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The metabolome of a cancer cell is likely to show changes after responding to an anticancer drug. These changes could be used to decide whether to continue treatment or, in the context of a drug trial, to indicate whether the drug is working and perhaps its mechanism of action. (Nuclear) magnetic resonance spectroscopy (NMR/MRS) methods can offer important insights into novel anticancer agents in order to accelerate the drug development process including time-course studies on the effect of a drug on its site of action (termed pharmacodynamics), in this case the cancer. In addition, some classes of anticancer agents currently under development (e.g. antiangiogenics) are designed to be used in combination with other drugs and will not cause tumour shrinkage when used as single agents in Phase 1 clinical trials. Thus NMR/MRS may have a special role in monitoring the pharmacodynamic actions of such drugs in early-phase clinical trials. This review focuses on the use of ex vivo NMR and in vivo MRS methods for monitoring the effect of some novel anticancer drugs on the cancer metabolome. Ex vivo NMR methods are complementary to in vivo measurements, as they can provide additional information and help in the interpretation of the in vivo data.

The standardization and reproducibility of techniques required to acquire anatomically localized 31P MR spectra non-invasively while studying tumors in cancer patients in a multi-institutional group at 1.5 T are reported. This initial group of patients was studied from 1995 to 2000 to test the feasibility of acquiring in vivo localized 31P MRS in clinical MR spectrometers. The cancers tested were non-Hodgkin's lymphomas, sarcomas of soft tissue and bone, breast carcinomas and head and neck carcinomas. The best accrual and spectral quality were achieved with the non-Hodgkin's lymphomas. The initial analysis of the spectral values of the sum of phosphoethanolamine plus phosphocholine normalized by the content of nucleotide triphosphates in a homogeneous sample of 32 NHL patients studied by in vivo (31)P MRS showed good reproducibility among different institutions. No statistical differences were found between the institution with the largest number of cases accrued and the rest of the multi-institutional NHL data (2.28 +/- 0.64, mean +/- standard error; n = 17, vs 2.08 +/- 0.14, n = 15). The preliminary data reported demonstrate that the institutions involved in this trial are obtaining reproducible 31P MR spectroscopic data non-invasively from human tumors. This is a fundamental prerequisite for the international cooperative group to be able to demonstrate the clinical value of the normalized determination of phosphoethanolamine plus phosphocholine by 31P MRS as predictor for treatment response in cancer patients., (Copyright 2006 John Wiley & Sons, Ltd.)

Vascular and angiogenic processes provide an important target for novel cancer therapeutics. Dynamic contrast-enhanced magnetic resonance imaging is being used increasingly to noninvasively monitor the action of these therapeutics in early-stage clinical trials. This publication reports the outcome of a workshop that considered the methodology and design of magnetic resonance studies, recommending how this new tool might best be used.

A multi-institutional group has been created to demonstrate the utility of in vivo 31P magnetic resonance spectroscopy (31P-MRS) to study human cancers in vivo. This review is concerned with the novel problems concerning quality control in this large multinational trial of 31P MRS. Our results show that the careful and systematic performance of the quality control tests depicted here (standardized dual 1H/31P tuned radiofrequency probe, quality control procedures, routine use of 1H irradiation while acquiring 31P MR signals) has ensured comparable results between the different institutions. In studies made in vitro, the root-mean-square error was 3.6 %, and in muscle of healthy volunteers in vivo the coefficients of variance for the ratios phosphocreatine/nucleotide-triphosphates, phosphocreatine/noise and nucleotide-triphosphate/noise were 12.2, 7.0 and 10.8 %, respectively. The standardization of the acquisition protocol for in vivo-localized 31P MR spectroscopy across the different institutions has resulted in comparable in vivo data, decreasing the possible problems related to a research study carried out under a multi-institutional setting., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

The aim was to determine if a specific inhibitor of vacuolar H(+)-ATPases (V-ATPases), Bafilomycin A1 (BFM), could increase the low extracellular pH (pHe) typical of solid tumours and thus inhibit their growth in vivo. BFM inhibited the proliferation of various human cells and rat pituitary GH3 tumour cells in vitro (IC50: 2.5-19.2 nM), and flow cytometry on GH3 cells showed a marked increase in S and G2M phases after 16-48 h, but no evidence of increased apoptosis. BFM caused significant inhibition of GH3 xenograft growth, and histomorphometry showed a 30% decrease in mitosis but no change in apoptosis. 31P-magnetic resonance spectroscopy (MRS) in vivo of GH3 xenografts showed that BFM increased pHe, but did not affect pHi, resulting in a decrease in the negative pH gradient (-delta pH). BFM decreased lactate formation suggesting a reduction in glycolysis. We suggest that BFM reduces extracellular H(+)-transport by inhibition of V-ATPases leading to an increase in pHe and decreased glycolysis, and thus reduced tumour cell proliferation. 19F-MRS in vivo showed that a smaller -delta pH was associated with decreased retention of 5-fluorouracil (5FU) which was consistent with our previous data in vivo implying the -delta pH controls tumour retention of 5 FU.

