Full title: Hyperpolarized carbon-13 Magnetic Resonance Spectroscopic Imaging: a clinical tool for studying tumour metabolism Abbreviated title: Hyperpolarized 13C MRSI for studying tumour metabolism Authors: Fulvio Zaccagna1, MD, James T Grist1, BSc, AMInstP, Surrin S Deen1, MBBS, Ramona Woitek1, MD, PhD, Laura M. T. Lechermann1, MSc, BSc, Mary A McLean2, PhD, Bristi Basu3, BM Bch, MRCP, FRCP, PhD and Ferdia A. Gallagher1, BA, BM BCh, MRCP, FRCR, PhD Affiliations 1 Department of Radiology, University of Cambridge, Cambridge, United Kingdom 2 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom 3 Department of Oncology, University of Cambridge, Cambridge, United Kingdom 28 Abstract Glucose metabolism in tumours is reprogrammed away from oxidative metabolism, even in the presence of oxygen. Non-invasive imaging techniques can probe these alterations in cancer metabolism providing tools to detect tumours and their response to therapy. Although Positron Emission Tomography with [18F]2-fluoro-2-deoxy-D-glucose (18F-FDG PET) is an established clinical tool to probe cancer metabolism, it has poor spatial resolution and soft tissue contrast, utilises ionizing radiation and only probes glucose uptake and phosphorylation and not further downstream metabolism. Magnetic Resonance Spectroscopy (MRS) has the capability to non-invasively detect and distinguish molecules within tissue but has low sensitivity and can only detect selected nuclei. Dynamic Nuclear Polarisation (DNP) is a technique which greatly increases the signal-to-noise ratio (SNR) achieved with MR by significantly increasing nuclear spin polarisation and this method has now been translated into human imaging. This review provides a brief overview of this process, also termed Hyperpolarized Carbon-13 Magnetic Resonance Spectroscopic Imaging (HP 13C-MRSI), its applications in pre-clinical imaging, an outline of the current human trials that are ongoing, as well as future potential applications in oncology. Introduction Tumours metabolise glucose very differently from normal tissue, generating lactate even in the presence of oxygen, a process known as the Warburg effect.1 Other metabolic pathways, such as amino acid utilisation and fatty acid synthesis, are also reprogrammed in tumours compared to normal tissue.2–4 A number of non-invasive imaging methods have been used to probe these alterations in cancer metabolism. This review will describe a new technique for studying this metabolism termed hyperpolarized carbon-13 MRSI.5 Positron Emission Tomography (PET) is an extremely sensitive technique for studying molecular processes without disrupting normal physiology, with tracer detection in the nano- to picomolar range.6,7 PET imaging in conjunction with [18F]2-fluoro-2-deoxy-D-glucose (18F-FDG), is an established clinical tool to probe altered glucose metabolism in cancer patients.8 Whole body 18F-FDG PET is superior to standard Computed Tomography for the assessment of tumour stage in many malignancies9, however it is limited by a relatively low clinical spatial resolution (of the order of 5 mm), poor soft tissue contrast, exposure of patients to ionizing radiation and the inability to discriminate between different metabolites or the compartment from which the signal arises e.g. extracellular vs intracellular.10,11 Magnetic Resonance Spectroscopy (MRS) may also play a useful role in probing cancer metabolism by detecting the resonant frequencies of nuclei within distinct molecules (such as that of hydrogen, 1H, or protons), therefore it can be used to non-invasively discriminate between metabolites.12,13 However, in comparison to PET, MR is a very insensitive technique utilising only a small number of the available nuclei to generate an image or spectrum. At clinical field strengths, only a few of the available hydrogen nuclei in every million are used to generate these images.14,15 This low sensitivity is due to the low polarisation of the nuclei at thermal equilibrium and consequently, only molecules at relatively high concentration can be probed with MRS. Despite the great potential of MRS as a tool to study biochemistry in vivo, it is not used routinely in the clinic due to both the low spatial and molecular sensitivity of the technique, and difficulty in subsequent data interpretation.10,13 Dynamic Nuclear Polarisation (DNP) is a technique based in solid state physics which greatly increases the signal-to-noise ratio (SNR) achieved on MR by significantly increasing the polarisation of nuclear spins and therefore enabling a much larger proportion of them to be utilised for imaging.16 In 2003 DNP was combined with an efficient method to transfer the substrate from the solid (~1 K) to liquid (~300 K) state, a process which is now known as ‘Dissolution DNP’. This technique opened up new possibilities for the method to be translated into biological systems such as cell studies and in vivo animal imaging.17 The