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Targeted chemotherapeutic agents often do not result in tumor shrinkage, so new biomarkers that correlate with clinical efficacy are needed. In this study, we investigated noninvasive imaging protocols to monitor responses to sorafenib, a multikinase inhibitor approved for treatment of renal cell and hepatocellular carcinoma. Healthy cells are impermeable to fumarate, so conversion of this metabolite to malate as detected by (13)C-magnetic resonance spectroscopy (MRS) has been suggested as one marker for cell death and treatment response in tumors. Diffusion MRI also has been suggested as a measure of therapy-induced cytotoxic edema because viable cells act as a diffusion barrier in tissue. For these reasons, we assessed sorafenib responses using hyperpolarized (13)C-fumarate, diffusion-weighted MRI (DW-MRI) in a xenograft model of human breast cancer in which daily administration of sorafenib was sufficient to stabilize tumor growth. We detected signals from fumarate and malate following intravenous administration of hyperpolarized fumarate with a progressive increase in the malate-to-fumarate (MA/FA) ratio at days 2 to 5 after sorafenib infusion. The apparent diffusion coefficient (ADC) measured by DW-MRI increased in the treated group consistent with cytotoxic edema. However, the MA/FA ratio was a more sensitive marker of therapeutic response than ADC, with 2.8-fold versus 1.3-fold changes, respectively, by day 5 of drug treatment. Histologic analyses confirmed cell death in the sorafenib-treated cohort. Notably, (13)C-pyruvate-to-lactate conversion was not affected by sorafenib in the breast cancer model examined. Our results illustrate how combining hyperpolarized substrates with DW-MRI can allow noninvasive monitoring of targeted therapeutic responses at relatively early times after drug administration.

Hyperpolarized (13)C magnetic resonance spectroscopy provides a unique opportunity to detect real-time metabolic fluxes as a means to measure metabolic treatment responses in vivo. Here, we show that pharmacologic inhibition of lactate dehydrogenase-A suppressed the conversion of hyperpolarized (13)C-pyruvate to lactate in murine xenografts of P493 human lymphoma. In contrast, a glutaminase inhibitor reduced conversion of (13)C-pyruvate to alanine without affecting conversion of pyruvate to lactate. These results illustrate the ability to monitor biomarkers for responses to antimetabolic therapy in real-time, paving the way for clinical development of imaging biomarkers to monitor metabolic pharmacodynamics., (©2013 AACR.)

Tumor choline metabolites have potential for use as diagnostic indicators of breast cancer phenotype and can be non-invasively monitored in vivo by MRS. Extract studies have determined that the principle diagnostic component of these peaks is phosphocholine (PCho), the biosynthetic precursor to the membrane phospholipid, phosphatidylcholine (PtdCho). The ability to resolve and quantify PCho in vivo would improve the accuracy of this putative diagnostic tool. In addition, determining the biochemical mechanisms underlying these metabolic perturbations will improve the understanding of breast cancer and may suggest potential molecular targets for drug development. Reported herein is the in vivo resolution and quantification of PCho and glycerophosphocholine (GPC) in breast cancer xenografts in SCID mice via image-guided 31P MRS, localized to a single voxel. Tumor metabolites are also detected using ex vivo extracts and high-resolution NMR spectroscopy and are quantified in the metastatic tumor line, MDA-mb-231. Also reported is the quantification of cytosolic and lipid metabolites in breast cells of differing cancer phenotype, and the identification of metabolites that differ among these cell lines. In cell extracts, PCho and the PtdCho breakdown products, lysophosphatidylcholine, GPC and glycerol 3-phosphate, are all raised in breast cancer lines relative to an immortalized non-malignant line. These metabolic differences are in direct agreement with differences in expression of genes encoding enzymes in the choline metabolic pathway. Results of this study are consistent with previous studies, which have concluded that increased choline uptake, increased choline kinase activity, and increased phosholipase-mediated turnover of PtdCho contribute to the observed increase in PCho in breast cancer. In addition, this study presents evidence suggesting a specific role for phospholipase A2-mediated PtdCho catabolism. Gene expression changes following taxane therapy are also reported and are consistent with previously reported changes in choline metabolites after the same therapy in the same tumor model.

