Tumour Metabolomics in Animal Models of Human Cancer - American

Cambridge, CB2 1QW, United Kingdom, and School of Sport and Exercise Sciences, ... MS techniques for animal tumor metabolomic work, as well as highlig...
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Tumour Metabolomics in Animal Models of Human Cancer Julian L. Griffin*,† and Risto A. Kauppinen‡ Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, United Kingdom, and School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom Received September 8, 2006

Multinuclear Nuclear Magnetic Resonance Spectroscopy (MRS) and mass spectrometry (MS) are the key analytical techniques used in an increasing manner to explore tumor metabolite profiles. Recent work has revealed that metabolite profiles in various tumor preparations (i.e., cultured cells, tissue specimens, and tumors in vivo) show strong correlations with tumor type, proliferation, metabolic activity, and cell death. These data are regarded as highly promising for tumor diagnosis as well as assessment of prognosis and treatment response in a clinical setting. In this pursuit, animal models of human cancer have played a central role. In this short account, we review the potentials of MRS and MS techniques for animal tumor metabolomic work, as well as highlight some interesting applications of these techniques for various animal tumor types. Keywords: Tumor • Brain • Metabolomics • NMR spectroscopy • Pattern recognition

Introduction

Analytical Techniques in Metabolomics

The use of mass spectrometry (MS) and Nuclear Magnetic Resonance Spectroscopy (MRS) as tools to monitor changes in tumor metabolism is not a new subject and significantly predates the concepts of metabolomics and metabonomics.1 However, perhaps what distinguishes metabolomic studies from those that were conducted previously is an attempt to consider metabolite changes in context of the global network of metabolic pathways present in a cell, tissue, or organism. This has produced a need for better analytical tools that can detect and quantify a wider range and number of metabolites. Furthermore, the use of multivariate statistics has become an integral part of the metabolomic/metabonomic approach to a disease.2,3 These developments have proved particularly useful in the study of cancer, both as an experimental tool4 and ultimately as a tool for diagnosis and monitoring treatment response.5 In this field, there is a drive to replace invasive histopathology with noninvasive MRS and Magnetic Resonance Imaging (MRI) techniques that can diagnose and monitor tumor metabolism readily in human patients at multiple time points. This minireview focuses on the use of metabolomic techniques and approaches to monitor tumor metabolism in some of the commonly studied animal models of human cancer. From these studies, the metabolic phenotyping of tumors has shown great potential in understanding metabolism during tumor growth and death.5,6 While ultimately it is MRS-based approaches which will be applied in vivo in human patients, to understand the importance of metabolic changes, MS-based approaches are being increasingly used.

Prior to any analysis, most metabolomic techniques require some sample preparation. For tissue and cell extracts, both water- and lipid-soluble metabolites can readily be extracted. With the use of acid and organic solvent extraction procedures, virtually all cellular and membranous pools of metabolites are extracted, irrespective of their participation in particular metabolic processes in situ. These facts constitute obvious differences between MRS of tissue extracts and detecting metabolites with the same technique in vivo. Despite this, it is a common observation that the acid-soluble metabolites represent the cytosolic pool of many metabolites; thus, these metabolite profiles may be regarded representative of the ‘active metabolome’. On the other hand, the lipid fraction often contains significant quantities of membranous lipids. Because of the need to limit technical variation across large metabolomic data sets, it is necessary to strictly adhere to Standard Operating Protocols (SOPs) to minimize this source of variation. A number of extraction procedures have been reported in the literature. Perchloric acid has been used widely to extract brain-derived material, including tumors, as this solvent rapidly quenches metabolism and is eliminated by precipitation with potassium. However, acid extraction increases the salt content of samples, which may reduce MS performance across many samples, and the procedure also oxidizes metabolites and proteins. An alternative to extract aqueous soluble metabolites is to employ a water/acetonitrile mixture, although the ability of this solvent mixture to quench metabolism quickly is questionable. The chloroform/methanol extraction is increasingly popular in metabolomics, as this allows the simultaneous extraction of both aqueous and lipidsoluble metabolites in one extraction procedure.7

* Author for correspondence: mole.bio.cam.ac.uk. † University of Cambridge. ‡ University of Birmingham.