technique, termed Hyperpolarized Carbon-13 Magnetic Resonance Spectroscopic Imaging (HP 13C-MRSI), has now been translated into humans.18 This review provides a brief overview of the technique, its applications in pre-clinical imaging, an outline of the current human trials that are ongoing as well as future potential applications. MR Spectroscopy and the benefits of metabolic imaging with MRI Conventional 1H-MRI largely measures the spatial distribution of water and fat molecules within tissue and has led to many clinical applications in neurological, cardiac, and oncological imaging.19–21 MR can also be used to provide information on the nature of other molecules by utilising the property of chemical shift to discriminate the resonant frequency of protons. MRS has been used to detect the steady state levels of diverse molecules including lactate, N-acetylaspartate (NAA) and glutamate.12,13 An alternative approach to the steady state measurement of tissue metabolites, is to probe dynamic changes in metabolites during enzymatic reactions, which can be assessed through the exogenous administration of labelled metabolites. A physiologically-active substrate labelled with a nucleus that can be detected with MRI, allows spectral discrimination between the substrate and its subsequent downstream metabolic products; when applied to imaging it allows the spatial distribution of both the substrate and the product to be detected. However, MRS requires long acquisition times which in turn makes it difficult to discriminate rapid changes in real-time metabolism in vivo. 13C is ideal for labelling endogenous metabolic molecules which are mostly carbon based, as its low natural abundance (1.1%) results in a low level of background noise. 13C-MRS acquisition from either a single voxel or a small number of voxels has been used to successfully analyse many physiological processes in organs such as in the brain, as well as pathological processes including cancer.22–24 Although 13C-MRS has been used to detect early changes in cancer metabolism as a biomarker for response to treatment23, its utility is limited by very low SNR as well as poor temporal and spatial resolution. Therefore the increase in SNR afforded by DNP has opened up the possibility of applying 13C-MRS in new ways, allowing for rapid acquisition of imaging to probe real-time metabolism.17,25 There are several methods that have been described for increasing nuclear polarisation and generically they are termed hyperpolarisation techniques; for example, there are a number of approaches for the hyperpolarisation of gases used in ventilation imaging.26–28 DNP was first postulated as a hyperpolarisation method by Overhauser in 195316, and has now been applied to 13C-labelled physiologically substrates which are rapidly transported and metabolised by the cells. To facilitate the process, labelled molecules are mixed with a free-radical containing agent, frozen in a glass state, and then cooled to approximately 1 Kelvin in a magnetic field e.g. 3.35 T or 5 T. At these extreme physical conditions the electron pool in the free radical becomes fully polarised and the transfer of polarisation from the electron pool to the nuclear pool is facilitated by microwave irradiation. Increasing the polarisation to levels required for in vivo imaging takes approximately two hours and following this the sample is removed from the field and quickly dissolved with superheated water.29,30 This hyperpolarized sample has an increase in signal of 10,000 to 100,000-fold and can be used to probe the real-time dynamics of enzymatic activity in vitro in cell culture or in vivo in animal models, using rapid spectroscopy or imaging.31–34 The term spectroscopic imaging is used where spectra are acquired from multiple voxels to generate an image of the metabolic distribution across the tissue. One of the challenges of the technique is that the signal from the hyperpolarized carbon is very transient and decays with a half-life that is commonly 20-30 s in vivo; therefore perfusion, transport and metabolism must occur during approximately 5 half-lives or 2-3 mins. This limits the number of in vivo molecules and reactions that can be probed with the technique and necessitates very rapid and efficient imaging strategies. Preclinical imaging To date, [1-13C]pyruvate has been the most widely used metabolite for hyperpolarized 13C MRSI, due to its relatively long polarisation half-life (the time it takes for a compound to lose half of its initial hyperpolarized signal), rapid distribution and uptake, and the central role it plays in many metabolic pathways (figure 1).35,36 Pyruvate is at an important metabolic crossroad between the formation of lactate on one hand, and entry into the tricarboxylic acid cycle (TCA) with the formation of carbon dioxide on the other. In cancer, the 13C label on [1-13C]pyruvate is predominantly exchanged to [1-13C]lactate via the enzyme