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

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

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

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

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

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

References

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

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

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

This post originally appeared in Bioquick News.

The 2018 annual meeting of the American Society for Exosomes and Microvesicles (ASEMV) was held October 20-24 in Baltimore, hard by the water’s edge in the Baltimore Marriott Waterfront Conference Center. ASEMV president, Stephen Gould, PhD, Professor of Biological Chemistry & Co-Director, Graduate Program in Biological Chemistry, Johns Hopkins, reported a record attendance of 250 scientists from the United States and around the world (Korea, Norway, Sweden, Canada, Australia, Japan, UK, Italy, Portugal, The Netherlands) at this historically intimate and highly interactive meeting that benefits greatly from having communal meals and no overlapping sessions. The five-day meeting featured over 100 podium presentations and myriad posters. The daily consecutive sessions typically ran from 8.30 in the morning to 9.30 in the evening, and were followed by two hours of poster viewing and interaction among researchers and with sponsors. The communal meals and poster sessions offered excellent opportunities for significant interaction amongst conference participants and also for interaction between attendees and the over 20 companies (see below) that were sponsors of the meeting. Dr. Gould highlighted the key role of these sponsors in enabling this very special meeting, and noted that this year featured record sponsorship, with almost triple the number of sponsors relative to the number for last year’s meeting at Asilomar in California. This impressive increase in sponsorship is a reflection of the recent explosion of research and interest in exosomes from many quarters of medicine and science.

Among the themes of this year’s meeting was the growing appreciation for the heterogeneity of exosomes/microvesicles in terms of content, surface markers, size, and function. The similarities between exosomes and viruses were discussed in a number of talks. The brain’s use of exosomes for cell-to-cell communication within the brain, and also to communicate beyond the brain, was highlighted in multiple presentations. One of these suggested the dual promise of extracellular microRNAs in the diagnosis and pathology of Alzheimer’s disease. The role of exosomes in metastasis, carrying information from primary cancer cells to sites of future metastasis, was discussed and presented as further strong support for the century-old “Seed & Soil” hypothesis advanced originally by London surgeon Stephen Paget in 1889 (https://en.wikipedia.org/wiki/Stephen_Paget). Dr. Paget’s original article was titled “Distribution of Secondary Growths in Cancer of the Breast” (Paget, 1889).

One presentation described EVs as epigenetic mediators of systemic communication in murine experimental sepsis and another, by sepsis expert Antonio De Maio, PhD, Professor and Member of the Biomedical Sciences Program at the University of California San Diego, described how phospholipids within EVs may contribute to the activation of target cells. Dr. De Maio, a graduate of the Central University of Venezuela in Caracas, had previously been Associate Professor and Research Director for the Division of Pediatric Surgery at Johns Hopkins, where he had also led the Committee for the Recruitment of Under-Represented Minorities to Graduate Programs. At UCSD, Dr. De Maio is also Director of the Initiative to Maximize Student Diversity Program at the university. At UCSD, Dr. De Maio’s laboratory focuses on the molecular and genetic bases of the response to injury.

Two presentations on tick exosomes and two on bacterial outer membrane vesicles highlighted the broad spectrum of exosome significance throughout the kingdoms of life. An opening night presentation suggested that vesicle-cloaked virus clusters are the optimal units for inter-organismal viral transmission.

The role of exosomes in glioblastoma was the subject of multiple presentations. Janusz Rak, MD, PhD, Senior Scientist in the Child Health and Development Program, and Professor, Department of Pediatrics, McGill University, began the Sunday morning sessions with a talk on the role of EVs in the evolution of glioma-initiating cells. Quantification of cancer EV populations using super-resolution microscopy was another highlight of Sunday morning talks.