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Metabolomics of Animal Tumor Models

Following the preparation of a tissue extract, this can then be analyzed by one of the global analytical techniques used to generate a metabolic profile or fingerprint. However, before conducting this analysis, one may want to consider exactly what information the study has been designed to collect. While global profiling by open platforms of a wide range of metabolites can be very useful for novel biomarker discovery and identifying new trends and effects in a particular study, these approaches are inherently less sensitive than ‘closed’ platforms, especially when particular classes of compounds of interest have been concentrated (e.g., on a solid-phase extraction cartridge). Below, the individual analytical tools are briefly discussed, particularly with reference to their uses to monitor tumor metabolism either directly or through extracts of excised tissues. Solution State MRS. High-resolution 1H MRS of cells or tissue extracts detects a wide range of metabolites in an unbiased manner with unambiguous identification possible, provided these metabolites are present at high enough concentrations. From brain tumor cells or biopsies, typically 25 metabolites can be identified and quantified by 1H MRS. 31P MRS of the extracts yields a further 10 metabolites from the same extracts. 13 C MRS is used to monitor the interconnection of metabolic pathways and their activities. Low endogenous background, wide chemical shift scale of the nucleus, and the ability to distinguish different metabolic pathways using isotopomeric analysis make it an attractive stable isotope for metabolic studies.8 In Vivo MRS Techniques. Spectroscopic signal localization in vivo is accomplished by means of field gradients and frequency selective rf-pulses analogously to the techniques used in MRI. The localized spectroscopy techniques allow a signal to be acquired either from a single volume or multiple volumes (so-called MRS imaging, MRSI) for any NMR nucleus. Tissue volumes required for acceptable signal-to-noise ratio are typically a few microliters for 1H MRS in high-field animal scanners and milliliters for modern human scanners. Tissue volumes for 31P and 13C MRS need to be an order of magnitude larger due to low inherent sensitivity of these nuclei. However, spectra can still be collected from volumes of most tumor xenocrafts or clinical tumors without contamination from surrounding nonmalignant tissue. The narrow chemical shift range for 1H nuclei leads to overlap of spectral lines, which can complicate the interpretation of these spectra. For instance, the CH3 resonance of lactate (Lac) and the CH2 resonances of lipids resonate at 1.33 and 1.20 ppm, respectively, and are difficult to separate in vivo. Spectral editing techniques can be used to resolve these peaks explicitly, and editing techniques can be exploited to expose many other metabolites in isolation, such as glutamate, glutamine,9 and glutathione.10 An option to improve sensitivity of 13 C MRS is to detect the protons that are bonded to 13C carbon using procedures such as proton-observed carbon-edited spectroscopy.11 In vivo MRS is less sensitive compared with solution state spectroscopy studies of biopsy samples, reducing the number of metabolites that can be detected. However, the fact that key metabolites of cell function, energetics, phospholipids, and glucose metabolisms are detected truly noninvasively makes MRS a powerful tool for in vivo applications. Furthermore, intracellular pH (pHi) and Mg2+ concentrations and temperature12 can be obtained from MR spectra.