lactate dehydrogenase (LDH) but some will also be exchanged to [1-13C]alanine via alanine aminotransferase (ALT), or irreversibly converted to 13CO2 via pyruvate dehydrogenase (PDH).33 The production of lactate in tumour tissue, even in the presence of oxygen, is termed the ‘Warburg effect’ and has been observed using HP 13C-MRSI in a variety of preclinical cancer models. There is evidence that the labelling of lactate correlates with tumour grade, with higher lactate labelling present in more aggressive tumours.1,37 HP 13C-MRSI has also been used to demonstrate a consistent decrease in the 13C exchange between [1-13C]pyruvate and [1-13C]lactate during tumour responses, regardless of treatment type in various in vitro and in vivo models, with changes seen as early as 24 hours after the initiation of therapy, and sometimes earlier than conventional imaging modalities such as diffusion-weighted imaging.34,38–42 HP 13C-MRSI may also have a role in the early detection of preneoplastic changes, as demonstrated in a pancreatic model evaluating LDH activity with increased HP 13C label exchange between 13C-pyruvate and endogenous lactate present with the onset of neoplastic transformation.43 Translation into human imaging The first human study using HP 13C-MRSI was completed in 2013 at the University of California San Francisco (UCSF).18 This study demonstrated the safety and feasibility of HP 13C-MRSI with [1-13C]pyruvate in thirty one male subjects with biopsy-proven prostate cancer. Patients were injected with hyperpolarized 13C-pyruvate. Signals from pyruvate and lactate were observed in the vasculature, tumour and normal prostate at the 3 dose levels considered (0.14, 0.28 and 0.43 mL/kg actual body weight of 230 mM pyruvate solution) and uptake in the prostate was observed approximately 20 seconds after injection. A few mild adverse events such as dysgeusia (5/31 patients) were reported but none of them were considered to be doselimiting toxicities. Two single events of dizziness and grade 2 diarrhoea reported in the phase 2 component, were attributed to concomitant medications. The highest dose tested, 0.43 mL/kg of 13Cpyruvate solution, showed the highest SNR for 13C-pyruvate as expected. The study also demonstrated an elevated 13C-lactate/13C-pyruvate ratio on HP 13C-MRSI in one patient where no abnormality was detectable on conventional 1H-MRI; this patient was subsequently shown to have a biopsy-proven low-grade tumour (figure 2). The study concluded that the data confirmed both the safety of the agent and that an elevated 13Clactate/13Cpyruvate may be seen in regions that were otherwise undetectable with standard methods, opening up the possibility of using metabolic imaging to detect occult tumours. This initial study required a sterile room for the production and hyperpolarisation of the 13C pyruvate prior to dissolution and injection. Wider application of the technology was made possible by the design of a new platform which utilises an integral, disposable and sterile fluid path or pharmacy kit (figure 3).29 The SPINlab hyperpolarizer, produced by GE Healthcare, uses a closed-cycle cryogenic system and has multi-sample capability (figure 4). The pharmacy kit is prefilled and sealed in a sterile environment, before being transferred to the clinical hyperpolarizer, thus enabling the hyperpolarisation of a pyruvate sample within a routine clinical environment such as a radiology department, whilst maintaining the sample sterility. Filled pharmacy kits can either be used on the day of production or stored frozen depending on the method that is used. Clinical imaging studies There are currently six sites that have undertaken clinical imaging with the technique, although several other facilities will commence human imaging in the near future. Current studies in human HP 13C-MRSI research are exploring pyruvate metabolism in a number of organs such as the brain, heart, liver and breast and in a variety of disease processes. Several of these studies are evaluating HP 13C-MRSI derived biomarkers in addition to tissue samples in order to understand and validate the biophysical nature of signal generation with HP 13C-MRSI. Another important aim is to establish the reproducibility and repeatability of the technique. A significant objective of the current clinical studies is to investigate HP 13C-MRSI as an imaging biomarker of early treatment response. Many of these studies are being run in parallel with other conventional imaging tests for measuring treatment response, as well as functional MR imaging techniques, such as dynamic contrast-enhanced (DCE) MRI. Changes in tumour metabolism in response to therapy have been shown to occur before anatomical changes can be detected and potentially could be earlier than functional imaging alterations. The use of the technique as a response biomarker has been demonstrated in several animal studies and is now being translated into