Unlocking the
Mysteries of
Extracellular RNA
Communication

Once thought to exist only inside cells, RNA is
known to travel outside of cells and play a role in newly
discovered mechanisms of cell-to-cell communication.

A recent study by Wei et al., 2017 catalogs the composition and characteristics of extracellular RNA (exRNA) secreted via three different routes from parent cells. The work provides novel insights into the biology of exRNA transport and intercellular communication, as well as the clinical potential of exRNA as a biomarker of disease.

The senior investigator of the study, Anna Krichevsky, Ph.D., at Brigham and Women’s Hospital and Harvard Medical School described the rationale for the study: “To understand the functions of exRNA complexes, we first have to define the exRNA repertoire with minimal bias.”

The exRNA Composition of Microvesicles, Exosomes, and Ribonucleoproteins (RNPs)
Dr. Krichevsky’s group sequenced RNA in microvesicles, exosomes, and extravesicular ribonucleoproteins (RNPs) isolated from glioma stem cells. They found that the majority of exRNA in all three fractions is noncoding. Although most exRNA studies focus on one class of noncoding RNA called microRNA (miRNA; 21-25 nucleotide molecules that repress gene expression), they reported that <10% of exRNA secreted by glioma stem cells is miRNA.

Comparing the profile of exRNA isolated from RNPs and extracellular vesicles (EVs) — including both exosomes and microvesicles, they found that RNPs contain higher amounts of noncoding cytoplasmic Y RNA and transfer RNA (tRNA) fragments.

Y RNA folds into a characteristic stem-loop structure and was originally found in protein-RNA complexes of individuals with autoimmune diseases. According to Dr. Krichevsky, “Despite abundant expression in all vertebrate cells, the physiological functions of Y RNA are only beginning to emerge.” Some evidence suggests that Y RNA plays a role in DNA replication and RNA quality control. Y RNA fragments may also be involved in cell death, ribosomal RNA maintenance, and histone gene expression.

tRNA is well known as a mediator of the translation of mRNA to protein. However, recent studies suggest that tRNA fragments found in exRNA are involved in regulating gene expression during cellular stress responses. Dr. Krichevsky discussed the implications of extracellular tRNA on cellular communication: “Based on the exRNA levels and biological functions of tRNA, we hypothesize that transferred tRNA transcripts can have a major impact on recipient cells.”

Along with noncoding RNA, a small proportion of gene-encoding messenger RNA (mRNA) was detected in extracellular vesicles and RNPs. Previous studies have also found extracellular mRNA; however, they did not determine if the mRNA transcripts were intact or fragmented. Dr. Krichevsky’s group was the first to show that short (<1000 nucleotides), endogenous full-length mRNAs can be packaged into exosomes, and microvesicles contain even longer mRNAs. Fragments of long mRNA transcripts were also present in exRNA.

Using exRNA as a Biomarker
Researchers are currently exploring the use of exRNAs as potential biomarkers for the diagnosis and monitoring of diseases. However, many exRNA biomarker studies have been limited in scope because they examined a heterogeneous pool of exRNA purified from an unfractionated collection of EV types.

By fractionating conditioned media from glioma stem cells into microvesicle, exosome, and RNP fractions, Dr. Krichevsky’s group was able to compare their RNA profiles with those of the parent cells. They found that the RNA content of microvesicles most closely resembled that of the parent cells, making this type of exRNA carrier a good candidate for disease biomarkers.

Dr. Krichevsky said, “We believe there is more intact mRNA in microvesicles, which we can consider for biomarkers. We can think about genes that are mutated. On the other hand, miRNAs are more enriched in the exosomes. It would be great if we could detect cancer mutations and non-coding RNA biomarkers in biofluids; then we would not need to do a biopsy.”

Conclusions
By developing novel experimental approaches to illustrate that exRNA composition differs by exRNA carrier, Krichevsky’s group has made significant contributions to exRNA research. Moreover, they highlighted that exRNA contains more than the well-studied miRNAs, including full-length mRNA molecules, Y RNA, and tRNA. Their data also indicate that the RNA profile of microvesicles is most similar to that of the cell of origin, including the presence of full-length mRNAs, making microvesicle exRNA a good candidate for some disease biomarkers.

