Journal Club

Targeting exosome biogenesis in cancer: Scientific Reports Top 100

This blog post originated as a press release from Tulane University.

Asim Abdel-Mageed, DVM, PhD, professor of urology and Marguerite Main Zimmerman Professor of Cancer Research at the Tulane School of Medicine, was recently honored by the journal Scientific Reports for authoring one of the top 100 accessed oncology papers for the journal in 2018.

His publication, “High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: a drug repurposing strategy for advanced cancer”, received 3,154 article views, placing it seventh on the list, which features authors from around the world whose papers highlight valuable research in oncology.

The article reveals the results of research supported by a $4.2 million National Institutes of Health grant awarded to Abdel-Mageed in 2014. His project involved using a rapid high-volume robotic screening technique to investigate drugs already approved by the Food and Drug Administration (FDA) to treat a large variety of diseases or conditions to see which, if any, could also be effective in preventing prostate cancer metastasis.

Targeting Metastasis

For cancer cells to spread to other places in the body — or metastasize — they need to communicate with resident and recruited cells, such as stem cells. One way they do this is through biomolecular messages delivered in exosome cargos. Exosomes are molecules that carry information from cell to cell. “They are routinely biosynthesized and released by cancer cells, including prostate cancer, and are implicated in cancer progression,” said Abdel-Mageed.

Currently there are no known drugs that selectively target and inhibit the biosynthesis and release of exosomes by tumor cells. To accelerate the discovery of effective drugs, Abdel-Mageed and his team, in partnership with investigators at the National Center for Advancing Translational Science (NCATS), investigated 4,580 known pharmacologically active compounds and found that 22 — including antibiotics, antifungal medicines and anti-inflammatory agents — were effective in preventing advanced prostate tumor cells from releasing exosomes or in blocking their production.

Future Work

Since the Scientific Reports publication, subsequent research by Abdel-Mageed’s team has further narrowed their investigation to five of these agents, and he hopes in the near future to receive additional funding to support this work.

“Drug repurposing is a golden opportunity,” said Abdel-Mageed. “Because drug discovery from concept to market takes an average time of 12 years, our identified drugs, which are already human approved, could be repurposed for the treatment of advanced prostate cancer within a relatively short period of time. It represents a quick way of adding an adjuvant therapy to existing therapies that might curb the progression of cancer.”

As a steering committee member of the National Institutes of Health Extracellular RNA Communication Consortium (ERCC), a summary of Abdel-Mageed’s study was also published as part of the ERCC leading-edge perspective paper in Cell.

05.13.19 by Bill Snyder

Discovery aids search for cancer biomarkers

This blog post originated as a press release from Vanderbilt University Medical Center.

A report by researchers at Vanderbilt University Medical Center has shattered conventional wisdom about how cells, including cancer cells, shed DNA into the bloodstream: they don’t do it by packaging the genetic material in tiny vesicles called exosomes.

Their findings, reported April 4 in the journal Cell, have important implications for the development of “liquid biopsies” that could speed cancer diagnosis and improve treatment by detecting tumor-specific genetic material in the blood.

“It’s been a big deal that there is supposedly DNA in exosomes … (and) you could isolate these exosomes in a relatively simple way,” said the paper’s first author, Dennis Jeppesen, PhD. The problem is exosomes don’t contain DNA.

“Exosomes are not your target,” said Jeppesen, a research fellow in the lab of Robert Coffey, Jr., MD, who is internationally known for his studies of colorectal cancer. Instead, Jeppesen and his colleagues propose a new model for how DNA is actively secreted by cells.

Research by Robert Coffey, MD, left, Dennis Jeppesen, PhD, and colleagues has revealed a new way cells shed DNA into the bloodstream.
(Photo by Steve Green / Vanderbilt University)

Greater precision in determining how DNA, RNA, and proteins are packaged and secreted from cells “is crucial for identification of biomarkers and design of future drug interventions,” the researchers concluded.

Coffey, Ingram Professor of Cancer Research in the Vanderbilt University School of Medicine, predicted the paper will “set the field on a firmer foundation” to understand what’s in exosomes and what’s not, and how exosomes might be used as biomarkers or therapeutic targets.

Coffey’s team is part a nationwide consortium funded by the National Institutes of Health (NIH) to study the role of extracellular RNA in diseases including diabetes, glaucoma, muscular dystrophy, and cancer.

Last month the NIH announced the publication of what it called a “landmark collection” of scientific papers in the Cell family of journals on the biology and possible clinical applications of extracellular RNA. Two of those papers, including the paper about exosomes, came from Coffey’s lab.

Virtually every cell in the body releases DNA, RNA, proteins, lipids, and other particles. These so-called “nanoparticles” are thought to be a way that cells communicate with each other. But they also might signal cancer to spread — or metastasize — to other parts of the body.

Cancer by its very nature is constantly evolving. That trait enables many tumors to escape or to become resistant to chemotherapy and other efforts to destroy them.

The genetic material released by cancer cells, however, may also reveal their points of vulnerability. A simple blood test that picks up these circulating cancer clues therefore could lead to earlier diagnoses and more effective treatments.

The problem is how to snag cancer-specific genetic material from the sea of circulating nanoparticles. Each milliliter of blood (it takes 5 milliliters to fill a teaspoon) contains a quadrillion (a thousand trillion) nanoparticles, and at least 100 million small extracellular vesicles.

Many of these vesicles are exosomes, which are known to carry extracellular RNA. Exosomes can be identified by the proteins (called antigens) that sprout from their surfaces. Identifying specific exosomes is one thing; determining what they carry is quite another.

The traditional way is to use high-speed centrifugation to spin exosomes out of a blood sample and into a “pellet” at the bottom of a test tube. Biochemical methods are then used to characterize the RNA, DNA, and proteins in the pellet.

The assumption was that the pellet contained only exosomes and their cargoes. But Jeppesen, who earned his PhD in Molecular Medicine at Aarhus University in Denmark, was skeptical. He decided to take a different approach.