Diffusion-weighted MRI is commonly used in the diagnosis and evaluation of ischemic stroke because of the rapid decrease observed in the apparent diffusion coefficient (ADC) of tissue water following ischemia. Although this observation has been clinically useful for many years, the biophysical mechanisms underlying the reduction of tissue ADC are still unknown. To help elucidate these mechanisms, we have employed a novel three-dimensional (3D) hollow-fiber bioreactor (HFBR) perfused cell culture system that enables cells to be grown to high density and studied via MRI and MRS. By infusing contrast media into the HFBR, signals from intracellular water and extracellular water are spectroscopically resolved and can be investigated individually. Diffusion measurements carried out on C6 glioma HFBR cell cultures indicate that ischemia-induced cellular swelling results in an increase in the ADC of intracellular water from 0.35 microm(2)/ms to approximately 0.5 microm(2)/ms (diffusion time = 25 ms)., ((c) 2008 Wiley-Liss, Inc.)

Choline-containing compounds (CCCs) are elevated in breast cancer, and detected in vivo by the (1)H MRS total choline (tCho) resonance (3.25 ppm) and the (31)P MRS phosphomonoester (PME) resonance (3.8 ppm). Both the tCho and PME resonances decrease early after initiation of successful therapy. The single major component of these composite resonances, phosphocholine (PCho), also responds to therapy by decreasing. The ability to resolve and quantify PCho in vivo would thus increase the sensitivity of this biomarker for early detection of therapeutic response. Herein, the in vivo resolution and quantification of PCho is reported in human mouse xenograft tumors of the human breast cancer cell lines MCF-7 and MDA-mb-231. Significant decreases in tumor PCho are observed within 2 to 4 d posttreatment with the antimicrotubule drug, docetaxel. To determine whether these decreases are a general tumor response or an intracellular metabolic response, high-resolution NMR spectroscopy was performed on extracts of cells treated with docetaxel. Significant decreases in intracellular PCho and increases in glycerophosphocholine (GPC) were observed. These decreases are coincident with other tumor and cellular responses such as tumor growth delay (TGD), cell-cycle arrest, and modes of cell death such as mitotic catastrophe, necrosis, and apoptosis, with mitotic catastrophe predominating.

Previous studies using MR spectroscopy have shown that the extracellular pH (pH(e)) of tumors is acidic compared to normal tissues. This has a number of important sequelae that favor the emergence of more aggressive and therapy-resistant tumors. New MRI methods based on pH-sensitive T1 relaxivity are an attractive alternative to previous spectroscopic methods, as they allow improvements in spatial and temporal resolution. Recently, pH-dependent GdDOTA-4AmP5- and a pH-independent analog, GdDOTP5-, were used to image renal pH in mice. The current study has used a similar approach to image pH(e) in rat gliomas. Significant differences were observed compared to the renal study. First, the relaxivity of GdDOTP5- was found to be affected by the higher extracellular protein content of tumors. Second, the pixel-by-pixel analysis of the GdDOTP5- and GdDOTA-4AmP5- pharmacokinetics showed significant dispersion, likely due to the temporal fluctuations in tumor perfusion. However, there was a robust correlation between the maximal enhancements produced by the two boluses. Therefore, to account for the local time-courses differences, pH(e) maps were calculated at the time of maximal enhancement in each pixel. Finally, the comparison of the pH(e) and the time to maximal intensity maps revealed an inverse relationship between pH(e) and tumor perfusion., (Copyright 2006 Wiley-Liss, Inc.)

The complexity of biologic tissues, with multiple compartments each with its own diffusion and relaxation properties, requires complex formalisms to model water signal in most magnetic resonance imaging or magnetic resonance spectroscopy experiments. In this article, we describe a magnetic susceptibility-induced shift in the resonance frequency of extracellular water by the introduction of a gadolinium contrast agent to medium perfusing a hollow fiber bioreactor. The frequency shift of the extracellular water (+185 Hz at 9.4 T) uncovers the intracellular water and allows direct measurement of motional and relaxation properties of the intracellular space. The proposed method provides a unique tool for understanding the mechanisms underlining diffusion and relaxation in the intracellular space.

