Epigenetic regulation established during development to maintain patterns of transcriptional expression and silencing for metabolism and other fundamental cell processes can be reprogrammed in cancer, providing a molecular mechanism for persistent alterations in phenotype. Metabolic deregulation and reprogramming are thus an emerging hallmark of cancer with opportunities for molecular classification as a critical preliminary step for precision therapeutic intervention. Yet, acquisition of therapy resistance against most conventional treatment regimens coupled with tumor relapse, continue to pose unsolved problems for precision healthcare, as exemplified in breast cancer where existing data informs both cancer genotype and phenotype. Furthermore, epigenetic reprograming of the metabolic milieu of cancer cells is among the most crucial determinants of therapeutic resistance and cancer relapse. Importantly, subtype-specific epigenetic-metabolic interplay profoundly affects malignant transformation, resistance to chemotherapy, and response to targeted therapies. In this review, we therefore prismatically dissect interconnected epigenetic and metabolic regulatory pathways and then integrate them into an observable cancer metabolism-therapy-resistance axis that may inform clinical intervention. Optimally coupling genome-wide analysis with an understanding of metabolic elements, epigenetic reprogramming, and their integration by metabolic profiling may decode missing molecular mechanisms at the level of individual tumors. The proposed approach of linking metabolic biochemistry back to genotype, epigenetics, and phenotype for specific tumors and their microenvironment may thus enable successful mechanistic targeting of epigenetic modifiers and oncometabolites despite tumor metabolic heterogeneity.

NMR spectroscopy has been applied to cells and tissues analysis since its beginnings, as early as 1950. We have attempted to gather here in a didactic fashion the broad diversity of data and ideas that emerged from NMR investigations on living cells. Covering a large proportion of the periodic table, NMR spectroscopy permits scrutiny of a great variety of atomic nuclei in all living organisms non-invasively. It has thus provided quantitative information on cellular atoms and their chemical environment, dynamics, or interactions. We will show that NMR studies have generated valuable knowledge on a vast array of cellular molecules and events, from water, salts, metabolites, cell walls, proteins, nucleic acids, drugs and drug targets, to pH, redox equilibria and chemical reactions. The characterization of such a multitude of objects at the atomic scale has thus shaped our mental representation of cellular life at multiple levels, together with major techniques like mass-spectrometry or microscopies. NMR studies on cells has accompanied the developments of MRI and metabolomics, and various subfields have flourished, coined with appealing names: fluxomics, foodomics, MRI and MRS (i.e. imaging and localized spectroscopy of living tissues, respectively), whole-cell NMR, on-cell ligand-based NMR, systems NMR, cellular structural biology, in-cell NMR… All these have not grown separately, but rather by reinforcing each other like a braided trunk. Hence, we try here to provide an analytical account of a large ensemble of intricately linked approaches, whose integration has been and will be key to their success. We present extensive overviews, firstly on the various types of information provided by NMR in a cellular environment (the "why", oriented towards a broad readership), and secondly on the employed NMR techniques and setups (the "how", where we discuss the past, current and future methods). Each subsection is constructed as a historical anthology, showing how the intrinsic properties of NMR spectroscopy and its developments structured the accessible knowledge on cellular phenomena. Using this systematic approach, we sought i) to make this review accessible to the broadest audience and ii) to highlight some early techniques that may find renewed interest. Finally, we present a brief discussion on what may be potential and desirable developments in the context of integrative studies in biology.

High magnetic fields expand our capability to use sodium MRI for biomedical applications. The central goal of this review is devoted to the unique features of sodium MRI in tumor animal models, mainly in glioma, performed at 9.4 and 21.1 T. The ability of sodium MRI to monitor tumor response to therapy was evaluated. It is noteworthy that sodium MRI can detect glioma response to chemotherapy earlier than diffusion MRI. Especially attractive is the ability of sodium MRI to predict tumor therapeutic resistance before therapy. The non-invasive prediction of tumor chemo-resistance by sodium MRI presents a potential to individualize strategies for cancer treatment. Specifics of sodium MRI and technical aspects of imaging are also presented.