reviews High-Resolution Magic Angle Spinning. One particularly useful tool for bridging the analytical gap between solution state and in vivo MRS is High-Resolution Magic Angle Spinning (HRMAS) MRS. By spinning a sample at the magic angle (54.7°) to the magnetic field, we can reduce the physical effects that lead to broad lines in solid tissues so as to produce spectra comparable to high-resolution spectra in the solution state.13 The tool has proven to be highly versatile in monitoring metabolism in tumors, especially where both lipid and aqueous metabolites need to be monitored alongside each other, such as assessing the contribution choline-containing metabolites (CCM) make to the spectra of brain tumors during apoptosis.14 Mass Spectrometry. MS-based approaches are inherently more sensitive than MRS techniques, providing access to lower concentration metabolites in the tumor metabolome. Most applications use prior chromatography with Gas Chromatography (GC) and Liquid Chromatography (LC) to initially separate metabolites in a tissue extract prior to analysis. GCbased approaches often require prior chemical derivatization to ensure that the components of the mixture are volatile, while LC-based approaches can analyze solutions directly, although this approach may suffer from ion suppression. The use of MS to monitor the metabolic profiles of brain tumors significantly predates the use of the term metabolomics. For example, Jellum and colleagues15 identified ∼160 peaks in GC-MS spectra from normal brain tissue, pituitary tumors, and brain tumors, and then used a pattern recognition approach to classify tissue into healthy and tumor. The sensitivity of MS-based approaches has also been used to monitor trace metabolites in excised tissue. For example, neurotransmitters in neuroctomas have been profiled, including acetylcholine and the metabolites of catecholamines by HPLC,16 while Olsen and colleagues17 have used Quadrupole Time-of-flight (Q-TOF) MS to detect morphine in glioma. MS has also been shown to be highly discriminatory for lipid metabolites, including ceramide metabolites, which vary in neuroblastoma cells during cell death.18 MS profiling of lipid metabolites has also been used to determine what components contribute to resonances that give rise to peaks in the in vivo 1 H MR spectra. Miller and co-workers19 demonstrated that the CCM peak detected in 18 brain tumors largely correlated with choline, phosphocholine (PC), and glycerophosphocholine (GPC), but not phosphatidylcholine. To investigate lipid metabolism within tumors, tandem MS approaches provide a unique insight into many classes of compounds. Sullards and colleagues20 have used this approach to monitor changes in sphingolipid metabolism in human glioma cell lines to correlate these profiles with either the induction or inhibition of apoptosis. Other types of mass spectrometry have also been used, including Fourier Transform Ion Cyclotron Resonance (FT-ICR) MS21 for accurate mass determination of metabolites, and matrix-assisted laser desorption ionization (MALDI)-MS22 for the detection of metabolites directly from tissue slices. Fourier Transform Infrared (FT-IR) and Raman Spectroscopy. Both techniques rely on vibration frequencies of metabolites to provide a fingerprint of metabolism in a tissue extract.23-26 While FT-IR spectroscopy is relatively poor at distinguishing metabolites which are chemically closely related, only being capable of distinguishing different functional groups, the approach is cheap, high-throughput, and hence useful as a first pass screening tool to identify bulk chemical changes. While only metabolites with a dipole are observed in FT-IR, Journal of Proteome Research • Vol. 6, No. 2, 2007 499

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Raman spectroscopy relies on detecting vibrations through light scattering from a laser and can detect a wider range of metabolites compared with FT-IR.