Using a technique called high-resolution density gradient fractionation, Jeppesen and his colleagues separated blood components based on their buoyant density. They found that exosomes floated at a relatively low density while higher-density proteins, including those that bind DNA, sank to a lower gradient.

“We showed this for multiple cancer cell lines,” he said. “We also see the same kind of thing in normal cells.”

To isolate and study exosomes apart from the traditional pellet, Jeppesen and his colleagues developed another technique they called direct immunoaffinity capture. They coated magnetic beads with a “capture antibody” that targeted one of the known proteins, or antigens, on the exosome surface.

That’s how they were able to determine that exosomes don’t carry DNA.

The DNA found in pellets must be secreted by the cell in other ways. One way, the Vanderbilt researchers reported, is through the formation of novel hybrid organelles termed amphisomes.

“We can actually see these amphisomes traffic to the cell surface,” Jeppesen said. “Now it’s possible to say with greater precision what’s in the exosome and what’s in these other vesicles. Now you have an idea of what is the target you’re looking for.”

05.02.19 by Graciela Gutierrez

Computational analysis reveals exRNA cargo types and their carriers across human biofluids

This blog post was adapted from a press release by the Baylor College of Medicine. See this related video from the ERCC Webinar Series for a discussion of the exceRpt pipeline used in the analysis presented here.

Scientists have improved their understanding of a new form of cell-cell communication that is based on extracellular RNA (exRNA). RNA, a molecule that was once thought to function only inside cells, is now known to participate in a cell-cell communication system that delivers messages throughout the body. To better understand this system, the Extracellular RNA Communication Consortium (ERCC), which includes researchers from Baylor College of Medicine, created the exRNA Atlas resource, the first detailed catalog of exRNAs in human bodily fluids. They also developed web-accessible computational tools other researchers can use to analyze exRNAs from their own data. The study (Murillo, Thistlethwaite, et al. 2019), published in the journal Cell, contributes the first ‘map of the terrain’ that will enable scientists to study the potential roles exRNA plays in health and disease.

“About 10 years ago, scientists began discovering a new communication system between cells that is mediated by exRNA,” said corresponding author Dr. Aleksandar Milosavljevic, professor of molecular and human genetics and co-director of the Computational and Integrative Biomedical Research Center at Baylor College of Medicine. “The system seems to work in normal physiological conditions, as well as in diseases such as cancer.”

The Milosavljevic lab worked with other members of the ERCC to analyze human exRNAs from 19 studies. They soon realized that the system was significantly more complex than initially assumed. Due to that unanticipated complexity, existing laboratory methods failed to reproducibly isolate exRNAs and their carriers. To help create the first map of this complex system of communication, Milosavljevic and his colleagues used computational tools to deconvolute the complex experimental data. Deconvolution refers to a mathematical method and a computational algorithm that separates complex information into components that are easier to interpret.

“Using computational deconvolution, we discovered six major types of exRNA cargo and their carriers that can be detected in bodily fluids, including serum, plasma, cerebrospinal fluid, saliva, and urine,” said co-first author Oscar D. Murillo, a graduate student in Baylor’s Molecular and Human Genetics Graduate Program working in the Milosavljevic lab. “The carriers act like molecular vessels moving their RNA cargo throughout the body. They include lipoproteins – one of the major carriers is High-Density Lipoprotein (HDL or the “good cholesterol”) – a variety of small protein-containing particles, and small vesicles, all of which can be taken up by cells.”

The researchers found that the computational method helps reveal biological signals that could not previously be detected in individual studies due to the naturally complex variation in the biological system. For example, in an exercise challenge study their computational approach revealed differences before and after exercise in the proportions of the exRNA cargo in HDL particles and vesicles in human plasma.

“Exercise increased a proportion of RNA molecules involved in regulating metabolism and muscle function, suggesting adaptive response of the organism to exercise challenge,” Milosavljevic said. “This finding opens the possibility that in other conditions, both in health or disease, the computational method might identify signals that could have physiological and clinical relevance.”

To help researchers around the world with their analyses, Murillo, Milosavljevic and their colleagues have made a computational tool available online (https://exRNA-Atlas.org).

“We anticipate that it will take a combination of scientific knowledge, enhanced experimental techniques to isolate cargo and carriers in bodily fluids, and advanced computational methods to deconvolute and interpret the complexity of the exRNA communication system,” Murillo said.

Other contributors to this work from Baylor College of Medicine include William Thistlethwaite, Matthew E. Roth, Sal Lakshmi Subramanian, Rocco Lucero, Neethu Sha, and Andrew R. Jackson. See the full article for details about the numerous other contributors from the consortium.

This work is part of the NIH Extracellular RNA Communication Consortium paper package and was supported by the NIH Common Fund Extracellular RNA Communication Program (grant U54 DA036134).

Reference
Murillo OD, Thistlethwaite W, et al. exRNA Atlas analysis reveals distinct extracellular RNA cargo types and their carriers present across human biofluids. (2019) Cell 177:463-477. doi: 10.1016/j.cell.2019.02.018. PMID: 30951672.

04.03.19 by Jason Alvarez

Cancer Exports Molecular Saboteurs to Remotely Disarm the Immune System

This blog post originated as a press release from UCSF.

Discovery May Help Explain Immunotherapy Resistance, Hints at New Therapies

Immunotherapy drugs known as checkpoint inhibitors have revolutionized cancer treatment: many patients with malignancies that until recently would have been considered untreatable are experiencing long-term remissions. But the majority of patients don’t respond to these drugs, and they work far better in some cancers than others, for reasons that have befuddled scientists. Now, UC San Francisco researchers have identified a surprising phenomenon that may explain why many cancers don’t respond to these drugs, and hints at new strategies to unleash the immune system against disease.

“In the best-case scenarios, like melanoma, only 20 to 30 percent of patients respond to immune checkpoint inhibitors, while in other cases, like prostate cancer, there is only a single-digit response rate,” said Robert Blelloch, MD, PhD, professor of urology at UCSF and senior author of the new study, published April 4 in Cell. “That means a majority of patients are not responding. We wanted to know why.”

