotide (NAD+) salvage pathway, FK866, and observed decreased viability in these cells. To understand the mechanism of action by which the inhibitor FK866 works, we used Raman imaging microscopy and identified that proteins and lipids are decreased upon treatment with the drug. Raman imaging showed a different distribution of lipids throughout the cell in the presence of the drug compared with the untreated cells. We employed nuclear magnetic resonance NMR spectroscopy and mass spectrometry to identify the classes of lipids altered. Our combined...
Cancer research, 2018
Label-free non-linear optical microscopy (NLOM) based on two-photon excited fluorescence (TPEF) from cofactors Nicotinamide Adenine Dinucleotide (NADH) and Flavin Adenine Dinucleotide (FAD+) is widely used for high-resolution cellular redox imaging. In this work, we combined three, label-free NLOM imaging methods to quantitatively characterize breast cancer cells and their relative invasive potential: 1) TPEF optical redox ratio (ORR = FAD+/NADH + FAD+), 2) coherent Raman scattering (CRS) of cellular lipids, and 3) second harmonic generation (SHG) of extracellular matrix (ECM) collagen. 3D spheroid models of primary mammary epithelial cells (PME) and breast cancer cell lines (T47D and MDA-MB-231) were characterized based on their unique ORR and lipid volume fraction signatures. Treatment with 17β-estradiol (E2) increased glycolysis in both PME and T47D ER+ breast cancer cells. However, PME cells displayed increased lipid content with no ECM effect, while T47D cells had decreased lip...
arXiv (Cornell University), 2019
In situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destruct the sample or introduce strong perturbations to the functions of target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a labelfree single-cell analysis platform with chemical specifity, and high-throughput capabilities. Cellular compartments such as lipid droplets, endoplasmic reticulum, and nuclei are seperated from the cytoplasm. Based on these chemical segmentations, 260 features from both morphology and molecular composition were generated and analyzed for each cell. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapy drug treatments. We unveiled lipid-facilitated protrusion as a metabolic marker for stress-resistant cancer cells through statistical analysis of thousands of cells. Our findings also demonstrate the potential of targeting lipid metabolism for selective treatment of starvation-resistant and chemotherapy-resistant cancers. These results highlight our SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity. Altered cell metabolism is recognized as one of the hallmarks of cancer (Hanahan and Weinberg, 2011). The reprogrammed metabolism, which is deployed by cancer cells to fulfill the demands of fast proliferation , offers new opportunities for diagnosis and treatment of cancer . However, challenges remain in the quantification of cell metabolism, one of which is the inter-or intratumoral heterogeneous metabolic profiles of cancer cells. Although the ensemble measurement of large number of cancer cells identifies specific metabolic features under certain condition, individual cancer cells might show significantly different metabolic response . This cell heterogeneity is considered one of the major causes of incomplete tumor remission and relapse . Such an inhomogeneous response of cells to environments and drugs cannot be addressed using conventional methods, such as biochemical assays or mass spectrometry, which are based on ensemble measurement of a cell population. High-efficiency imaging tools which can quantify metabolic features of individual cells with subcellular information for a large number of cells become essential. Flow cytometry is one of the commonly used technologies for high-throughput single-cell analysis, which generates statistical information of a cell population .
Optical imaging of metabolic dynamics in animals
Nature Communications, 2018
Direct visualization of metabolic dynamics in living animals with high spatial and temporal resolution is essential to understanding many biological processes. Here we introduce a platform that combines deuterium oxide (D 2 O) probing with stimulated Raman scattering (DO-SRS) microscopy to image in situ metabolic activities. Enzymatic incorporation of D 2 O-derived deuterium into macromolecules generates carbon–deuterium (C–D) bonds, which track biosynthesis in tissues and can be imaged by SRS in situ. Within the broad vibrational spectra of C–D bonds, we discover lipid-, protein-, and DNA-specific Raman shifts and develop spectral unmixing methods to obtain C–D signals with macromolecular selectivity. DO-SRS microscopy enables us to probe de novo lipogenesis in animals, image protein biosynthesis without tissue bias, and simultaneously visualize lipid and protein metabolism and reveal their different dynamics. DO-SRS microscopy, being noninvasive, universally applicable, and cost-effective, can be adapted to a broad range of biological systems to study development, tissue homeostasis, aging, and tumor heterogeneity.
