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British Journal of Radiology (2003) 76, S111-S117
© 2003 British Institute of Radiology
doi: 10.1259/bjr/50577981
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Molecular imaging using magnetic resonance: new tools for the development of tumour therapy

K M Brindle, BA, MA, DPhil

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK


   Abstract
Top
 Abstract
Introduction
 MRI in the laboratory
 Molecular imaging using MRI
 Concluding remarks
 References
 
Molecular imaging – the exploitation of specific molecules as the source of image contrast – promises new insights into disease processes in the laboratory and since the imaging modalities employed are applicable clinically, can be used to translate this knowledge into new diagnostics and treatments in the clinic. This brief review focuses on the use of MR-based molecular imaging techniques for developing tumour therapy. As examples, methods for detecting drug-induced tumour cell apoptosis; the response of tumours and their susceptibilities to an antivascular drug; early signs of tumour immune rejection and methods for detecting immune cell infiltration of tumours are described.


   Introduction
Top
 Abstract
 Introduction
MRI in the laboratory
 Molecular imaging using MRI
 Concluding remarks
 References
 
Despite many recent advances in non-invasive imaging technologies, such as MRI and positron emission tomography (PET), we are still limited in our ability to detect tumours at their very early stages of development, to monitor their invasion and metastasis and to assess their responses to therapy. The advent of "molecular imaging", the coupling of these imaging technologies with specific molecular probes designed to detect tumour-specific markers, is set to radically change this situation, and to revolutionize our approach to the detection and treatment of cancer [1–4].

While the focus of attention in molecular imaging has been on the potential clinical applications, these techniques are also being used to great effect in the laboratory, where their use in the characterization of the growing number of animal models of cancer will be invaluable in addressing important questions about tumourigenesis, and in developing new disease treatments [3, 4]. Of course these laboratory-based studies form the basis of the clinical applications. Indeed molecular imaging sits at the interface between our growing understanding of the molecular basis of the disease and the translation of this knowledge to new treatments in the clinic. Molecular imaging should increase both the specificity and sensitivity of tumour detection. It will allow us to exploit the information coming out of genome sequencing to develop imaging methods that are targeted at specific molecular features of the diseased tissue. The technology has tremendous potential for the early detection of tumours, characterization of the disease process, understanding of the underlying biochemistry and evaluation of subsequent treatment. Detection of tumours, for example, could be improved by using more specific parameters, such as detection of tumour cell surface markers. When combined with new targeted therapies, such as gene therapy, these imaging methods could allow assessment of therapeutic effectiveness long before gross morphological changes, such as tumour shrinkage, occur.


   MRI in the laboratory
Top
 Abstract
 Introduction
 MRI in the laboratory
Molecular imaging using MRI
 Concluding remarks
 References
 
MRI is being used increasingly by biologists for mouse phenotyping. A common approach to elucidating the function of an unknown gene is to modify gene function and then determine the consequent effects on the phenotype of the organism. This approach is often termed functional genomics. Since, for many genes, the effect on phenotype can be very subtle, it requires methods that are comprehensive, both in scope and detail, for describing the resultant phenotype. With the completion of the mouse genome sequence, we can expect a deluge of mouse gene-knockout and gene-knock-down mutants from functional genomics studies, in which MRI will be used to characterize the resulting changes in tissue morphology. At the field strengths commonly used for imaging in the laboratory, the technique can give relatively high-resolution three-dimensional (3D) images in a relatively short space of time. For example, with an acquisition time of 14.5 h, an isotropic image resolution of 110 µm was obtained from a whole fixed mouse at 2 T, 50 µm from the mouse abdomen at 7.1 T and 25 µm from an isolated fixed kidney [5]. These images can then be sectioned by the biologist in silico in order to examine the features of interest. Since the technique is non-invasive it allows longitudinal studies and it can also be used to guide subsequent histological sectioning of tissues obtained post mortem. This could save a considerable amount of time since not all tissues would need to be sectioned, only those identified by MRI as being abnormal. MRI even has utility in imaging fixed tissues ex vivo. For example, in a mouse model of polycystic kidney disease, we showed that we could image oedema in the spinal cord of fixed embryos (Figure 1Go) [6]. This was difficult to determine using conventional histological sectioning methods.



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  		Figure 1. 1H MR images from fixed mouse embryos. The top image is from a Pkd1 knockout embryo and the bottom is from a control. Regions of oedema and haemorrhage in the knockout are marked by arrows. Figure taken, with permission, from [6].