Animal Models for Tumours Brain Tumors. The use of MRS to profile metabolites in tumor cells and tissues has been applied to a wide range of human tumors for a number of years, with the approach being particularly useful at generating new hypotheses that link growth characteristics of a tumor to metabolism. For example, Bhakoo and colleagues27 examined the process of immortalization in primary rat Schwann cells, noting that an increase in the PC/GPC ratio correlated with this process. The ability to quantify both aqueous soluble and lipid metabolites simultaneously in intact tissue has made HRMAS 1 H NMR spectroscopy a versatile tool for investigating the accumulation of polyunsaturated fatty acids (PUFA) during apoptosis in glioma in rats28 (Figure 1). Using a combination of MRI and 1H MRS, Griffin and colleagues followed the progression of apoptosis in these tumors and were able to show that the metabolic changes were the same in the center of the tumor where significant cell clearance had occurred compared with the whole tumor (Figure 1A,B). However, HRMAS 1H NMR spectroscopy provided much better resolution and hence the definitive identification of a greater number of metabolites. This approach demonstrated a profound accumulation of PUFAassociated resonances across the time course investigated (Figure 1C). When the line widths of these lipids at different temperatures and the spinning rates and measuring diffusion rates for the lipids were examined, it was established that these metabolites were found in cytoplasmic vesicles seen by electron microscopy (EM). The extra resolution obtained using HRMAS 1 H MRS compared with MRS in vivo has also been applied to separate peaks from choline, GPC, PC, taurine (Tau), and myoinositol, all these metabolite peaks contributing to the in vivo 1 H NMR peak of CCM.14,29 These MRS approaches have also demonstrated the presence of nucleotide peaks from adenosine and uridine nucleotides in glioma samples ex vivo, suggesting DNA laddering might be observable using MRS. In Vivo Approaches of Brain Tumor Models. Animal models have great impact for assessment of brain tumor perfusion, angiogenesis, metabolism, and response to therapy.30 Brain tumor models for glial tumors, such as F98,31 C6,32 and BT4C33 xenocrafts and neuroblastoma tumors,34 have been used for in vivo MRS work. 1H MRS has shown that spectra from these brain tumors are characterized by the absence of N-acetyl aspartate, low creatine, and high CCMs resonances. F98 gliomas are further characterized by an increased glycine concentration alongside a decrease in glucose. Both C632,35 and BT4C33,36 tumors display resonances from mobile lipids present in cytoplasmic vesicles.32,36,37 The detection of lipid resonances in 1H MR spectra has been correlated with nonviable tumors due to either the process of necrosis35 or apoptosis.28,33 Saturated lipid resonances have been associated with necrosis,32,35 while PUFA resonances increase in glioma during apoptosis, indicating that cell death mechanisms may differently influence NMR detectability of cellular lipids.5 Furthermore, Larech et al.32 have used diffusion 1 H MRS to measure the droplet diameters to be ∼4.5 µm, matching closely with EM quantification. Recent 1H MRSI data have show heterogeneity in the spatial distribution of metabolites within C6 gliomas.35,38 The metabolite patterns associated with ‘proliferating’ tumors are distinct 500

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Figure 1. T2-weighted MRI (A) and in vivo 1H NMR spectra (B) from a BT4C glioma. T2-images and 1H NMR spectra were acquired from a tumor 8 days after initiation of ganciclovir (GVC) treatment to induce apoptosis in the tumor. In (i), a spectrum from the entire volume (128 averages) and in (ii) from the T2-hyperintense core (256 averages) are shown. (C) HRMAS 1H NMR spectra from tumor samples as a function of GVC treatment. Spectra were acquired from normal parietal cortex (Cortex) and tumor samples from days 0-8 of GVC treatment. Key: 1, CHdCH; 2, choline-containing compounds; 3, CHdCHCH2CHdCH; 4, CH2CHdCH; 5, CH2CH2CH2; and 6, CH2CH3. PtdCholine, phosphatidylcholine; PCholine, phosphocholine. Reprinted with permission from ref 28. Copyright 2003 The American Association for Cancer Research.

from ‘nonviable’ ones. The former tissue type is characterized by high CCM and Lac, whereas the latter by strong 1H resonances from saturated lipids.35 Interestingly, there is also a spatial match between 1H MR-detectable metabolites and extracellular pH (pHe) so that regions with high Lac and CCM concentrations show close to normal pHe, and this pattern is thought to reflect rapid washout of Lac from viable tumors. In contrast, nonviable parts of tumors, which by 1H MRSI display lipids, show acidic pHe.38 In addition to studies based on 1H MRS, 13C MRS is a powerful technique for metabolic assessment of tumors, because both glycolytic and oxidative metabolism of glucose can be estimated in the same experiment. The switch from oxidative to ‘anabolic’ glucose metabolism (involving glucose carbon shunting for nucleic acid synthesis) is one of the characteristics of cancer cells.39 Terpstra et al. have used this approach to show that, in C6 tumors, the Lac pool is metabolically active.40