In malignant tissue, a protein called PD-L1 functions as an “invisibility cloak”: by displaying PD-L1 on their surfaces, cancer cells protect themselves from attacks by the immune system. Some of the most successful immunotherapies work by interfering with PD-L1 or with its receptor, PD-1, which resides on immune cells. When the interaction between PD-L1 and PD-1 is blocked, tumors lose their ability to hide from the immune system and become vulnerable to anti-cancer immune attacks.

One reason that some tumors may be resistant to these treatments is that they do not produce PD-L1, meaning that there is nowhere for existing checkpoint inhibitors to act — that is, they may avoid the immune system using other checkpoint proteins yet to be discovered. Scientists have previously shown the PD-L1 protein to be present at low levels, or completely absent, in tumor cells of prostate cancer patients, potentially explaining their resistance to the therapy.

But in their new paper Blelloch’s group is suggesting a very different answer to this puzzle: PD-L1 is being mass-produced by these tumors, they found, but instead of displaying the protein on their surface, cancer cells export PD-L1 in molecular freighters known as exosomes. These PD-L1–packed exosomes sprout from cancer cells and travel through the lymphatic system or bloodstream to lymph nodes, the sites where immune cells are activated to protect the body. There, the PD-L1 proteins act as itinerant molecular saboteurs, remotely disarming immune cells and preventing them from locating tumors to mount an anti-cancer offensive.

So rather than shutting down the immune response at the tumor surface, exosomal PD-L1 can inhibit immune cells before they even arrive there. And unlike PD-L1 found on the tumor’s surface, exosomal PD-L1, for unclear reasons, is resistant to existing checkpoint inhibitors.

“The standard model says that PD-L1 acts on immune cells that travel to the tumor niche, where they encounter this immune-suppressing protein,” Blelloch said. “Our data suggests that this isn’t true for many immunotherapy-resistant tumors. These tumors evade the immune system by delivering exosomal PD-L1 to lymph nodes, where they inhibit the activation of immune cells remotely. These findings represent a break from dogma.”

Blelloch’s group decided to explore exosomes when they noticed something strange that suggested the standard model of PD-L1 presentation was flawed. Like scientists that came before, they found low levels of PD-L1 protein in resistant cancers. But when they looked at messenger RNA (mRNA), the molecular precursor of all proteins, they observed an odd discrepancy: there was far too much PD-L1 mRNA for the scant amount of PD-L1 protein that they measured in the cells.

“We saw the difference between mRNA and protein levels and wanted to figure out what was happening,” Blelloch said. “Our experiments also showed that the protein was in fact being made at some point, and that it wasn’t being degraded. That’s when we looked at exosomes and found the missing PD-L1.”

Exosomal PD-L1 Hampers Immune Response, Promotes Cancer Growth

To show that exosomal PD-L1 was responsible for imparting immune invisibility, the researchers turned to a mouse prostate cancer model that’s resistant to checkpoint inhibitors. When they transplanted these cancer cells into healthy mice, tumors rapidly sprouted. But when the scientists used the gene-editing tool CRISPR to delete two genes required for exosome production, the edited cancer cells were unable to form tumors in genetically identical mice. Though both edited and unedited cells were producing PD-L1, only those unable to create exosomes were visible and vulnerable to the immune system when PD-L1 was blocked.

“The importance of this discovery was immediately evident,” said postdoctoral fellow Mauro Poggio, PhD, lead author of the new study. “Currently in the clinic, there are no drugs available that are capable of counteracting the destructive power of exosomal PD-L1, so understanding the biology of exosomal PD-L1 is the first fundamental step that might lead to novel therapeutic approaches for patients.”

In a complementary experiment, the same CRISPR-edited cancer cells were transplanted into healthy mice, immediately followed by a series of injections of exosomes carrying PD-L1. Unable to produce exosomes, the CRISPR-edited cancer cells should have fallen victim to the immune system. Instead, the injected exosomes were able to neutralize the immune response on behalf of the cancer, which allowed the exosome-deficient cancer cells to form tumors.

To figure out how exosomal PD-L1 was interfering with the immune system, the researchers inspected the lymph nodes of mice that received either CRISPR-edited or unadulterated cancer cells. Mice that received the edited cells showed increased immune cell proliferation and had higher numbers of activated immune cells in their lymph nodes, the central command hubs of the immune system.

In a separate mouse model — a colorectal cancer that’s only partially responsive to immunotherapy — the researchers identified two distinct pools of PD-L1: one on the surface of tumor cells that’s sensitive to PD-L1 inhibitors, and another in exosomes that’s resistant. When they treated the cancer with a combination therapy that involved both preventing exosome formation and administering PD-L1 inhibitors, the mice survived longer than those treated with either approach alone.

“These data from two very different cancer models suggest a novel therapeutic approach, where suppressing the release of PD-L1 in exosomes, either alone or in combination with current checkpoint inhibitors, could overcome resistance in a large fraction of patients currently resistant to treatment with checkpoint inhibitors alone,” Blelloch said.

Exosome-Deficient Tumor Cells Can Act as ‘Vaccine’ Against Immune Resistance

In a surprising result from the new paper, the researchers found that they could use CRISPR-edited, exosome-deficient cancer cells to induce an anti-cancer immune response that targeted tumors that normally resist immune attack.

The researchers first transplanted CRISPR-edited cancer cells unable to produce exosomes into normal mice and waited 90 days. They then transplanted unedited — and presumably immune-evading — cancer cells into the same mice. After having exposed the immune system to the CRISPR-edited, exosome-deficient cancer cells, the unedited cells were no longer invisible. Instead of ignoring these cells, the immune system mounted a vigorous response that targeted these formerly immune-evading cancer cells and prevented them from proliferating.

“The immune system develops an anti-tumor memory after being exposed to cancer cells that can’t produce exosomal PD-L1. Once the immune system has developed memory, it is no longer sensitive to this form of PD-L1 and thus targets exosomal PD-L1–producing cancer cells as well,” Blelloch said.

Another surprising result was achieved when both unedited and CRISPR-edited, exosome-deficient cancer cells were simultaneously transplanted into opposite sides of the same mouse. Though they were introduced at the same time, the CRISPR-edited cells proved dominant — they were able to activate the immune system, which then launched an attack that destroyed the unedited, supposedly immune-resistant tumors growing on the other side.