 
The advantage of using MRI in these studies, as in the clinic, is that it gives very good delineation of soft tissues. However, as biologists, what we are interested in is, not only the tissue morphology, but also the underlying tissue biochemistry and cell physiology. Studies of tissue biochemistry, using MRI, have traditionally been the province of the spectroscopists. Since the mid-1970s it was known that MR spectra could be obtained from intact biological systems. 31P MR spectroscopy (MRS), in particular, was used in the laboratory to study cellular energetics in tissues like the heart and brain [7] and this work was subsequently translated through to clinical application [8]. However, the problem with MRS is that it is relatively insensitive, which means that patient examination times can be long and "image" resolution is poor. Usually spectra are obtained, using localized spectroscopy techniques, from relatively large volumes of tissue and thus, in the case of a tumour for example, the spectra may be contaminated with signals from normal tissue, if indeed the tumour is detectable at all. As a consequence of these limitations, MRS has not found widespread clinical application. What we need is a technique that has the sensitivity and resolution of MRI, but which can also report on the underlying tissue biochemistry. Such a technique would be useful in the lab, but more importantly could be used in the clinic. This is where molecular imaging comes in.


   Molecular imaging using MRI
Top
 Abstract
 Introduction
 MRI in the laboratory
 Molecular imaging using MRI
Concluding remarks
 References
 
MRI has a number of important advantages over other non-invasive imaging modalities; it has relatively high spatial resolution (when compared with PET and SPECT), it has very good sample penetration (when compared with optical imaging methods) and it is already widely used in the clinic. A drawback is its low sensitivity compared with these other methods, and this requires the development of powerful signal amplification strategies. Nevertheless there are several recent examples of molecular imaging using MRI which have demonstrated the potential of the methodology [3, 4, 9]. In this brief review I will describe the possible applications of molecular imaging using MRI in cancer research, focusing predominantly on examples from my own laboratory, in the belief that these are both representative and illustrative of what can be done.

Detection of tumour cell apoptosis
There is already evidence, both from the laboratory and the clinic, that an early apoptotic response to therapy is a good prognostic indicator for treatment outcome [10–13]. The introduction into the clinic of a non-invasive imaging method for detecting tumour cell apoptosis (programmed cell death) would have a significant and immediate impact on the management of cancer patients. Such a method could be used in Phase I clinical trials of a new drug to provide an early indication of efficacy, and could be used subsequently in the clinic for the selection of drug and treatment regimens for individual patients. This would allow the clinician to abandon those treatments that are not working, at an early stage, and to try alternative approaches. This could improve outcome while reducing patient suffering and financial costs. For these reasons we have, over a number of years, been developing non-invasive MR-based methods for apoptosis detection in tumours. We showed that there were two metabolic markers of the process, fructose-1,6-bisphosphate and cytidine diphosphocholine (CDP)-choline, that could be detected using 31P MRS [14, 15]. Fructose-1,6-bisphosphate is an intermediate in the glycolytic pathway and accumulates in apoptotic cells because of the accompanying activation of the enzyme poly-ADP-ribose polymerase (PARP). PARP uses as a substrate NAD+ and in apoptosis, can substantially deplete the cell of this coenzyme. As a consequence there is a block in the glycolytic pathway at the level of the enzyme glyceraldehyde 3-phosphate dehydrogenase and an accumulation of upstream intermediates in the pathway, including fructose-1,6-bisphosphate. This biochemistry was already known in the 1980s, however the demonstration that CDP-choline accumulates in apoptotic cells was novel. CDP-choline is an intermediate in the Kennedy pathway of phosphatidylcholine biosynthesis, and we went on to show that during apoptosis this pathway is inhibited at the level of the enzyme choline phosphotransferase (CPT) [15]. Inhibition of CPT, which is probably due to the cellular acidification that occurs early in the apoptotic process, results in the accumulation of its substrate CDP-choline.

However the problem with these 31P MRS measurements is that they are relatively insensitive and therefore lack both temporal and spatial resolution. These techniques are unlikely, for example, to be able to detect apoptosis in a tumour when the percentage of apoptotic cells is relatively low (5–10% on average of the cellular mass of a solid tumour). 1H MRS is more sensitive and has been used in a brain tumour model to detect therapy-induced apoptosis, through the increase in mobile lipid resonances that occurs in apoptotic cells [16]. These signals arise predominantly from cytoplasmic lipid droplets [17]. Why these droplets accumulate in apoptotic cells is not clear but it may be the result of phospholipase activation, or alternatively it could be due to the inhibition of CPT. Inhibition of CPT, as well as leading to the accumulation of CDP-choline, will also result in the accumulation of its co-substrate, diacylglycerol, which could then be converted to triacylgycerol, which is a major constituent of the droplets [18]. However the specificity of this 1H MRS lipid signature for apoptotic cells remains to be established, as does the sensitivity of the technique for detecting relatively low levels of apoptosis. Furthermore the presence of lipid deposits in the body, which also give rise to lipid signals, could obscure the increases in tumour lipid signals.