humans (figure 5).34,39,42,44 An exciting area for future research is how changes in metabolism measured by HP 13C-MRSI following treatment relate to circulating tumour biomarkers such as cell-free DNA45,46, as this would increase the ability to non-invasively interrogate tumour activity and responses to therapies, and spare patients the complications associated with serial biopsies. Potentially, HP 13C-MRSI may provide a specific pharmacodynamic downstream imaging biomarker of both conventional and novel agents, and could also be used to detect early changes following therapy which are predictive of long-term response. This could therefore provide a novel non-invasive precision medicine approach to drug development, permitting early cessation of ineffective drugs and providing a cost saving in terms of patient time and toxicities. The future of the technique MRI hardware developments for hyperpolarisation Despite the large increase in signal-to-noise that is generated by DNP, both the hardware and acquisition sequences required have to be optimised to avail of this increased signal. Dedicated 13C coils are needed for the technology: current research being undertaken aims to optimize coil design and pulse sequences to maximize the signal acquired. For example, the use of multiple-channel coil arrays can enable parallel imaging techniques to accelerate acquisition while retaining sensitivity.47 Calibrationless methods have been shown to overcome the limitations of the short-lived hyperpolarized species in this context.48 There have been several different imaging schemes proposed for the acquisition of hyperpolarized 13C data.49–51 A key component of most sequences is the use of single shot, multiple time point acquisition to acquire dynamic, metabolite-specific data.51 The differences mainly involve encoding efficiency, gradient demand and artefact behaviour: non-Cartesian trajectories are more efficient but are prone to artefacts.50 The choice of the optimal sequence depends on the spatial and temporal resolution desired and the number of different chemical species to be resolved. Future developments in this area will involve assessing and improving intersite reproducibility of the data acquisition methods. Pharmacy requirements and developments in formulation The current approach for the consumables for the clinical polariser requires a local sterile production facility to fill pharmacy kits which can be costly to establish. A more practical approach may be the development of a large, centralised, filling facility where pharmacy kits could be frozen and transported to other sites. There are a number of country-specific regulatory issues that need to be addressed for the production and transport of these filled pharmacy kits at each site which needs to be considered before developing a programme of research locally. If hyperpolarized 13C-MRSI is to be more widely used, it needs to be accessible for non-specialist centres. To improve availability, there is ongoing research in the area of long-lived polarised states and transport of pre-polarised molecules. Although this area is still in the early stages of development and some way from clinical translation, it could potentially open up the possibility of hyperpolarizing molecules centrally and transporting them to the imaging site without the need for a local polariser. New hyperpolarized tracers Hyperpolarized 13C-labelled tracers offer unique opportunities to probe many metabolic pathways. Although many hyperpolarized tracers have been developed, very few have the necessary biological, chemical and physical properties required for successful translation into human imaging. Table 1 summaries some of the tracers which have clinical potential and provide biologically useful information. As discussed above, [1-13C]pyruvate has been used to probe LDH and PDH activity and similarly, [1-13C]lactate can be used to probe LDH; however [1-13C]pyruvate cannot be used to probe the tricarboxylic acid (TCA) cycle as the label is not incorporated into acetyl CoA52: [1-13C]pyruvate oxidation mediated by PDH releases the hyperpolarized 13C as 13CO2 and therefore the production of acetyl CoA and incorporation into the Krebs cycle cannot be studied. In contrast, [2-13C]pyruvate allows downstream metabolites such as acetyl carnitine, citrate and glutamate to be detected.53,54 [2-13C]pyruvate can assess metabolism in tissues where PDH activity is high such as the heart, by probing oxidative metabolism through the Krebs cycle rather than anaerobic glycolysis and lactate formation.54 13C-Urea has been utilised to probe tissue perfusion but unlike conventional perfusion tracers, is a small endogenous molecule.55,56 A number of potential issues has been identified with gadolinium-based contrast agents recently such as accumulation in the brain with repeated use;57,58 consequently, hyperpolarized urea may provide an alternative method to image