Imaging in 4-D

Just a few short decades ago, cell biologists--essentially relegated to the tissue culture equivalent of Flatland--couldn't imagine working in three dimensions without sacrificing their subjects, much less having the ability to view the impact of their work in real time, over time. Now, state-of-the-art imaging technologies and new biological reagents and probes are sending biologists and other scientists on fantastic voyages into the molecular world of living animals to watch how cancer develops, grows, and spreads. "This isn't just three-dimensional biology--this is four-dimensional analysis," enthuses molecular biologist Harvey R. Herschman, director of basic research at the Jonsson Comprehensive Cancer Center, University of California, Los Angeles. UCLA is home to one of three new In vivo Cellular and Molecular Imaging Centers, or ICMICs, established early this year with the support of grants from the National Cancer Institute (NCI). The other two centers are at Massachusetts General Hospital/Harvard Medical School in Boston and Memorial Sloan-Kettering Cancer Center in New York. "The focus today is really on molecular--in imaging, biology, genetics, pharmacology, and medicine," says UCLA's Michael E. Phelps, who co-developed positron emission tomography (PET) in the mid-1970s1,2 and serves as chair of the department of molecular and medical pharmacology at UCLA School of Medicine. "With the sequencing of the genomes and the proteomes taking us to a very fundamental molecular characterization of how cells function and how they fail in disease--to the point of discovering and understanding a cell's initial coded instructions--the evidence is clear that the majority of diseases occur by something that changes the instructions. The goal of molecular imaging is to see cellular function, and in the ICMIC studies to see very specifically how cancer cells function." "The 'big promise' of ICMIC is to provide for the development of noninvasive imaging paradigms that can ... assess specific molecular/signaling pathways in tissue [tumors] that are important in the development and treatment of disease processes [cancer] ... [and] assess gene therapy trials by monitoring the delivery and targeting of the vector to specific tissues, as well as monitoring the magnitude and persistence of 'therapeutic gene' expression," adds neurologist Ronald G. Blasberg, head of the NeuroOncology PET program and a principal investigator (P.I.) of the ICMIC at Sloan-Kettering. The ultimate goal is to translate these noninvasive imaging techniques to patient care. In fact, imaging technology has the potential to improve cancer treatment in a number of ways. For starters, by allowing for more rapid and thorough evaluation of benefits and limitations of experimental therapies, such as gene therapy, the technology will give physicians the capacity to render faster diagnoses and be able to make more informed decisions about effective treatments or even preventives. Eventually, the discoveries revealed through imaging technology will empower researchers with the knowledge to develop designer drugs and gene therapy interfaces that could guide transgene targeting and tissue destruction with a precision never before possible. The input of all kinds of specialists will be required to achieve that goal; therefore, the ICMIC concept was designed to produce a multidisciplinary paradigm. Together with the technology and innovations, this paradigm is intended to create a potent approach that promises to have a profound impact on cancer research. "The ICMIC programs will have a direct impact on cancer treatment and diagnosis in the future," projects Blasberg. "These programs will provide new research opportunities that will further our understanding of cancer, cancer progression, and response to therapies targeted to specific molecular processes." While imaging technology has improved dramatically during the last decade, one of the problems in the biological imaging sciences has always been the lack of funding, notes radiologist-chemist Ralph Weissleder, director of the Center for Molecular Imaging Research, and P.I. of the Harvard ICMIC. Weissleder also is co-director of the NFCR Center for Molecular Analysis and Imaging, an effort--established last November with a $1 million grant from the National Foundation for Cancer Research--aimed at developing noninvasive, early diagnosis methods. "It was always sort of a cat-and-mouse game, "says Weissleder," but now that there's some money and people are being recruited, things are really beginning to happen." The ICMIC concept originated "with the insight and the vision of NCI director Richard Klausner," says Blasberg, who worked at the National Institutes of Health for more than 20 years before arriving at Sloan-Kettering in 1990. As part of Klausner's vision to explore areas of "extraordinary opportunity," the NCI recognized about three years ago the "great untapped potential" that imaging technology holds for cancer research, he adds. The NCI leadership envisioned "that imaging will become very important in understanding basic biological properties of cancer and cancer treatment--and that we will need these very powerful techniques to move cancer research forward," confirms John M. Hoffman, chief of the molecular imaging branch in the NCI's biomedical imaging program. "Eventually, we do expect to be able to visualize with imaging technologies the molecular signatures of a cancer." The ICMICs (www.nci.nih.gov/bip/ICMICs.htm) are designed to broaden not only the knowledge of cancer per se, but molecular and cellular biology as well. In addition, the institute is also providing "planning grants" to 16 other institutions. And, adds Hoffman, the NCI intends to fund the establishment of ICMICs at six more institutions by 2005. Imaging: A Technology Whose Time Has Come In vivo cellular and molecular imaging is a technology