ARC is repurposed retrotransposon Gag protein that mediates intercellular RNA transfer in brain

Paul Worley, MD, Professor of Neurology at Johns Hopkins and an expert on the molecular basis of learning and memory, with a focus on cellular mechanisms that support synapse-specific plasticity, opened the Sunday evening session with a highly stimulating discussion of how the neuronal gene ARC encodes a repurposed retrotransposon Gag protein that mediates intercellular RNA transfer. Dr. Worley described evidence suggesting that Gag retroelements have been repurposed during evolution to mediate intercellular communication in the nervous system. Previous work had shown that the neuronal gene ARC is essential for long-lasting information storage in the mammalian brain and mediates various forms of synaptic plasticity. ARC has been implicated in neurodevelopmental disorders. It has been shown that ARC self-assembles into virus-like capsids that encapsulate RNA. Endogenous ARC protein is released from neurons in EVs that mediate the transfer of ARC mRNA into new target cells, where it can undergo activity-dependent translation. Purified ARC capsids are endocytosed and are able to transfer ARC mRNA into the cytoplasm of neurons. ARC exhibits similar molecular properties to retroviral Gag proteins. Evolutionary analysis has indicated that ARC is derived from a vertebrate lineage of Ty3/gypsy retrotransposons, which are also ancestors to retroviruses.

Sensational Tuesday evening

Tuesday evening featured a number of riveting presentations in a sensational session moderated by Xandra Breakefield, PhD, Professor of Neurology, Harvard Medical School, and Geneticist, Massachusetts General Hospital. A presentation on the use of machine learning-assisted histopathology to categorize large oncosomes held the audience spell-bound. Another suggested that EVs serve as delivery vehicles for LINE-1 retrotransposons.

Dr. Tushar Patel, Dean of Research at the Mayo Clinic-Jacksonville and an expert on liver cancer and liver transplants, had opened the session with a discussion of how biological nanoparticles might serve as therapeutic agents.

Other presentations in this session included ones on tools for live monitoring of exosome release from single cells, on the detection of mutant KRAS and TP53 DNA in circulating exosomes from healthy individuals and patients with pancreatic cancer, and on how an infected cell tolerates its viral pathogen using the exosomal pathway.

Beach Boys performance can’t distract ASEMV attendees

The attendees’ profound interest in exosomes was indicated on the opening evening of the meeting. At the outset, in outlining the logistics of the meeting, ASEMV president Dr. Gould explained that a late-breaking room change for Saturday night’s opening session had been occasioned by concern that the original room might be too noisy due to a performance taking place downstairs by The Beach Boys. The Beach Boys? Many thought that Dr. Gould was joking. But yes, the real Beach Boys were actually playing at a benefit event just downstairs from the ASEMV opening session, and yet, such was the audience’s interest in exosomes that no one moved. This reporter, however, could not resist checking out the iconic band after the opening ASEMV session had ended, and the photo here was taken of The Beach Boys who were indeed playing just downstairs. One of the original band members, Mike Love, was playing keyboard and singing. The Beach Boys performance was the highlight of a gala evening sponsored by Chimes (https://chimes.org/), a Baltimore-based international not-for-profit organization dedicated to assisting people with intellectual and behavioral challenges to achieve their fullest potential. It was an awesome backdrop to what would be an awesome ASEMV meeting.

Nearby International Human Virology meeting features major session on “Exosomes in health & disease”

And one further note is that the Institute for Human Virology (IHV), headed by legendary HIV virologist Dr. Robert Gallo, was holding its 20th International Meeting in the Four Seasons Hotel, right next to the Marriott where the ASEMV meeting was held.

Further indication of the exploding interest in exosomes was that the IHV meeting held a major session on exosomes this year. Titled “Exosomes in Health and Disease,” this session was listed second among nine sessions called out for special attention on the IHV meeting web page (http://www.ihv.org/ihvmeeting/). Areas of emphasis in this session ranged from cytokines in EVs to mechanisms of EVs in viral transmission.

Chairpersons of the IHV exosome session were Robert Gallo, MD, Director, Institute of Human Virology, University of Maryland School of Medicine, US, and Leonid Margolis, PhD, Senior Investigator, National Institute of Child Health and Human Development, US.