Characteristic of the tumor microenvironment are fluctuating gradients of reduced nutrient levels and released lactate. A fundamental issue is how tumor cells modulate their metabolic activity when both glucose and glutamine levels become limiting in the presence of high exogenous lactate. For functional analyses, the activities of pyruvate kinase, lactate dehydrogenase (LDH) and plasma membrane NADH oxidase (NOX) as well as cell growth were measured in breast cancer MCF-7 cells cultured in medium containing various concentrations of these metabolites. After 3 days at glucose concentrations below 2.5 mM, cell number was higher with 0.1 mM than with 1.0 mM glutamine, indicating that the glucose/glutamine balance is important for growth. On the other hand, NOX activity increased with increasing glucose >2.5 mM, but only with low glutamine (0.1 mM). Pyruvate kinase activity also increased, with LDH activity remaining 2-3-fold lower. Here NOX could have a complementary role in reoxidizing NADH for glycolysis. Exogenous lactate supported cell survival at limiting concentrations of glucose and glutamine while increasing NOX and pyruvate kinase activities as well as NADH levels. It is proposed that lactate supports cell survival by fuelling gluconeogenesis and/or the TCA cycle in mitochondria, from where NADH could be shuttled to the cytosol and reoxidized by NOX. Cell survival and the metabolic phenotype are thus interrelated to the dynamics of NADH and plasma membrane NOX activity, which are regulated by the balance of glucose/glutamine levels, in conjunction with lactate in a precarious tumor microenvironment.

We aimed to characterize the (31)P magnetic resonance spectra of various ovarian cancer cell lines exhibiting differences in cytotoxic drug resistance. We examined the metabolic profile of three different ovarian cancer cell lines, OC238, A2780, and A2780-cisplatin resistant (A2780cisR), including their response to various cytotoxic drugs (paclitaxel, cisplatin, and carboplatin) by (31)P magnetic resonance spectroscopy (MRS) in vitro. In the OC238 cell line, there were higher levels of phosphorylcholine, phosphodiesters, and uridine diphosphosugar (UDPS) + nicotinamide adenine dinucleotide phosphate (NADP). In A2780 and A2780cisR cell lines, phosphocreatine gave a high signal, which was absent in the OC238 cell line. In the OC238 cell line, a significant decrease in the glycerophosphoethanolamine, glycerophosphocholine, NADP, and UDPS signals was detected following cytotoxic drug treatment, mainly in response to paclitaxel. A significant increase in the glycerophosphocholine signal was detected following exposure to paclitaxel in both A2780 and A2780cisR cell lines. NADP and UDPS signals increased in response to all drugs in the A2780 cell line; however, in the cisplatin-resistant cell line A2780cisR, no significant change in those signals was detected following cisplatin treatment. We conclude that different ovarian cancer cell lines show characteristic (31)P MRS fingerprints and specific metabolic changes in response to cytotoxic drug treatment.

MR-compatible bioartificial liver (BAL) studies have been performed for 30 years and are reviewed. There are two types of study: (i) metabolism and drug studies using multinuclear MRS; primarily short-term (< 8 h) studies; (ii) the use of multinuclear MRS and MRI to noninvasively define the features and functions of BAL systems for long-term liver tissue engineering. In the latter, these systems often undergo not only modification of the perfusion system, but also the construction of MR radiofrequency probes around the bioreactor. We present novel MR-compatible BALs and the use of multinuclear MRS ((13)C, (19)F, (31)P) for the noninvasive monitoring of their growth, metabolism and viability, as well as (1)H MRI methods for the determination of flow profiles, diffusion, cell distribution, quality assurance and bioreactor integrity. Finally, a simple flexible coil design and circuit, and life support system, are described that can make almost any BAL MR-compatible.

Ex vivo and in vivo applications of magnetic resonance spectroscopy have been developed which aid in distinguishing malignant from normal tissues. Studies of breast, colon, cervix, oesophageal and prostate cancer reveal both the successes and failings of present technology. Verification that these non-invasive tests might supplant conventional histology in obtaining spatial diagnostic and chemical prognostic information remains for the time being illusive.

