Journal Club

Therapeutic exosomes and Huntington’s disease
Extracellular vesicles, specifically exosomes, are currently being explored as therapeutic delivery systems for disease-targeting RNA molecules. In a talk at the ERCC9 conference, Reka A. Haraszti, M.D., a researcher in Dr. Anastasia Khvorova’s group at the University of Massachusetts Medical School, described how exosomes could be used to treat Huntington’s disease, a progressive neurodegenerative disorder. There are currently no effective therapies for this illness, which is caused by a mutation in the Huntingtin gene. Exosomes capable of transporting molecular payloads designed to silence the defective Huntingtin gene represent a potential therapy for this fatal disease.

Comparison of exosome production methods
Technical challenges in the large-scale production of exosomes currently limit their utility for disease treatment. To address this issue, Dr. Haraszti and Dr. Khvorova’s group teamed up with MassBiologics to develop and compare two different exosome production methods for yield and therapeutic efficacy of the exosomes. They utilized Tangential Flow Filtration (TFF) and ultracentrifugation to isolate exosomes from the conditioned media of cultured mesenchymal stem cells.

In TFF, conditioned media is continuously swept along the surface of a filter while a downward pressure is applied to force molecules through the filter. This process is like shaking a sifter to concentrate large particles blocking the holes in the filter, allowing smaller particles to pass through. In contrast, ultracentrifugation works by placing the conditioned media in a column of viscous fluid and spinning rapidly to separate extracellular vesicles in the media by their differing densities.

The researchers found that isolation by TFF resulted in 10-100 times more exosomes than ultracentrifugation. TFF-generated exosomes were also more heterogenous and contained 10 times more protein.

Exosomes produced by TFF and ultracentrifugation were also studied for their therapeutic potential in Huntington’s disease. After purification, exosomes were loaded with small interfering RNA (siRNA) molecules that could silence the expression of the mutant Huntingtin gene in target cells that take up the exosomes.

In cell cultures of primary neurons, TFF-generated exosomes showed greater inhibition of Huntingtin expression than those generated by ultracentrifugation. Moreover, TFF-generated exosomes infused into the brains of mice suppressed Huntingtin gene expression in vivo.

Future for therapies using exosomes isolated by Tangential Flow Filtration
The findings of Dr. Haraszti’s group indicate that Tangential Flow Filtration can generate a higher yield of exosomes for clinical use than older methods. Further, TFF-generated exosomes were effective gene therapy agents in experimental models of Huntington’s disease, and hold promise as delivery systems for clinical treatments.
 
 
Related Mini-conference

There is an upcoming mini-conference on EV manufacturing and isolation, a topic closely related to the research described here. The in-person conference is in Gainesville, Florida, but a webcast will also be available for those who want to participate remotely.
 
 
References
1. Haraszti RA, et al. Loading of extracellular vesicles with chemically stabilized hydrophobic siRNAs for the treatment of disease in the central nervous system. Bio-protocol (2017) 7: e2338. doi: 10.21769/BioProtoc.2338
2. Schwartz L., and Seeley K. Introduction to tangential flow filtration for laboratory and process development applications. Retrieved from https://laboratory.pall.com/content/dam/pall/laboratory/literature-library/non-gated/id-34212.pdf
3. Sunkara V, Woo HK, and Cho YK. Emerging techniques in the isolation and characterization of extracellular vesicles and their roles in cancer diagnostics and prognostics. Analyst (2016) 141: 371-81. doi: 10.1039/c5an01775k
4. Synder Filtration. “Characterization of polymeric, porous membranes: UF/MF & solute rejection measurements.” Retrieved from https://synderfiltration.com/learning-center/articles/membranes/characterization-of-polymeric-porous-membranes/

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.


Scientists from the ERCC have joined forces to create a CSF Consortium to pool resources and establish standard practices in the study of cerebrospinal fluid (CSF).

