Illinois researchers developed a method to detect microRNA cancer markers with single-molecule resolution, a technique that could be used for liquid biopsies.
From left: Taylor Canady, postdoctoral scholar; Andrew Smith, professor of bioengineering; Nantao Li, graduate student; Lucas Smith, postdoctoral scholar; and Brian Cunningham – professor of Electrical and Computer Engineering; director of Micro and Nanotechnology Laboratory. Photo by L. Brian Stauffer
Thanks to the University of Illinois News Bureau for allowing us to share this article here.
CHAMPAIGN, Ill. — A fast, inexpensive yet sensitive technique to detect cancer markers is bringing researchers closer to a liquid biopsy – a test using a small sample of blood or serum to detect cancer, rather than the invasive tissue sampling routinely used for diagnosis.
Researchers at the University of Illinois developed a method to capture and count cancer-associated microRNAs, or tiny bits of messenger molecules that are exuded from cells and can be detected in blood or serum, with single-molecule resolution. The team published its results in the Proceedings of the National Academy of Science.
Thanks to Eileen Leahy from Elsevier and Chhavi Chauhan, Director of Scientific Outreach for the Journal of Molecular Diagnostics, for sharing this post here.
A novel non-invasive technique may detect human papilloma virus-16, the strain associated with oropharyngeal cancer, in saliva samples, reports The Journal of Molecular Diagnostics.
Philadelphia, December 13, 2019 – Unfortunately, cancers that occur in the back of the mouth and upper throat are often not diagnosed until they become advanced, partly because their location makes them difficult to see during routine clinical exams. A report in The Journal of Molecular Diagnostics, published by Elsevier, describes the use of acoustofluidics, a new non-invasive method that analyzes saliva for the presence of human papilloma virus (HPV)-16, the pathogenic strain associated with oropharyngeal cancers (OPCs). This novel technique detected OPC in whole saliva in 40 percent of patients tested and 80 percent of co published by Elsevier, describes the use of acoustofluidics, a new non-invasive method that analyzes saliva for the presence of human papilloma virus (HPV)-16, the pathogenic strain associated with oropharyngeal cancers (OPCs). This novel technique detected OPC in whole saliva in 40 percent of patients tested and 80 percent of confirmed OPC patients.
“OPC has an approximate incidence of 115,000 cases per year worldwide and is one of the fastest-rising cancers in Western countries due to increasing HPV-related incidence, especially in younger patients. It is paramount that surveillance methods are developed to improve early detection and outcomes,” explained co-lead investigator Tony Jun Huang, PhD, Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA.
“Considering these factors, the successful detection of HPV from salivary exosomes isolated by our acoustofluidic platform offers distinct advantages, including early detection, risk assessment, and screening,” added Dr. Huang. This technique may also help physicians predict which patients will respond well to radiation therapy or achieve longer progression-free survival.
Malignant gliomas are highly aggressive brain tumors. Surgical removal and chemoradiation of the tumor are the standard of care. Recently, the U.S. Food and Drug Administration (FDA) approved a compound called 5-aminolevulinic acid (5-ALA) as an imaging agent to aid in differentiating tumor from normal tissue during surgery. 5-ALA is a precursor in the heme biosynthesis pathway, which is inefficient in glioma cells because their strongly rewired metabolism does not rely on heme. When patients with malignant glioma ingest 5-ALA prior to surgery, the glioma cells fluoresce pink under a blue light due to their preferential uptake and conversion of 5-ALA to the final precursor in heme biosynthesis, the fluorescent molecule protoporphyrin IX (PpIX). We sought to investigate whether extracellular vesicles (EVs) released from PpIX-enriched glioma cells would fluoresce and be detectable in the blood of these patients.
I am a scientist at Scripps Research Institute in La Jolla, California working in the lab of Professor Hollis Cline. A thirst for knowledge is what originally attracted me to science. The potential to contribute, even in a small way, to alleviating suffering drives that thirst and passion even more.
Human biology has always fascinated me. Imagine for a moment how the human body is created. It starts with a single cell that multiplies to create a complex organism of trillions of cells. The human brain alone is estimated to contain more than 150 billion cells, 86 billion neurons and about an equal number of non-neuronal cells, all of a wide variety of specializations. It is mind boggling to imagine that a few founder cells contain the programming information that, through a series of cell fate decisions, produces a complex organ like the brain. What kind of communication and logistics are required to orchestrate the development and function of this behemoth?
This blog originated as a press release from the University of Sussex.
New research by scientists at the University of Sussex could be the first step towards developing a blood test to diagnose the most aggressive type of brain tumour, known as Glioblastoma.
A team from Professor Georgios Giamas’ lab at the University of Sussex has identified novel biomarkers within bodily fluids, which signal the presence of the tumour. Dr. Giamas is Professor of Cancer Cell Signalling in the School of Life Sciences.
Cancer biomarkers are molecules that are either exclusively found or over-expressed in cancer cells, as compared to ‘normal’, healthy cells. Biomarkers can be considered as biological signatures for a disease, as they indicate the presence of cancer in the body.
In a new paper published in the Nature journal Communications Biology, Professor Giamas and his team describe particular biomarkers that are associated with extracellular vesicles – small ’packages’ released by cells into bodily fluids so cells can communicate with each other.
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.
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.
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) Cell177:463-477. doi: 10.1016/j.cell.2019.02.018. PMID: 30951672.
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.
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.