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Metabolomics of Animal Tumor Models 13C

Further, diffusion MRS measurements suggest that significant pool of Lac is intracellular and, thus, directly available for metabolism.41 The C6 glioma showed very slow rate of glutamate C4 labeling, indicating low oxidative glucose metabolism, a finding that is consistent with the metabolic hypothesis of Boros and colleagues.39 This demonstrates the versatility of 13C MRS as a tool for understanding tumor metabolism in vivo. Models for Connective Tissue Tumors. The radiationinduced fibrosarcoma (RIF-1) tumor is a commonly used animal tumor model for both MRS and MRI work. 31P MRS of RIF-1 tumors showed that the phosphocreatine-to-inorganic phosphate (PCr/Pi) ratio decreased and Pi/ATP increased in the fast growth phase of this tumor type. During radiation42 or cyclophosphamide treatment,43 these ratios change in opposite directions to growth and are associated with alkalinization of pHi. Radiation has also been shown to result in increased GPC and phosphoethanolamine (PE) concentrations in RIF-1 tumors, reflecting membrane phospholipid breakdown.44 Koutcher et al.45 found that the PCr/Pi ratio decreased during growth of radiation-sensitive fibrosarcomas, but these indices did not change in radio-resistant mammary carcinoma. These observations were thought to be due to varying degrees of hypoxia, a factor determining radiation sensitivity, and are encouraging for the use of 31P MRS to predict treatment response. 19F MRS has been used together with 31P MRS to monitor 5-fluorouracil (5-FU) therapy in RIF-1 tumors.46 19F MRS allowed monitoring of both the uptake of 5-FU and intratumor metabolism of 5-FU to toxic fluoronucleotides. These processes were correlated with cell death revealed by decrease in total MR-observable phosphates. McSheehy et al.47 have recently shown with 19F MRS that 5-FU uptake by RIF-1 tumors is increased by carbogen breathing leading to improved treatment response. Concurrent 31P MRS and MRI indicated that high 5-FU uptake was due to both acidification of pHe and blood flow changes upon carbogen exposure. Finally, it has been shown that Lac48 and CCM49 concentrations decrease in RIF-1 tumors treated with cytotoxic drugs. These multinuclear MRS studies on RIF-1 tumors demonstrate the metabolic patterns that may have value for diagnosis, prognosis, and treatment monitoring. Lymphoma Models. 31P MRS data of a murine T-cell lymphoma has shown that alkalinization of pHi during anti-cancer drug treatment was an indicator of positive response.50 Furthermore, apoptotic murine lymphoma xenocrafts are characterized by decreases in the phosphomonoester-to-Pi ratio, and histological recovery from radiation-enhanced apoptosis was associated with increase in β-ATP/ Pi.51 These early observations provided encouraging evidence that MRS may be able to pick up metabolic changes during ongoing apoptosis in vivo. 31P MRS cell studies provided further evidence for metabolic changes during apoptosis, including accumulation of fructose1,6-bisphosphate and CDP-choline.52 These responses were due to NADH depletion52 and inhibition of choline phosphotransferase.53 The link between 1H MRS detectable lipids with apoptosis was originally demonstrated in lymphoma cells,54 following the earlier detection of neutral lipids in the 1H MRS of myeloma cells.55 Blankenberg and co-workers54 found that both CH2 and CH3 lipid signal intensities and their ratio increase in apoptotic, but not in necrotic, cells. The 1H MRS lipid response during apoptosis has been confirmed in several cell lines.5 A recent paper by Schmidt et al. showed that EL-4 lymphomas grown