whose time has come. Today's models of imaging instruments such as PET, magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and cutting edge optical imaging systems are taking much clearer, more detailed pictures of organs and tissues, allowing researchers to probe deeper than ever before. "PET, for example, allows you to look in all three dimensions, giving you accurate three-dimensional representations. That spatial information, and doing that in real time for gene expression, unlocks the fourth dimension," explains Herschman. MicroPET images of a mouse with 3 tumors illustrates that fluoropenciclovir (FPCV) accumulates in greater quantities in cells expressing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter gene as compared to a wild type HSV1-tk reporter. While the process takes from a few seconds to minutes for each image, "a sort of movie develops," elaborates physician-scientist Sanjiv S. (Sam) Gambhir of the UCLA ICMIC. "That's what opens the door to the molecular world and into the fourth dimension, where you can watch how things are changing. And to visualize physiological, cellular, or molecular processes in living tissue is to actually see things like blood flow, oxygen consumption, and glucose metabolism in real time, as they happen in the cells of a living body." NCI's Hoffman agrees: "We really are going beyond the era of anatomy here, and into the next realm." Each of the initial three ICMIC institutions--which were selected in a peer-review process--has experience in multidisciplinary projects in the context of their molecular imaging programs and each brings its own expertise and a host of achievements to the table. Researchers at Sloan-Kettering and UCLA, for example, have independently shown that reporter gene expression can be imaged,3-5 and both teams are now working on the development of new models for studying gene therapy and basic cancer biology using reporter genes. On the imaging instrument front, UCLA has introduced MicroPET for small animal (mouse) models,6 and researchers at Massachusetts General have created optical near infrared fluorescent probes, which they have used to show in vivo imaging of enzyme activity.7 They have also used TAT peptide derivatized magnetic nanoparticles to track and recover progenitor cells in vivo.8 Multidisciplinary Interaction One of the major goals of these core facilities is to provide a place where scientists can do their research without having to 'reinvent the wheel' learning radioisotope design and production, scanning instrumentation, and data analysis. "These services are here so that our biologists can have a strong infrastructure and support that allows them to use this without having to go through a three-year learning curve, and that's very important," points out Herschman. Even so, as the NCI recognized the power of imaging, it also realized that a "scientific gulf" existed between the basic scientists, who discover new cancer genes and intracellular pathways, and imaging scientists, who focus on noninvasive approaches to transform discoveries into understanding. For its mission to be successful, the institute saw the need for a strong collaboration among biologists, radiologists, chemists, physician-researchers, instrumentation scientists, neuroscientists, and various other specialists. The NCI mandated that, in addition to the four primary research projects, each ICMIC would provide for a series of one- or two-year revolving pilot or developmental projects and establish a career development program to cross-train younger scientists in this multidisciplinary realm. Both the developmental projects and the career development program are designed to motivate multidisciplinary interaction and to elicit a sharing of knowledge, says Weissleder. "The main reason for the pilot or developmental projects is to attract other scientists from within, as well as visiting scientists from other institutions, to either use imaging technologies or to develop new things, new projects--and that is critical," he says. "There's so much knowledge out there that sometimes it's more of a question of harnessing what has already been developed in other fields for other applications." As for the career development aspect, at Massachusetts General, for example, the primary focus is to train junior staff. "Say a molecular biologist comes in and wants to be trained in imaging or an imaging person comes in and he gets cross-trained--they get two years break to learn the other discipline," Weissleder explains. Despite an unspoken competition among these institutions, "the work is tremendously exciting and the end results are sure to be far-reaching," says Herschman. The one thing about which everyone agrees is the crucial role the NCI is playing. "Most places are unable to bring biologists and imaging scientists together," says Gambhir, director of the Crump Institute for Molecular Imaging at UCLA. "They realize it's an important thing to do, and [they] haven't been able to do it because, without this kind of grant incentive, it's hard. Actually, this kind of research couldn't happen without multidisciplinary input and the NCI deserves to be thanked for supporting the technology and this approach in the way that it is." Brave New World The importance of imaging to cancer research--not to mention basic research--seems like a no-brainer. But, it has been a long time coming. Although technologies such as PET have been around since the 1970s, the worlds of imaging and biology rarely, if ever, crossed. "There's been a big gap between the molecular cell biology community--the people who do, for example, mouse models of disease--and the people who developed imaging technologies, because the imaging technologies were primarily being used either at the microscopic level by cell biologists or at the human level by clinicians," Herschman notes. "There wasn't much in-between." MicroPET images of