These results suggest that even the temporary inhibition of the release of PD-L1 in exosomes could lead to long-term, body-wide suppression of tumor growth. Furthermore, they hint at the possibility of a new kind of immunotherapy, one in which a patient’s cancer cells can be edited and reintroduced in order to activate the immune system and goad it into attacking immune-resistant cancers. Suppressing the release of PD-L1 in exosomes or the introduction of the “tumor cell vaccine” devised by the Blelloch team may one day offer hope to patients whose tumors don’t respond to today’s treatment options.

“Much more needs to be uncovered about PD-L1’s function in cancer,” Poggio said. “We are just scratching the surface of what could be a new mechanism that, if blocked, has the potential to suppress many aggressive tumors that don’t currently respond to treatment.”

Authors: Additional authors on the paper include TJ Hu, Chien-Chun Pai, Brandon Chu, Cassandra D. Belair, Anthony Chang, Ursula E. Lang, Qi Fu, and Lawrence Fong of UCSF; Elizabeth Montabana of UC Berkeley.

Funding: Research was supported by the National Institutes of Health Common Fund Extracellular RNA Consortium, the George and Judy Marcus Innovation Fund, and an NIH training grant.

Conflicts: The authors declare no competing financial interests.

About UCSF: UC San Francisco (UCSF) is a leading university dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care. It includes top-ranked graduate schools of dentistry, medicine, nursing and pharmacy; a graduate division with nationally renowned programs in basic, biomedical, translational and population sciences; and a preeminent biomedical research enterprise. It also includes UCSF Health, which comprises three top-ranked hospitals – UCSF Medical Center and UCSF Benioff Children’s Hospitals in San Francisco and Oakland – as well as Langley Porter Psychiatric Hospital and Clinics, UCSF Benioff Children’s Physicians and the UCSF Faculty Practice. UCSF Health has affiliations with hospitals and health organizations throughout the Bay Area. UCSF faculty also provide all physician care at the public Zuckerberg San Francisco General Hospital and Trauma Center, and the SF VA Medical Center. The UCSF Fresno Medical Education Program is a major branch of the University of California, San Francisco’s School of Medicine. Please visit ucsf.edu/news.

03.12.19 by Steven M. Jay

A large (scale) advance for small RNA therapeutics

This commentary originally appeared as an Editor’s Choice in Science Translational Medicine. Thanks to STM and Steven Jay for permission to reprint here.

Abstract
Combining established techniques enables large-scale production of potentially therapeutic extracellular vesicles enriched with specific miRNAs.

Extracellular vesicles (EVs) are currently being intensively studied for their therapeutic potential following promising clinical results and recent regulatory approvals of cell-based therapies. However, for the excitement surrounding EVs to ultimately yield useful therapies, critical challenges remain to be overcome. Specifically, although microRNA (miRNA) is often cited as a critical component of EV therapeutic activity, specific miRNA amounts in native EVs can be quite low (far less than one miRNA per one EV on average in many cases), raising concern about the potency of EV-based therapies. Further, scalable biomanufacturing of therapeutic EVs is nontrivial and could present a barrier to translation.

To address these issues, Yoo et al. used a combination of established, commercially available technologies to define a method for producing large quantities of EVs enriched with specific miRNAs. First, they used lentiviral vectors to generate stable HEK293 cell lines capable of producing EVs with more than 2000-fold enrichment of specific miRNAs. Then, a hollow fiber bioreactor was employed for continuous production of EVs from the same stable cell lines for up to 30 days, with additional gains in miRNA levels observed compared with EVs harvested from cells grown in conventional cell culture flasks. Last, tangential flow filtration was used to concentrate miRNA-enriched EVs by ~200-fold without precipitate formation. To validate the potential therapeutic utility of EVs produced through this scheme, miR-133a-3p–enriched EVs were injected intraperitoneally in mice. The result was an increase in the level of circulating miR-133a-3p after four hours. The broad applicability of the techniques used in this process suggests that it could be used to increase blood levels of any desired miRNA via EV association.

Further optimization of this method will be necessary to enable production of EVs from different primary cell types, and this production scheme still contains potential manufacturing bottlenecks, such as lentiviral transfection. The ultimate therapeutic potential of miRNA delivery via EVs produced by the process still remains to be established. However, the general approach described is widely applicable to platform production of miRNA-enriched EVs. More importantly, all the technologies employed are commercially available and should be within reach for a majority of academic labs and small companies to access or acquire. Thus, this process could serve as an important template for advancing research and overcoming the lack of method standardization in development of EV therapeutics, taking the entire field closer to clinical translation.

Highlighted Article
K. Yoo, N. Li, V. Makani, R. Singh, A. Atala, & B. Lu. Large-scale preparation of extracellular vesicles enriched with specific microRNA. Tissue Eng. Part C: Methods (2018) 24: 637-644. doi: 10.1089/ten.TEC.2018.0249 PMID: 30306827.

02.26.19 by University of Sheffield

miRNA secreted through astrocyte EVs causes neuronal network degeneration in ALS

This post originated as a press release from the University of Sheffield.

Motor neurone disease (MND), also known as Amyotrophic Lateral Sclerosis (ALS), is a devastating neurogenerative disorder that affects the nerves – motor neurones – in the brain and spinal cord that tell your muscles what to do. The messages from these nerves gradually stop reaching the muscles, leading them to weaken, stiffen and eventually waste. The progressive disease affects a patient’s ability to walk, talk, eat and breathe. MND affects 5,000 adults in the UK and 16,000 in the US, and there is currently no cure.

Scientists from the University of Sheffield have identified new messenger molecules shuttled between cells which could help to protect the survival of neurones – potentially leading to new treatments for MND. The pioneering research has discovered the role of a small molecule which can regulate large signalling cascades and significantly improve the survival of neurones – something which will help pave the way to identify and develop new therapies for neurodegenerative diseases.

