February 1, 2014 (Vol. 34, No. 3)

Neil McKenna, Ph.D.

The discipline of in vivo imaging encompasses a variety of reagents, instruments, and software platforms. These tools provide for the noninvasive visual monitoring of processes in living tissues, in both preclinical models and human subjects.

In vivo imaging is a critical component of the drug development pipeline, affording researchers the ability to monitor—in real time—the efficacy of a drug candidate with respect to critical indices specific to the disease for which it is being developed. In the clinic, in vivo imaging provides physicians a way to evaluate the progress of lines of therapy without the complications and costs of surgical interventions.

At the recent “World Molecular Imaging Congress” in Savannah, GA, the presenters included a variety of commercial and academic groups. They showcased the diversity of approaches under development to address specific challenges and obstacles in in vivo imaging.

“Preclinical in vivo imaging allows researchers to study and characterize the longitudinal progression of diseases such as cancer, and their response to therapies in small animal models of disease,” said Rob Sandler, svp of marketing at AspectImaging. Bioluminescence, the process by which living organisms generate and emit light, is a mainstay of in vivo imaging because of its high sensitivity, low operating cost, and high throughput.

By generating transgenic cell lines that express luciferase and are viable in living animals, it is possible to generate animal models in which the progress of the disease can be monitored with high sensitivity using bioluminescence imaging (BLI) techniques. “The luminescent signal is created upon introduction of the substrate into the animal, allowing for interaction of the enzyme produced by the transgenic cells and the luciferin substrate,” noted Sandler.

Although BLI is popular, it typically generates two-dimensional, surface-weighted planar images, which tend to limit the accuracy with which the actual source of the luminescent signal (and the associated viable cell population) can be located. “To understand the true nature and nuances of the etiology of a given disease, three-dimensional (3D) imaging tools that allow for the true location and characterization of the disease are greatly preferable,” Sandler pointed out.

In partnership with inviCRO, AspectImaging is developing a tomographic software platform, LumiQuant™, that generates 3D, BLI-generated images and co-registers them with magnetic resonance (MR)-based anatomical structures. “The complimentary nature of BLI and MR provides 3D anatomical, morphological, and cellular information on the biological processes in the disease model being studied,” asserted Sandler.

While he cautioned that BLI is not approved for clinical use because of the genetic modification involved, Sandler stressed that combined 3D luminescent-MR images represent a potentially powerful approach to providing more accurate preclinical data for drug development efforts.

Lanthanide Probes

Homeostasis in living cells depends in large part upon striking a balance between production of reactive oxygen species (ROS) by oxidative processes such as oxidative phosphorylation in mitochondria, and the scavenging of ROS by antioxidant species such as glutathione. Oxidation by ROS has been linked to compromised stability and improper folding of proteins, and increased levels of these species are associated with a variety of pathological states, including cancer, inflammatory conditions, and neurodegenerative disorders.

“Localization and quantification of ROS in vivo can provide important diagnostic information for these diseases,” said Rajendra Singh, Ph.D., senior director of reagents/assay R&D at PerkinElmer. Still, the very short lifetimes of ROS, coupled with the presence of antioxidants in living systems, greatly complicates their detection in vivo, especially in deep tissues.

Dr. Singh addressed this issue in his discussion of lanthanide probes. He reported that the chemiluminescent properties of lanthanide acceptor beads could be harnessed to develop a highly sensitive probe for ROS detection by noninvasive optical imaging.

“A chemiluminescent reaction between singlet oxygen in the ROS and an electron-rich thioxene moiety in the acceptor bead generates a high-energy dioxetane,” explained Dr. Singh. Decomposition of the dioxetane results in the emission of light with a wavelength of approximately 340 nm, which would be absorbed by surrounding body tissues and is therefore unsuitable for imaging applications.

Dr. Singh described a hybrid technique that addresses this issue by combining sensitive luminescent detection with fluorescence to shift the wavelength, thereby overcoming interferences associated with the lower wavelength of the chemiluminescent reaction alone: “When appropriately chelated, lanthanides such as europium and terbium can be excited at 340 nm to emit in the green to red wavelengths and obviate the interference issues.”

Nailing the chemistry is only one part of the puzzle however, and Dr. Singh’s group is currently addressing the challenge of routing the probes to specific tissues and organs in which ROS are produced. “The liver is a storehouse of redox enzymes, and nonspecific signals in this organ are a problem,” remarked Dr. Singh. “We have conjugated the acceptor particles with specific antibodies to direct them to macrophages, which in turn target inflamed and diseased organs.”