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subcutaneously display strong increase in H MRS lipids as expected. However, the CH2/CH3 ratio did not change at all.56 The latter observation might be due to differential contributions of lipids to the macromolecular resonances in vitro and in vivo.57 Liver Tumors Models. The effects of hypoxia-induced factor1β (HIF-1β, a constituent of the transcription factor HIF-1) deficiency on tumor metabolism and growth have been analyzed in vivo and in vitro using MRS in liver tumors.58,59 HIF-1β is upregulated in several cancer types as a consequence of hypoxia, resulting in the increased expression of proteins involved in glucose transport, glycolysis, and growth factors to counteract hypoxia. When a multinuclear MRS-based metabolomic approach was used, the mutant tumors had a 5-fold decrease in total ATP content and a 3-fold increase in the ratio of phosphodiester-to-Pi, despite similar vascularity in normal and mutant tumors. Thus, HIF-1β appears to largely increase the rate of glycolysis rather than modify the vascularity of the tumors. When 1H MRS was used to observe metabolic changes in the hepatoma, the reduced rate of glycolysis was also correlated with a declined capability to synthesize nucleotides in these tumors. In this manner, metabolomic studies of this type could be used to identify metabolic pathways that could be targeted therapeutically, to undermine the bioenergetic status of the tumor, a factor that may be useful in anti-cancer drug design. A number of metabolic surrogate makers of tumor growth and death have been detected in animal models of liver cancer. Tau has been found to increase during liver metastasis.60 Hypotaurine is thought to be an antioxidant and may protect cells from free-radical damage, while Tau is an important osmoregulatory compound. Alanine, in conjunction with Lac, has also been detected to increase in growing hepatoma and is thought to provide a good marker of tissue hypoxia. Finally, CCMs increase in concentration during apoptosis of hepatoma58,59 as well as during tumor cell growth in chemically induced hepatocellular carcinoma,61 while deficiency of choline in the diet induces a reduction in PUFA prior to liver cancer.62 To date, the majority of studies have focused on the use of MRS as the primary analytical tool to investigate metabolism in liver tumors. However, as mentioned previously, MS-based approaches allow the detection of a wider range of metabolites. Figure 2 shows some data from a pilot study to investigate metabolic markers of nongenotoxic induction of liver cancer by Phenobarbital. While MRS allows the detection of ∼20 metabolites, MS is capable of detecting 5-6 times as many metabolites. Colon Cancer Models. 1H MR metabolic profiling of a human colorectal xenocraft arising from HT-29 cell line has shown that Tau, Cr, PC, PE, glycogen, and glucose are promising discriminating compounds against abdominal host tissues.60 MR spectral patterns, however, failed to separate metastatic and nonmetastatic forms of colorectal tumors. A large majority of colon tumors contain no 1H MRS detectable lipids, unlike healthy mucosa.63 A 13C MRS study of human colon cancer cells grown on microcarriers demonstrated high rates for aerobic Lac production and pentose phosphate cycle.64 This is consistent with high endogenous Lac in colon xenocrafts as quantified by multiplequantum-coherence 1H MRS in vivo.65 31P MRS has shown inherent variation in energy states, pHi, and pHe in murine colon tumor models derived from distinct cell lines.66 These factors provided predictive power for response to 5-FU treatJournal of Proteome Research • Vol. 6, No. 2, 2007 501

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Figure 2. Preliminary results from a study investigating early stage metabolic markers in liver tissue of nongenotoxic carcinogenicity induced by Phenobarbital exposure in rats. (A) A typical high resolution 1H NMR spectrum of the aqueous fraction of an extract of liver tissue. (B) The total ion chromatogram from the GC-MS analysis of the same fraction following trimethylsiylation of metabolites. (C) Principal components analysis of the GC-MS data for three exposure levels of Phenobarbital compared with the control group. Personal communication: Waterman, C. L., Cottrell, L. A., Waterfield, C. J., and Griffin, J. L. Key: (9) control; (b) 50 ppm; (() 500 ppm; (2) 1000 ppm.

