July 1, 2016 (Vol. 36, No. 13)


Subcellular imaging technologies have come a long way. Conventional microscopy—the use of upright microscopes to detect small changes in light transmission through the cell—has been augmented by phase-contrast microscopy and fluorescence microscopy. Phase-contrast microscopy improves images by measuring changes in the refractive index within the cell. Fluorescence microscopy provides even better contrast enhancement through the use of fluorescent probes that bind to specific molecules on and in the cell.

Technological advances have led to the development of imaging systems that integrate advanced instrumentation and refined workflows. Such imaging systems, which are capable of high throughput and low error rates, include BioTek’s Cytation Cell Imaging Multi-Mode Readers. The Cytation 3 and Cytation 5 platforms incorporate both charge-coupled device (CCD) and photomultiplier tube (PMT) technology.

To model metastasis, scientists at BioTek Instruments used tumor spheroids that incorporated MDA-MB-231 cells and primary fibroblasts. Incubated and treated tumor spheroids underwent imaging that entailed the drawing of object masks: (A) day 1, untreated; (B) day 5, untreated; (C) day 1, treated with 250 µM oridonin; (D) day 5, treated with 250 µM oridonin. These 4× brightfield images were captured with the company’s Cytation 5 Cell Imaging Multi-Mode Reader.

“One can think of the Cytation as two instruments in one,” says Peter Banks, Ph.D., scientific director. “Below the microplate-loading mechanism is an inverted digital microscope using CCD-based detection for brightfield, color brightfield, phase-contrast, and fluorescence microscopies. Above the microplate-loading mechanism is a hybrid microplate reader system using multimode PMT-based detectors.”

Cytation has been evaluated in studies involving complex cell models including a tumor spheroid, an in vitro surrogate model for metastasis. For example, Dr. Banks and colleagues examined the role CXCR4 inhibitors can play in CXCR4-mediated tumor metastasis. Tumor metastasis was quantified by brightfield microscopy and the Cytation imaging software.

“Any sort of assay that mimics metastasis is of huge worth to oncology drug discovery,” asserts Dr. Banks. “People don’t die from having a mutation in their cells. They die from tumor metastasis.” Any compound that inhibits this process should be a good drug candidate to investigate as an antimetastatic drug.

Looking at research imperatives more broadly, Dr. Banks and others see multiparametric analysis of disease as driving the trends in imaging advancement. “Imaging methods are one of the better tools for doing phenotypic assays,” he explains, “you can quantify many changes to cellular structure and function using imaging through the use of various probes or just by label-free detection.”

Overall, what is being observed is a move toward combining target-based science, made popular in the last few decades, and classical phenotypic assays. “This is a more balanced approach,” comments Dr. Banks. “In certain diseases, we have a strong knowledge of disease mechanism of action, so it makes sense to perform drug discovery through target-based approaches. On the other hand, in diseases where mechanism of action is doubtful or unknown, like many forms of cancer, I expect phenotypic approaches to be more successful.”

Novel PET Imaging Detectors

Meanwhile, at the David Geffen School of Medicine at the University of California, Los Angeles (UCLA), Anna Wu, Ph.D., professor and chair of the department of molecular and medical pharmacology, has been engineering antibodies for both therapeutic and imaging purposes. Antibodies meant for imaging, she points out, reflect a slightly different set of engineering characteristics.

Dr. Wu works with colleagues at UCLA and ImaginAb (a company she co-founded in 2007 and now serves as chief scientist) to engineer labeled antibodies. Because Dr. Wu in interested in advancing positron emission tomography (PET) imaging, she is engineering antibodies that incorporate positron-emitting radionucleotides.

“Each imaging modality has its strengths and weaknesses,” notes Dr. Wu. “Magnetic resonance imaging (MRI) gives you anatomical and physiological imaging, but it doesn’t have the sensitivity of PET. PET has a higher resolution and sensitivity compared to single-photon emission computed tomography (SPECT), but it has lower resolution than MRI or CT.”

