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1 Molecular Imaging: Applying Systems Biology to Medical Imaging Charles Xiao Bo Yan Abstract—Conventional medical imaging technologies are imperfect because they do not provide information at the genetic or molecular level. Thanks to research in systems biology, a large number of potential targets for the development of molecular imaging were discovered, which makes possible the extraction of information at the molecular level, such as gene expression or protein-protein interactions. The three imaging paradigms used in molecular imaging are MRI, radionuclide imaging and optical imaging. Direct imaging and indirect imaging are the two major technics employed. Imaging probes are used in both technics to provide better image contrast. Molecular imaging enables medical imaging at high sensitivity and specificity, which improves diagnostic accuracy and reduces the need for biopsy. It is also a tool that could accelerate systems biology research and improve drug development. Index Terms—Molecular Imaging, Systems Biology, Medical Imaging, Imaging Probes. I. INTRODUCTION M imaging is an indispensable tool for the diagnosis, treatment guidance and follow-up of many diseases nowadays. However, a number of weaknesses associated with the current medical imaging modalities (technological methods) make their usefulness limited in many circumstances. This is mainly due to the fact that most imaging modalities only provide information at the anatomical level, which is insufficient for diseases at the genetic or molecular level. For instance, in cancer diagnosis and followup, it is inappropriate to merely use the physical shape and size of the tumour for deciding whether it is benign or not and how to treat it. Instead, its type, distribution, gene expression and cellular functions are the driving decision parameters for setting the method of treatment, if it should be treated at all [1]. Another problem with current imaging technologies is that their spatial resolution are typically greater than 2 mm wide, which means that tumours containing fewer than 500,000 cells are likely to pass undetected [1]. This paper discusses how molecular imaging shows the promise to solve many problems that exist in medical imaging, including the ones mentioned above, how research in systems biology has made possible the development of molecular imaging, and how molecular imaging will in turn become a very useful tool to EDICAL study biological processes from a systems biology perspective. Systems biology is a research paradigm that studies the genetic and metabolic pathways as a whole system instead of studying the individual elements in the system. In systems biology, more emphasis is put on the interrelationships among the components rather than the isolated behaviour of each component. Through an iterative refinement of the mathematical model of the system by systematically perturbing and monitoring its components and by reconciling the experimentally observed responses with the model’s predictions, systems biology reveals many relevant data about the interactions and workings of the pathways that were unknown before [2]. Molecular imaging could be broadly defined as the in vivo imaging, characterization, and measurement of biological processes at the cellular and molecular level [1]. Therefore, it allows processes such as gene expression and protein-protein interactions to be imaged. It results from the convergence of the disciplines of medical imaging and cellular biology. Research in cellular biology using a systems biology approach has led to the discovery of a large number of potential targets for imaging probes (probes are discussed in more detail later), which plays a vital role in producing the image contrast needed to obtain information at a cellular or molecular level. The promises made by molecular imaging are numerous. Molecular imaging will greatly reduce the need for animal tissue sampling or human biopsy when studying the progression of a disease or the follow-up of a gene therapy. It has the advantage of being non-invasive, and because it could be repeated many times (unlike the case of biopsy), it provides both spatial and temporal dimensions to the understanding of the gene expression of the disease or therapy [3]. The fact that molecular imaging detects changes at the cellular level makes it much more sensitive than conventional medical imaging. This means that cancerous tumours are detected at a much earlier stage, and combined with image-guided therapy, the disease could be treated right at the time of recognition [4]. Finally, if advances in systems biology provides better understanding of the human genome, molecular imaging will change medical imaging from diagnosis of diseases to the prediction and prevention of diseases. 