An artificial tumor method was developed to study cells inside the sensitive volume of an NMR spectrometer during growth and apoptosis. The tumor was composed of a 50:50 mixture of tightly packed porous-collagen and nonporous-polystyrene microspheres. The porous collagen served as a growth surface for the tumor cells, and the nonporous polystyrene served as a structural support to limit compression of the packed bed during perfusion. The microspheres were held between two porous polyethylene discs that were tightly sealed inside the NMR perfusion chamber. The new method was evaluated with two cell types: a mouse mammary tumor line (EMT6/SF) and a human glioma line (SF188). The results indicate that for both lines, approximately 10(9) metabolically active cells could be sustained for at least 1 week in the 12-cm(3) artificial tumor. Further, cells undergoing chemotherapy-induced apoptosis (which is known to cause detachment of cells from their surroundings) were retained in the artificial tumor. In preliminary 31P NMR studies, glioma cells treated with temozolomide (TMZ) exhibited reduced phosphocholine (PCh) levels relative to glycerophosphocholine (GPC) and diphosphodiester (DPDE) levels. They also exhibited sharply reduced oxygen consumption and TCA cycle 13C labeling, while they retained glycolytic activity. These metabolic changes are consistent with those that would be expected during mitochondrially-mediated apoptosis.

The discovery of the phenomenon of nuclear magnetic resonance occurred just 60 years ago. The profusion of subsequent discoveries in this domain has led to the development of magnetic resonance spectroscopy - refined as an analytical tool to discern molecular structure - and magnetic resonance imaging, a cornerstone of modern radiology. Observable alterations in cellular structure and metabolism can be discerned using the non-destructive chemical analysis of magnetic resonance spectroscopy in vitro or in vivo. Differences may thus be discerned between malignant and normal tissues.

A method was developed for obtaining high signal-to-noise 13C NMR spectra of intracellular compounds in metabolically active cultured cells. The method allows TCA cycle labeling kinetics to be determined in real time without significant oxygen transport limitations. Cells were immobilized on the surface of nonporous microcarriers that were either uncoated or coated with polypeptides and used in a 12-cm3 packed bed. The methods were tested with two EMT6 mouse mammary tumor cell lines, one strongly adherent and the other moderately adherent, and a weakly adherent mouse insulinoma line (betaHC9). For both EMT6 lines, NTP and oxygen consumption measurements indicated that the number of cells in the spectrometer ranged from 6 x 10(8) to 1 x 10(9). During infusion of [1-13C]glucose, labeling in C-4 glutamate (indicative of flux into the first half of the TCA cycle) could be detected with 15-min resolution. However, labeling for C-3 and C-2 glutamate (indicative of complete TCA cycle activity) was fivefold lower and difficult to quantify. To increase TCA cycle labeling, cells were infused with medium containing [1,6-13C2]glucose. A 2.5-fold increase was observed in C-4 glutamate labeling and C-3 and C-2 glutamate labeling could be monitored with 30-min resolution. Citrate synthase activity was indirectly detected in real time, as [3,4-13C2]glutamate was formed from [2-13C]oxaloacetate and [2-13C]acetate (of acetyl-CoA). Cell mass levels observed with betaHC9 cells were somewhat lower. However, the 13C S/N was sufficient to allow real-time monitoring of the response of intracellular metabolite labeling to a step change in glucose and a combined glutamine/serum pulse.

For the past 70 years the dominant perception of cancer metabolism has been that it is fuelled mainly by glucose (via aerobic glycolysis) and glutamine. Consequently, investigations into the diagnosis, treatment and the basic metabolism of cancer cells have been directed by this perception. However, the data on cancer metabolism are equivocal, and in this study we have sought to clarify the issue. Using an innovative system we have measured the total ATP turnover of the MCF-7 breast cancer cell line, the contributions to this turnover by oxidative and glycolytic ATP production and the contributions to the oxidative component by glucose, lactate, glutamine, palmitate and oleate. The total ATP turnover over approx. 5 days was 26.8 micromol of ATP.10(7) cells(-1).h(-1). ATP production was 80% oxidative and 20% glycolytic. Contributions to the oxidative component were approx. 10% glucose, 14% glutamine, 7% palmitate, 4% oleate and 65% from unidentified sources. The contribution by glucose (glycolysis and oxidation) to total ATP turnover was 28.8%, glutamine contributed 10.7% and glucose and glutamine combined contributed 40%. Glucose and glutamine are significant fuels, but they account for less than half of the total ATP turnover. The contribution of aerobic glycolysis is not different from that in a variety of other non-transformed cell types.

