exRNA Research
in Cell Press

Cell Press recently published 18 articles from the ERCC, a capstone to 5 years of research during Stage 1.

Tulane University

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.

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.”

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.