ment in colon tumors administered either with or without carbogen breathing. It was shown that the pretreatment low energy state and intratumor acidity, as well as a retained pH gradient across the plasma membrane during carbogen exposure, were correlated with long-lasting growth arrest by the drug.66 Prostate Cancer Models. The interest shown in the MRS analysis of animal models of prostate cancer does not match the clinical applications of both 1H MRS and MRSI to this disease.67 Indeed, relatively little has been published from MRS of prostate xenocrafts in animal models, yet outside the brain, prostate has been a common target organ for 1H MRS in humans.67 Kiessling et al. used an ortotopic Dunning rat prostate tumor model and observed high CCM/Cr ratio and a broad lipid peak between 2.0 and 2.2 ppm, which all responded to radiation therapy.68 Recently, genetically modified mice models have been introduced for MRS and MRI assessment of malignancy.69 In the transgenic adenocarcinoma mouse prostate (TRAMP) model, the CCM/citrate ratio was not different from that determined in wild-type animals. This observation is surprising when compared with human data which shows a grossly elevated ratio due to the absence or low concentration of citrate in prostate cancer tissue.67 Interestingly, in a transgenic mouse harboring both PB-ErbB-2∆ and Pten+/- genotypes, 1H MRS showed elevated CCM/citrate ratio in ventral prostate consistent with malignant histology 69 These observations are intriguing, and the new genetic models for cancer warrant more detailed MRS studies on the involvement of 502

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metabolic abnormalities in cell transformation and dedifferentiation in situ.

Future Directions The application of metabolomics/metabonomic techniques to the study of cancer in animal models of disease will benefit greatly from improvements in technology. A great deal of these techniques has been applied to study human cancers. Both 31P and 1H MRS has been shown to provide both diagnostic and prognostic information in cancers such as breast,70,71 brain,72-74 head and neck,75 lymphomas,76 liver,77,78 and prostate.79,80 It is evident that data from clinical MRS for oncology applications are rapidly accumulating following the development of techniques in animal models. Genetically modified mouse lines for cancer research are one example of recent breakthroughs which have opened new avenues to exploit metabolomic approaches scrutinizing the role of metabolic aberrations on cell transformation and dedifferentiation in the tumor syndromes.81 In MRS, this has led to an increase in static magnetic field strengths for in vivo applications and the use of cryoprobes and microcoil probes to improve sensitivity for in vitro analyses. One recent advance which appears particularly appropriate for monitoring tumor metabolism is nuclear hyperpolarization techniques for 13C containing metabolic substrates.82 This approach is expected to allow tumor oxidative and anabolic metabolisms to be monitored without radioactivity in vivo. Because of the hyperpolarized substrates, in terms of concen-

Metabolomics of Animal Tumor Models

tration, MRS will be more sensitive than positron emission tomography, and adding the explicit chemical specificity of MRS to this, MRS is likely to enable detailed functional metabolic assessment of tumors in vivo. These MRS techniques may render enzymatic and metabolic drug targets, such as those involved in glucose carbon shunting for nucleic acid synthesis,83 for noninvasive assessment in vivo. The sensitivity and resolution capabilities of mass spectrometers are also increasing rapidly. In addition, better software solutions are being developed to ensure the robust analysis of the large multivariate data sets produced by these approaches. In addition to improvements in the detection and quantification of ions within the mass spectrometer, improvements are also being made in the approaches used to ionize metabolites. For example, MALDI-MS produces an image of a tissue section which represents certain metabolites and could be used as a metabolomic alternative to histology. This is already being used in cancer cell proteomics as well as certain metabolomic experiments.84 Finally, on the use of animal models of human disease, there is an ethical desire to reduce the number of animals needed in a study. In addition to metabolomics defining a wide range of metabolites, proteomics and transcriptomics can similarly be applied to the same tissue to provide a complete snapshot of the system, reducing the need for follow-up studies in the future.

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