A localized 2D correlation spectroscopic sequence (L-COSY) was implemented and applied in human breast cancer in vivo to evaluate the water to fat (both saturated and unsaturated) ratios and also to identify choline. Being in agreement with the conventional 1D magnetic resonance spectroscopy (MRS) results, elevated water to lipids ratios were found in breast cancers and choline was observed only in a few cancer patients.

To assess the potential clinical utility of in-vivo 31P magnetic resonance spectroscopy (MRS) in patients with various malignant and benign breast lesions.

Seventeen patients with untreated primary malignant breast lesions (group I), eight patients with untreated benign breast lesions (group II) and seven normal breasts (group III) were included in this study. In-vivo 31P MRS was performed using a 1.5 Tesla MR scanner. Because of the characteristics of the coil, the volume of the tumor had to exceed 12 cc (3 x 2 x 2 cm), with a superoinferior diameter at least 3 cm. Mean and standard deviations of each metabolite were calculated and metabolite ratios, such as PME/PCr, PDE/PCr, T-ATP/PCr and PCr/T-ATP were calculated and statistically analyzed.

Significant differences in PME were noted between groups I and III (p=0.0213), and between groups II and III (p=0.0213). The metabolite ratios which showed significant differences were PME/PCr (between groups II and III) (p=0.0201), PDE/PCr (between groups I and III, and between groups II and III) (p=0.0172), T-ATP/PCr (between groups II and III) (p=0.0287), and PCr/T-ATP (between groups II and III) (p=0.0287). There were no significant parameters between groups I and II.

In-vivo 31P MRS is not helpful for establishing a differential diagnosis between benign and malignant breast lesions, at least with relatively large lesions greater than 3 cm in one or more dimensions.

Hepatocyte growth factor/scatter factor (HGF/SF) is a paracrine growth factor which increases cellular motility and has also been implicated in tumor development and progression and in angiogenesis. Little is known about the metabolic alteration induced in cells following Met-HGF/SF signal transduction. The hypothesis that HGF/SF alters the energy metabolism of cancer cells was investigated in perfused DA3 murine mammary cancer cells by nuclear magnetic resonance (NMR) spectroscopy, oxygen and glucose consumption assays and confocal laser scanning microscopy (CLSM). 31P NMR demonstrated that HGF/SF induced remarkable alterations in phospholipid metabolites, and enhanced the rate of glucose phosphorylation (P < .05). 13C NMR measurements, using [13C1]-glucose-enriched medium, showed that HGS/SF reduced the steady state levels of glucose and elevated those of lactate (P < .05). In addition, HGF/SF treatment increased oxygen consumption from 0.58+/-0.02 to 0.71+/-0.03 micromol/hour per milligram protein (P < .05). However, it decreased CO2 levels, and attenuated pH decrease. The mechanisms of these unexpected effects were delineated by CLSM, using NAD(P)H fluorescence measurements, which showed that HGF/SF increased the oxidation of the mitochondrial NAD system. We propose that concomitant with induction of ruffling, HGF/SF enhances both the glycolytic and oxidative phosphorylation pathways of energy production.

Glucosamine (GlcN) modulates fluoropyrimidine metabolism and enhances cytotoxicity of 5-fluorouridine (FUrd), but not of 5-fluorouracil (FUra), in human tumor models. To elucidate the underlying metabolic differences between FUra and FUrd, by the use of 19F and 31P NMR spectroscopy we studied these drugs in multicell tumor spheroids (MTS) formed by human colon carcinoma cells HT-29. This experimental system allowed detailed kinetic measurements of anabolic intracellular phosphates and fluorophosphates over periods of up to 2 days. Time-dependent NMR data were reduced and interpreted by the use of nonlinear compartmental models which yielded numerical values for the empirical rate constants characterizing mass transfer among the compartments. An analysis of these rate constants indicated qualitative and quantitative differences in the metabolism of FUra and FUrd and in the effects of GlcN on these drugs. The enhanced generation of FUDP-hexoses was a predicted effect of GlcN, but inhibited formation of fluorouridine diphosphates and fluorouridine triphosphates in FUra-treated MTS, and the magnitude of stimulation of fluoropyrimidine incorporation into macromolecules in FUrd-treated MTS were not predicted.