Attempts to detect tumour cell death in vivo using MRI have focused on the use of diffusion weighted 1H MRI to detect changes in the apparent diffusion coefficient (ADC) of water. The observed increase in water ADC following therapy was shown to be directly related to the number of cells killed and is thought to reflect liberation of water into the extracellular space due to cell necrosis ([19] and references cited therein). However, in a rat glioma model a therapy-induced change in water ADC was shown to be concurrent with a reduction in tumour volume and is therefore limited as an early marker of tumour cell death [20]. The same study, however, showed that T1 relaxation contrast in the rotating frame changed even when tumour cell volumes were still increasing but significant apoptosis was taking place, suggesting that this might provide an early indicator of apoptosis.

Our approach to detecting apoptosis using 1H MRI, was to adopt a molecular imaging approach that had already been tried with nuclear imaging, and which was well established in the field of optical microscopy. A relatively early event in apoptosis is the transfer of phosphatidylserine (PtdylSer) from the inner leaflet of the plasma membrane to the outer leaflet, where it serves as a signal to promote engulfment by neighbouring phagocytic cells [21]. Annexin V, labelled with a fluorescent probe such as fluorescein, which binds to PtdylSer on the surface of apoptotic cells, has long been used as a reagent for detecting apoptotic cells, either by fluorescence microscopy or by flow cytometry [22]. The protein has also been labelled with 99Tcm and used to detect apoptotic cells non-invasively in vivo, using radionuclide imaging techniques [23, 24]. We used a protein, the C2 domain of synaptotagmin, which like annexin V binds to PtdylSer. We showed that the fluorescently-labelled protein would bind to apoptotic cells and went on to show that the protein tagged with a paramagnetic label, superparamagnetic iron oxide (SPIO) particles, allowed detection of apoptotic cells, in vitro and in vivo, using MRI techniques [25] (see Figure 2Go). These experiments have demonstrated proof of concept, the apoptotic cells bind enough of the labelled protein to allow their detection using MRI. However, this system has a number of significant limitations. Although SPIO particles are very sensitive to MR detection (see below), they are detected by their effects on T2* and thus give negative contrast in the MR image. In an irregular structure like a tumour, where there are often hypointense regions in the image due, for example, to haemorrhage, this can make detection of the SPIO label difficult. Furthermore, SPIO particles are relatively large, the ones we used were between 20 nm and 30 nm in diameter, and this can limit extravasation of the material into the tumour interstitium, and consequently access to the apoptotic tumour cells. Fortunately tumours tend to have very leaky vasculature [26] making this less of a problem in this case. For these reasons we have been investigating the use of dendrimers as vehicles for generating high relaxivity Gd3+-based contrast agents [27], which give T1-dependent contrast and thus increase signal intensity in T1 weighted images. Tissue Gd3+ concentrations of the order of 10–4 M are required to give detectable changes in image contrast [1] and therefore a Gd3+-based reagent must carry a lot of Gd3+ ions. Dendrimers are highly branched, monodisperse macromolecules which can be synthesized with a strictly defined size and surface chemistry [28]. We have been using polyamidoamines (PAMAM) based on an ethylenediamine core and which are built-up by successive reactions with methyl acrylate and ethylenediamine. We have used a generation 2 dendrimer, which has a molecular mass of 3252 and 16 surface terminal amino groups and a generation 6 dendrimer, which has a molecular mass of 57972 and 256 surface amino groups. The amino groups can be crossed linked to Gd3+-chelates, to proteins and to fluorescent probes. Thus we can generate protein-targeted contrast agents which, by cross-linking many Gd3+-chelates to their surface, can have high relaxivity and which can also be fluorescently labelled to allow optical detection as well. By choosing the generation of dendrimer we can select the properties that we require from the agent. For example a large reagent will have good relaxivity but may show poor extravasation, and thus access to the tissue, and will tend to have a long plasma half-life [27]. The latter property may lead to toxicity problems. By contrast, a smaller dendrimer will have better access to the tissue and its shorter plasma half-life means that tissue of interest could be imaged, when the agent has cleared from the plasma, without interference from unbound agent in the plasma and tissue. With targeted contrast agents it is obviously important that the agent binds to the target of interest, but it is as important that the unbound agent is cleared from the tissue. However the problem with a smaller dendrimer is that it may not have sufficient sensitivity to allow detection and therefore a compromise may need to be struck between size and relaxivity. The availability of dendrimers from generations 0 (516 Da, 4 terminal amino groups) to 10 (933492 Da, 4096 amino groups) makes this possible.