Approximately 10 per cent of MND cases are inherited, but the remaining 90 per cent of MND cases are caused by complex genetic and environmental interactions which are currently not well understood – this is known as sporadic MND. The most common known genetic cause of MND is a mutation of the C9orf72 gene.

Although MND affects the survival of neurones, other supporting cell types such as astrocytes – star-shaped glial cells in the brain and spinal cord – play an important role in the progression of the disease. Normally responsible for keeping the neurones protected and nourished, astrocytes can become toxic in MND. In a healthy organism, these cells release pockets of vesicles containing messages to communicate with other cells. In MND, these extracellular vesicles (EVs) can contain toxic factors – no longer supporting the neurones but instead contributing to their death.

The new research, led by Dr Laura Ferraiuolo from the University of Sheffield’s Insitute of Translational Neuroscience (SITraN) found that when the micro-RNA molecule – which can regulate large signalling cascades – is introduced to an astrocyte-motor neurone culture, the survival of neurones was significantly improved.

The micro-RNA identified in the study, called miR-494-3p, regulates genes involved in maintaining the health and strength of neurones axons. Researchers also found miR-494-3p was significantly depleted in cells derived from patients with sporadic MND.

Dr Ferraiuolo from SITraN and lead author of the study said: “When an artificial form of miR-494-3p was introduced to the astrocyte-motor neuron culture, the survival of neurons was significantly improved.

“The study shows that restoring depleted micro-RNAs can improve cell survival. The results not only shed more light on the mechanisms of this complex disease, but they hold massive potential for the identification and development of new therapies for ALS and other neurodegenerative diseases.”

The research, in collaboration with Dr Guillaume Hautbergue’s team at SITraN and Dr Stuart Hunt’s lab in the University of Sheffield’s Dental School, is published in the Journal EBioMedicine (published by The Lancet).

The study was funded by the Thierry Latran Fondation and the Academy of Medical Sciences.

Reference
Varcianna A, et al. Micro-RNAs secreted through astrocyte-derived extracellular vesicles cause neuronal network degeneration in C9orf72 ALS. EBioMedicine. AOP 2019 Jan 30. doi: 10.1016/j.ebiom.2018.11.067 PMID: 30711519.

02.06.19 by Jeff Hansen

Protease enzymes in exosomes from activated white blood cells cause destruction in the COPD lung

This blog post originated as a press release from the University of Alabama at Birmingham.

University of Alabama at Birmingham researchers have found a novel, previously unreported pathogenic entity that is a fundamental link between chronic inflammation and tissue destruction in the lungs of patients with chronic obstructive pulmonary disease, or COPD. COPD is the fourth-leading cause of death in the world.

This pathogenic entity — exosomes from activated polymorphonuclear leukocytes, or PMNs — caused COPD damage when the small, subcellular particles, collected from purified PMNs, were instilled into the lungs of healthy mice. Remarkably, the UAB researchers also collected exosomes from the lung fluids of human patients with COPD and the lung fluids of neonatal ICU babies with the lung disease bronchopulmonary dysplasia; when those human-derived exosomes were instilled into the lungs of healthy mice, they also caused COPD lung damage. Damage was primarily from PMN-derived exosomes from the human lungs.

“This report seems to provide the first evidence of the capability of a defined non-infectious subcellular entity to recapitulate disease phenotype when transferred from human to mouse,” said J. Edwin Blalock, Ph.D., professor of pulmonary, allergy and critical care medicine in the UAB Department of Medicine. “I think this could be a very profound discovery. A lot of what we have found here will apply in other tissues, depending on the disease.”

Other diseases marked by immune cell inflammation and tissue destruction include heart attacks, metastatic cancer, and chronic kidney disease. The activated PMN exosomes may also contribute to lung damage in other lung diseases that have excessive PMN-driven inflammation, such as cystic fibrosis. The study is reported in the journal Cell.

“These findings highlight a novel role of the innate immune response in chronic lung diseases and could be used for the development of new diagnostics and therapeutics for COPD and possibly cystic fibrosis,” said James Kiley, Ph.D., director of the Division of Lung Diseases at the National Heart, Lung, and Blood Institute, part of the National Institutes of Health.

Background
COPD, a smoking-associated disease, is marked by PMN-driven inflammation in the lungs. Damage to the lung tissue leads to airway obstruction, shortness of breath, and respiratory failure. PMN immune cells, also known as neutrophils, are part of the body’s white blood cell defense against infections and tissue damage. They comprise 60 percent of the body’s white blood cells, or about 2.5 billion PMNs in each pint of blood. PMNs are voracious eaters of microbes or damaged human cells after activation by a signal of infection.

All cells shed exosomes. These tiny extracellular membrane-bound vesicles can be mediators of cell-to-cell communication, and they can ferry a diverse cargo of proteins, lipids, and nucleic acids from cell to cell. The UAB research focused on a recently found third role for exosomes — the ability to harbor protease enzymes.

Activated PMNs are known to release neutrophil elastase, or NE, a protease that can degrade type I collagen and elastin. The collagen and elastin proteins help form the extracellular matrix that glues cells together. In the lungs, the extracellular matrix and lung cells are sheets of tissue that help form the tiny alveoli, where the lung exchanges oxygen and carbon dioxide. In COPD, the damaged alveoli enlarge, reducing oxygen exchange and forcing the heart to pump harder to push blood through the lungs.

NE and other proteases from PMNs can attack microbes. Healthy lungs are protected by anti-proteases that can inhibit the proteases. Normally, NE is inhibited by a robust barrier of alpha1-antitrypsin in the lung.

The research
Blalock and fellow researchers investigated whether NE might exist in an exosomal form and whether such exosomes might bypass alpha1-antitrypsin inhibition to contribute to inflammatory lung disease.

They found that exosomes from quiescent PMNs did not cause COPD when transferred to healthy mice. In contrast, exosomes from activated PMNs did cause COPD, as measured by histologic changes of the alveoli, increased pulmonary resistance and enlargement of the right heart ventricle that pumps blood to the lung.