One of the goals of the ERCC is not only to understand the fundamental biology of extracellular RNA (exRNA), but to develop exRNA-based biomarkers of disease. When such biomarkers have been found, studied, and cleared for clinical use, liquid biopsy of blood and other biofluids can enable earlier disease detection and less invasive tracking of disease progression. For neurological disorders, drawing CSF from the spinal cord has clear benefits over a more invasive brain biopsy. Progress in our technical understanding of how to accurately assess biomarkers in CSF will increase our basic understanding and promote clinical advancements in the diagnosis and treatment of neurological disease. Unfortunately, there are many inconsistencies between the processing of CSF in current studies. Data replication is often difficult, in large part due to variability across laboratories and institutions in protocols for sample isolation, purification, and analysis. Thus, the CSF Consortium, spearheaded by Dr. Fred Hochberg (https://fredhhochbergmd.com), was designed to be a resource for researchers to help minimize these discrepancies.

The CSF consortium plan calls for CSF researchers and clinicians to work together to improve standard practices. A major focus is transparency through open sharing of their work. Researchers are encouraged to establish collaborations, share in-depth details of experimental designs and reagents (including batch/lot numbers), and release any details of in-house protocol modifications. Working with the same biosamples shared through the Virtual Biorepository (VBR) enables multiple labs to compare and synchronize their protocols with one source of variability removed. The expectation is that sharing of detailed information will enable future researchers to avoid common pitfalls and plan their own experiments appropriately. Ultimately, the goal is to have open-access information available from each stage of every project: from biofluid, RNA, and extracellular vesicle (EV) collection, isolation, and storage to downstream analyses such as RT-qPCR and RNA sequencing.

If you are a CSF researcher, please contact us so that we can work with you as well!

Highlights of recent CSF Consortium efforts
Saugstad et al. (2017) recently demonstrated the strength of the CSF consortium. In a collaboration between three institutions (UC San Diego, Oregon Health & Science University, and the Translational Genomics Research Institute), researchers worked together to characterize the EV and RNA composition of identical pools of CSF at each institute from patients with five different neurological disorders. This work in parallel allowed the groups to identify potential sources of variability in protocols including sample preparation, RNA isolation, and quantification of RNA via RNA sequencing and RT-qPCR. The study identified changes in EVs and RNA in the disease CSF samples and detected an enrichment of microRNAs and mRNAs related to disease in both EV and total RNA. The paper highlights the importance of stringent standard operating procedures, including the use of common standard sample collection and data analysis protocols across institutions.

In other work, Figueroa et al., 2017 performed a multi-institutional study of RNA extracted from CSF-derived EVs of patients with glioblastoma (GBM), a very aggressive form of brain cancer. (See this related blog on glioblastoma.) A key diagnostic biomarker in classical GBM is the functional status of the Epidermal Growth Factor Receptor (EGFR). This cell-surface receptor is the starting point of a series of signaling pathways related to cell growth. When its expression surges or it folds incorrectly, the result is cells with hyper-active signaling that never stop growing. This study involved the development of a liquid biopsy that scans RNA extracted from CSF EVs for tumor-associated amplifications and mutations in EGFR. The test has very high specificity and fair sensitivity: it almost never incorrectly flags a healthy patient as having GBM and correctly identifies almost two thirds of GBM sufferers. The clinical standard for diagnosis of GBM is magnetic resonance imaging (MRI), which correctly classifies most brain tumors, but in too many cases incorrectly suggests that healthy brain tissue might be cancerous. The complementarity of highly sensitive MRI and highly specific RNA liquid biopsy argues that updating the standard of care to include collection of CSF and brain images at the same time would better separate healthy from diseased brain tissue.

The CSF Consortium is casting its nets wider in its fight against glioblastoma, looking at molecules beyond EGFR in the attempt to develop an RNA-based diagnosis tool for GBM. Akers et al., 2017 developed a diagnostic panel of 9 miRNA biomarkers by analyzing the EV RNA from 135 CSF samples in 3 cohorts, followed by validation of the miRNA panel in 60 CSF samples from 2 cohorts. The researchers found that even with that fairly large sample size, the miRNA profiles in lumbar and cisternal CSF — fluid collected from the spine or the base of the neck, respectively — are significantly different, which is problematic, since cisternal CSF is much more difficult to collect. On the other hand, they also found that RNAs extracted from raw CSF had a similar profile and diagnostic power as RNAs extracted from vesicles after an initial EV purification step, which might simplify the translation of this biomarker research into the clinic.

References
Akers, J.C. et al. A cerebrospinal fluid microRNA signature as biomarker for glioblastoma. Oncotarget (2017) 8: 68769-68779.
Figueroa, J.M et al. Detection of wtEGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients. Neuro. Oncol. (2017) Advance online publication. doi: 10.1093/neuonc/nox085
Saugstad, J.A. et al. Analysis of extracellular RNA in cerebrospinal fluid. J. Extracellular Vesicles (2017) 6: 1317577.

