February 1, 2012 (Vol. 32, No. 3)

In vivo imaging, an umbrella term for a spectrum of technologies that facilitate visualization and quantitation of functional and anatomical events within a whole animal or person, is an area of intensive research and development. Many companies and academic centers are actively engaged in developing and refining methodologies to monitor a wide range of parameters, from larger scale measurements such as tumor size and morphology to subtle but critical indices at the cellular level, such as metabolism, subcellular dynamics, and motility.

Using these techniques, instead of more invasive methods, data points can be amassed on a much earlier time scale in a variety of contexts (e.g., monitoring the efficacy of drug candidates in clinical trials). Techniques commonly employed in in vivo imaging, depending upon the application, include magnetic resonance imaging (MRI), x-ray computed tomography (CT), positron emission tomography (PET), optical imaging, and more recently photo acoustic imaging (PAI).

GEN spoke to a number of scientists from companies and research laboratories, several of whom will present at this month’s Wellcome Trust “Mouse Models and Disease” conference about their in vivo imaging programs.

A major focus of research the laboratory of Kevin Brindle, Ph.D., at the University of Cambridge, is to develop imaging methods that guide therapy by allowing for the earlier evaluation of the efficacy of a drug in a particular patient in a clinical trial.

“Assessing the efficacy of an antitumor drug, for example, relies on tracking a decrease in tumor volume over time—a period of days or even weeks—and some cytostatic treatments don’t even have an impact in terms of tumor size,” said Dr. Brindle.

MRI is commonly used in the clinical setting to visualize internal structures. In a collaboration with GE Healthcare, Dr. Brindle’s lab is developing a novel MRI-based approach that boasts a 10,000-fold increase in sensitivity over conventional MRI methodologies.

The novel technique detects small carbon metabolites, which are present at a much lower concentration than water, which is the imaging target for conventional MRI.

“We take radiolabelled derivatives of these metabolites, such as pyruvate, and supercool them to just above absolute zero in order to align the spins in a field and give us a fabulous increase in sensitivity,” explained Dr. Brindle.

The rate at which individual cells metabolize the compound can then be correlated with the effect of the drug on those cells’ metabolism. “When we treat a tumor, we see that its metabolism is slowed,” he continued. “In some cases we can obtain very rapid feedback on drug action, on the order of 24–48 hours.”

Getting a jump on tumor cell response in this manner affords clinicians the opportunity to alter or adjust therapies much sooner than would normally be possible, which ultimately translates to improved therapeutic practice.

Dr. Brindle listed several advantages that his lab’s adaptation of MRI holds over PET, which is widely used in the clinical setting, particularly in the diagnosis of tumors of the prostate and brain.

“The glucose surrogate typically used in PET, fluorodeoxyglucose, isn’t taken up well by the prostate and is, in fact, preferentially taken up by the kidney, which limits the use of PET in prostate cancer,” he said.

Citing the high background glucose levels in the brain, Dr. Brindle went on to point out that it can be difficult to discern specific brain signals using PET.

Finally, the increased radioactive hazard that PET-based techniques carry limits the number of repeat exams that a clinician might run during early evaluation of a patient.

Dr. Brindle’s adaptation of MRI is currently being used in a prostate cancer trial under way at the University of California, San Francisco. It it is also slated for trials of glioma, lymphoma, and breast cancer.

“Its potential application isn’t limited to oncology,” he emphasized. “We are planning studies in cardiac imaging and kidney disease, so this could potentially have widespread application in the clinic.”

Innovative Techniques

Research at Animascope focuses on two main subdisciplines of imaging.

“One of our projects is aimed at developing innovative nuclear and optical imaging tracers for oncology and diabetes in the biomarker field,” said Arnaud Briat, Ph.D., CSO. “In the equipment field, Animascope is setting up a collaborative consortium to develop a new generation CT scanner based on an exclusive license recently acquired by Animascope in the preclinical, veterinary and human clinical fields.”

Animascope is also developing another project that aims to replace current invasive methodologies by x-ray imaging technology in the toxicology field.

“Longitudinal whole-system datasets generated by noninvasive imaging provide a platform for investigating the time course relationship between target, disease state, and therapeutic. This data can be extremely valuable in interpreting early clinical results,” noted Dr. Briat.

Soluble T-cell receptors (TCRs) are an entirely novel class of targeting agent for cancer and diabetes since they zero in on antigens not accessible to monoclonal antibodies. A recent TCR-based Animascope research project has garnered a Eureka label from the High Level Group of the EU’s European Innovation Institute, which recognizes groundbreaking research applications with significant potential for commercial success.

The application, ANIDIAG, is based on leveraging a SPECT (Single Photon Emission Computed Tomography) imaging platform to detect radiolabelled TCRs, which were developed by Immunocore, that have greatly enhanced affinity for target antigens and which have potential as therapeutic agents in cancer and type I diabetes.

Spatial Coregistration

Noninvasive in vivo imaging in animals has two useful utilities, according to University of California at Davis pathologist Alexander Borowsky, M.D., an expert in breast cancer. He makes extensive use of mouse models as a precursor to trials and therapy in humans.