Magnetic resonance spectroscopy (MRS) has been used for more than two decades to interrogate metabolite distributions in living cells and tissues. Techniques have been developed that allow multiple spectra to be obtained simultaneously with individual volume elements as small as 1 uL of tissue (i.e., 1 x 1 x 1 mm(3)). The most common modern applications of in vivo MRS use endogenous signals from (1)H, (31)P, or (23)Na. Important contributions have also been made using exogenous compounds containing (19)F, (13)C, or (17)O. MRS has been used to investigate cardiac and skeletal muscle energetics, neurobiology, and cancer. This review focuses on the latter applications, with specific reference to the measurement of tissue choline, which has proven to be a tumor biomarker that is significantly affected by anticancer therapies.

Acid-base balance is altered in a variety of common pathologies, including COPD, ischemia, renal failure, and cancer. Because of robust cellular pH homeostatic mechanisms, most of the pathological alterations in pH are expressed as changes in the extracellular, systemic pH. There are data to indicate that altered pH is not simply an epiphenomenon of metabolic or physiologic imbalance but that chronic pH alterations can have important sequelae. MRSI and MRI measurements indicate that pH gradients of up to 1.0 pH unit can exit within 1-cm distance. Although measurement of blood pH can indicate systemic problems, it cannot pinpoint the lesion or quantitatively assess the magnitude of excursion from normal pHe. Hence, there is a need to develop pHe measurement methods with high spatiotemporal resolution. The two major approaches being investigated include magnetization transfer methods and relaxation methods. pH-dependent MT effects can observed with endogenous signals or exogenously applied CEST agents. While endogenous signals have the advantage of being fully noninvasive and relatively straightforward to apply, they lack a full biophysical characterization and dynamic range that might be afforded by future CEST agents. pH-dependent relaxivity also requires the injection or infusion of exogenous contrast reagents. In both MT and relaxographic approaches, the magnitude of the effect, and, thus, the ability to quantify pHe, depends on a spatially and temporally varying concentration of the CR. A number of approaches have been proposed to solve this problem and, once it is solved, pH imaging methods will be applicable to human clinical pathologies.

The microenvironment within tumors is significantly different from that in normal tissues. A major difference is seen in the chaotic vasculature of tumors, which results in unbalanced blood supply and significant perfusion heterogeneities. As a consequence, many regions within tumors are transiently or chronically hypoxic. This exacerbates tumor cells' natural tendency to overproduce acids, resulting in very acidic pH values. The hypoxia and acidity of tumors have important consequences for antitumor therapy and can contribute to the progression of tumors to a more aggressive metastatic phenotype. Over the past decade, techniques have emerged that allow the interrogation of the tumor microenvironment with high resolution and molecularly specific probes. Techniques are available to interrogate perfusion, vascular distribution, pH, and pO(2) nondestructively in living tissues with relatively high precision. Studies employing these methods have provided new insights into the causes and consequences of the hostile tumor microenvironment. Furthermore, it is quite exciting that there are emerging techniques that generate tumor image contrast via ill-defined mechanisms. Elucidation of these mechanisms will yield further insights into the tumor microenvironment. This review attempts to identify techniques and their application to tumor biology, with an emphasis on nuclear magnetic resonance (NMR) approaches. Examples are also discussed using electron MR, optical, and radionuclear imaging techniques., (Copyright 2002 Wiley-Liss, Inc.)