Speakers and topics included Xandra Breakefield, PhD, Professor of Neurology, Harvard Medical School / Genetist, Massachusetts General Hospital, “Extracellular Vesicle As Advance Forces in Cancer;” Dr. Margolis, “Not All Soluble Cytokines Are Soluble: Cytokines in Extracellular Vesicles Mediate Cell-Cell Communications;” Fatah Kashanchi, PhD, Former Director of Research, George Mason University, “Presence of HIV-1 RNA in Extracellular Vesicles from HIV-1 cART-Treated Cells;” Ayuko Hoshino, PhD, Instructor of Molecular Biology in Pediatrics, Weill Cornell Medical College, “Exosomal Protein Signatures: Mechanistic Insights and Biomarker Potential;” and Yoel Sadovsky, MD, Executive Director, Magee-Womens Research Institute, University of Pittsburgh, “Placental Exosomes in Maternal-Placental-Fetal Communication and Viral Resistance.”

Sponsors of ASEMV 2018 annual meeting

Sponsors of the ASEMV 2018 annual meeting included Particle Metrix, System Biosciences (SBI), iZON, Caris Life Sciences, nanoView Diagnostics, WAKO, ReNeuron, Norgen Biotek Corporation, Millipore Sigma, Beckman-Coulter, Wyatt Technology, AcouSort, Spectradyne, Fiber Cell Systems, ONI, abcam, Ceres Nano, Nanostics Precision Health, cellex, HansaBioMed Life Sciences, Lonza, and NanoTech.

Reference
Paget S. The distribution of secondary growths in cancer of the breast. The Lancet 133: 571-573. doi: 10.1016/S0140-6736(00)49915-0


Image: Kelsey Burke

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

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

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

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

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

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

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

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

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

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

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

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

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

 

Reference

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


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

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

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

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

This post originated as a press release from Linköping University.

The waste-management system of the cell appears to play an important role in the spread of Alzheimer’s disease in the brain. A new study, published in the prestigious scientific journal Acta Neuropathologica, has focused on small membrane-covered droplets known as exosomes. It was long believed that the main task of exosomes was to help the cell to get rid of waste products. In simple terms, they were thought of as the cell’s rubbish bags. However, our understanding of exosomes has increased, and we now know that cells throughout the body use exosomes to transmit information. It’s now known that the exosomes can contain both proteins and genetic material, which other cells can absorb.

The Linköping researchers have shown in the new study that exosomes can also transport toxic aggregates of the protein amyloid beta, and in this way spread the disease to new neurons. Aggregated amyloid beta is one of the main findings in the brains of patients with Alzheimer’s disease, the other being aggregates of the protein tau. As time passes, they form ever-increasing deposits in the brain, which coincides with the death of nerve cells. The cognitive functions of a person with Alzheimer’s disease gradually deteriorate as new parts of the brain are affected.

“The spread of the disease follows the way in which parts of the brain are anatomically connected. It seems reasonable to assume that the disease is spread through the connections in the brain, and there has long been speculation about how this spread takes place at the cellular level,” says Martin Hallbeck, associate professor in the Department of Clinical and Experimental Medicine at Linköping University and senior consultant of clinical pathology at Linköping University Hospital.

Cells became diseased
In a collaboration with researchers at Uppsala University, he and his co-workers have investigated exosomes in brain tissue from deceased persons. The research team at Linköping University found more amyloid beta in exosomes from brains affected by Alzheimer’s disease than in healthy controls. Furthermore, the researchers purified exosomes from the brains from people with Alzheimer’s disease, and investigated whether they could be absorbed by cells cultured in the laboratory.

“Interestingly, exosomes from patients were absorbed by cultured neurons, and subsequently passed on to new cells. The cells that absorbed exosomes that contained amyloid beta became diseased,” says Dr. Hallbeck.