“This investigation reveals an entirely unappreciated aspect of the interplay between inflammation, proteolysis, and matrix remodeling with far-reaching implications for future research.”
J. Edwin Blalock

The activated PMN exosomes were covered with enzymatically active surface-bound NE, while quiescent PMN exosomes had none. This surface NE was resistant to alpha1-antitrypsin inhibition; the exosomes from activated PMNs degraded collagen, they caused emphysema when put into mouse lungs, and they carried the PMN cell-surface markers CD63 and CD66b that identify them as coming from PMNs. Human COPD lung-derived exosomes carrying those PMN cell-surface markers conferred COPD to mice.

A very large dose of purified NE — enough to overwhelm the alpha1-antitrypsin barrier — can cause alveolar enlargement in mice. Because the exosome-bound NE was protected against apha1-antitrypsin inhibition, researchers found that the dose of activated PMN exosomes needed to cause the same damage as purified NE was 10,000 times less.

The activated PMN exosomes had another cause for their aggressive proteolysis — they carried integrin Mac-1 on their surface. Integrin Mac-1 allowed the exosomes to bind directly to collagen fibrils, a second mechanism besides protected NE for why the proteolytic exosomes exert an outsized degradative capacity in relation to their size and protease load.

“This investigation reveals an entirely unappreciated aspect of the interplay between inflammation, proteolysis and matrix remodeling with far-reaching implications for future research,” Blalock said. “Our report significantly expands the biological repertoire of the exosome, demonstrating potent biological effects of these particles ex cellula.”

Looking ahead
The study also suggests therapeutic strategies to interrupt pathogenic aspects of PMN exosome function: 1) disrupting the ionic binding of the NE to the exosome, to dislodge the NE and make it susceptible to alpha1-antitrypsin; 2) inhibiting the exosomal integrin Mac-1 to block collagen binding; and 3) directly inhibiting the exosomal NE with small-molecule compounds.

Blalock is also interested in another big question — exosome activity in healthy smokers.

“Only one in seven or one in eight smokers gets COPD,” he said. “It would be an amazing outcome if we found activated PMN exosomes in a subpopulation of people who smoke.” Those people could then be warned of the risk they faced.

This Cell study took six years of work.

Significant research was done by co-first authors Kristopher Genschmer, Ph.D., and Derek W. Russell, M.D., who were NIH T32 grant trainees with Blalock. Both are assistant professors in the UAB Division of Pulmonary, Allergy and Critical Care Medicine. Amit Gaggar, M.D., Ph.D., a professor of pulmonary, allergy and critical care medicine, is co-senior author with Blalock, and he is a former trainee who did his Ph.D. with Blalock. Co-author Charitharth Vivek Lal, M.D., assistant professor in the UAB Pediatrics Division of Neonatology, is the physician who collected the lung fluid from neonates and performed all of the bronchopulmonary dysplasia work.

Dr. Amit Gaggar, MD, PhD (Associate Professor, Pulmonary/Allergy/Critical Care; Director, UAB Cystic Fibrosis Inflammation Group; Co-Director, Pulmonary Biospecimen Sample Repository)

Co-authors with Genschmer, Russell, Gaggar, Lal and Blalock of the paper “Activated PMN exosomes: Pathogenic entities causing matrix destruction and disease in the lung” are Tomasz Szul, Mojtaba Abdul Roda, Xin Xu, Liliana Viera, Tarek H. Abdalla, Robert W. King, J. Michael Wells and Mark T. Dransfield, UAB Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine; Preston E. Bratcher, National Jewish Medical Center, Denver, Colorado; Brett D. Noerager, University of Montevallo, Montevallo, Alabama; Gabriel Rezonzew, UAB Department of Pediatrics; Brian S. Dobosh, Camilla Margaroli and Rabindra Tirouvanziam, Department of Pediatrics, Emory University, Atlanta, Georgia; and Carmel M. McNicholas, UAB Department of Cell, Developmental and Integrative Biology.

This study was supported by National Institutes of Health grants HL135710, HL077783, HL114439, HL110950, HL126596, HL102371, HL126603, HL123940, HL105346-07 and HL105346-05; American Heart Association grant 17SDG32720009; and Veterans Affairs grant BX001756.

Blalock is a distinguished professor in the UAB School of Medicine, and he holds the Nancy E. Dunlap, M.D., Endowed Chair in Pulmonary Disease.

Reference
Genschmer KR, Russell DW, et al. Activated PMN Exosomes: Pathogenic Entities Causing Matrix Destruction and Disease in the Lung. Cell (2019) 176: 113-126. doi: 10.1016/j.cell.2018.12.002. PMID: 30633902.

01.30.19 by Myotonic Dystrophy Foundation

Non-invasive biomarkers for Muscular Dystrophy

This blog post comes from the Myotonic Dystrophy Foundation.

Pharmacodynamic Biomarkers and DM
There is now strong support for the concept that a panel of splicing events may serve as a pharmacodynamic biomarker for go/no go decisions in drug development for myotonic dystrophy type 1 (DM1) and Duchenne muscular dystrophy (DMD). Data establishing splicing event sensitivity to free MBNL levels has converged with the natural history of alternative splicing patterns in DM patients to yield a subset of splicing events with the sensitivity and reproducibility to evaluate candidate therapeutics in early stage clinical trials. Quantitative pharmacodynamic biomarkers are invaluable in de-risking industry drug discovery and development, as they facilitate early stage assessment of molecular target engagement and modulation and may inform dose ranging studies. The only caveat is the dependence of these measures upon repeated muscle biopsies (a risk reduced, but not eliminated, by more tolerable needle biopsies). The identification and validation of a non-invasive assay of patient splicing status would be a valuable step forward for clinical trials in DM.

Early Support for a Non-Invasive Biomarker for DM1
Dr. Thurman Wheeler and colleagues at Massachusetts General, Harvard Medical, and Boston Children’s have explored the concept that a subset of extracellular RNAs (exRNAs) released into blood or urine may: (a) reflect alternative splicing status in DM-affected tissues and (b) thereby serve as an easily accessible pharmacodynamic biomarker platform for DM1 (Antoury et al., 2018). These studies were supported in part by a grant to facilitate “Development of Biomarkers for Myotonic Studies” from Myotonic Dystrophy Foundation/Wyck Foundation.