Nm23 genes are involved in the control of the metastatic potential of breast carcinoma cells. To understand the impact of nm23 genes on tumor physiology and metabolism, a 31P nuclear magnetic resonance (NMR) spectroscopic study was performed on tumors formed in the mammary fat pad of severe combined immunodeficiency mice by MDA-MB-435 human breast carcinoma cells transfected with cDNA encoding wild type nm23-H1 and nm23-H2 proteins. Tumors formed by MDA-MB-435 cells transfected with vector alone were used as controls. All transgene tumors exhibited significantly higher levels of phosphodiester (PDE) compounds relative to phosphomonoester (PME) compounds in vivo compared with control tumors. Similar differences in PDE and PME also were observed for spectra obtained from cells growing in culture. Intracellular pH was significantly lower and extracellular pH was significantly higher for transgene tumors compared with control tumors. Histologic analysis of lung sections confirmed reductions in incidence, number, and size of metastatic nodules for animals bearing transgene tumors. These results suggest that nm23 genes may affect suppression of metastasis through phospholipid-mediated signaling and cellular pH regulation.

Quantification of metabolite or drug concentrations in living tissues requires determination of intra- and extracellular volumes. This study demonstrates how this can be achieved non-invasively by 31P magnetic resonance spectroscopy (MRS) employing dimethyl methylphosphonate (DMMP) as a marker of total water space, 3-aminopropylphosphonate (3-APP) as a marker of extracellular space and P and 3-APP as markers of intracellular pH (pH) and extracellular pH (pHe) respectively. The MRS measurements of the tumour volumes were validated by classic radiolabelling methods using 3H2O and [14C]inulin as markers of total and extracellular space respectively. The extracellular volume fraction measured by radiolabelling of RIF-1 tumours was 23 +/- 0.83% (mean +/- s.e.m. n = 9), not significantly different (P > 0.1) from that found by MRS (27 +/- 2.9%, n = 9, London, and 35 +/- 6.7, n = 14, Baltimore). In untreated RIF-1 tumours, pH was about 0.2 units higher than pHe (P < 0.01). 5-Fluorouracil (5FU) treatment (165 mg kg(-1)) caused no significant changes in either pHe or per cent extracellular volume. However significant increases in pH, 48 h after treatment (P < 0.01) correlated with decreased tumour size and improved bioenergetic status [NTP/inorganic phosphate (Pi) ratio]. This study shows the feasibility of an MR method (verified by a 'gold standard') for studying the effects of drug treatment on intra- and extracellular spaces and pH in solid tumours in vivo.

The purpose of this study was to study the metabolic events during a slow acidosis in three different cell lines by combining 31P magnetic resonance spectroscopy and hollow fiber bioreactor technology. The rate of change in intracellular pH, glycerophosphorylcholine (GPC), phophorylcholine (PCho), and nucleoside-triphosphate (NTP) levels were measured during 8 h of acidosis and 16 h of recovery in EPO, EAT, and RN1a cells, three cultured mammalians cell lines. Our results show a significant increase in GPC levels to 330 +/- 21.540 +/- 25, and 220 +/- 21% of their initial value correlated to a decrease of PCho levels to 57 +/- 14.58 +/- 17 and 45 +/- 15% of their initial value in EAT, RN1a, and EPO cells, respectively. These changes are discussed in terms of perturbation of energetic metabolism in cells undergoing a slow acidosis.

In vivo magnetic resonance spectroscopy (MRS) has revealed that phosphomonoesters (PME) such as phosphocholine (PCho) and phosphoethanolamine (PEth) are elevated in tumors and rapidly proliferating tissues. The regulation of PME levels and their relationship to proliferation are not well known. In the present study, we investigated the regulation of PCho and PEth levels in rat glioma cells grown in vivo and in vitro using 31P and 13C MRS. However, the ability of cells to produce choline endogenously is variable. To fully understand regulation of PCho levels, it is necessary to characterize the activity of the endogenous pathway, if it exists. This was first investigated by following the metabolic fate of 13C-labeled methionine of 9L glioma tumors in vivo. Our results indicate that there is a significant amount of de novo choline synthesis in vivo. However, similar experiments performed in vitro using cells cultured in bioreactors indicated that glioma cells themselves are unable to synthesize choline de novo, suggesting that the in vivo results were due to the involvement of extra-tumoral organs, e.g., liver. Further in vitro experiments demonstrated that the uptake and phosphorylation of physiologically relevant concentrations of exogenous choline is very active in these systems. Thus, it appears that the exogenous pathway for PCho biosynthesis predominates and regulates PCho levels in glioma cells. Our results also demonstrate that PCho levels are lowest, and PEth levels are highest, in non-proliferating cells. These observations indicate that there is a decrease in the biosynthesis of PCho concomitant with a reduction in culture growth. The source of the increased PEth is, as yet, undefined.

