Year: 2016

MicroRNAs in Human Cerebrospinal Fluid as Biomarkers for Alzheimer’s Disease

Alzheimer’s Disease (AD) accounts for a large number of dementia cases resulting in impaired memory, thinking, and behavior. Risk factors for AD include age and family history, but unfortunately there is not yet a definitive way to predict if an individual will develop the disease. There are reference biomarkers that can indicate a higher risk of developing AD, such as APOE genotype. Carriers of the APOE4 allele, present in ~20% of the population, are at increased risk for AD. Cerebrospinal fluid (CSF) is a body fluid found in the brain and spine that cushions and protects the brain from injury. CSF protein biomarkers, such as Aβ42, tau and phospho-tau, are important in screening for brain disease, but these reference markers often lack the sensitivity and specificity necessary for clinical utility.

Extracellular RNA, specifically microRNA (miRNA), has been found in CSF and may serve as a useful resource for improved AD biomarkers. In a recently published study, the Saugstad lab from Oregon Health and Science University examined CSF from a large group of living donors to identify unique miRNA biomarkers enriched in AD patients. In the study, miRNA expression levels from 50 AD and 49 control subjects were assessed using TaqMan Low Density Arrays containing probes for 754 validated miRNAs. Each miRNA was given a “Multitest Score” combining the results of four statistical tests, and miRNAs that passed two or more of the tests were considered for further analyses.

Two statistical tests, log-rank and logistic regression, were used to identify candidates that were twice as likely to be associated with AD status as not. The other tests were two variants of random forest classifier, CART and CHAID, designed to select biomarker candidates able to reliably distinguish AD from non-AD status when grouped with random subsets of other miRNAs. 36 miRNA biomarker candidates were identified by at least two of these analyses. The researchers found that linear combinations of subsets of miRNA, and the addition of ApoE genotyping status, further increased the sensitivity and specificity of AD detection (Figure 1).

Figure 1. CSF miRNA biomarkers and APOE genotype predict AD status better together. AUC – Area Under the Curve; higher AUC indicates higher predictive power.

Reprinted with permission from IOS Press.

This study shows the potential use of miRNAs isolated from CSF as AD biomarkers. The stringent statistical analyses and large sample size together provided strength to these initial studies. These 36 candidate biomarkers are currently being tested in further validation studies in CSF from a new group of 120 donors, which will also include APOE genotyping and Aβ42 and tau protein levels. Ultimately, a combination of miRNA CSF biomarkers with existing reference biomarkers (APOE, Aβ42, tau) may provide a specific and sensitive tool for the diagnosis of AD in the clinic.

Citation:
MicroRNAs in Human Cerebrospinal Fluid as Biomarkers for Alzheimer’s Disease
Lusardi T, Phillips J, Wiedrick, J, Harrington C, Lind B, Lapidus J, Quinn J, Saugstad J. Journal of Alzheimer’s Disease (2017) 55: 1223-1233. doi: 10.3233/JAD-160835

11.29.16 by Rahul Mitra

Ubiquitous release of exosomal tumor suppressor miR-6126 from ovarian cancer cells

Cancer cells actively reprogram gene expression to promote their ability to produce tumors. One way this reprogramming is carried out is by subverting the main routes of cell-to-cell communication by loading exosomes (vesicles that bud off from cells) with specific miRNAs that either promote or suppress tumors and then releasing them into the tumor microenvironment. In a Cancer Research paper published online recently (Kanlikilicer et al., 2016), we found that miR-6126, a miRNA that was reported to be correlated with better overall survival in high-grade serous ovarian cancer patients, is ubiquitously removed from ovarian cancer cells via exosomes.

miR-6126 is ubiquitously removed from ovarian cancer cells via exosomes

We found that miR-6126 suppresses tumors by directly targeting integrin ß1, a key regulator of cancer cell metastasis. Treatment of orthotopic mouse models of ovarian cancer with miR-6126 reduced tumor growth, proliferating cells, and microvessel density. Our findings provide new insights into the role of exosomes in mediating tumor progression and suggest a new therapeutic approach to disrupt the origin and growth of tumors.