“Firstly, it allows us to use one mouse as its own control, which circumvents any issues in mouse-to-mouse variability,” he explained. “Secondly, compared to other technologies, it is immediately translatable to the clinic.”

Dr. Borowsky highlighted a recent paper in PNAS in which small-animal high-resolution in vivo PET-imaging was demonstrated as a viable surrogate for more invasive standard histological measurements of tumor progression. The latter by their nature, he pointed out, cannot shed light on more subtle indices of tumor growth.

“A great innovation in this study was what we call spatial coregistration,” said Dr. Borowsky. He defined this concept as “using high-resolution histology to achieve careful, anatomically registered validation of the imaging signals.”

The technique has been combined with MRI to achieve more detailed characterization of amyloid plaques in mouse models of Alzheimer disease, which, since they contain iron, can generate confounding data in MRI studies.

Relying on this two-pronged strategy—in vivo data collection and ex vivo validation—Dr. Borowsky’s longer-term goal is to assemble a catalog of associative data that correlates histology and microanatomy with the images collected in the live animal. “This is a problematic task, particularly in cancer,” explained Dr. Borowsky.

Tumors are often a composite of many different cellular densities, angiogenic states, differentiation grades, and other variables. Dr. Borowsky is hopeful for the future, although, “it’s a matter of bringing the bioengineering behind the imaging platforms to a more sophisticated level that can address these subtleties.

“Down the road I think you’re going to see an increasing convergence between the microscope slide and what can be achieved with in vivo imaging. I see plenty of space for more molecular and physiologically-based imaging methods, particularly in the area of optical imaging, that will gradually supplant methodologies with limited resolution, such as PET.”

IVIS Platform

While MRI, PET, and x-ray are well-established types of in vivo imaging that offer the most clinical translational opportunities in preclinical models and patient imaging, optical imaging is gaining in importance. The IVIS platform initially developed by Caliper Life Sciences, which was recently acquired by PerkinElmer, takes advantage of fluorescent and bioluminescent reporters to facilitate noninvasive longitudinal monitoring of a variety of processes in living animals, including cellular trafficking and disease expression.

“Caliper’s platform is applicable to a variety of therapeutic contexts, such as oncology, inflammation and metabolic disease, neuroscience and stem cell biology,” maintained Anna Christensen, product manager, life sciences and technology at PerkinElmer.

The IVIS optical platform has grown to incorporate multispectral fluorescence imaging, 3-D tomography, fast kinetic imaging, integrated x-ray, and microCT.

“Quantum FX microCT is a dedicated high-resolution longitudinal imaging platform for CT-focused applications and offers seamless co-registration with functional optical datasets,” added Christensen.

On the translational side, the IVIS platform has already been leveraged in the progression of over 15 drugs into the clinic with many more in development, continued Christensen.

“Optical imaging is quickly gaining acceptance within the clinic as a diagnostic tool for biopsy profiling and margin identification of resected tumors,” she observed.

“Image-guided optical imaging can help surgeons not only define cleaner tumor margins intra-operatively, but identify metastatic lesions not visible with the naked eye all within the same surgical procedure.”

PerkinElmer’s IVIS platform takes advantage of fluorescent and bioluminescent reporters to facilitate noninvasive longitudinal monitoring of a variety of processes in living animals. A. MDA-MB-231-luc2 mammary tumor cell line implanted into the mammary fat pad. B. Metastasis into the left ancillary lymph node. C. Ex vivo confirmation of lymph node burden. D. Multibiomarker characterization of breast tumor. Blue: DAPI; Green: Phospho-p-44/42 (Erk1/Erk2); Red: Phospho-Akt; Yellow: Phospho-S6.

Novel In Vivo X-Ray Imaging System

Officials at Carestream Molecular Imaging report that early adopters of the company’s In Vivo Xtreme instrument are carrying out research to study multiple pathways simultaneously within the same small animal.

The pathways under investigation are myeloperoxidase (MPO) activity as it relates to inflammation and various apoptotic responses to the exposure of anionic membranes.

The new high-resolution optical/x-ray system was specifically designed for applications in the preclinical, small animal research market and that require high sensitivity luminescence, fluorescence, radioisotopic, and radiographic imaging.

In one application, the scientists have been studying a brain injury mouse model using luminol to longitudinally image neutrophil and macrophage activity in vivo, while simultaneously studying apoptosis and necrosis with a Zinc-DPA-NIR fluorophore conjugate. These probes have been previously validated in the literature to target MPO and anionic membranes respectively.

The researchers relied on Xtreme’s x-ray signal to localize MPO activity and cell death utilizing bone structure in the skull of the mouse as landmarks. Using this same model, if researchers needed to quantify changes in brain metabolism, they could also image the subject using 18F-FDG in Carestream’s Albira PET/SPECT/CT system, according to a Carestream Molecular Imaging spokesperson.

He added that the Xtreme, when paired with Albira, provides researchers with a combination of seven modalities for experiments requiring both high-throughput 2-D optical imaging and quantitative 3-D tomographic imaging.

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