The research team initially found that > 30 transcripts that are alternatively spliced in DM1 muscle biopsies were detectable in human blood and urine samples; follow-up studies confirmed the presence of RNAs in extracellular fluids/exosomal particles. Normalized DMPK expression levels in urine from DM1 patients, by droplet digital PCR, were ~50% of unaffected controls. Assessments of DM1-established alternative splicing events showed that a subset (10/33) also occurred in urine exRNA, including being conserved in longitudinal (6-26 month) studies of the same patients. Assessments of alternative splicing events in blood exRNA did not yield the same value.

Using principal component analysis of 10 alternative splicing events observed in urine exRNA, the research team then generated a putative composite biomarker panel for DM1. The ensuing predictive model of alternative splicing in DM1 proved to be 100% accurate in comparisons of training and independent validation data sets to distinguish DM1 from unaffected controls and in distinguishing disease status of subsequently enrolled subjects. The research team also linked alternative splicing patterns in urine exRNA to variation in DM1 clinical phenotypes, suggesting that modeling of urine exRNA alternative splicing may allow both the tracking of disease progression and the impact of candidate therapeutics.

Finally, to address questions as to the source of urine exRNA, the team assessed alternative splicing in urinary tract cells of DM1 mouse models (the ubiquitous Mbnl1 ko and the tissue-specific HSALR). While kidney and bladder cells of the Mbnl1 ko reflected patterns in skeletal muscle, assessments of the same tissues in the HSALR showed no differences from control mice. These data strongly suggested that the exRNAs assessed in urine reflect exosomes released from urinary tract cells. Some of the alternatively spliced transcripts in urine exRNA also were shown to be altered by antisense oligonucleotide drugs previously shown to correct splicing patterns in DM1 mouse models. The research team’s parallel studies of Duchenne muscular dystrophy also supported the concept that urine exRNA has utility as a pharmacodynamic biomarker in drug intervention studies.

Towards a Non-Invasive Biomarker for DM1
Taken together, these data provide compelling proof of concept that a panel of alternative splicing events assessed in urine may serve as a robust composite biomarker of DM1 progression and as a tool for assessment of candidate therapeutics. A non-invasive biomarker such as this would greatly extend the ability to perform repeated measurements in longitudinal natural history studies (as a disease progression biomarker) and in interventional clinical trials (as patient stratification and pharmacodynamic biomarkers), including making assessment of pediatric DM1 patient cohorts feasible. Although it is not essential to formally qualify a biomarker, existing regulatory agency guidance documents (see References below) provide a valuable evidentiary framework for moving non-invasive biomarker work towards an accepted clinical tool for DM1.

References
Antoury L, Hu N, Balaj L, Das S, Georghiou S, Darras B, Clark T, Breakefield XO, Wheeler TM. Analysis of extracellular mRNA in human urine reveals splice variant biomarkers of muscular dystrophies. Nat Commun. (2018) 9: 3906. doi: 10.1038/s41467-018-06206-0. PMID: 30254196

Framework for Defining Evidentiary Criteria for Biomarker Qualification. Foundation for the National Institutes of Health (FNIH) Evidentiary Criteria Writing Group. October 2016. (announcement) (pdf)

Guidance for Industry and FDA Staff: Qualification Process for Drug Development Tools. (pdf)

12.04.18 by Kenneth Witwer

MISEV2018: Update to the Minimal Information for Studies of Extracellular Vesicles

Updated guidelines on Minimal Information for Studies of Extracellular Vesicles have now been published in the Journal of Extracellular Vesicles (JEV, Taylor & Francis) as MISEV2018.

The original MISEV2014 guidelines were released in 2014 by the Board of Directors of the International Society for Extracellular Vesicles (ISEV) to provide guidance in standardization of protocols and reporting in the EV field. Accumulating more than 800 citations since its release, the MISEV2014 guidelines have achieved the aim of becoming a guiding standard for researchers. A 2016 survey of ISEV members reaffirmed the need for guidelines and recommended that they be updated regularly…but with broad community input to accommodate and shape the quickly developing field.

MISEV2018 updates the topics of nomenclature, separation, characterization, and functional analysis, integrating the contributions of over 380 ISEV members, a strong tribute to the commitment of ISEV members. A two-page checklist summarizing the main points is also included.

So what’s new? MISEV2018 recommends the use of ‘extracellular vesicle’ as the preferred generic terminology for use in publications, in part due to challenges in confirming the biogenesis mechanisms of exosomes, microvesicles, and other particles, and in part due to the vague and varied uses of other terms. Separation and concentration options are now many and diverse; researchers should pick the methods most fit for downstream purpose and, more importantly, report these clearly and accurately. The EV-TRACK database (van Deun et al., Nature Methods, 2017) is supported as a means to record these details in order to improve clarity and reproducibility. To establish presence of EVs, examples of EV-enriched markers are provided, but the need for “negative” (better: “depleted”) markers is also highlighted. MISEV2018 adds topology as a recommended form of EV characterization, for example identifying where in or on a vesicle your favorite protein or RNA resides. It also recommends functional analysis of the ‘non-EV’ fractions to confirm EV-specific function (or not!). An appreciation of EV heterogeneity is included with a reminder that ‘larger EVs matter’ and a request to explore a range of EV subtypes in functional studies. Finally, although some of the specific details contained in MISEV2018 are focused on mammalian components, it is appreciated that the guidelines are applicable to non-mammalian and non-eukaryote research.

Please contact the corresponding authors, Clotilde Théry and Kenneth Witwer with any questions or comments.

For more information on the process of writing and publishing MISEV2018, see this white paper and Witwer et al., J. Extracell. Vesicles, 2017.

References

Lotvall J, Hill AF, Hochberg F, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the international society for extracellular vesicles. J. Extracell. Vesicles. (2014) 3: 26913. doi:10.1080/20013078.2018.1535750. PMID:25536934.

Witwer KW, Soekmadji C, Hill AF, et al. Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility. J. Extracell. Vesicles. (2017) 6: 1396823. doi:10.1080/20013078.2017.1396823. PMID:29184626.