Citation:
Ubiquitous release of exosomal tumor suppressor miR-6126 from ovarian cancer cells
Pinar Kanlikilicer, Mohammed Saber, Recep Bayraktar, Rahul Mitra, Cristina Ivan, Burcu Aslan, Xinna Zhang, Justyna Filant, Andreia M Silva, Cristian Rodriguez-Aguayo, Emine Bayraktar, Martin Pichler, Bulent Ozpolat, George A Calin, Anil K. Sood and Gabriel Lopez-Berestein*
Cancer Res. October 14, 2016 doi: 10.1158/0008-5472.CAN-16-0714
*Corresponding author

11.22.16 by Francesco Russo

The hunt for fatal myocardial infarction biomarkers

The concept of a hype cycle is a well-established business concept, in which novel ideas are said to have an initial wave of hype followed by disillusionment. Only after that, the novel concept takes off and become truly useful entering a so-called plateau of productivity. In biomedical science, the field of microRNAs (miRNAs) certainly had a peak of interest in the end of the last decade. This led by high impact publications (1) and characterization of both novel miRNA-entities as well as their associations to a broad range of diseases. Nonetheless, no clear pharmaceutical successes emerged: miRNA targets are being pursued as therapeutic targets, but none have as of yet successfully made it through clinical trials (2). Likewise the use of miRNA-based treatment strategies targeting regular mRNA is an area of interest (3). In this editorial we focus on a third aspect of miRNAs: the use of miRNAs as prognostic biomarkers in disease, asking the question if miRNAs are now entering this plateau of productivity in which actual benefit will be seen.

We focus on the recent paper by Bye et al.: “Circulating microRNAs predict future fatal myocardial infarction in healthy individuals – The HUNT study” (https://www.ncbi.nlm.nih.gov/pubmed/27192016).

The hunt for fatal myocardial infarction biomarkers: predictive circulating microRNAs.
Russo F, Rizzo M, Belling K, Brunak S, Folkersen L.

Ann Transl Med. 2016 Oct;4(Suppl 1):S1.

PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27867969

Journal: https://atm.amegroups.com/article/view/11314

11.02.16 by Andrew Neff

Extracellular Oncogenes as Biological Effectors and Biomarkers

As part of the Extracellular RNA Communication Consortium (ERCC) seminar series, on 30 June 2016, Dr. Janusz Rak from McGill University presented his work on “Extracellular Oncogenes as Biological Effectors and Biomarkers.”

When focusing on a single cancer cell, we consider how particular mutations alter the cell’s properties, causing it to grow, replicate, evade immune detection, and do all the other things cancer cells can do. But it’s clear that cancer is more than a collection of cells operating autonomously. Perhaps the most interesting and frightening part of cancer is that tumors are complex, heterogeneous entities that operate and interact with each other and with their environment in characteristic ways. Researchers have been studying cancer cell signaling pathways for years. An example is paracrine interactions with neighboring cells that induce irregular vascular growth. What is new and exciting about Dr. Rak’s work is the focus on oncogenes as cargo in extracellular vesicles (EVs), which is a nontraditional means of communication between cancer cells and from cancer to non-cancer cells.

Dr. Rak has followed several interesting lines of research. He addresses fundamental questions; for example:

With which cell types do cancer-cell-derived EVs (CCEV) interact?
Are tumor-associated mutant proteins found on or in CCEVs?
Are these mutant proteins then expressed on recipient cells?

Preliminary work in these areas has led to the question: Can cancer cells transform non-cancer cells (i.e. make them cancer-like) via EV-mediated transfer of oncogenic proteins? And is this process complete and permanent? Numerous lines of inquiry, from in vitro to mouse studies, lead to the conclusion that the answer is probably no. Dr. Rak argues that such a finding is not surprising: if horizontal oncogenic transformation were occurring regularly between cell types, we would see more cancer patients with mixed tumor types. Secondly, phylogenetic approaches have been used to trace tumors back to single cell ancestors, another argument against the model of horizontal transformation. The current leading hypothesis is that CCEVs, rather than inducing oncogenic transformation, play a modulatory role, regulating interactions between cancer cells and between cancer and host cells but without changing their identity.

A better understanding of cancer EV biology has promise for improving cancer therapy and diagnosis. CCEVs and their oncogene cargo may represent interesting and novel pharmaceutical targets. They also constitute a unique reservoir of mutant molecules in blood, far away from an inaccessible tumor. Efforts are underway to advance cancer diagnosis through detection of CCEVs, particularly in tumor classes like glioblastoma multiforme (GBM) where no other methods of early detection are available. Research in humans has indicated that GBM patients can be identified by EVs in blood serum. As research such as Dr. Rak’s progresses, and EV biology is further elucidated, we will continue moving towards a future where we can utilize EVs in clinical practice to improve health outcomes.

09.09.16 by Keren Regev, Radhika Raheja and Roopali Gandhi

Serum microRNAs as biomarkers in Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). Currently, magnetic resonance imaging (MRI) is the most commonly used method to diagnose and monitor MS, but there is a poor correlation between MRI disease measures and clinical disability or disease progression in MS. MRI is also an expensive tool that might carry potential risks due to brain accumulation of contrast material (Kanda et al., 2015). In the last few years, a lot of effort has been invested in the identification of biomarkers for MS; however, to date, few of these findings have proven clinically useful. Thus, there is a strong unmet clinical need for objective body fluid biomarkers to assist in early diagnosis, predicting long-term prognosis, monitoring treatment response, and predicting potential adverse effects in MS.