Now, at the beginning of a new century, 80 years after Warburg's Nobel prize winning discoveries, we are beginning to make sense of the underlying causes of the well known metabolic phenotype of tumor cells. Building on decades of research to understand the interrelationships between respiration and glycolysis in cancer, the tumor metabolic phenotype can now begin to be understood in a genomic context. With the discovery of hypoxia inducible factor-1 (HIF-1), which is widely overexpressed across a broad range of cancers, modern molecular tools have allowed us to put together the pattern of events that might explain the metabolic differences between tumor and normal cells. HIF-1 controls cellular and systemic responses to oxygen availability and coordinates up-regulation of genes involved in many pathways concerned with tumour growth and metabolism including angiogenesis, glucose and energy metabolism, cellular proliferation, differentiation and viability, apoptosis, pH regulation and matrix metabolism. These findings begin to explain how glucose uptake and glycolysis could be up-regulated in cancer cells (through binding to a core DNA recognition sequence) in a co-ordinated and constitutive fashion that may also allow us to elucidate new targets for tumor therapy.

An MRI method is described for demonstrating improved oxygenation of human tumors and normal tissues during carbogen inhalation (95% O2, 5% CO2). T2*-weighted gradient-echo imaging was performed before, during, and after carbogen breathing in 47 tumor patients and 13 male volunteers. Analysis of artifacts and signal intensity was performed. Thirty-six successful tumor examinations were obtained. Twenty showed significant whole-tumor signal increases (mean 21.0%, range 6.5-82.4%), and one decreased (-26.5 +/- 8.0%). Patterns of signal change were heterogeneous in responding tumors. Five of 13 normal prostate glands (four volunteers and nine patients with nonprostatic tumors) showed significant enhancement (mean 11.4%, range 8.4-14.0%). An increase in brain signal was seen in 11 of 13 assessable patients (mean 8.0 +/- 3.7%, range 5.0-11.7%). T2*-weighted tumor MRI during carbogen breathing is possible in humans. High failure rates occurred due to respiratory distress. Significant enhancement was seen in 56%, suggesting improved tissue oxygenation and blood flow, which could identify these patients as more likely to benefit from carbogen radiosensitization., (Copyright 2001 Wiley-Liss, Inc.)

31P MRS studies have shown that the intracellular compartment ot tumours is kept near neutrality, whereas the interstitial fluid is acidic (pH 6.5-6.8). Why is this compartment acidic? Balance studies confirm that tumours produce excessive lactic acid, although less than usually supposed, but this cannot be the whole story, since Tannock and co-workers have shown interstitial acidity in glycolysis-deficient tumours. Another major acid load is caused by hydration of CO2 molecules to carbonic acid, catalysed by carbonic anhydrase. The distance that H+ must diffuse from cancer cells to capillaries is further than in normal tissue and this will increase acidification near the cells. We show that previous quantitative models based on simple H+ diffusion are unsatisfactory. This is because most H+ ions cross the interstitial space bound to buffers such as inorganic phosphate. Although these protonated buffers (i.e. conjugate acids) diffuse much more slowly than H+ ions they carry most of the protons, so the pH predicted by this model is closer to neutrality for a given proton production rate than that predicted by the dissolved H+ model. We have developed a mathematical model of this carrier-mediated system that predicts pHe values as low as those observed in some tumours.

Tumour cells have a lower extracellular pH (pHe) than normal cells; this is an intrinsic feature of the tumour phenotype, caused by alterations either in acid export from the tumour cells or in clearance of extracellular acid. Low pHe benefits tumour cells because it promotes invasiveness, whereas a high intracellular pH (pHi) gives them a competitive advantage over normal cells for growth. Molecular genetic approaches have revealed hypoxia-induced coordinated upregulation of glycolysis, a potentially important mechanism for establishing the metabolic phenotype of tumours. Understanding tumour acidity opens up new opportunities for therapy.

A consequence of metabolism in any tissue is the formation of hydrogen ions, which are actively transported out of the cell. However, although most solid tumors maintain their intracellular pH (pHi) within a narrow range to provide a favorable environment for various intracellular activities, their extracellular pH (pHe) is on average about 0.2 pH units more acid. It is important to understand the relationship between tumor metabolism and pH, and how it differs from that of normal tissue and to ask the question: How does an understanding of pH and tumor metabolism affect strategies for therapeutic approaches? Although, in vitro, isolated cell experiments have shown positive correlations between pHi and pHe the relationship is complex and somewhat dependent on experimental conditions. Measurement of pHi in solid tumors by non-invasive Magnetic Resonance Spectroscopy (MRS) has been possible for some time now and recently several specific markers for measuring pHe have become available. As a result we have been able to study the relationship between pHi and pHe in vivo in several different solid tumor types. In only one tumor type (HT29 xenografts) was there a significant correlation between pHi and pHe; in 3 other tumor types (RIF-1 in mice, GH3 prolactinomas and H9618a in rats) there was no correlation. A significant correlation between pHi and NTP/Pi ratios was seen across all tumor types. Theoretical considerations of causes of tumor acidity, hypotheses to explain extracellular acidity and the possibility that low pHe might be an intrinsic feature of the tumor phenotype and not merely the consequence of metabolic activity have been discussed. In addition the consquences for concepts of treatment based on pH are considered.

