Seeking Tiny Vesicles For New Medical Diagnostics

The cells in our bodies constantly release information that doctors could use to diagnose disease. Some of this information comes in the form of tiny extracellular vesicles called exosomes. Every day, both healthy and diseased cells produce thousands of these lipid-membrane-enclosed packets. Exosomes end up in every type of biological fluid and carry proteins, nucleic acids, and sugars from their cells of origin.

These cellular calling cards provide snapshots of the cells they came from. The 30- to 100-nm-diameter packets protect their biomolecular cargo from degradation in biofluids. This means exosomes could provide more information about disease states than the free-floating biomolecules being targeted by many current medical diagnostics. The vesicles aren’t easy to fish out of biofluids normally collected by doctors, but some researchers have started to harness exosomes to develop diagnostics for diseases as disparate as cancer and Alz­heimer’s disease.

Biologists think that cells use exosomes to communicate with one another. Cells generate the biomolecule-filled packages as part of the same pathway that generates lysosomes, the intracellular vesicles involved in breaking down biomolecules. Exosomes aggregate inside larger organelles called multivesicular bodies that can then fuse with the cell membrane to release the exosomes into the body. Alternatively, the multivesicular body can mature into a lysosome.

When researchers isolate exosomes from biofluids, they often grab another type of vesicle that cells produce. These so-called microvesicles are 200 to 1,000 nm in diameter and consist of fragments of cell membrane that get shed into the body. Scientists don’t understand the biological function of these microvesicles.

Separating the different types of extracellular vesicles remains a challenge, and there is some debate over whether doing so matters in developing new diagnostics.

“When we analyze extracelluar vesicles derived from clinical specimens, we don’t really know the biogenic mechanisms by which those vesicles arose,” says Clark Chen, a neurosurgeon at the University of California, San Diego, who is developing vesicle-based diagnostics for monitoring treatment response in brain cancer patients. Because of that lack of information on origin, Chen prefers to refer to the mixtures as extracellular vesicles, rather than exosomes.

Richard R. Drake, an exosome researcher at the Medical University of South Carolina Hollings Cancer Center, in Charleston, isn’t convinced that it matters for diagnostic purposes whether people are measuring exosomes or a mix of vesicles. “In a clinical chemistry workflow, you need something you can quickly isolate and rapidly and efficiently analyze,” he says. “For diagnostics, separating vesicles by their size and trying to determine their origin is pretty complex.”

But no matter what they’re called, these extracellular vesicles are exciting to diagnostics developers for what they could reveal about the cells that produce them. Unlike free-floating biomolecules, exosomes can be traced back to the tissue that produced them. So instead of serving solely as a marker for cancer, exosomes could indicate the presence of liver cancer, for example.

Also unlike individual biomolecules, exosomes—and their cargo—are quite stable. Fresh plasma samples and those that have been refrigerated for several weeks yield the same amount of exosomes, says Douglas D. Taylor, a pioneer in the study of exosomes and chief scientific officer at Exosome Sciences, in Monmouth Junction, N.J.

But little is known about how long exosomes circulate in the body. So researchers analyzing exosomes don’t know if they indicate cellular events currently happening or ones that occurred in the past.

Animal studies have suggested that the vesicles survive only minutes, but those studies were based on injecting exosomes into animals. It’s possible those exosomes were victims of the immune system attacking the vesicles as something foreign, says Alan M. Ezrin, chief executive officer of NX PharmaGen, a company that is developing exosome-based diagnostics for cancer and pregnancy complications.

The company has been running an experiment in patients to try to get a better answer. Working with brain surgeons, NX PharmaGen scientists analyzed exosomes from patients who had taken Gliolan, which becomes fluorescent in cancer cells and is used to help surgeons visualize glioblastoma tumors during surgery.

In all the patients studied so far, the tumor-derived exosomes also fluoresce. Also, this fluorescence is still present in subsequent blood draws days and weeks after surgeons remove the tumor—the source of the exosomes. “They stay around a lot longer than anybody expected,” Ezrin says.

Perhaps because of their stability, isolating exosomes from other materials in biofluids, such as other cell components, turns out to be a pretty big hurdle to using exosomes as diagnostics. The typical isolation procedure starts with hours of ultracentrifugation, in which the sample is spun at forces up to hundreds of thousands times as great as that of gravity. These high levels of force are needed to compel the exosomes to fall out of suspension so researchers can harvest them. Other isolation methods have been developed, but they all tend to be labor-intensive.

The laborious isolation procedures aren’t appropriate for large-scale analyses in clinical labs, Ezrin says, and they hamper a detailed understanding of exosomes. One big problem is that intense ultracentrifugation protocols might trap other biological materials between the clumped exosomes, producing a sample that doesn’t contain solely the desired vesicles. “As we look across the field, what’s drastically missing is rigorous quality control over the isolation of these things so we can understand what we’re looking at,” Ezrin says.

Despite that lack of understanding, researchers continue to forge ahead to develop exosome-based diagnostics. And they are doing so for many different diseases and in every biofluid imaginable.

Plasma has the greatest number and diversity of exosomes. But exosomes are also found in urine, saliva, and cerebrospinal fluid. In those cases, the exosomes mostly come from cells close to the fluid.

For example, Peter Yuen, a researcher at the National Institute of Diabetes & Digestive & Kidney Diseases, part of the National Institutes of Health, is using exosomes in urine to find biomarkers of kidney disease. At first, he and his coworkers wanted to find exosomes that indicate a patient has the disease. But they quickly realized that the lengthy isolation methods put exosomes at a competitive disadvantage against other types of diagnostic biomarkers.

So they tried to find exosomes that provided another useful piece of information. “Exosomes have the potential to contain more prognostic information” about the trajectory of a disease than do other biomarkers, Yuen says.