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  		Figure 2. Sequential 1H MR spin echo images (echo time=30 ms) obtained from an apoptotic tumour following injection of the C2-SPIO (superparamagnetic iron oxide) contrast agent. The images were acquired over a period of nearly 2 h. The region of negative contrast, marked by the arrow, was coincident with a region of apoptotic cells observed by histological analysis of a corresponding tumour section obtained post mortem. Figure modified, with permission, from [25].

 
Imaging vascular function in tumours
A tumour can not grow beyond 1–2 mm in diameter without stimulating the growth of blood vessels into the tumour mass, to provide it with essential nutrients such as glucose and in particular, oxygen. As well as allowing growth, vascularization of the tumour also allows it to metastasise [29, 30]. Inhibition of this process, which is known as angiogenesis, can inhibit tumour growth and metastasis and in some cases, results in tumour regression. Since, in a normal individual, there is very little angiogenesis taking place outside of the tumour, the specificity of an antiangiogenic drug is ensured. As a consequence there has been considerable interest in the tumour vasculature as a therapeutic target. This has included the development of antiangiogenic drugs, which inhibit vessel growth, and antivascular drugs, which cause acute shutdown of tumour vasculature [31]. The perceived principal advantage of these drugs over conventional chemotherapy, which is targeted at the tumour cells, is that they are unlikely to be affected by acquired drug resistance (ADR). ADR, which is observed in about 30% of all cancer patients undergoing conventional chemotherapy, depends in part on the genetic instability, heterogeneity and high mutational rate of tumour cells. Endothelial cells, by contrast, are relatively homogeneous, genetically stable and have a low mutational rate [32]. However, further work is required to show that long-term antiangiogenic therapy does not lead to the development of drug resistance [30]. MRI has already demonstrated its potential for the non-invasive monitoring of tumour angiogenesis and perfusion and the response of these parameters to therapy, both in the laboratory and the clinic [31]. A recent special issue of NMR in Biomedicine, which was dedicated to imaging of tumour vascular function using MR, illustrates what can be done [33].

For a number of years we have been working with an antivascular drug, combretastatin. This drug, which is a natural product, inhibits the proliferation of endothelial cells [34] but has no apparent effect on quiescent endothelial cells or on tumour cells. Using dynamic contrast agent enhanced MRI (DCE-MRI) and localized 31P MRS we showed that the drug, at 1/10th of its maximum tolerated dose, could induce severe disruption of tumour blood flow and haemorrhaging within 2 h of drug treatment [35]. This rapid response was assumed to be the result of a drug-induced change in endothelial cell shape [34]. DCE-MRI techniques similar to these were used subsequently in a Phase I clinical trial of the drug, in order to get an early indication of drug efficacy [36]. Further work on a panel of murine tumours and two human tumour xenografts showed that susceptibility to combretastatin correlated with vascular permeability, as determined using DCE-MRI with a macromolecular contrast agent [37]. Since vessel permeability measured using MRI has been shown to be a surrogate marker for vascular endothelial growth factor (VEGF) expression this suggests that the responsive tumours have higher levels of VEGF. This would accord with the observations that combretastatin is effective only on proliferating endothelial cells and that VEGF is one of the principle growth factors responsible for stimulating endothelial cell proliferation and angiogenesis in tumours. More recently we have shown that treatment of a tumour with a VEGF-receptor tyrosine kinase inhibitor can inhibit its response to combretastatin [38]. As well as increasing our understanding of how this drug works, these experiments suggest that MRI measurements of vascular permeability could be used in the clinic to assess the possible responsiveness of a tumour to this and possibly other antivascular drugs.

Vascular permeability can be used as a surrogate marker for tumour angiogenesis [39, 40]. However angiogenesis can be imaged more directly by using a contrast agent targeted to angiogenic endothelium. Antibodies that recognize the integrin {alpha}v{beta}3, a molecular marker that is characteristic of angiogenic endothelium, have been conjugated to paramagnetic labels (Gd-containing liposomes and Gd-perfluorocarbon nanoparticles) and used to image angiogenic vessels in vivo [41, 42].

Changes in vascular function could also be used as a surrogate marker for tumour responses to other forms of therapy. For example Su et al [43] showed, using DCE-MRI, that there was a reduction in vascular volume in tumours undergoing immune rejection in the rat. In contrast we showed that there was an increase in the vascular volume of an immunogenic lymphoma undergoing immune rejection in the mouse, which preceded the subsequent reduction in tumour volume [44]. Whatever the explanation for the difference between these two studies, they have shown that it may be possible in patients to pick up early responses to tumour immunotherapy using clinically applicable DCE-MRI techniques.