The researchers treated the cultured neurons with various substances that prevent exosomes from being formed, released, or absorbed by other cells. They were able to reduce the spread of the aggregated amyloid beta between cells by disrupting the mechanism in these ways. The methods used in these laboratory experiments are not yet suitable for treating patients, but the discovery is important in principle.

“Our study demonstrates that it is possible to influence this pathway, and possibly develop drugs that could prevent the spreading. The findings also open up the possibility of diagnosing Alzheimer’s disease in new ways, by measuring the exosomes,” says Martin Hallbeck.

The research has received financial support from donors that include the Swedish Research Council, the Swedish Alzheimer’s Foundation, and the Swedish Brain Foundation.

Sinha MS, Ansell-Schultz A, Civitelli L, Hildesjö C, Larsson M, Lannfelt L, Ingelsson M & Hallbeck M. Alzheimer disease pathology propagation by exosomes containing toxic amyloid-beta oligomers. Acta Neuropathologica AOP 13 June 2018. doi: 10.1007/s00401-018-1868-1

Translation by George Farrants.

Despite being one of the earliest known classes of non-coding RNA molecules, tranfer RNAs (tRNAs) are still notoriously difficult to study. The challenge is largely due to this molecule’s secondary structure, chemical modifications to its constituent nucleotides (see figure), and the multiplicity of tRNA genes. As the number of non-coding RNA datasets proliferates, it is becoming increasingly important for tRNA genes to be accurately annotated. In a recent study, Thomas Tuschl from Rockefeller University and colleagues tackled this problem by developing a new protocol for sequencing tRNAs. The new method enabled them to assemble an atlas of human tRNAs for other researchers to use in analyzing their non-coding RNA data.

Hydro-tRNA Sequencing
Transfer RNAs have thermodynamically stable secondary and tertiary structures, and their constituent nucleotides are highly modified by RNA editing. Both of these characteristics are problematic for traditional RNA sequencing methods. The key to the Tuschl lab’s protocol, called hydro-tRNA sequencing (hydro-tRNAseq), is a partial alkaline hydrolysis step that breaks the 60-100 nucleotide-long tRNA into smaller fragments with fewer RNA modifications. These fragments, 19-35 nucleotides in size, have weaker secondary structure and fewer RNA modifications per fragment than the parent tRNA.

Applying the method to short RNA extracted from human embryonic kidney (HEK293) cells resulted in an increase in the fraction of reads mapped to tRNA between 2% and 40%, depending on the depth of sequencing. The short fragment length also improved read accuracy per base compared to standard tRNA sequencing.

To develop a thorough and representative reference set of human tRNAs, the HEK293 dataset was subjected to iterative cycles of mapping to existing reference tRNAs followed by manual curation. In each round, all transcripts with an error distance (number of mismatches, insertions, and deletions) of 1-2 from a given tRNA were kept as candidate reference sequences if they could be attributed to a tRNA isoacceptor (i.e. a different tRNA that binds to the same amino acid). If not, assuming that other mismatches were caused by misidentifying a modified base, transcripts with more than 10% mismatches compared to reference were expanded into a set of all possible combinations of RNA modifications and included in the reference pool (see figure). This mapping and selection process was repeated until there were no longer any modified positions left with a mismatch frequency over 10% compared to reference.

Candidate pre-tRNA genes were obtained by mapping the final tRNA reference sequences back to the genome. Altogether, this analysis was able to account for 93% of the 114 million reads in the deepest library of HEK293 cells’ tRNAs.

tRNA Modification Sites

tRNA Modification Sites
The team identified sites of modification from the high frequency of mismatches during mapping caused by read errors there during reverse transcription. Here the reference nucleotide is at ring center, known modification outside the ring, and frequency of each nucleotide read at that site inside the ring.
Source: Cell Reports