a nude mouse with 4 tumors, 3 of which (A,B,C) express different levels of the bicistronic construct pCMV-D2R-IRES-HSV1-sr39tk, and the fourth tumor serves a control (D). Beyond that, adds Gambhir, "the science is very difficult." Developing a probe that could find a gene is equivalent, he says, "to finding a drug that can treat a disease." But therein lies the path to what may well be the greatest reward of imaging. "The real focus of the ICMICs is not so much on the imaging technology now, but on the application, and the application at the forefront is reporter systems," says Blasberg. "We have adopted what has been established by our molecular biology colleagues--namely using reporter genes and reporter systems. Molecular imaging is not new. There's a whole tradition and history that has preceded this. What is new is the language and the focus. We are now entering the next phase. What we and the UCLA team have shown is that you can image endogenous gene expression with biological probes. Now the exciting part comes. We can report, with radiotracers, the level of the transgene expression or the level of the reporter system." "What we've done is to make the imaging gene 'generalizable'--it doesn't matter what gene someone wants to use, all we have to do is link," Gambhir continues. "We link their gene to our imaging gene, and we can find our imaging gene because we have probes that can find it. Once we find our imaging gene, we can indirectly determine what's going on with a gene therapy gene because the two are linked." According to Herschman, the focus at UCLA has been on building a model system in animals. "What we've done over the last couple of years is develop this model system where we transfer gene therapy vectors that target tumor cells and then ask whether or not they get to the tumors," he says. "Now we're in the stage of doing translational studies with experimental animals, with various mouse tumor models--breast, prostate, leukemia, and hepatomas or liver tumors." At each of the ICMICs, the application of noninvasive reporter gene imaging is being expanded to include the imaging of specific cell processes at a molecular level. At Sloan-Kettering, recent studies include imaging the expression of p53. "Since it's an important gene in the development of cancer and one that is mutated in a whole variety of cancers, it's important to know the endogenous expression of p53 and influence of that expression," Blasberg says. "If you put in a promoter element that is sensitive to p53, you can determine that. The basic process of transcriptional activation is one thing that has been done now and has been imaged and will eventually, very shortly, be translated into the clinic." All three institutions are preparing translational studies outside ICMIC grants that will utilize their reporter systems in ongoing human cancer trials (See "Trans-lational Research," page 15). Meanwhile, the progress in the development of reporter probes and imaging instruments for small animals, along with communication among those who have been introduced to this new multidisciplinary science, is already proving to be a boon for imaging coast to coast. "There has been an enormous burst of interest," Herschman says. "There is just this hoard of basic scientists who have been using animal models who couldn't spell PET three years ago and who are now really interested in using it because it's a new tool that gives them the ability to look at gene expression in this longitudinal way." Expectations High But Realistic Looking to the future, when these five-year grants come to an end in 2006, the investigators have high--yet realistic--expectations. "I would hope that the funded imaging centers would solidify their research, extend it, and become competitive enough to be funded through other mechanisms," says Weissleder. Chances are this will happen, he adds, because much of what is cutting edge right now, will be "fairly routine" in about five years. "What is and will emerge from these projects are important steps for science in general," Gambhir offers. "These are basic tools we're building--chemicals, assays, reporter genes, reporter probes, and the instruments. They will form a toolkit for other biologists, so a few years down the road, biologists will be able to reach into that bag, easily pull out a set of turn-key and easy to use tools to accelerate their research." Phelps goes further: "As I see it, the most important objective is that we establish the role of molecular imaging to guide the molecular diagnosis and the cure of cancer in mice. We can then, by a commonality of the methods that we develop and prove in the mouse, say, 'Okay, now let's go do it in people.'" A.J.S. Rayl (ajsrayl@loop.com) is a contributing editor for The Scientist. References 1. M. Terpogossian et al., "A positron emission transaxial tomograph for nuclear imaging (PETT)," Radiology, 114:89-98, 1975. 2. M.E. Phelps, "Application of annihilation coincidence detection to transaxial reconstruction tomography," Nuclear Medicine, 16:210-24, 1975. 3. J. Tjuvajev, et al., "Imaging the expression of transfected genes in vivo," Cancer Research, 55:6126-6132, 1995. (First of series) 4. U.S. Patent #5703056, Noninvasive Imaging of Gene Transfer, issued Dec. 30, 1997. 5. Y. Yu et al., "Quantification of target gene expression by imaging reporter gene expression in living animals," Nature Medicine, 6:933-7, August 2000. 6. S.R. Cherry, S.S. Gambhir, "Positron emission tomography in animal research," Institute for Laboratory Animal Research Journal, in press. 7. R. Weissleder, et al., "In vivo imaging of enzyme activity with amplifiable near infrared fluorescent probes," Nature Biotech, 17:375-8, 1999. 8. M. Lewin et al., "Tat peptide derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells," Nature Biotech, 18:410-4, 2000.

Imaging in 4-D

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