Glioblastoma multiforme is the most common type of malignant brain tumor. These tumors actively divide and send invasive cells throughout the brain. This migration complicates patient treatment, because simply removing the tumor does not clear the brain of migrating cells, which can initiate new tumors elsewhere. This problem has nearly halted progress in prognosis or treatment of glioblastoma over the past 20 years, and has afflicted many of our most important political figures, including Ted Kennedy and John McCain. This astonishing lack of improvement in treatment has motivated Dr. Xandra Breakefield and her team to explore an alternative pathway for treatment. Dr. Breakefield discussed her work in a recent Wednesday afternoon lecture at NIH.

Saboteurs

To start their investigation, Dr. Breakefield gathered a “fresh” sample of glioblastoma multiforme that came straight out of the operating room. The idea of studying a fresh sample was novel; previously, samples were a few hours old or from mouse models. Examining fresh samples in a living condition revealed that glioblastomas are extremely physically active, constantly extending protrusions from the surface of the tumor cells. These protrusions turn into extracellular vesicles (EVs) of many sizes, released at the rate of about 10,000 vesicles per cell per day. When profiled, these EVs were found to contain RNAs, enzymes, transcription factors, other proteins, and many other cellular components. The team hypothesized that the EVs released by the tumor must somehow promote tumor progression.

Using a fluorescent tag that labels both cell and vesicle membranes, they tracked the tumor EVs in the living brain of mice and found that many of them are taken up by surrounding healthy myeloid cells — microglia and macrophages. Macrophages are the primary form of defense in the central nervous system. Normally functioning microglia are sentinels, warriors, and nurturers. As sentinels, they actively scan the brain for damage, then rush in to nurture injured areas to repair the damage. In their warrior role they kill and eat invading cells. Ironically, a higher density of microglia and macrophages in a tumor results in a worse prognosis. They are attracted to but then subjugated by the glioblastoma, and are coerced into an abnormal function of supporting tumor growth. Understanding how glioblastoma sabotages these immune cells and co-opts them to support tumor growth is key to ultimately finding a cure for the disease.

miR-21

Glioblastomas and their EVs typically have very high levels of miR-21, a microRNA affiliated with various cancer pathways. Dr. Breakefield and her team found that microglia grown near a glioblastoma and exposed to its EVs had higher levels of miR-21, higher proliferation rates, and lower expression of proteins from pathways involved in sensing and attacking invaders. They hypothesized that miR-21 transferred from the tumor to surrounding benign microglia by EVs plays a key role in recruiting and transforming them. Then they asked whether microglia that have taken up many tumor vesicles have a different phenotype than microglia that haven’t taken up as many. To test this question, the Breakefield lab implanted fluorescently (GFP) labelled gliomas into the brains of mice and developed a flow cytometry method to sort cells extracted from that environment based on their level of GFP. The more tumor vesicles the surrounding normal cells have taken up, the more GFP they should have. The researchers used single-cell RNA sequencing to compare the expression profile of mRNAs in microglia and macrophages with high vs. low levels of GFP, indicating uptake of tumor vesicles. They found significant differences in mRNA expression in brain microglia but not in macrophages.

The genetic pathways responsible for microglia’s sentinel role have been called the “microglial sensome” (Hickman et al., 2013). The mRNA for most sensome genes were down-regulated in microglia that had taken up more tumor EVs – thus, the tumor EVs “blinded” the microglia. Pathways for immune suppression were up-regulated – showing that uptake of the tumor EVs compromised the microglias’ warrior function. Finally, pathways for tissue repair were up-regulated – indicating that the tumor EVs co-opted the microglias’ nurturing function to support the tumor instead of normal brain cells.

Biomarkers

EVs are a promising biomarker for earlier diagnosis of disease. They are released by all cells and found in all biofluids. They can contain a wide variety of different information, including cell specific proteins and RNAs.