The application of new gradient, high-resolution, magic angle spinning (MAS) 1H nuclear magnetic resonance (NMR) spectroscopy to the study of intact undifferentiated and differentiated NIH 3T3 F442A cells demonstrated improved spectral resolution and sensitivity compared with static studies. MAS of cells permits the detection and quantitation of many cellular metabolites that are not clearly resolved in nonspinning measurements and provides an improved visibility of phospholipids. Gradient, MAS enables the use of diffusion weighting for compartment assignment and the determination of mobility for many metabolites which are incompletely resolved using static techniques. The smaller, undifferentiated preadipocytes show no microscopic evidence of cell lysis after 2 h of MAS at 3.5 kHz and 82% of these cells remain viable by trypan blue exclusion. In contrast, 15-19% of the larger, lipid-laden differentiated adipocytes were found to suffer some degree of cell lysis with MAS. This new method is an attractive alternative to either nonspinning perfusion or extraction techniques for NMR studies of cells.

Tumor necrosis factor-alpha inhibited growth of cultured MCF-7 human breast cancer cells in a dose dependent manner. Tumor necrosis factor-alpha also markedly increased glucose consumption, and its cytotoxicity was modified by glucose concentrations in the growth medium; higher glucose levels were associated with increased cell survival. However, when the cells were perfused in physiological conditions, very high levels of tumor necrosis factor-alpha (200 ng/ml) in the perfusion solution had no inhibitory effects. Moreover, tumor necrosis factor-alpha had no effects on 31P nuclear magnetic resonance spectra of the perfused cells. In the traditional growth inhibition assays, cells are incubated for several days with a drug, a situation where their metabolism is altered due to the depletion of nutrients, the accumulation of toxic waste materials and pH changes. Perfusion experiments are more relevant to in vivo conditions, and may be used for studying metabolic processes and the mechanisms of action of therapeutic agents.

Lonidamine (LND) is a relatively new anti-cancer drug, and several clinical trials have indicated that it may be effective in combinations with other therapeutic modalities. LND is classified within the metabolic inhibitor agents. Multidrug resistance (MDR) phenomenon is often associated with increased energy requirements, and enhanced glycolysis rate. These studies were performed to delineate the mechanism of action of LND on MDR human breast cancer cells, and to investigate whether LND as a single agent, or in combination with another anti-metabolism drug, 2-deoxyglucose (2-DG), may be useful against MDR tumors. The effects of LND on intact perfused drug-sensitive (WT) and 33-fold resistant to Adriamycin (Adr) MCF-7 cells, embedded in alginate micro capsules, were continuously monitored by 31P and 13C nuclear magnetic resonance (NMR) spectroscopy. 31P NMR studies showed that LND induced intracellular acidification and depletion of NTP in both WT and Adr cells. However, pH and NTP levels decreased less in the Adr cells than in the WT cells (p < 0.05 for both parameters). 13C NMR demonstrated that LND inhibited lactate transport, and lactate signals were elevated in both cell lines. However, the intracellular lactate levels increased to a greater extent in the WT than in the Adr cells (p < 0.05). There were major differences in the effects of LND on metabolism between sensitive and resistant cells. While LND enhanced glucose uptake in the WT cells, and its administration was followed by continuous increase of lactate signal, both processes were not affected by LND in the Adr cells. 2-DG is a glucose analogue that inhibits both cellular uptake and utilization of glucose, leading to cell starvation. Combined treatment with LND and 2-DG yielded at best additive, but not synergistic, cellular toxicity, and the metabolic effects of LND were attenuated by 2-DG. These results showed that the principal mechanism of action of LND is inhibition of lactate transport leading to intracellular lactate accumulation and acidification in both WT and Adr cells. The Adr cells were only 2-fold resistant to LND (compared to the WT cells), and since cellular uptake of alkaloid chemotherapy is improved in acidic environment, LND may have a role in the treatment protocols of MDR tumors, especially when given as the initial means for induction of intracellular acidification.