2 II. MOLECULAR IMAGING MODALITIES A. Magnetic Resonance Imaging Magnetic resonance imaging (MRI) is based on the phenomenon of nuclear magnetic resonance. The nuclei of atoms that have an uneven number of nucleons have a nuclear spin and a magnetic moment. When placed in an external magnetic field, the magnetic moments of each atom will align themselves with the external field. If a pulse at radio frequency is applied to the atoms, their equilibrium will be disturbed. Once the pulse is removed, the atoms will fall back to their equilibrium state and in doing so, emit a signal whose frequency is equal to the atoms’ characteristic resonance frequency. Because each element has its own resonance frequency, and because the amplitude of the signal received is proportional to the number of atoms with the same resonance frequency, the signal received could be used to construct a image of the composition of the target. In molecular imaging, paramagnetic material is typically used to enhance the signal and provide a good contrast between the anatomical image and the molecular image. B. Radionuclide Imaging Radionuclide imaging encompasses all modalities that are based on the detection of photons emitted by radioactive atoms. The most commonly used ones are positron emission tomography (PET) and single photon emission computed tomography (SPECT). In positron imaging, positrons are emitted from nuclei of proton-rich isotopes, which travels up to a few millimetres and eventually interact with electrons. Annihilation occurs, and the mass of the electron and positron is converted into two photons at gamma ray frequency. The photons travel outward from the site of annihilation at ~180° to one another. In SPECT, only a single photon at gamma ray frequency is emitted from the decay of the radioactive isotope. Scintillation crystals such as bismuth germanate or lutetium oxyorthosilicate are used to capture the gamma ray photons [5]. In molecular imaging, radionuclide tracers are used to provide the image contrast. Fig. 1 shows a diagram explaining the basic concepts of PET and SPECT. C. Optical Imaging Optical imaging makes use of fluorescent or bioluminescent proteins to create internal biological light. For conciseness, only the basic concept of bioluminescent imaging (BLI) is presented here. To illustrate, consider the example of luciferase, which is a class of enzymes that emit light (bioluminescence) in the presence of oxygen and a substrate. The light from these enzyme reactions typically has very broad emission spectra that frequently extend beyond 600 nm. The red components of the emission spectra are the most useful for imaging because of the relatively low absorption by tissues at these wavelengths. Hemoglobin is the primary absorber of light in vivo and the hemoglobin absorption spectrum significantly decreases above 600 nm, and the absorption due to water content begins to rise at 900 nm. This defines the spectral window for optically-based imaging modalities. Detectors based on charge coupled device (CCD) cameras are used for BLI [6]. In molecular imaging, the bioluminescent proteins are usually produced within the cell, which will be discussed in more detail in the next section. III. MOLECULAR IMAGING TECHNICS Fig. 1. Schematic illustrating single photon emission computed tomography (SPECT) and positron emission tomography (PET). (a) In SPECT, a single photon is produced as the isotope decays and this single photon must be detected through rotating detectors using a collimator. (b) In PET, annihilation eventually occurs with the positron and electron to produce two high energy (511 keV) gamma rays at ~180° that are detected using a circular ring of detectors [5]. A. Direct Imaging The molecular imaging technics could be mainly divided into two categories, direct imaging and indirect imaging. Of the two categories, indirect imaging is more widely used for reasons that will be explained later. In both cases, imaging probes are employed to aid in the targeting of specific cell features or gene expressions. Imaging probes are often molecules that are designed to bind to specific target molecules such as receptors, mRNAs or intracellular proteins. In some circumstances, the imaging probes could even be proteins produced within the cells through the expression of a transduced gene (genetic material that has been transferred over). The labeling of the imaging probes allows them to be detected and imaged and the labeling type depends on the imaging modality used. For radionuclide imaging, the probes are radiolabeled (tagged with a radioactive tracer). For magnetic resonance imaging, paramagnetic tracers are used to tag the probes. In the case of optical imaging, the probes are usually fluorescent or bioluminescent proteins that are encoded by transduced gene [6]. The direct imaging approach relies on a proportional relationship between the concentration of the imaging probes 3 and the level of gene expression in