Dr. Breakefield was interested in finding biomarkers for glioblastoma that could be found in the bloodstream or other accessible biofluids. She targeted EGFRvIII, a deletion in the EGFR gene quite common in glioblastomas, and IDH1/2, a single point mutation that results in lower grade tumors with better prognosis. Her team is examining cerebrospinal fluid (CSF) and serum from patients to identify the presence or absence of EGFRvIII with promising success. Examining exRNA and free circulating DNA in plasma, her team was able to differentiate IDH1/2 mutant carriers from healthy volunteers. That work is being developed into a clinical tool for earlier diagnosis of low-grade gliomas which have a better prognosis and different treatment options than non-IDH1/2 gliomas.

Therapy EVs are proven, effective therapies for a variety of diseases. They can be obtained from many cell types and protect their fragile molecular cargo when administered into the body. They can also be efficiently taken up by specific target cells in vivo. EVs have been used therapeutically in immune modulation, tissue repair, and antigen presentation for vaccination.

After working on glioblastomas for an extended period, Dr. Breakefield decided to study schwannomas, a related but benign class of tumor. There are three types of hereditary diseases that stem from schwannomas: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), and schwannomatosis. These diseases cause motor dysfunction, pain, and potential hearing loss.

In NF2, tumors form along nerve fibers, including those at the base of the spine. The current treatment is surgical removal of the tumor, but that can cause irreparable nerve damage. Because these tumors are not malignant, reducing their size could be an effective alternative treatment.

Dr. Breakefield and colleagues made a model system using human Schwann cells from an NF2 patient. They implanted those cells in the sciatic nerve of mice, forming tumors. They then created an adenovirus-associated virus (AAV) vector containing the pro-inflammatory enzyme caspase 1 attached to a promoter (P0) exclusively active in Schwann cells. They then injected this vector into the tumors to try to shrink them.

In this model system, the tumor grew when the AAV-GFP was injected. It regressed when AAV-P0-ICE was injected. P0-ICE creates a “bystander” effect, in which cells surrounding the injected cell are also killed. Surprisingly, Schwann cells appeared completely normal after treatment with P0-ICE, while the tumor (schwannoma) shrunk after injection of the tumor with AAV-P0-ICE. Although the EVs’ role in this finding is not known, Dr. Breakefield theorizes that caspase-1 is incorporated into the EVs and transferred to surrounding schwannoma cells.

There is still much to be discovered about EVs’ roles in tumors, both benign and malignant. We have not yet found glioblastoma’s Achilles’ heel, but further research may yet do so. Dr. Breakefield looks forward to continuing her work and potentially creating better treatment options for those afflicted by these devastating tumors. The previously unrecognized role of the tumor-derived EVs in transferring genetic information and co-opting other cells in their microenviroment may be an important key.

Early detection of viral infections is extremely important for control of disease transmission, prompt initiation of treatment, and prevention of infection-related complications. Because of our hypothesis that viral DNA, messenger RNA, and proteins cannot be detected in all infected individuals, we wanted to determine whether detection of exogenous miRNAs encoded by viruses represents a more sensitive assay of the true prevalence of infection by viruses including latent Kaposi Sarcoma Herpes Virus (KSHV) and Epstein Barr Virus (EBV). Therefore, we measured plasma miRNAs using RT-qPCR and compared it to the current standard method for detection of viral infection, an enzyme-linked immunosorbent assay (ELISA) of blood plasma, which detects antibodies generated by the host against the infecting virus. Our study population was 214 Caucasian patients from the United States and Romania, separated into four independent patient cohorts. We examined a total of 300 plasma samples from this population. This study enabled us to develop an approach to detect infection by KSHV using multiplexed RT-qPCR of multiple viral miRNAs.

We found that our method had clear advantages over the current ELISA-based approach. It detected a significantly higher prevalence of KSHV infection than that determined by seropositivity, with the difference most pronounced in immuno-depressed patients. When applied to EBV, our new method based on plasma viral miRNA quantification proved that EBV infection is ubiquitous. This strategy has the potential to become a gold standard method in clinical practice to detect latency of viruses and viremia — viral infection of the bloodstream — in both general and immune-compromised populations.

Detecting viral infection by amplifying viral miRNA has clear advantages over the current ELISA-based approach.