Théry C, Kenneth W Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles. (2018) 7: 1535750. doi:10.1080/20013078.2018.1535750.

10.22.18 by Karen Kreeger

Cancer cells send out ‘drones’ to battle the immune system from afar

Image: Kelsey Burke

This post originated as a press release from the University of Pennsylvania.

Cancerous tumors are more than a lump of cells growing out of control; they participate in active combat with the immune system for their own survival. Being able to evade the immune system is indeed a hallmark of cancer. Now, researchers from the University of Pennsylvania show that, to assist in the fight, cancer cells release biological “drones,” small vesicles called exosomes circulating in the blood and armed with the protein PD-L1, which causes T cells to tire before they have a chance to reach the tumor and do battle.

The work, published in the journal Nature (Chen et al., 2018), is a collaboration between Wei Guo of Penn’s School of Arts and Sciences and Xiaowei Xu of the Perelman School of Medicine. While primarily focused on metastatic melanoma, the team found that breast and lung cancer also release the PD-L1-carrying exosomes.

The research offers a paradigm-shifting picture of how cancers take a systemic approach to suppressing the immune system. In addition, it also points to a new way to predict which cancer patients will respond to certain checkpoint inhibitor drugs, which disrupt immune suppression to fight tumors, and a means of tracking the effectiveness of such therapies.

“Immunotherapies are life-saving for many patients with metastatic melanoma, but about 70 percent of these patients don’t respond,” says Guo, a professor of biology. “These treatments are costly and have toxic side effects, so it would be very helpful to know which patients are going to respond. Identifying a biomarker in the bloodstream could potentially help make early predictions about which patients will respond and, later on, could offer patients and their doctors a way to monitor how well their treatment is working.”

“Exosomes are tiny lipid-encapsulated vesicles with the diameter less than one-hundredth of a red blood cell. What we have found with these circulating exosomes is truly remarkable,” says Xu. “We collected blood samples from melanoma patients treated with anti-PD1 checkpoint inhibitor therapy. This type of liquid biopsy assay allows us to monitor tumor-related immune suppression with time.”

One of the most successful innovations in cancer therapy has been the use of checkpoint inhibitor drugs, which are designed to block attempts by cancer cells to suppress the immune system to allow tumors to thrive and spread. One of the primary targets for this class of drugs is PD-1, a protein on the surface of T cells. Tumor cells express PD-L1, which interacts with PD-1, effectively turning off that cell’s anti-cancer response. Blocking that interaction using checkpoint inhibitors reinvigorates T cells, allowing them to unleash their cancer-killing power on the tumor.

While it was known that cancer cells carried PD-L1 on their surface, Guo, Xu and colleagues found, in the new work, that exosomes from human melanoma cells also carried PD-L1 on their surface. Exosomal PD-L1 can directly bind to and inhibit T cell functions. Identifying the exosomal PD-L1 secreted by tumor cells provides a major update to the immune checkpoint mechanism, and offers novel insight into tumor immune evasion.

“Essentially exosomes secreted by melanoma cells are immunosuppressive.” Guo says. “We propose a model in which these exosomes act like drones to fight against T cells in circulation, even before the T cells get near to the tumor.”

Since a single tumor cell is able to secrete many copies of exosomes, the interaction between the PD-L1 exosomes and T cells provides a systemic and highly effective means to suppress anti-tumor immunity in the whole body. This may help explain why cancer patients have weakened immune systems.

Because exosomes circulate in the bloodstream, they present an accessible way of monitoring the cancer-T cell battle through a blood test, compared to a traditional, more-invasive tumor biopsy. After an acute phase of treatment, the researchers envision such a test as a way to monitor how well the drugs are keeping cancer cells in check.

By measuring pre-treatment levels of PD-L1, oncologists may be able to predict the extent of tumor burden in a patient and associate that with treatment outcome. In addition, a blood test could measure the effectiveness of a treatment. For example, levels of exosomal PD-L1 could indicate the level of T cell invigoration by immune checkpoint inhibitors.

“In the future, I think we will begin to think about cancers as a chronic disease, like diabetes,” says Guo. “And just as diabetes patients use glucometers to measure their sugar levels, it’s possible that monitoring PD-L1 and other biomarkers on the circulating exosomes could be a way for clinicians and cancer patients to keep tabs on treatment. It’s another step toward precision and personalized medicine.”

Reference

Chen G. et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature (2018) 560: 382–386. doi: 10.1038/s41586-018-0392-8 PMID: 30089911

Guo and Xu coauthored the work with Penn’s Gang Chen, Alexander C. Huang, Wei Zhang, Min Wu, Jiegang Yang, Beike Wang, Honghong Sun, Wenqun Zhong, Bin Wu, Xiaoming Liu, Lei Guan, Tin Li, Shujing Liu, Ruifeng Yang, Youtao Lu, Liyun Dong, Suzanne McGettigan, Ravi Radhakrishnan, Junhyong Kim, Youhai H. Chen, Giorgos C. Karakousis, Tara C. Gangadhar, Lynn M. Schuchter, and E. John Wherry, as well as collaborators from Wuhan University, The Wistar Institute, Xi’an Jiaotong University, the University of Texas MD Anderson Cancer Center, and the Mayo Clinic.

The research was supported by the National Institutes of Health (GM111128, GM085146, AI105343, AI108545, AI082630, and AI117950), Parker Institute for Cancer Immunotherapy, American Heart Association, Tara Miller Melanoma Foundation, University of Pennsylvania, Wistar Institute, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, CAST Foundation, and NSFC Foundation.

Wei Guo is a professor of biology in the School of Arts and Sciences, and Xiaowei Xu is a professor of pathology and laboratory medicine and of dermatology in the Perelman School of Medicine at the University of Pennsylvania.

Wei Guo, Xiaowei Xu, and Gang Chen are listed as inventors on a patient owned by the University of Pennsylvania related to this work. Guo and Xu serve on the Scientific Advisory Board and have equities in Exo Bio, a company that has licensed the patent from the University of Pennsylvania.