the target cells or tissues [7]. An example of direct imaging would be the use of RASONs, or radiolabeled antisense oligonucleotides probes, that have been developed to directly image endogenous (within the cell) gene expression at the transcriptional level (replication of DNA into mRNA). RASON sequences can be made complementary to a small segment of target mRNA or DNA, and could potentially target any specific mRNA or DNA sequence. In this context, imaging specific mRNAs with RASONs produces “direct” images of specific moleculargenetic events [3]. Another example of direct imaging is the imaging of a tumour by using an engineered transferrin receptor that is transduced and expressed in a tumour cell line, which becomes detectable when a paramagnetic transferrin ligand imaging probe is injected [1]. B. Indirect Imaging Most indirect molecular imaging paradigms involve a reporter gene coupled to a specific gene under study. Each reporter gene has a complementary reporter probe. Imaging the level of reporter gene product activity through probe accumulation provides indirect information that reflects the level of expression of the gene under study [3]. In other words, the expression of the reporter gene, which is being imaged, infers the expression of the coupled gene under study. Fig. 2 illustrates the general paradigm used in indirect imaging. In this case, the gene under study is the promoter gene. It is coupled with a reporter gene that encodes an enzyme capable of trapping its complementary reporter probe within the cell so as to amplify the signal emitted by the reporter probe. The reporter could very well encode a receptor or a biased carrier protein, which will serve just as Fig. 2. Schematic illustrating an indirect imaging approach. A reporter gene is introduced into the cell, which is driven by a promoter of choice. Transcription of the imaging reporter gene with subsequent translation of the mRNA leads to an enzyme. This enzyme can selectively trap an imaging reporter probe. The imaging reporter probe will not be trapped in those cells in which there is no expression of the imaging reporter gene. Note that it is also possible for the imaging reporter gene to encode for an intracellular and/or cell surface receptor. This receptor would then bind the imaging reporter probe (a ligand). Levels of the trapped probe can be related to levels of imaging reporter gene expression in either approach. [5] well the function of accumulating imaging probes for signal amplification. There has been many reports of successful application of indirect imaging. For example, Blasberg et al. mentioned that his group has been able to image the transcriptional regulation of the p53 gene [3]. In this case, a retroviral vector (Cisp53/TKeGFP) was generated, where the gene under study is the Cis-p53 and the reporter gene is TKeGFP, which encodes enhanced green fluorescent protein (GFP) (hence the imaging modality is optical). DNA damage-induced upregulation (increased response due to stimulus) of p53 transcriptional activity was demonstrated and correlated with the expression of p53-dependent downstream genes. In another instance, indirect imaging was used to monitor the effect of gene therapy in breast cancers in animal experiments [5]. Her2/neu, the gene linked to the overexpression and chemotherapy-resistance of breast cancer, was suppressed by a transcriptional adenovirus regulator. The imaging of the gene therapy to verify the location, magnitude and duration of the gene expression is very useful to aid in the optimization of the therapy [5]. C. Signal Amplification Methods Signal amplification methods for molecular imaging are necessary to increase the signal strengths of the imaging probes. This is because imaging probes are often injected at minimal dosage and do not provide enough signal strength when present in low concentration. Some concepts of signal amplification have already been alluded to previously. Three molecular imaging signal amplification methods are currently used: trapping, gene expression and activation. Trapping refers to the process of confining imaging probes within the cell or on the cell surface by means of one-way receptors, one-way carrier proteins, or enzymatic reactions that convert the probes. The accumulation of imaging probes over time enhances the signal strength emitted by the probes. In gene expression method, imaging probes can be inducibly expressed, such as by the genetic expression of a green fluorescent protein (GFP) gene, which results in thousands of GFP copies per cell. The activation method is used where imaging probes may be in a dormant phase, and switched on in response to interaction with a target molecule, enzyme, or receptor. In practice, multiple methods could be used in conjunction. For example, a dormant gene of a one-way membrane carrier protein can be turned-on in a target cell (such as a