Fuentes-Mattei E, Giza DE, Shimizu M, Ivan C, Manning JT, Tudor S, Ciccone M, Kargin OA, Zhang X, Mur P, do Amaral NS, Chen M, Tarrand JJ, Lupu F, Ferrajoli A, Keating MJ, Vasilescu C, Yeung SJ, Calin GA. Plasma viral miRNAs indicate a high prevalence of occult viral infections. EBioMedicine. (2017) 20:182-192. doi: 10.1016/j.ebiom.2017.04.018. Pubmed: 28465156.

Extracellular vesicles, such as exosomes and microvesicles, are small vesicular particles that are constantly being produced and shed by cells. Due to their natural origin, and their ability to efficiently deliver their cargo to target cells and alter biological functions, exosomes attracted researchers to study their potential use as drug delivery systems. In the past few years, numerous studies have reported the effective use of exosomes to deliver therapeutic cargo ranging from miRNA, siRNA and even small molecule drugs in both in vitro cell models and in vivo animal models. However, since exosomes are cell-derived vesicles, it is unclear how these natural carriers of biomolecules may induce immune responses or induce toxicity either in animal models of disease or eventually in humans as we progress toward clinical evaluation of exosomes in healthy volunteers or in patients. Furthermore, what can we conclude about the presence or lack of immunogenicity or toxicity in our animal models as we work toward delivery of exosomes in humans?

Our lab has been studying the production of therapeutic exosomes using genetically engineered HEK293T cells for treatment of hepatocellular carcinoma (HCC). We developed engineered HEK293T cells that endogenously package miR-199a-3p, a miR commonly downregulated in HCC, into exosomes, and we are evaluating these and also exosomes exogenously loaded with therapeutic miRs in vitro and in vivo. Although demonstrating in vivo efficacy is a major milestone for all drug development efforts, understanding the potential toxicities and immunogenic responses associated with exosome therapy is equally important. The ability to identify and characterize adverse responses in preclinical models is critical to the drug development process and a necessary component of an Investigational New Drug (IND) application. Therefore, approaches to characterizing potential toxicities and immune responses induced by exosomes will be a necessary component of any effort to develop therapeutic exosomes.

In our article, “Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells,” that was just published online in the Journal of Extracellular Vesicles, we provide a general template process for comprehensively evaluating toxicity and immunogenicity of therapeutic exosomes in preclinical animal models. We started by dosing mice with wild type or engineered HEK293T-derived exosomes over a period of three weeks. Mice received 10 doses via intraperitoneal and intravenous routes of injection, and blood samples were collected at various times throughout the 3-week study. Animals were euthanized 24 hours after the last dose, and blood and all organs were collected from each animal for gross necropsy and evaluation of various markers of immune response and potential exosome-induced toxicity markers.

This study demonstrates one approach to immunogenicity and toxicity evaluations of human-derived exosomes in mice, and it highlights some of the variables that must be considered during these evaluations. For example, what are appropriate animal models in which to study immunogenicity and toxicity? What doses and dose regimens should be evaluated? How might the cell type from which the exosomes were harvested impact immunogenicity and toxicity? Just as with efficacy evaluations in animals, comprehensive study of these other factors will be necessary for safely moving therapeutic exosomes into human trials.

Acute liver failure is a potentially fatal consequence of severe liver injury. Liver transplantation may be necessary for survival if the liver injury exceeds the ability of the liver to regenerate (also called fulminant liver failure). New therapeutic interventions are needed to enhance tissue regeneration and improve the outcome of acute or fulminant liver failure. Previous studies have reported that mesenchymal stem cell (MSC) transfusions can improve function in liver facing acute failure. Stem cells can grow into multiple cell types and may support the replacement of functional liver tissue. In addition, paracrine effects — signalling between nearby cells — resulting from the release of soluble factors and extracellular vesicles (EV) may contribute to some of the beneficial effects observed with stem cell therapies.

A new study by Haga et al. reports on the beneficial effects of EV derived from stem cells. EV were isolated using classical ultracentrifugation methods. To mimic liver injury, D-galactosamine and recombinant tumor necrosis factor-α were injected into male mice. Subsequently, systemic administration of EV was shown to result in a dramatic improvement in survival. Whereas control animals receiving placebo showed complete lethality within 12 hours of D-galactosamine/TNF-α injection, the mice injected with mouse stem-cell-derived EV had a 57% survival at 24 hrs. When human MSC-EV were administered, a 37.5% survival was noted. Also noteworthy, survival was observed even with EV that had been cryopreserved. The EV reduced hepatic inflammation, likely by protecting the hepatocytes from apoptosis and recruiting Kupffer cells that protect from liver injury. The figure shows an overview of this process. Some of the beneficial effects were shown to be mediated by Y-RNA-1, a long non-coding RNA that is enriched within MSC-EV.