Dimethyl methylphosphonate (DMMP), when added to a suspension of erythrocytes, has been reported to have a lower frequency chemical shift inside of cells than outside. This work further investigates the same phenomenon in hollow-fiber bioreactor cultures of six mammalian cell lines and describes the application of DMMP as a measure of intra- versus extracellular volumes in mammalian cell cultures. No toxic effects of the DMMP were observed at the concentrations used here. The dependence of the shift of intracellular DMMP on intracellular protein content was shown to be similar for cultured mammalian and red blood cells. Also consistent with shifts in erythrocytes, an increase in the intracellular protein concentration due to a reduction in cultured cell volume increased the magnitude of the shift to lower frequency. Longitudinal relaxation (T1) values for intra- and extracellular DMMP were measured so that partially saturated DMMP peaks in 31P NMR spectra of mammalian cell cultures can be corrected to give the relative volumes of the intra- and extracellular compartments; this information provides a relative measure of culture growth. Intracellular volume measured by this method can also be used to quantify intracellular metabolites such as ATP during the growth of the culture. To explore the mechanism behind the intracellular shift, we have also addressed the three possible contributions to the chemical shift of DMMP: hydrogen-bonding interactions, magnetic susceptibility, and ionic strength. Data is presented which eliminates the latter two mechanisms and strongly supports the hypothesis that the observed intracellular shift is due to a reduction in hydrogen bonding between water and DMMP in the cytoplasm.

MR spectroscopy of cultured cells allows non-invasive analyses of the metabolism of cells with specific phenotypes under defined conditions. This technique can be used to investigate the intracellular metabolism of cells or extended to critically evaluate phenomena observed by in vivo MRS. In this paper, a cell maintenance system is described which allows MR analyses with unparalleled spectral resolution, S/N and stability. This system consists of a 25 mm diameter hollow fiber bioreactor and a supporting circuit. The hollow fiber reactor was chosen because it yields a large filling factor which can be perfused through defined volumes. The fibers were 300 microns diameter microporous (0.2 micron) cellulose acetate/cellulose nitrate membranes with high porosity, which allow bulk convective flow throughout the extracapillary space. This flow (Starling flow) is necessary to disrupt steady-state gradients in substrates and waste products. In many respects, the design of the supporting circuit is more important than the bioreactor itself, since it provides the reactor with the proper chemical and physical environment. Hence, this circuit can be applied to a variety of bioreactor configurations. The circuit consists of a hollow fiber oxygenator and a bleed-and-feed system housed in a temperature-controlled cabinet. Culture of mammalian cells in this reactor yields 31P spectra which have excellent spectral and temporal resolution. At confluence, endogenous 31P line widths were typically < 10 Hz (at 162 MHz) and well resolved spectra were obtained in < 30 s.

Cell volume regulation in the face of osmotic stress is a fundamental homeostatic activity, and is most critical in brain, which is spatially constrained. Despite the importance of this phenomenon, little is known about volume regulation in the brain, primarily because of the cellular heterogeneity in the tissue. We describe here simultaneous in vivo 31P nuclear magnetic resonance (NMR) measurements of cell volume, intracellular pH and phosphate metabolites during early responses to hyperosmotic stress in C6 glioma cells perfused in NMR-compatible bioreactors. Cell volume was measured using dimethyl methylphosphonate (DMMP) as a probe which has an intracellular NMR resonance shifted upfield from the extracellular resonance. The sensitivity of these measurements allowed 31P NMR spectra to be collected every 30 s. Following an increase in osmolarity from 320 to 480 mOsm by addition of NaCl to the perfusate, C6 glioma cells shrank to 67% of their original volume. We also observed a simultaneous increase of intracellular pH coincident with the decrease in cell volume. The signals from ATP decreased by 10%, but those from phosphocreatine (PCr) increased by 31% after hyperosmotic shock. However, correcting the ATP signals for the decrease in cell volume indicated that its intracellular concentrations increased after treatment. Signals from glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) were not changed significantly. This is the first in vivo report of early cellular responses monitored by NMR spectroscopy following hyperosmotic shock in cultured cells.