tumor), producing mRNA coding for the protein. Gene expression produces thousands of membrane-bound carrier proteins, which allows for the internalization and trapping of extracellular imaging probes [1]. D. Direct vs. Indirect Imaging Compared to indirect imaging, the advantage of direct imaging is that highly specific images can be obtained and that the delivery of the imaging probes does not require the 4 transduction of a coupled reporter gene. However, because the imaging probe is target specific, a drawback of the direct imaging approach is that a specific probe needs to be developed for each molecular target. The development and validation of both the sensitivity and specificity of the probe could become as resource consuming as the development of a new drug [3]. On the other hand, for indirect imaging, once a reporter-gene and reporter probe pair has been fully tested and validated, it could potentially be coupled to any gene and image the expression of the gene under study indirectly. This is the main reason for which indirect imaging has the potential to be more widely adopted, especially in animal studies. However, indirect imaging applications to human patients are currently limited due to the necessity of transducing target tissue with the reporter gene. Each new imaging probe and each new vector of coupled genes requires extensive and timeconsuming safety testing prior to government approval for human administration [3]. IV. APPLICATIONS OF MOLECULAR IMAGING A. Medical Imaging As suggested previously, molecular imaging greatly enhances the usefulness of medical imaging thanks to its higher sensitivity and specificity. In cancer diagnosis, the higher sensitivity of molecular imaging lowers the tumour detection threshold and its specificity to receptor status and gene expression reduces the need for invasive evaluation using biopsy [1]. In addition, gene therapy effectiveness could be immediately evaluated and followed on an ongoing basis. Similarly, tracking of tumour cells will allow the metastasis (spreading) of cancerous tumours to be closely monitored. Finally, image-guided therapy will expand thanks to the better detection of smaller regions of disease [1]. B. Systems Biology Research Molecular imaging is also a valuable tool for research in systems biology, because it offers the possibility to monitor in vivo the detailed location, magnitude, and time variation of gene expression or protein-protein interactions with high sensitivity in animals and humans [5]. Studies of weak promoters, post-transcriptional regulation of gene expression, protein-protein interactions and many others have been reported [3]. C. Drug Development In drug development, molecular imaging is used to study in vivo pharmacodynamics (effect of drug on living organism), concentrating on looking at how drugs act and what are the downstream effects of the drugs on tumours and normal tissue. It is also possible to study in vivo pharmacokinetics (whether the drugs are actually reaching the tumours) and look at drug uptake and retention. This is especially important now for more targeted therapies where the plasma drug concentration does not necessarily reflect tumour drug concentration [8]. Another area of application is pre-Phase I, where molecules are injected at one-thousandth of the therapeutic starting dose. Using molecular imaging one can actually screen the chemical compounds, look at the upregulated (stimulated) genes in normal tissues, before testing on human subject. This improves the quality of decision making, allows decisions to be made at a much earlier stage and reduce the cost of development [8]. V. CONCLUSION Molecular imaging greatly improves the conventional medical imaging modalities thanks to its high sensitivity and specificity. However, its research and development is still at its infancy and although it holds great promises, its accuracy is still being questioned by many researchers today and its application hasn’t found its way into clinical practice on human patients. Nevertheless, the potential it holds for improvements in medical technologies of the twenty first century is too great to be ignored and its development will for sure raise new questions about the appropriateness of current practice. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] D. A. Benaron, “The future of cancer imaging,” Cancer and Metastasis Reviews, vol. 21, 2002, pp. 45-78. H., Kitano, “Looking beyond the details: a rise in system-oriented approaches in genetics and molecular biology,” Current Genetics, vol. 41, 2002, pp.1-10. R. G. Blasberg and J. G. Tjuvajev, “In vivo molecular-genetic imaging,” J. of Cellular Biochemistry Supplement, vol. 39, 2002, pp. 172-183. C. M. C. Tempany and B. J. McNeil, “Advances in biomedical imaging,” JAMA, vol. 285, Feb. 2001, no. 5, pp. 562-567. F. Berger and S. S. 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