The Added Power of SSB PAR-CLIP
Though hydro-tRNAseq greatly improved the reference dataset of human tRNAs, there was still a risk that it alone would miss pre-tRNAs expressed at low levels or processed quickly into mature tRNA. Previous efforts to assay that ephemeral population employed ChIP-seq of POLR3, the polymerase that transcribes all tRNA genes, but doing so assumed that polymerase binding always led to expression and complete processing. The Tuschl lab focused instead on SSB, a protein that binds to the 3′ end of pre-tRNAs, immunoprecipitating tRNAs crosslinked to SSB using a method called PAR-CLIP. As predicted, almost half of the reads from their SSB PAR-CLIP experiments mapped to pre-tRNAs. Combining SSB PAR-CLIP with hydro-tRNAseq allowed the team to better identify mature and pre-tRNAs with improved, accurate, nucleotide-level resolution.

This study supplies the community with several new and useful resources. Hydro-tRNAseq provides a new method to overcome many of the struggles of tRNA sequencing analyses. Combining this method with SSB PAR-CLIP enabled the construction of a comprehensive atlas of pre-tRNAs and mature tRNAs in humans. This methodology can now be applied to study the tRNA complement in other species to further dissect tRNA biology.

Reference
Tasos Gogakos T, Brown M, Garzia A, Meyer C, Hafner M, & Tuschl T. Characterizing Expression and Processing of Precursor and Mature Human tRNAs by Hydro-tRNAseq and PAR-CLIP. Cell Reports (2017) 20: 1463-1475. doi: 10.1016/j.celrep.2017.07.029

(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.


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.

Dr. Alissa Weaver, Vanderbilt University professor and Extracellular RNA Communication consortium (ERCC) member, will be inducted as an AAAS Fellow this Saturday, February 18, 2016. Dr. Weaver joins Dr. James Patton, also of Vanderbilt, and Dr. David Wong of UCLA as consortium members who are also current AAAS Fellows. This honor is bestowed upon her for her contributions to the field of cancer biology and studies of extracellular vesicles (EVs) in cell motility and cancer metastasis.

Alissa Weaver

Dr. Weaver’s academic career began at Stanford University where she double majored in Biology and Political Science. Always aspiring to be a physician, she then attended medical school at the University of Virginia, Charlottesville. However, along the way, she realized that she missed the academics of a PhD. “When I was in medical school, I realized that I really missed thinking about scientific discovery and was not being taught to do research,” she explained. “I really wanted to have the formal training of getting a PhD so I applied for the program from medical school.” After completing her MD/PhD at UVA, she traveled to Washington University, Saint Louis for 5 years where she did a Laboratory Medicine residency and a postdoctoral fellowship in the Department of Cell Biology and Physiology with Dr. John Cooper.

Finally in 2003, she accepted a faculty position at Vanderbilt University where she now remains as a full time researcher. Her lab focuses on all aspects of extracellular vesicles. The interest originally stemmed from her investigations of cell invasion, migration and cancer metastasis. The lab’s focus shifted as they learned that many of the secreted molecules that facilitated invasion were transported by EVs.

Part of the invasive nature of cancer cells in metastasis involves structures called invadopodia, actin-based protrusions of the plasma membrane that facilitate degradation of the extracellular matrix. For cells to invade, they secrete matrix-degrading proteinases. Work in Weaver’s lab demonstrated that not only were these proteinases carried by EVs but that hot spots for their secretion actually aligned with invadopodia.

Specifically, Weaver’s lab established that invadopodia are important sites for the docking and secretion of exosomes. Exosomes are extracellular vesicles secreted from many different cell types. They originate from multivesicular bodies (MVB), which are mature endosomes that contain many smaller vesicles. Secretion of exosomes occurs when these MVBs fuse with the cell membrane, releasing the molecules contained inside. Though normal cells may use environmental cues to regulate exosome secretion, cancerous cells constitutively turn it on.

Exosome cargoes mediate invadopodia biogenesis, stability, and activity

Exosome cargoes mediate invadopodia biogenesis, stability, and activity.
Source: Hoshino, et al. Cell Rep 2013

“One of the big questions we are working on is the cell biological aspects of these vesicles,” Weaver explained. “How they are made, how cargo gets sorted there, and what does that mean for their biological function after they are secreted? So that is where our work with the ERCC comes in.”