tissue perfusion with a physiologically relevant molecule.52 13C-Urea could also be used to study tissue oxygenation as has been shown in both the healthy and diabetic rat kidney.59,60 Hyperpolarized [1,4-13C2]fumarate has been proposed as a positive contrast agent for detecting successful treatment response and tissue necrosis. [1,4-13C2]fumarate is exchanged to [1,4-13C2]malate in the presence of the enzyme fumarase. The intracellular enzyme is released into the extracellular space following necrosis allowing enzymatic conversion of fumarate to malate (figure 6).61 This has been demonstrated in tumours and in acute kidney injury.31 One important advantage of HP 13C-MRSI over PET imaging is that it allows multiple probes to be injected either sequentially or simultaneously so that several metabolic pathways can be studied at a single time point. Although dual PET probe injection has also been undertaken, the modelling required to differentiate these probes is complicated by perfusion, metabolism and excretion and is often impractical; furthermore, conversion into metabolic products cannot be detected.62 In comparison, HP 13C-MRSI has the advantage of discriminating probes and their metabolites by virtue of differences in chemical shift: therefore, metabolism of molecules injected simultaneously can be directly compared. This has been demonstrated in several pre-clinical models and has the potential to be applied to humans in the future.38 Image analysis and quantification Robust quantification of HP 13C-MRSI will be important for the repeatability and reproducibility of the technique. Given that the technique probes exchange or flux between two metabolic pools, there are many analogies between the approaches taken for HP 13C-MRSI and those used in PET and DCE-MRI. Several methods have been proposed to reliably and reproducibly quantify the pyruvate-lactate exchange reaction: kinetic parameters can be fitted to model-based approaches such as the two-site exchange model which are the most accurate; simpler model-free approaches such as the area under the curve (AUC) of the lactate-to-pyruvate ratio and the lactate time-to-peak may adequately represent the reaction and may be more suitable for widespread clinical use.34,63,64 Further challenges of the technique include the segmentation of tumour from normal tissue which is compounded by the low resolution and SNR of the data. However, by fully utilising the 5-dimensional nature of HP 13C MRSI – including spatial, temporal and spectral information – segmenting areas of high metabolic exchange from low exchange can be greatly facilitated and these approaches could be used for automated and reproducible delineation of tumour regions in the future65. Hyperpolarized 13C-MRSI with PET/MRI Since its introduction in clinical practice, PET/CT has found many clinical applications due to the combination of metabolic (PET) and anatomical (CT) information.66 18F-FDG PET/CT is a powerful tool for staging and treatment response monitoring in a wide range of cancers such as lung cancer and lymphoma.8,67 The recent creation of hybrid systems combining PET and MRI have capitalized on the inherent advantages of MRI over CT such as the lack of ionizing radiation, functional imaging capabilities, spectroscopy and improved soft-tissue contrast.11,68 Several clinical applications have been proposed for PET/MRI69,70: in oncology, examples include head and neck cancers, liver tumours and pelvic malignancies which may significantly benefit from the hybrid approach due to improvements in co-registration and reduction in movement artefact.71–75 PET/MRI has also demonstrated potential in organs such as the lung where traditionally MRI has only had a limited role, highlighting the potential of the combination of those two techniques.76–78 PET/MRI could provide a powerful tool for imaging tumour metabolism. While 18F-FDG is a highly sensitive measure of metabolism and has many applications, it simply measures glucose uptake and phosphorylation and therefore provides only an indirect measure of the Warburg effect.79–81 In contrast, hyperpolarized 13C-MRSI is capable of monitoring the uptake of 13C-pyruvate and its conversion into 13C-lactate (or 13C-alanine and 13C-bicarbonate) enabling real-time in vivo imaging of metabolism and a more direct measure of the Warburg effect.82–84 Combining PET/MRI with 13C-MRSI provides the opportunity to study cancer metabolism in conjunction with other MR measures of tumour structure, function and heterogeneity.85 A recent study reported the simultaneous acquisition of 18F-FDG PET and 13C-MRSI using a PET/MRI system in 10 dogs.86 This demonstrated that, although there is a strong correlation between uptake of FDG and pyruvate conversion to lactate, there are areas of mismatch between the two techniques confirming the potential added value of combining the two techniques.86,87 A wide range of tracers in addition to 