NMR has many potential and realized applications to the study of physiology. A large number of nuclei can be observed non-destructively in samples ranging from molecules to man. The ensuing chapters detail four areas into which NMR spectroscopic analyses have developed.

Commercial exploitation of the fruits of recombinant DNA and cell fusion technologies is significantly limited by the lack of fundamental metabolic information on the cell lines of interest, whether these are plant, animal, insect, or microbial cells. NMR can help to provide this information and thereby improve bioreactor design and operation. However, in the case of on-line NMR of dense cell culture devices for metabolic studies, these devices are inherently heterogeneous bioreactors. To ensure that the metabolic information generated is reliable, a number of precautions should be taken. These are the same precautions that should be taken to ensure that commercial bioreactors operate in a reaction-controlled regime. Therefore, reactor engineering methodologies, particularly diffusion and reaction analyses and reaction monitoring by whole-cell NMR must go hand in hand, each extending, complementing, and validating the other.

Human amniotic fluid (HAF) is a dynamic system whose characteristics depend on continuous interchanges between fetal and maternal circulations. HAF reflects not only the environment of the fetus but may also provide information about fetal development or pathology. The concentration of HAF constituents varies with gestational age and pathological states. The number of the compounds currently implicated in fetal developmental pathology are relatively few. Currently used assay methods are not adequate to totally explain or predict the complex biochemistry of the fetus. The purpose of this work was to investigate HAF with NMR spectroscopy. In the present study HAF was obtained from 47 women undergoing routine amniocentesis. Cells were separated for karyological analysis and the supernatant was acid-extracted, lyophilized and re-suspended in D20 resulting in a concentration increase over native fluid. 1H NMR spectra were obtained at 360 MHz and 60 MHz. Eighteen compounds including several amino acids, were identified using parallel reference and standard addition protocols. NMR spectroscopy detected compounds of known clinical importance including glucose, leucine, isoleucine, lactate and creatinine. In conclusion, we have demonstrated that a number of physiologically relevant compounds are readily observable in HAF using 1H NMR spectroscopy. This technique can currently provide valuable information regarding HAF composition and has the potential of being used in vivo in the future.

The 31P NMR pH indicator, methylphosphonate, has been extensively characterized, and the uncertainty in pH determination by its chemical shift has been analyzed. The pKa decreases by 0.003 pH unit/degrees C and 0.06 pH unit/100 mM ionic strength. The pKa appears not to be sensitive to Ca2+ but is sensitive to Mg2+, resulting in an uncertainty of +/- 0.05 pH unit. Substituting 300 mM Na+ for 300 mM K+ causes the pKa to decrease by 0.1 pH unit. Taking the effects of temperature, ionic strength, and cation identity into account, the overall estimated uncertainty in pH determination can be as high as +/- 0.1 pH unit. Methylphosphonate was tested as a pH indicator in Ehrlich ascites tumor cells. Our data indicate that both the unchanged and monoanion forms of methyl phosphonate are very permeable, rendering this compound unsuitable as a pH indicator in this system. However, the sensitivity of this compound's chemical shift to pH and the relative insensitivity to other parameters suggest that phosphonates, as a group, may be applicable as pH indicators by 31P NMR.

Natural abundance carbon-13 nuclear magnetic resonances (NMR) from human arm and rat tissues have been observed in vivo. These signals arise primarily from triglycerides in fatty tissue. Carbon-13 NMR was also used to follow, in a living rat, the conversion of C-1-labeled glucose, which was introduced into the stomach, to C-1-labeled liver glycogen. The carbon-13 sensitivity and resolution obtained shows that natural abundance carbon-13 NMR will be valuable in the study of disorders in fat metabolism, and that experiments with substrates labeled with carbon-13 can be used to study carbohydrate metabolism in vivo.