Tracking cell movements in vivo using MRI
In the tumour model that we used to investigate possible MRI-detectable surrogate markers for immune rejection we, and others, have demonstrated that successful immune rejection is accompanied by significant infiltration of the tumour by immune cells, including CD4+ and CD8+ T-cells and macrophages [44]. Since, in this model, escape from immune rejection results from the CD8+ T-cells leaving the tumour site to go to adjacent lymph nodes [45], we have been exploring the possibility of tracking the immune cells in real-time with MRI by tagging them with a paramagnetic label. Over the past 10 years there have been numerous studies demonstrating the potential of MRI to track labelled cells in vivo. This has included dynamic tracking of T-cells to a site of inflammation in the rat testicle [46] and immune cell infiltration in the pancreas of an animal model to Type I diabetes [47]. This field has received added impetus from the rapidly growing interest in "cell therapy", where damaged tissues are repaired or regenerated by implantation of autologous or allogeneic cells. Our growing understanding of the biology of embryonic and adult stem cells and the conditions required to induce them to differentiate into a particular tissue is making this approach a very realistic prospect [48]. Development of these cell-based therapies will be assisted by techniques that can be used to track cell migration post implantation. MRI has been used to track paramagnetically-labelled stem cells in the brain and spinal cord of the rat [49–51] and efficient uptake of a paramagnetic label has been demonstrated in human haematopoietic and mesenchymal stems cells [52]. An example of our own studies in the brain and the spinal cord are shown in Figure 3Go. The overwhelming majority of cell labels have been based on complexes of iron and dextran. These paramagnetic labels have been shown in most cases to be readily and rapidly taken up by cells in an endocytic process and they have little or no effect on cell viability, proliferation and most importantly on cell function. An important feature of these labels is that they can allow the detection of single cells [53]. Although MR image resolutions are rarely comparable with cell dimensions, these iron-dextran complexes distort the magnetic field far beyond the boundaries of the cell and thus the effect of their presence is massively amplified in T2* weighted MR images. Their only drawback is that they give negative contrast. While this might not be a problem when trying to detect the presence of labelled cells in a regular structure, like that of the brain or the spinal cord, it makes detection of labelled cells in an irregular structure, like a tumour, much more difficult.



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  		Figure 3. 1H MR image of a rat brain obtained ex vivo, 7 days after the injection of superparamagnetic iron oxide (SPIO)-labelled oligodendrocytes (right side of image) and unlabelled cells on the contralateral side. Figure modified, with permission, from [49].

 

   Concluding remarks
Top
 Abstract
 Introduction
 MRI in the laboratory
 Molecular imaging using MRI
 Concluding remarks
References
 
The field of molecular imaging is somewhat like the field of fluorescence microscopy was 20 years ago. Here the development of fluorescent probe molecules, most notably those for detecting intracellular Ca2+ [54], and the subsequent introduction of green fluorescent protein (GFP), which can be used to tag proteins in the cell [55], have revolutionized this field. Fluorescence microscopy is now used routinely in the lab to map ions, proteins, gene expression, protein mobility and protein–protein interactions in intact cells or in thin sections of tissues. The same sort of experiments are now being developed for the imaging modalities used in "molecular imaging", such as PET, MRI and fluorescence imaging in the near infrared. These developments mean that these same investigations can now be carried out deep inside intact tissues [4]. In the field of MR, for example, we can map the distribution of small molecules (e.g. contrast agents), pH, using pH-sensitive contrast agents [56–58], proteins, cells and gene expression [59, 60]. Even receptor imaging, which traditionally has been thought to require very sensitive imaging modalities such as PET, may be accessible to MRI. Recently the Her-2/neu receptor on breast cancer cells was imaged using a monoclonal antibody conjugated to iron oxide nanoparticles [61]. These techniques will undoubtedly be of great value for studies of basic science in the laboratory, however they also have tremendous potential for the clinic where they offer new opportunities for diagnosing disease and guiding therapy.


   Acknowledgments
 
The work in my laboratory is supported by Cancer Research UK, the MRC and the BBSRC. I would like to thank the members of my lab, both past and present, for their input into the work described here and also past and current collaborators including Silvio Aime (Turin), David Parker (Durham) and Robin Franklin (Cambridge).


   References
Top
 Abstract
 Introduction
 MRI in the laboratory
 Molecular imaging using MRI
 Concluding remarks
 References
 

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