Circulating miRNAs have been detected in several body fluids (Cortez et al., 2011) where they are highly stable as they are resistant to circulating ribonucleases (Mitchell et al., 2008). Their stability, along with the development of sensitive methods for their detection and quantification (Guerau-de-Arellano M. et al., 2012), makes them ideal candidates for biomarkers. We previously reported changes in circulating plasma miRNAs in MS patients (Gandhi R. et al., 2013). In a new study, our group investigated serum miRNAs as biomarkers in MS as part of an NCATS-funded UH2 initiative. We found that several serum miRNAs were differentially expressed in MS, were associated with disease stage, and correlated with disability.

Study Design (Figure 1): Serum from 296 participants including patients with MS, other neurologic diseases (Alzheimer’s disease and amyotrophic lateral sclerosis), inflammatory diseases (rheumatoid arthritis and asthma), and healthy controls (HC) were tested. miRNA profiles were determined using LNA (locked nucleic acid) based qPCR. MS patients were categorized according to disease stage and disability. In the discovery phase, 652 miRNAs were measured from the serum of 26 MS patients and 20 healthy controls. Those miRNAs from the discovery set that were significantly differentially expressed (p <0.05) in cases vs controls were validated using qPCR in 58 MS patients and 30 healthy controls.

Serum miRNA biomarkers in MS - Figure 1

Note: Results in the current study were normalized to the four most stably expressed miRNA across all the subjects. We agree with other blogs posted on exRNA.org suggesting that there is an immediate need to identify reference miRNA/exRNA that could be used for data normalization.

Figure 2: Differentially expressed circulating miRNAs as biomarkers in Multiple Sclerosis (MS). Up to top five miRNAs with p<0.05 are represented for each group comparison; a) MS, b) relapsing remitting MS (RRMS) and secondary progressive (SPMS) compared to the healthy control (HC), c) RRMS vs. SPMS, and d) the correlation of miRNA with the expanded disease severity scale (EDSS).

Results: We found 7 miRNAs (p<0.05 in both discovery phase and validation) that differentiate MS patients from healthy controls; miR-320a up-regulation was the most significantly changing serum miRNA in MS patients. We found 8 miRNAs that differentiated relapsing-remitting MS (RRMS) from HC. Among these, miR-484 up-regulation in RRMS patients showed the strongest association. When comparing secondary progressive MS (SPMS) patients to HC, 34 miRNAs significantly differentiated between the groups in both phases, with miR-320a up-regulation showing the strongest link. We also identified two miRNAs linked to disease progression, with miR-27a-3p being the most significant. Ten miRNAs correlated with degree of disability according to the Kurtzke Expanded Disability Status Scale (EDSS), of which miR-199a-5p had the strongest correlation with disability. Of the 15 unique miRNAs we identified in the different group comparisons, 12 have previously been reported to be associated with MS, but not in serum. Kegg Pathway Analysis showed that significant and differentially expressed miRNAs target important immune functions and are related to the maintenance of neuronal homeostasis. For example, miR-27a-3p, the strongest miRNA to distinguish RRMS from SPMS and progressive MS (PMS) (up-regulated in the relapsing form as compared to the progressive forms) shows a strong link to both the neurotrophin signaling pathway and the T cell receptor signaling pathway. Other studies have shown that miR-27a-3p targets multiple proteins of intracellular signaling networks that regulate the activity of NF-κB and MAPKs 6. As a consequence, miR-27a inhibits differentiation of Th1 and Th17 cells and promotes the accumulation of Tr1 and Treg cells (Min S. et al., 2012). It has also been shown that miRa-27-3p is up-regulated in MS active brain lesions and that the level of miR-27a-3p in CSF is reduced in patients with dementia due to Alzheimer’s disease (AD) (Frigerio C.S. et al., 2013). Of all the miRNAs, miR-486-5p was identified in the largest number of comparisons. It correlates with EDSS and is up-regulated in MS compared to HC, to other neurological diseases, and to other inflammatory diseases. This particular miRNA was found to be associated with TGF-beta signaling pathways and is a known tumor suppressor (Oh H.K. et al., 2011). miR-320a has been previously described to be highly expressed in B cells of MS patients and was suggested to contribute to increased blood-brain barrier permeability due to regulation of MMP-9 (Aung L.L. et al., 2015). Pathway analysis links this miRNA to cell-to-cell adhesion pathways, another indication that it may be linked to blood-brain barrier permeability.

The current study is the most comprehensive evaluation to date of the role of serum miRNAs as biomarkers in MS, with the largest sample size and employing two independent cohort designs. One limitation of our study is that participant subject samples were collected from a single MS center. Further external validation of our results will require investigating samples from patients at other centers. We are currently performing such multicenter studies, which may also increase the power of our results. A second limitation of our study is the relatively small number of participants who contributed to each group comparison. Future work will require larger sample sizes to ensure that we have sufficient power to detect miRNAs with smaller effect sizes. Although miRNAs have been studied in cells and the CNS of MS patients, ours is the first comprehensive investigation of serum miRNAs.