She hopes that working with the scientists of the consortium, they can understand how RNA and RNA binding proteins are trafficked into vesicles. Last year, in a paper published in Cell Reports, her group demonstrated one possible mechanism for the sorting of microRNAs into EVs. They demonstrated that Argonaute 2 (Ago2), part of the RISC machinery that binds to miRNAs, is transported in microvesicles and exosomes. Organization of Ago2 into exosomes is regulated by KRAS-MEK signaling. Dr. Weaver highlighted the study in a blog here mid-last year.

Despite these initial findings, Dr. Weaver admits it is difficult to determine how important extracellular RNA and miRNAs are in regulating cancer metastasis. “I honestly don’t think we know yet, and I think that the field is just now really trying to figure out what are the cargo components that are driving all of these phenotypes we have been trying to characterize so well.” She elaborated, “I think for both the protein and the RNA, the next big step for the field is trying to pin individual EV functions back to specific cargo molecules.”

Asked to reflect on her AAAS fellowship, Dr. Weaver turned the focus on her colleagues in the consortium. “I continue to be very impressed by the quality of investigators and the research being done by the ERCC. I mean really top people who are driving forward what I think is a tough problem.” She and fellow AAAS fellows Dr. Patton and Dr. Wong are, as Dr. Weaver pointed out, “just a small snapshot of fabulous investigators that are part of the consortium.”

Immunology 2016
Immunology 2016

Extracellular RNA was a hot topic of discussion at Immunology 2016, the annual meeting of the American Association of Immunologists (AAI), held at the Washington State Convention Center in Seattle, Washington May 13-17th, 2016. The National Cancer Institute (NCI) sponsored a symposium on “Extracellular RNA in the Immune System”, co-chaired by Dr. Kevin Howcroft (Division of Cancer Biology, Cancer Immunology, Hematology, and Etiology Branch, NCI) and K. Mark Ansel (University of California San Francisco – your faithful blogger). Four invited speakers presented and participated in lively discussion with an audience of gathered experts and curious newcomers to the field of extracellular RNA.

Dr. Gyongyi Szabo (University of Massachusetts) opened the symposium with a presentation of her laboratory’s work on extracellular vesicles and miRNAs in innate immune cell communication in the liver. Alcohol exposure induces liver inflammation, marked by release of pro-inflammatory cytokines and activation of myeloid cells, including Kupffer cells, the resident macrophages of the liver. In a mouse model, alcohol consumption increased expression of miR-155 in both macrophages and hepatocytes via TLR4 and NFκB-driven transcription. Inhibition or genetic deletion of miR-155 in this model blunted macrophage activation and cytokine production. Exosomes loaded with miR-155 mimetics could be delivered to hepatocytes and other liver cells to correct some of the defects observed in miR-155-deficient animals. Remarkably, endogenous miR-155 and miR-122 were elevated in serum collected after controlled “binge-drinking” in human study subjects, and these exosomes also conveyed information to cultured monocytes, altering their production of TNF and IL-1. Together these data suggest that extracellular communication between hepatocytes and innate immune cells via exosomal miRNAs regulates inflammation in response to alcohol consumption.

The theme of regulation of inflammatory responses by miRNA-containing exosomes was extended by Dr. Ryan O’Connell (University of Utah). His pioneering work on miR-155 and miR-146 demonstrated their opposing roles in inflammatory processes mediated by various cell types in several tissues and disease settings. Recent work in his laboratory showed that both of these miRNAs are released by bone-marrow-derived dendritic cells in a fashion dependent on Rab27 and neutral sphingomyelinase (N-SMase) activity, and that these miRNAs could be exchanged between cells separated by a filter that prevents cell-cell contact. Transferred miR-146a reduced recipient cells’ response to bacterial lipopolysaccharide, a classical innate immune stimulant in vitro and in vivo. In addition, transferred miR-155 was found to directly repress the 3’ UTR of target genes in recipient cells, supporting the possibility that functional miRNA transfer via exosomes could be used as a therapeutic modality for regulating inflammation. Getting these miRNAs to the right cell types in vivo remains an important challenge to bringing this technology to the clinic.