Dr. Wu has chosen PET because it fits well with her antibody fragments (called minibodies and diabodies) in a process coined immunoPET. She favors PET because it can, she says, be combined with the specificity of antibodies.

“One of the key features of antibodies is they have very long circulating half-lives,” she points out. “Antibodies stay in the blood for days to weeks.” To match the half-lives of antibodies, she and others have been testing longer-lived positron emitters, such as zirconium-89. “The fluorine-18 everyone uses has a two-hour half-life, so it’s a challenge to make a fragment that targets and clears quickly enough to image.” Zirconium-89, in contrast, has a half-life that is a little over three days.

According to Dr. Wu, the use of PET brings certain caveats to mind. For example, you can only image one target at a time through this method, which is why she advocates multimodal approaches. PET can also be rather expensive. “But on the other hand,” says Dr. Wu, newer cancer treatments, such as checkpoint inhibitors, can cost between $100,000 and $200,000 per year, “and they might be ineffective and very toxic.” Her immunoPET antibodies could be used to guide the use of expensive therapies.

Dr. Wu’s laboratory has published on roughly 8 to 10 cancer targets using this system. They have also begun to target immune cells using standard CD markers. “We started with CD8,” recalls Dr. Wu. In 2015,  in an article that appeared in the Journal of Nuclear Medicine, Dr. Wu and colleagues showed both CD4 and CD8 T cell reconstitution in the bone marrow via PET. “You can watch the repopulation of either CD4 or CD8 compartments in the mouse,” she details, “and you can see the spleen and lymph nodes filling out over time.”

Dr. Wu hopes to see these techniques used in the tracking of immunological disease responses. “When you talk about patient-optimized treatments, you have to have a way of looking at the whole patient,” she posits. “Our company is based on the belief that these kinds of very specific imaging agents will be invaluable to that effort.”

Novel MRI Imaging Detectors

A short way across town, Julia Ljubimova, M.D., Ph.D., director of the Nanomedicine Research Center, department of neurosurgery at Cedars-Sinai Medical Center, firmly believes that the future of imaging is in MRI. She has several multimillion dollar grants from the NIH and the NCI to develop MRI-detectable tumor biomarkers and therapeutics.

Dr. Ljubimova and colleagues are working on imaging technologies that incorporate nontoxic, biodegradable nanobioconjugates. These constructs are able to cross the blood-brain barrier (BBB) using receptor-mediated transcytosis. “Our nanoplatform,” she explains, “is a natural polymer that we obtain from a one-celled organism.”

The platform—which consists of a polymalic acid scaffold, a target monoclonal antibody or peptides, and gadolinium enhancement reagent—was designed by German chemist Eggehard Holler, Ph.D., now a professor in the same department as Ljubimova Dr. at Cedar-Sinai. “We developed this for MRI,” explains Dr. Ljubimova, “because MRI is the method that is widely spread in almost every hospital.” PET, she says, is more expensive, requires radionucleotides, and “requires clinical chemists that will synthesize things 24 hours prior to the procedure.”

Her focus is on neurological disorders, neuro-oncology, and metastasizing cancers of the brain. “If you are thinking about using a nanoplatform to image the brain,” she points out, “you have to recognize, number one, that it has to go through the blood-brain barrier.” It also has to be nontoxic and easily cleared from the system. Her nanoplatform, she claims, degrades into carbon dioxide and water within 12 hours.

According to Dr. Ljubimova, current technologies are not as targeted as they should be. For example, brain imaging enhancements can be caused by inflammation, metastatic disease, primary brain tumors, or a combination of these factors. “Inflammation you have to treat with antibiotics, metastatic disease you treat with one drug, and primary brain cancer you treat with another,” she continues.