Stem-cell-derived extracellular vesicles repair tissue after acute liver failure

The beneficial effects of stem cells, mediated through EV and their RNA content, in severe injury models provides new avenues for investigation of the pathophysiology of liver injury and inflammation. These observations provide a very compelling justification for the future use of MSC-EV as therapeutics for severe liver injury.

Reference:
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T. Stem Cells Transl Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.

The role of extracellular vesicles (EVs) in cancer has recently become a promising area of research. A primary function of EVs is to deliver molecules from a donor cell to regulate cellular processes in a target cell. Little research has investigated what effect departing EVs may have on the donor cell.

In cancer, EVs from tumor cells can deviate from their original purpose. Altering vesicular content could benefit tumors, for example by increasing tumor proliferation or strengthening drug resistance.
 

Exosomal Packaging
Could donor tumor cells selectively shunt cancer-fighting molecules into secreted vesicles to escape their effects? Teng et al. thought this might be the case. They hypothesized that tumor cells specifically secrete tumor suppressor miRNA, namely miR-193a, into exosomes, a class of EV secreted via the endocytic membrane transport pathway, while oncogenic miRNAs are kept.

To test this theory, they validated miR-193a’s function in a mouse model. Specifically, they found that miR-193a targets Caprin1, a cell-cycle-associated protein, and arrests the cell cycle in phase G1. Thus, secreting miR-193a from the cell in exosomes would restart the cell cycle and enable a tumor cell to proliferate.

After studying miR-193a’s primary function, the authors explored what might facilitate its secretion. They showed that MVP (major vault protein) complexes with miR-193a and that knock-down of MVP leads to higher levels of miR-193a in the cell and less miR-193a in exosomes. They concluded that MVP mediates the sorting of miR-193a into exosomes. They also found that a higher level of MVP in the cell correlates with lower levels of miR-193a and higher levels of Caprin1, indicating that MVP aids in cell proliferation. Lastly, the researchers determined in a mouse model that export of miR-193a by MVP promotes metastasis of colon cancer to the liver.

Figure 1 shows a model of these interactions. In the pre-metastatic cell, miR-193a is freely expressed. In the tumor metastatic cell, MVP has complexed with miR-193a, driving it into exosomes to be secreted.

Figure 1: Model for the mechanism of colon cancer metastasis to the liver. Tumor suppressor mir-193a is sorted into exosomes by Major Vault Protein (MVP) and then secreted from the cell.

Figure 1: Model for the mechanism of colon cancer metastasis to the liver. Tumor suppressor mir-193a is sorted into exosomes by Major Vault Protein (MVP) and then secreted from the cell.


 

Cancer Biomarker
This finding implicates exosomal miR-193a as a potential biomarker for colon cancer. Could higher levels of exosomal miR-193a indicate a more aggressive disease? To answer these questions, the authors applied their findings in the clinical setting. Teng et al. examined the livers of mice with metastatic colon cancer, performing an extensive characterization of the levels of tumor suppressive and oncogenic miRNAs in normal and tumor cells and in the exosomes secreted by both. From that extensive analysis, they chose three miRNAs upregulated (miR-193a, miR-126 and miR-148a) and one miRNA downregulated (miR-196b) in exosomes from tumor cells and examined the levels of those miRNAs in exosomes purified from the blood (plasma) of 40 colon cancer patients, 15 with metastasis to the liver and 25 without. They found the same upregulation and downregulation of the 4 miRNAs in the patient population, with higher levels in the population with liver metastasis.

This exciting study identifies potential biomarkers for colon cancer. The two main findings highlight the importance of exosomes in cancer proliferation. First, the authors found that tumor suppressing miRNAs are packed into exosomes, while oncogenic miRNAs remain in the cell. Next, they found that there are higher amounts of tumor suppressing miRNAs in tumor-derived exosomes relative to exosomes from healthy cells. They also found that MVP acts as a mediator of these differences between metastatic and healthy cells. Teng et al. believe that once we develop a dependable method to purify exosomes, scientists may uncover further roles for exosomes in cancer progression.