In addition to exosomes, high density lipoprotein (HDL) particles carry miRNAs and other extracellular RNAs in blood. Abnormal pro-inflammatory HDL is associated with systemic lupus erythematosus (SLE). Dani Michell (Vanderbilt University), a postdoctoral fellow in Kasey Vickers’ laboratory, discussed her work, conducted in collaboration with Amy Major’s laboratory, on miRNAs in HDL in SLE. HDL from subjects with SLE contained increased levels of miR-22-3p and miR-192-5p compared with HDL from healthy control subjects. Blocking miR-22 with locked nucleic acid inhibitors in vivo reduced spleen size and interferon production, and affected some clinical features in a mouse model of lupus. Experiments aimed at defining source and recipient cells in this system indicated that monocytes are much better than T lymphocytes at taking up HDL-associated miRNAs. It will be interesting to learn how HDL-associated miRNAs regain gene regulatory function in recipient cells.

The final presentation focused on lymphocytes as source cells for naturally occurring exRNAs in body fluids. Immuno-compromised mice with a mutation that specifically blocks lymphocyte development exhibit altered serum extracellular miRNA profiles. In support of the idea that lymphocytes themselves are an important source of ex-miRNAs, the most reduced exRNA species detected was miR-150, a miRNA highly expressed by lymphocytes. Activated T lymphocytes secrete vesicles that are enriched for tRNA fragments and miRNAs including miR-150. Rigorous purification revealed that these vesicles have characteristics of exosomes, including defined density, size, and protein markers including the tetraspanin CD9. Cellular fractionation also revealed tRNA fragment and miRNA enrichment in membrane fractions containing multivesicular bodies. Whether these extracellular lymphocyte-derived RNAs mediate cell-to-cell communication or not, signal-mediated reduction of cellular miRNAs certainly alters gene regulation in activated T lymphocytes. Thus, exRNA secretion may have important roles in regulating inflammatory processes in both source and recipient cells.

These topics will certainly remain on the mind of immunologists that attended the exRNA symposium — at least until Immunology 2017, to be held in Washington DC next May.

Non-coding RNAs (ncRNAs), for example microRNAs (miRNAs), are frequently dysregulated in cancer and other diseases, and have shown great potential as tissue-based markers for cancer classification and prognostication. ncRNAs are present in membrane-bound vesicles, such as exosomes, in extracellular human body fluids. Circulating miRNAs are also present in human plasma and serum and cofractionate with the Argonaute2 (Ago2) protein and high-density lipoprotein (HDL). Since miRNAs and other ncRNAs circulate in the bloodstream in highly stable forms, they may be used as blood-based biomarkers for cancer and other diseases. A knowledge base of non-invasive biomarkers is a fundamental tool for biomedical research in this field.

In 2012, miRandola was developed as the first database of circulating extracellular miRNAs (Russo et al., 2012). miRandola is a comprehensive, manually curated collection and classification of circulating extracellular miRNAs. We recently updated miRandola with 271 papers, 2695 entries, 673 miRNAs and 12 long non-coding RNAs. The future direction of the database is to be a resource for all potential non-invasive circulating nucleic acid biomarkers.

miRandola_schema

miRandola is the first online resource which gathers all the available data on circulating RNAs into one environment (see Figure). It represents a useful reference tool for anyone investigating the role of extracellular RNAs as biomarkers, as well as their physiological function and their involvement in pathologies.

The database is constantly updated as soon as new data is available, and the online submission system is a crucial feature which helps to ensure that the system is always up-to-date. We are working on a second version of the database to increase the amount of data and to improve usability. miRandola is available online at http://mirandola.iit.cnr.it/.