In an interview, Dr. Krichevsky discussed the importance of this study: “Our work changed the way people thought about exRNA by showing them the exRNA in numbers, which helps appreciate their heterogeneity and the overall impact. The field is shifting from focusing on a specific extracellular miRNA to now considering that there are thousands of different RNAs present in extracellular complexes.”

References
Wei, Z. et al. Coding and noncoding landscape of extracellular RNA released by human glioma stem cells. Nature Communications (2017) 8:1145. doi:10.1038/s41467-017-01196-x

(This blog first appeared as a press release from Ohio State University.)

Principal investigator Peixuan Guo, PhD, Sylvan G. Frank Endowed Chair professor of the OSU College of Pharmacy and a member of the OSUCCC – James Translational Therapeutics Program.

  • Therapies based on RNA, such as small interfering RNA, hold great promise for cancer treatment but delivering these agents to their targets in cancer cells has been a problem.
  • A new study shows that attaching antibody-like RNA nanoparticles to microvesicles can deliver effective RNA therapeutics specifically to cancer cells.
  • The researchers are now working to adapt the technology for use in the clinic.

Columbus, Ohio – A new study shows that attaching antibody-like RNA nanoparticles to microvesicles can deliver effective RNA therapeutics such as small interfering RNA (siRNA) specifically to cancer cells. Researchers used RNA nanotechnology to apply the RNA nanoparticles and control their orientation to produce microscopic, therapy-loaded extracellular vesicles that successfully targeted three types of cancer in animal models.

The findings, reported in the journal Nature Nanotechnology, could lead to a new generation of anticancer drugs that use siRNA, microRNA and other RNA-interference technologies.

The study was led by researchers at Ohio State’s College of Pharmacy; the Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC – James).

“Therapies that use siRNA and RNA interference technologies are poised to transform cancer therapy,” says the principal investigator Peixuan Guo, PhD, Sylvan G. Frank Endowed Chair professor of the College of Pharmacy and a member of the OSUCCC – James Translational Therapeutics Program. “But clinical trials evaluating these agents have failed one after another due to the inability to deliver the agents directly to cancer cells in the human body.”

Guo noted that even when agents did reach and enter cancer cells, they were trapped in internal vesicles called endosomes and rendered ineffective.

“Our findings solve two major problems that impede these promising anticancer treatments: targeted delivery of the vesicles to tumor cells and freeing the therapeutic from the endosome traps after it is taken up by cancer cells. In this study, cancers stopped growing after systemic injection of these particles into animal models with tumors derived from human patients.” Guo says. “We’re working now to translate this technology into clinical applications.”

Guo and his colleagues produced extracellular microvesicles (exosomes) that display antibody-like RNA molecules called aptamers that bind with a surface marker that is overexpressed by each of three tumor types:

  • To inhibit prostate cancer, vesicles were designed to bind to prostate-specific membrane antigen (PSMA);
  • To inhibit breast cancer, vesicles were designed to bind to epidermal growth factor receptor (EGFR);
  • To inhibit a colorectal cancer graft of human origin, vesicles were designed to bind to folate receptors.

All vesicles were loaded with a small interfering RNA for down-regulating the survivin gene as a test therapy. The survivin gene inhibits apoptosis and is overexpressed in many cancer types.

Key findings include:

  • Vesicles targeting the prostate-specific membrane antigen completely inhibited prostate-cancer growth in an animal model with no observed toxicity.
  • Vesicles targeting EGFR inhibited breast cancer growth in an animal model.
  • Vesicles targeting folate receptors significantly suppressed tumor growth of human patient-derived colorectal cancer in an animal model.

“Overall, our study suggests that RNA nanotechnology can be used to program natural extracellular vesicles for delivery of interfering RNAs specifically to cancer cells,” Guo says.