18F-FDG could be used with this combined hyperpolarized 13C-MRSI/PET approach such as the amino acid analogues 11C-methyl-L-methionine (MET) and O-(2-[18F]fluoroethyl)-L-tyrosine (FET) in brain tumours for diagnosis and therapy assessment.88 This combination could be important in the management of some cancers, and certain population groups where longitudinal monitoring is required or where dose reduction is important such as children or women of reproductive age.86 The technique will be translated into humans shortly. As HP 13C-MRSI is in its infancy and rapidly evolving, the projected long-term costs of the technology are difficult to accurately evaluate. Hardware costs will reduce in time if there is increased demand for hyperpolarisation approaches. The authors believe that in the future the costs for undertaking such studies could be similar to those currently for PET imaging. These costs could be defrayed by the clinical benefits and treatment cost savings if the technique is shown to have unique applications, particularly if ineffective but expensive therapies can be discontinued. Given that MRI is more widely available than PET in most hospitals, HP 13C-MRSI could become an important additional clinical tool if unique applications are revealed. Conclusion HP 13C-MRSI can be used to probe tissue metabolism non-invasively in vivo. This may have important implications for the management of cancer patients in the future. 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The use of amino acid PET and conventional MRI for monitoring of brain tumor therapy. NeuroImage Clin. 2017;13:386–94. Tables Tracer Name Biological Relevance [1-13C]Pyruvate LDH and PDH activity [2-13C]Pyruvate LDH, PDH, TCA activity [1-13C]Lactate LDH activity [1-13C]Acetate TCA activity and fatty acid oxidation [1-13C]Glutamine Glutaminolysis [13C]Urea Tissue perfusion [1,4-13C2]Fumarate Cellular necrosis Table 1: Some of the hyperpolarised tracers with clinical potential which are currently being investigated and their biological relevance. Figures Figure 1: Diagram showing the metabolic fate of glucose and pyruvate. Abbreviations: LDH - lactate dehydrogenase, ALT - alanine transaminase, PDH - pyruvate dehydrogenase, CA - carbonic anhydrase, PC - pyruvate carboxylase, CAT - carnitine acyltransferase, CS - citrate synthase, IDH - isocitrate dehydrogenase, GLDH - glutamate dehydrogenase, OGDC – oxoglutarate (-ketoglutarate) dehydrogenase complex, SCS - succinyl coenzyme A synthetase, SDH – succinate dehydrogenase (part of succinate-coenzyme Q reductase), FH - fumarate hydratase, MDH - malate dehydrogenase, AST - aspartate transaminase. Pyruvate, lactate and fumarate has been highlighted in bold. Figure 2: Hyperpolarized pyruvate metabolism in human prostate cancer. Images from a patient with bilateral biopsy-proven Gleason grade 3+3 prostate cancer. (a) Axial T2 weighted image and (b) apparent diffusion coefficient map showing a peripheral zone tumour (red arrows). (c) False colour metabolic map superimposed over the proton image showing voxels with an elevated ratio of hyperpolarized [1-13C]lactate/[1-13C]pyruvate bilaterally in the prostate and highlighted in pink, obtained after administration of hyperpolarized [1-13C]pyruvate. Adapted from Nelson et al18 with permission. Figure 3: Pharmacy kit for clinical polarisation. The kit contains pyruvate sealed within a closed sterile environment. The kit includes: a sample vial, the concentric tube assembly, the multi-position inlet/outlet valve, a syringe for dissolution medium, a filter and a receiver unit. Figure 4: (a) Schematic of cryostat, magnet and sorption pump. The 3.35 T magnet is located within a helium-filled cryostat and suspended within an external vacuum vessel. Reproduced from Ardenkjaer-Larsen et al29 with permission. (b) Photograph of the SPINlab Diamond Polariser for clinical use in Cambridge (GE Healthcare). This clinical SPINlab uses a 5T magnet. Figure 5: 13C spectroscopic imaging of a murine lymphoma tumour before and after drug treatment. Colour maps representing [1-13C]lactate and [1-13C]pyruvate peak intensities obtained from 13C chemical-shift imaging in the same mouse before and after treatment with the chemotherapeutic agent etoposide. The proton grayscale images define the tumour margins (indicated in white). Reproduced from Day et al34 with permission. Figure 6: Transverse images from (a) untreated and (b) etoposide-treated mice with implanted lymphoma tumours. The 1H image shows the anatomical location of the tumour, outlined in white. The false-colour maps superimposed on the proton images demonstrate the spatial distribution of the total hyperpolarized 13C malate and 13C fumarate signals. The colour scale indicates the relative signal intensity compared with the maximum intensity in each image. Reproduced from Gallagher et al61 with permission. image1.jpeg image2.jpeg image3.jpeg image4.jpeg image5.tiff image6.jpeg