Viral Nanoparticles

A novel class of biomolecular agents consisting of viral nanoparticles (VNPs) is capable of harnessing the natural circulatory and targeting properties of viruses for the development of therapeutics, vaccines, and imaging tools. “As a biological imaging platform, plant viruses possess a number of distinct advantages over other particles, primarily because they are biocompatible and biodegradable,” said John Lewis, Ph.D., associate professor of oncology at the University of Alberta. “Furthermore, they are not pathogenic in humans, and are less likely to induce undesirable side-effects.”

Dr. Lewis’ work focuses specifically on the cowpea mosaic virus (CPMV), a small plant icosahedral virus that is composed of 60 identical copies of an asymmetric protein unit assembled around a bipartite single-stranded RNA genome. The structure of the virus makes it highly amenable to the attachment of dyes and other functional groups, to the extent that each virion contains a total of 300 chemically addressable sites.

Dr. Lewis’ group was the first to describe the use of CPMV-based VNPs as a tool for vascular imaging in vivo, and more recently his group has been focusing on their use as an imaging platform for monitoring angiogenesis in cancer. “We have identified a subset of blood vessels that express EGFL7, a molecule which is highly specific for sites of active angiogenesis, such as healing wounds, the pregnant uterus, and tumors,” reported Dr. Lewis. “EGFL7 expression is associated with poor prognosis in several cancers including prostate cancer, hepatocellular carcinoma, and glioma.”

At the meeting, Dr. Lewis described the development of fluorescein dye-labeled VNPs containing EGFL7-homing peptides for specific labeling of tumors and their associated blood vessels in a mouse xenograft model of human prostate cancer. “We determined that a click chemistry approach, where we attach the peptides to the distal end of a polyethyleneglycol polymer, was best in terms of efficiency and binding to the target,” asserted Dr. Lewis.

Confocal imaging and flow cytometry showed that the EGFL7-targeted VNPs specifically labeled tumor and endothelial cells. This result suggests that the VNPs have potential as in vivo imaging agents to monitor the impact of angiogenesis inhibitors as cancer therapeutics.

Researchers at the University of Alberta have shown the the cowpea mosaic virus (CPMV) is highly amenable to the attachment of dyes and other functional groups. They have been developing CPMV-based nanoparticles as an imaging platform for monitoring angiogenesis in cancer.

Gold Nanorods

The plasmon-resonant absorption and scattering in the near-infrared region of gold nanorods makes them attractive probes for both in vitro and in vivo imaging. “Gold nanorods allow us to visualize proteins and other macromolecules in tumor tissues by optical microscopy,” said Raghuraman Kannan, Ph.D., an assistant professor of radiology and an assistant professor of biological engineering at the University of Missouri. Dr. Kannan leads a team that includes Gerald Arthur, M.D., a research assistant professor in the department of pathology, and graduate student Chuck Caldwell.

There are several significant advantages to using gold-nanorod-based reagents in the development of tumor biomarkers, including improved quantification of biomarkers due to the one-to-one relationship between the visual signal produced by the gold nanorod and the peptide that binds to the protein.

Overexpression of the epidermal growth factor receptor (EGFR), a cell surface receptor for members of the EGF family of signaling peptides, has been linked to the development of cancer. As such, EGFR represents a promising biomarker candidate for a variety of neoplastic conditions.

“Many targeted anticancer therapeutic approaches are aimed at the inhibition of EGFR,” explained Dr. Kannan. “For these to be effective, precise determination of the EGFR expression levels within the cell membrane is important, because only EGFR-dependent (EGFR-positive) tumors will respond to these approaches.”

At the meeting, Mr. Caldwell presented work in which PEG-modified gold nanorods were used to visualize the highly specific binding of EGFR-targeting peptides to cells in human colorectal and lung tumors. “We are currently developing a gold nanorod based EGFR detection kit as a companion diagnostic to qualify patients for individualized chemotherapy,” added Dr. Kannan.

An Immunocytokine Probe

Immunotherapy refers to a host of strategies aimed at marshalling components of the body’s own immune system to target tumors for destruction. “Successful immunotherapeutic approaches require orthotopic, fully immunocompetent models for better translation,” said Christian Gerdes, Ph.D., of the pharma research and early development group at Switzerland-based Roche Glycart.

“Such models must incorporate sophisticated live imaging techniques for therapeutic biodistribution and targeting, and in vivo cell-to-cell interaction monitoring,” asserted Dr. Gerdes. He emphasized the importance of in vivo imaging in collecting accurate information that can help identify which patients are most likely to respond to a particular drug treatment.

At the meeting, work from Dr. Gerdes’ team was described in which a novel immunoctyokine targeting CEACAM5, a cell adhesion molecule implicated in the development of a variety of human cancers, was labeled using a far-red fluorescent dye. Using fluorescence and bioluminescence imaging, the team was able to closely monitor the therapeutic effects of the immunocytokine in a mouse model of metastatic liver cancer.