Funding from the National Institutes of Health/National Cancer Institute (grants TR000875 and CA207946, CA186100, CA197706, CA177558 and CA195573) supported this research.

Other researchers involved in this study were Fengmei Pi, Daniel W. Binzel, Zhefeng Li, Hui Li, Farzin Haque, Shaoying Wang and Carlo M. Croce, The Ohio State University Wexner Medical Center; Meiyan Sun and Bin Guo, University of Houston; Piotr Rychahou and B. Mark Evers, University of Kentucky; and Tae Jin Lee, now at University of Texas.

About the OSUCCC – James
The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute strives to create a cancer-free world by integrating scientific research with excellence in education and patient-centered care, a strategy that leads to better methods of prevention, detection and treatment. Ohio State is one of only 49 National Cancer Institute (NCI)-designated Comprehensive Cancer Centers and one of only a few centers funded by the NCI to conduct both phase I and phase II clinical trials on novel anticancer drugs sponsored by the NCI. As the cancer program’s 308-bed adult patient-care component, The James is one of the top cancer hospitals in the nation as ranked by U.S. News & World Report and has achieved Magnet designation, the highest honor an organization can receive for quality patient care and professional nursing practice. At 21 floors with more than 1.1 million square feet, The James is a transformational facility that fosters collaboration and integration of cancer research and clinical cancer care.

Quantitative measurements of the number, size, and cargo of extracellular vesicles (EVs) are essential to both basic research on how EVs are produced and function, and to application of this knowledge to the development of EV-based biomarkers and therapeutics. Flow cytometry is a popular method for analyzing EVs, but their small size and dim signals have made this a challenge using the conventional flow cytometry approaches developed for analysis of cells (1). Moreover, established flow cytometry calibrators, standards, and experimental design considerations for cell studies are not regularly used in EV studies. As a result, there is significant variation in instrument set up, sample preparation, and data reporting for flow cytometric measurements of EVs. These issues are increasingly appreciated (1-5), but much needs to be done to develop consensus on best practices. To address these issues, members of the International Society for Extracellular Vesicles (ISEV), the International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Hemostasis (ISTH) are participating in a tri-Society Working Group, which includes several ERCC members, to improve the reporting of methods and results for FC-based EV measurements.

Reporting of EV Measurement Methods

A flow cytometer is an instrument, not a method. An EV analysis method that uses a flow cytometer involves many instrument setup, sample preparation, and data analysis decisions, including: 1) what signal to use for EV detection (light scatter or fluorescence); 2) how to resolve single EVs from the simultaneous occurrence of many EVs in the laser at the same time (aka coincidence or “swarm”); 3) how to gate the data to focus on EVs versus background events (without introducing artifacts or mis-representing the data); 4) how to estimate the size of the particles detected; 5) how to estimate the brightness of the particles detected; 6) how to verify that the particles detected are EVs and not other particles present in the sample, to name just some of the many decisions involved.

Several years ago, ISAC developed and introduced the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) (6), a set of guidelines to promote the sharing, reproducibility, and proper interpretation of flow cytometry data. These guidelines were developed with cell analysis, and particularly high parameter immunophenotyping, in mind, but they also apply to multiparameter EV analysis. However, there are several additional details about an EV measurement that are essential to include. The ISEV-ISAC-ISTH EV FC Working Group has been conducting a series of standardization studies to develop a consensus on the essential elements of an FC-based EV measurement that should be reported. These studies will be reported, along with the consensus reporting guidelines, in a paper planned for the coming year.

Standards and Calibrators for EV Analysis

Standards and calibration are essential components of any analytical method. These standards, and their use, are well established for flow cytometry and include 1) counting beads that can be used to calibrate sample flow rates for reporting of absolute particle concentrations, 2) fluorescence intensity standards that enable particle brightness to be expressed in NIST-traceable absolute units of mean equivalent soluble fluorochromes (MESF) (7) or equivalent reference fluorochromes (ERF) (8); 3) antibody-capture standards that can be used to estimate antibody binding in immunofluorescence measurements; and 4) NIST-traceable particle size standards.