To get this prognosis information, Yuen and coworkers were interested in two types of regulatory molecules that end up in exosomes: microRNAs and transcription factors. A transcription factor can control many different genes in a cell, so its presence can indicate the activity of multiple genetic pathways. “We thought of this as a way to get insight into what decision-making processes cells are undergoing,” Yuen says. “That might give us a hint of where the disease is headed.”

Urine exosomes are also a promising source of biomarkers for prostate cancer. The company Exosome Diagnostics, based in Cambridge, Mass., plans to launch a urine-based exosome test for high-grade prostate cancer next year.

Prostate cancer is “one of the few cancers that is being overdiagnosed today, leading to unnecessary biopsies,” says Johan Skog, chief scientific officer at the company. The firm hopes to reduce the number of those invasive tests by identifying patients most likely to need treatment.

Their test is geared toward men with prostate-specific antigen (PSA) levels in the so-called gray zone between 2 and 10 ng/mL of blood. At those concentrations, PSA is poorly predictive of whether a man has cancer and how severe that cancer is. For the test, the company isolates exosomes from the urine and analyzes them for three messenger RNA biomarkers associated with prostate cancer.

At the American Urological Association meeting in May, Exosome Diagnostics reported the results of a study with more than 1,000 patients. The test improved the ability to predict high-grade prostate cancer, identifying men who require aggressive treatment.

South Carolina’s Drake focuses his research on urine markers of prostate cancer and other genitourinary tract diseases, such as bladder cancer and kidney disease. For prostate cancer, he works with urine samples obtained during a digital rectal exam, in which a urologist massages the prostate to force prostatic fluid into the urethra.

Whereas other researchers tend to look for nucleic acids or proteins, Drake is interested in glycans, glycoproteins, and lipids. He uses matrix-assisted laser desorption ionization mass spectrometry to profile the lipid chain lengths in exosomes from different tissues and different disease states. At this point, he’s still looking for potential biomarkers.

When it comes to proteins found in exosomes, researchers think there are a number that are common to all exosomes. These markers can help improve isolation of exosomes by distinguishing them from the other vesicles that researchers worry about. But markers specific for disease exosomes have been harder to come by.

Now, Raghu Kalluri of the MD Anderson Cancer Center in Houston and coworkers have found a marker—a proteoglycan called glypican-1—on cancer exosomes but not on normal ones (Nature 2015, DOI: 10.1038/nature14581). Glypican-1 allows them to both identify cancer exosomes and isolate them with beads labeled with antibodies for the proteoglycan. Then researchers can analyze the exosomes’ contents, looking at the DNA, RNA, microRNA, and proteins inside, to determine the type of cancer, Kalluri says.

In a study with more than 250 patients, Kalluri and coworkers used the level of exosomes with glypican-1 to differentiate healthy individuals from those with various stages of pancreatic cancer. Only cancer exosomes carried DNA or mRNA transcripts with the KRAS mutation, which is known to be a driver of most pancreatic cancers.

Kalluri thinks that screening for glypican-1 exosomes in populations at high risk for particular cancers might serve as a way to detect cancers at early stages.

The molecular content of exosomes also offers a means to isolate the vesicles. Researchers can use antibodies that recognize molecules on the exosome surface to snag their vesicles of choice. The target molecules can be generic for all exosomes, specific for exosomes from certain tissue types, or specific for particular diseases.

For example, Exosomics Siena, located in Italy, uses metabolic tumor markers to identify cancer exosomes, says Natasa Zarovni, head of R&D at the company. These markers are linked to metabolic changes that are hallmarks of solid tumors.

The company is currently seeking European regulatory approval for a test based on quantification of these exosomes. “We are proposing a generic exosome-based tumor test for monitoring populations that have high risk of developing cancer,” Zarovni says.

Exosome Sciences also uses an immunoaffinity approach to isolate exosomes. Their method relies on sugar-binding proteins called lectins to pull exosomes out of biofluids, Taylor says. They have been able to pinpoint lectins that, alone or in combination, bind specific types of exosomes.

The company is using this strategy to develop a test for recurrent or metastatic cancer in patients who have had tumors surgically removed, Taylor says. In these patients, the only source of cancer exosomes would be from metastasized tumors. So detecting cancer exosomes could indicate that the cancer has come back or is still lingering.

The company is now testing to see how early they can detect recurrent head and neck cancer, Taylor says. “Head and neck patients have about a 50% recurrence rate in the first year,” he says. “That’s an ideal group where we can do a small number of patients and yet have a significant number of recurrences.”

Cancer isn’t the only disease that exosome researchers are targeting. Edward J. Goetzl, a professor at UC San Francisco and principal scientist at NanoSomiX, a California-based biotech company, is working on an exosome-based diagnostic for Alzheimer’s disease. He’s looking for proteins in neuron-derived exosomes that can identify individuals with mild cognitive impairment who will go on to develop Alzheimer’s disease.

To pull out neuron-specific exosomes, he uses an antibody for a neuronal protein called L1CAM. He then examines the isolated exosomes for a panel of proteins that have been found in brain lesions of Alzheimer’s patients. Working with Dimitrios Kapogiannis of the National Institute on Aging, Goetzl is getting ready to test this strategy in archived samples from 400 known Alzheimer’s patients in NIH’s Baltimore Longitudinal Study of Aging.

Despite these early successes, challenges remain for the use of exosomes for diagnostics. In particular, Ezrin hopes researchers will institute improved quality control so they can better characterize exactly what they are studying when they analyze exosomes isolated from specimen samples. Then they can be sure they’re asking questions that exosomes can answer.

“We’re getting answers every day, but I’m not certain we’ve defined the questions to maximize the opportunity” that exosomes present, Ezrin says. “We’ve got to better understand the questions we’re asking.”