Targeted imaging studies are very important for developing a strategy of treatment. “For brain, it would be easier to have a molecular MRI diagnosis,” she says. “It is not only brain problems we can solve,” with their MRI approach, “but with breast (cancer and others) it is easier to do a biopsy.”

“Our technology is very flexible,” she insists, “We have a precise nanodrug that targets pathological conditions in the brain. It’s like a key in a locker. If the normal cell doesn’t have the marker, then our drug doesn’t go to this cell.” Of note, these nanobioconjugates can be used for both imaging and therapy. For targeted therapy, the same scaffold can be used with a different chemical attached, one that is potentially deadly to the tumor.

“My major interest always was molecular cancer biomarkers,” states Dr. Ljubimova. “It would be revolutionary if MRI technology could not only give us a radiological signal of enhancement in the brain or lung, but also be based on specific molecular markers, which are constantly changing during the tumor progression and important for treatment modulation.”

Imaging Care

Perhaps, however, the answer does not lie in either MRI imaging or PET, but in a combination of technologies with a focus on improving patient outcomes. One place where advancement in imaging is apparent is within university core facilities. At the University of Notre Dame, 19 core facilities provide resources and instrument support for internal and external clients, part of a growing trend to provide expensive technologies to more researchers.

The Bruker Albira, a PET/SPECT/CT imaging system, in operation at the University of Notre Dame. The trimodal system has allowed Notre Dame researchers to conduct preclinical studies in oncology that have advanced the development of novel radiotracers and therapeutics.

“We offer our services to the entire university,” says Sarah Chapman, assistant director of biological imaging and manager of the Notre Dame Integrated Imaging Facility (NDIIF). “Actually, we are a CTSI [Clinical and Translational Science Institute]-approved facility, so researchers at Indiana University, Purdue, and Notre Dame can use this facility at the internal rate.” The facility contains instrumentation for magnetic resonance (MR), SPECT, PET, and bioluminescence imaging, as well as electron microscopy and traditional optical microscopy.

At Notre Dame, the instruments being used are all built by Bruker. Todd Sasser, Ph.D., field applications scientist for Bruker’s preclinical imaging division, works closely with the NDIIF to support its Bruker products, which include an Albira PET/SPECT/CT imager. “It’s an interesting time,” notes Dr. Sasser. “The optical market has become more and more sensitive. We are almost at the theoretical limit of resolution for PET imagers, based on the physics of the positron.”

The Albira II, which came out in 2010, has already been supplanted by a newer model called the Albira Si, which has a few unique differences from its predecessor, but is named for its use of silicon (Si) PMTs, which, as Dr. Sasser explains, pave the way for MR integration. “Standard PMTs,” notes Dr. Sasser, “are not compatible with MR fields without shielding.”

Currently, animals must be manually transferred from one instrument to another for multimodal imaging. In the NDIIF at Notre Dame, animals are moved from a Bruker benchtop 1 tesla MR machine called the ICON, to the Albira II, to a bioluminescence detector called the Xtreme if multimodal imaging is desired. To do this, technicians place animals in a holding and monitoring apparatus called a bed.

“This is actually my favorite,” says Chapman of a special “multimodal” animal bed. “It actually fits on the Albira, and you have a chamber so that it fits on the Xtreme.” An animal bed that fits all instruments is the only way to achieve perfect overlay, says Chapman, because it ensures that the animal never moves.

Last year, Bruker introduced the PET/MR 3T, which uses the same PMT detectors as the Albira Si to create a platform that combines PET and MR imaging. “This is really one of the big revolutions in PET technology,” asserts Dr. Sasser. “With traditional PMT detectors, any electric field would throw off the PET detection.”

The new instrument will allow two modalities in one device, promising combinatorial examinations of soft tissues and spectroscopy. It represents a technological paradigm that has already reached clinical practice, and it may lead currently competing ideas to merge.

Ian Clift Ph.D. is a Scientific Communications Consultant, Biomedical Associates and Clinical Assistant Professor, Indiana University


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