Most of these standards and calibrators, and their methods of use, can be applied to EV measurements, with some caveats and cautions. Particle size standards, in particular, are often mis-used in FC-based EV measurements due to a lack of understanding of the effect on light scatter of refractive index (RI), which is different for polystyrene, silica, and lipids. With care, however, these differences can be used in conjunction with Mie scattering theory to enable estimates of EV size based on FC light scatter measurements. Commercially available fluorescence intensity and antibody-capture standards are generally designed for cell measurements, and tend to be brighter than EVs, but still have value for facilitating comparison of measurements between labs or instruments. EV-scaled intensity and antibody-binding standards will be a useful addition to the EV analysis toolbox, and are in development by several groups and companies.

A major unmet need is for EV standards, which will have use not only in FC-based EV measurements, but across the EV field. This is a challenging prospect, as an ideal EV standard will reflect not only the size and number of EVs, but also cargo, including surface molecules (for immunophenotyping) and intra-vesicular cargo, including nucleic acids, soluble proteins, and small molecules. Moreover, EVs are themselves quite diverse, raising the question of what type of EV, if any, might represent a universal standard. EV preparations for various cultured cell lines are commercially available from a number of sources but, in general, these have not been subjected to rigorous, independent characterization of these essential features or their uniformity, stability, or reproducibility. Such characterization is essential for validation of any putative standard and may be the subject of future activities by the ISEV-ISAC-ISTH EV FC Working Group.

Conclusions and Prospects

As EV research expands to impact every area of biology, issues with rigor and reproducibility are front and center. Translating observations made in the basic research lab into mechanistic understanding of EV actions and clinically actionable knowledge requires robust and validated analytical methods. Careful attention to the description of methods, standardization and calibration of analytical instrument and methods, and reporting of results are essential. Community efforts by the ERCC and relevant international societies will be key to helping researchers maximize the value of their work to the broader community.

In future blog posts we will discuss the controversial issue of whether to use light scatter or fluorescence to detect EVs, as well as new EV detection methods we’ve developed using fluorogenic membrane probes.

References

1. Nolan JP. Flow cytometry of extracellular vesicles: potential, pitfalls, and prospects. Curr. Protoc. Cytom. (2015) 73:13.14.1-13.14.16. PMID: 26132176. doi: 10.1002/0471142956.cy1314s73.
2. Chandler WL. Measurement of microvesicle levels in human blood using flow cytometry. Cytometry B Clin. Cytom. (2016) 90:326-336. PMID: 26606416. doi: 10.1002/cyto.b.21343.
3. Coumans FA, et al. Methodological guidelines to study extracellular vesicles. Circ. Res. (2017) 120:1632-1648. PMID: 28495994. doi: 10.1161/CIRCRESAHA.117.309417.
4. Nolan JP, Duggan E. Analysis of individual extracellular vesicles by flow cytometry. Methods Mol. Biol. (2018) 1678:79-92. PMID: 29071676. doi: 10.1007/978-1-4939-7346-0_5.
5. Nolan JP, Jones JC. Detection of platelet vesicles by flow cytometry. Platelets (2017) 28:256-262. PMID: 28277059. doi: 10.1080/09537104.2017.1280602.
6. Lee JA, et al. MIFlowCyt: the minimum information about a flow cytometry experiment. Cytometry A. (2008) 73:926-930. PMID: 18752282 doi: 10.1002/cyto.a.20623.
7. Wang L, Gaigalas AK, Abbasi F, Marti GE, Vogt RF, Schwartz A. Quantitating fluorescence intensity from fluorophores: practical use of MESF values. J. Res. Natl. Inst. Stand. Technol. (2002) 107:339-354. PMID: 27446735. doi: 10.6028/jres.107.027.
8. Wang L, Gaigalas AK. Development of multicolor flow cytometry calibration standards: Assignment of equivalent reference fluorophores (ERF) unit. J. Res. Natl. Inst. Stand. Technol. (2011) 116:671-83. PMID: 26989591. doi: 10.6028/jres.116.012.