J Biomed Opt, 2014
Measurement of endogenous free and bound NAD(P)H relative concentrations in living cells is a useful method for monitoring aspects of cellular metabolism, because the NADH∕NAD þ reduction-oxidation pair is crucial for electron transfer through the mitochondrial electron transport chain. Variations of free and bound NAD(P)H ratio are also implicated in cellular bioenergetic and biosynthetic metabolic changes accompanying cancer. This study uses two-photon fluorescence lifetime imaging microscopy (FLIM) to investigate metabolic changes in MCF10A premalignant breast cancer cells treated with a range of glycolysis inhibitors: namely, 2 deoxy-D-glucose, oxythiamine, lonidamine, and 4-(chloromethyl) benzoyl chloride, as well as the mitochondrial membrane uncoupling agent carbonyl cyanide m-chlorophenylhydrazone. Through systematic analysis of FLIM data from control and treated cancer cells, we observed that all glycolytic inhibitors apart from lonidamine had a slightly decreased metabolic rate and that the presence of serum in the culture medium generally marginally protected cells from the effect of inhibitors. Direct production of glycolytic L-lactate was also measured in both treated and control cells. The combination of these two techniques gave valuable insights into cell metabolism and indicated that FLIM was more sensitive than traditional biochemical methods, as it directly measured metabolic changes within cells as compared to quantification of lactate secreted by metabolically active cells.
Journal of Photochemistry and Photobiology B: Biology, 2009
Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., freeto-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed noninvasive assay, we estimate the average NADH concentration in cancer cells (168 ± 49 μM) to be ~ 1.8 fold higher than in breast normal cells (99 ± 37 μM). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, timeresolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18 ± 0.08) and enzyme-bound (0.82 ± 0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different from those in breast cancer cells (free: 0.25 ± 0.08; bound: 0.75 ± 0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as in conventional biochemical assays.
Proteins are one of the major components of biological systems, and understanding their metabolism is critical to study various biochemical processes in living systems. Despite extensive efforts to study protein metabolism such as autoradiography, mass spectrometry, and fluorescence microscopy, visualizing the spatial distribution of overall protein metabolism in mammals at subcellular resolution is still challenging. A recent study from our group reported imaging newly synthesized proteins in cultured mammalian cells, tissues, or even in mice using stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (dAA). However, our previous method of dAA administration via drinking water, albeit convenient, is insufficient for in vivo studies. This is due to poor labeling efficiency and limited access to many important organs such as the brain, pancreas, or tumor. In this study, we have significantly improved and optimized the in vivo administration method by intra-carotid arterial injection of dAA in mice and obtained imaging contrast of protein metabolic activity in many more organs and tissues, such as cerebral and cerebellar cortex and hippocampal regions in the mouse brain. We also imaged newly formed proteins in the choroid plexus and pancreas at different time points, illustrating the metabolic dynamics of proteins in these important secretory organs. In addition, we visualized the metabolic heterogeneity of protein synthesis in colon tumor xenografts, which can be used to distinguish tumor and normal tissues. In summary, this combination of a new dAA administration technique and SRS imaging platform demonstrates an effective tool for the in vivo study of complex protein metabolism in mammals, in both physiological and pathological states.
Scientific Reports
Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. The effectiveness of traditional therapies for GBM is limited and therefore new therapies are highly desired. Previous studies show that lipid metabolism reprogramming may be a potential therapeutic target in GBM. This study aims to evaluate the therapeutic potential of free fatty acid-induced lipotoxicity for the suppression of glioma growth. U87 glioma cells are treated with three fatty acids (FAs): palmitic acid (PA), oleic acid (OA), and eicosapentaenoic acid (EPA). Uptake of the FAs and formation of lipid droplets (LDs) are imaged and quantified using a lab-built stimulated Raman scattering (SRS) microscope. Our results show that a supply of 200 µM PA, OA, and EPA leads to efficient LDs accumulation in glioma cells. We find that inhibition of triglycerides (TAGs) synthesis depletes LDs and enhances lipotoxicity, which is evidenced by the reduced cell proliferation rates. In particular, our results suggest...
Cancer Research, 2014
Alterations in the balance between different metabolic pathways used to meet cellular bioenergetic and biosynthetic demands are considered hallmarks of cancer. Optical imaging relying on endogenous fluorescence has been used as a noninvasive approach to assess tissue metabolic changes during cancer development. However, quantitative correlations of optical assessments with variations in the concentration of relevant metabolites or in the specific metabolic pathways that are involved have been lacking. In this study, we use high-resolution, depth-resolved imaging, relying entirely on endogenous two-photon excited fluorescence in combination with invasive biochemical assays and mass spectrometry to demonstrate the sensitivity and quantitative nature of optical redox ratio tissue assessments. We identify significant differences in the optical redox ratio of live, engineered normal and precancerous squamous epithelial tissues. We establish that while decreases in the optical redox ratio...