The application of functional magnetic resonance (MR) imaging techniques to the measurement of oxygenation and blood flow in tumors is described. Gradient recalled echo MR imaging (GRE-MRI) offers a real-time noninvasive method for monitoring tumor response to vasomodulators such as carbogen (95% O2/5% CO2) breathing in attempts to overcome tumor hypoxia and improve treatment efficacy. Although the response is tumor-type dependent, increases in signal intensity of up to 100% have been observed in several animal tumor types. Responses are also seen in human tumors. The observed increases in GRE-MRI signal intensity are due to a combination of a reduction of deoxyhemoglobin in the blood causing changes in the MR imaging relaxation time T2* and changes in blood flow and may also reflect the capillary density. Thus, the magnitude of the GRE image intensity change gives an indication of the potential response of an individual tumor to treatments that aim to improve tissue oxygenation and therefore how the tumor may respond to therapy. In addition, carbogen breathing by the host has been shown to increase the uptake and efficacy of chemotherapeutic agents in animal tumors., (Copyright 1998 W.B. Saunders Company.)

Nuclear magnetic resonance spectroscopy (MRS) offers a non-invasive approach for studying tumour biochemistry and physiology. This review highlights NMR nuclei (31P, 1H, 19F, 13C, 2H) that have been observed in both pre-clinical and clinical spectroscopic studies of cancer.

Purpose: Gradient-Recalled Echo (GRE) Magnetic Resonance Imaging (MRI), which detects changes in blood vessel deoxyhaemoglobin content, has been investigated as a noninvasive monitor of changes in human tumor oxygenation and blood flow, in response to carbogen (95% O2, 5% CO2) breathing., Methods and Materials: GRE images (TE = 60 ms, TR = 200 ms, alpha = 40 degrees, 256[2] matrix) were acquired from 31 patients with primary and metastatic disease, prior to and during carbogen breathing. Three patients underwent a follow-up examination after radiotherapy., Results: Seventeen out of 34 tumors showed enhanced image intensity, consistent with an improvement in tumor oxygenation and blood flow, while 11 showed no response; 6 studies were technical failures. In one patient a metastatic node that had eluded orthodox investigation was visualized. A reduction in response was observed in the three patients studied postradiotherapy., Conclusion: This method, which can be performed on a standard clinical MRI instrument, provides a noninvasive measurement of tumor response to oxygenation/blood flow modification. In principle, this should enable the clinician to optimize treatment protocols, such as carbogen breathing, for individual radiotherapy patients.

It is well known that low levels of tissue oxygen (pO2) protect tumour cells from ionising radiation and some chemotherapeutic agents. Thus, numerous studies have been aimed at developing methods to measure tissue oxygenation. An initial discussion of some of the traditional methods for measuring oxygenation is included, followed by a discussion of magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) methods for measuring tumour and normal tissue oxygenation. The latter methods are of interest because of the non-invasive nature of magnetic resonance (MR). Some of the MR methods described herein include: 31P MRS, 1H MRS and MRI, and 19F MRS and MRI. Each method is detailed, including a brief assessment of its ability to measure tumour oxygenation and its potential for clinical application.

This review discusses the relationship between tumour oxygenation status, tumour cell death and the 31P MRS parameters associated with cellular energy metabolism (phosphocreatine, nucleoside triphosphates and Pi). The presence of cells dying by apoptosis, and during mitosis would be unlikely to affect the 31P spectrum directly since they represent only a small fraction of tumour cells and remain energized until phagocytosed. Histologically necrotic cells also probably contribute nothing to the 31P spectrum. Instead, the spectrum appears to reflect the degree of hypoxia of the remaining viable cells, and the metabolic alterations required to sustain ATP synthesis as the oxygen supply diminishes. The biochemical theory developed to account for the 31P spectra of acutely hypoxic tissues does not apply to chronically hypoxic tumours. The concentrations of free ADP and Pi have major roles in the control of oxidative phosphorylation and glycolysis, as in normal tissues, but the precise relationships are still obscure. Cell-killing following therapy may indirectly affect 31P MRS parameters via changes in oxygen concentration brought about by an improvement in tumour blood flow and alterations in oxygen consumption rates and diffusion distances.

31p nuclear magnetic resonance (NMR) spectra have been obtained from animal and human tumours grown in laboratory rodents. The tumour cells are only slightly more acid than the surrounding muscle but they tend to have large Pi and sugar phosphate peaks, suggesting anoxia, and large but variable phosphodiester peaks. The results indicate that NMR will be an important tool for studying tumours in the laboratory, and for their diagnosis, assessment, and monitoring in clinical practice.