Conclusions: Our findings identify circulating serum miRNAs (Figure 2) as potential biomarkers to diagnose and monitor disease status in MS. These findings are now being tested using patient samples obtained from other international MS centers. We are now investigating the role of miRNA as biomarkers for disease prognosis and treatment response in MS.

Acknowledgements: This study is a highly collaborative project, and I thank my whole team at the Ann Romney Center for Neurologic Diseases & MS Center for their contribution. The grant TR000890 is supported by the NIH Common Fund, through the Office of Strategic Coordination / Office of the NIH Director.

08.10.16 by Andrew Neff

Fetal Bovine Serum RNA interferes with cell-culture-derived extracellular RNA

Cell culture is a staple of modern biology, and Fetal Bovine Serum (FBS) is an essential component of many cell culture protocols. A specific use for FBS is to supply nutrients to cells and to stimulate their growth. Another role of FBS in cell culture research is to represent the complexities and functionality of endogenous biological environments; however, precisely this complexity has long been a potential confounding factor for researchers. For example, cytokines in FBS can lead to the stimulation of cells, thus producing unintended experimental environments. Despite these disadvantages, FBS retains a prominent role in modern cell culture, with estimated sales as high as 700,000 liters per year. Because of its ubiquity in cell culture research, it is critical to investigate how the components of FBS may be influencing experiments and downstream analysis.

Variability and uncertainty in the composition of FBS is especially problematic for studies that evaluate cellular secretions. For example, to successfully determine the array of RNA secreted by cultured cells, we need to know the extent to which the medium is contaminated by exogenous RNA. Additionally, extracellular RNA (exRNA) is not only found distributed freely throughout the liquid medium, but it is also often found packaged inside of extracellular vesicles (EVs) or lipoprotein complexes. Therefore, in a paper released online yesterday, Wei et al. evaluated how the RNA composition of FBS might be confounding research.

The authors first evaluated exogenous RNA contamination. They grew cultures of a cell type known not to express a particular RNA, then evaluated the presence of that RNA in the culture media. If that RNA was found, its origin was probably the media itself. For example, the authors demonstrated that miR-122, a liver-specific miRNA, is present in media from cultured glioma cells, suggesting that its source is likely FBS itself. They then attempted to deplete RNA from FBS via ultracentrifugation, but despite a 24 hour spin at 100,000g, about 75% of total RNA remained in the supernatant. This result has also been found by researchers attempting to deplete FBS of RNA-containing EVs and emphasizes the difficulty of producing media truly free from contaminating RNA.

These results led the authors to ask whether existing studies have wrongly attributed the presence of exRNA to a particular experimental procedure or cell type, when it should be recognized as a component of the FBS in the cell culture media. To answer this question, the authors first broadly profiled the RNA composition of FBS using RNA sequencing. They determined that between 9% and 22% of FBS RNA mapped to the human genome, depending on the stringency of the mapping algorithm and FBS preparation. They also checked for the presence of bovine-specific RNA in existing human cell culture exRNA datasets, finding levels as high as 17%, with samples from exosomes (a type of EV) containing particularly high levels. Finally, they demonstrated experimentally that bovine-specific transcripts are taken up into cells, interfering not only with exRNA analysis but also with intracellular RNA studies.

Moving forward, a significant remaining issue is deciding how to treat conserved RNA known to be present in both FBS and the cell line under study. Switching from FBS to purely chemically defined media can help with this problem, but it is not possible for all cell types and experimental conditions. Alternatively, a quantitative analysis of the chemical composition of the media might make it possible to estimate which RNAs are secreted by the cells of interest by filtering out known FBS RNAs from the total RNA pool.

This research cautions us to be careful in the design and interpretation of experiments to identify extracellular RNAs that use FBS in culture media. The paper, Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA, released in Scientific Reports yesterday, is authored by Zhiyun Wei, Arsen O. Batagov, David R. F. Carter, and Anna M. Krichevsky.

08.01.16 by Mark Ansel

Extracellular RNA in the Immune System symposium ignites interest at 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.

07.19.16 by Jeff Franklin, James N. Higginbotham and Robert J. Coffey

Fluorescence-Activated Vesicle Sorting (FAVS) uncovers the heterogeneous nature of EGFR in exosomal subsets

Secreted RNAs leave the intracellular environment by associating with diverse vesicular and protein components. Secreted vesicles are heterogeneous and follow various routes of egress from the cell (1). Subclasses of such vesicles contain distinct cell surface proteins (2). In order to fully understand the diversity of vesicles that contain RNA, it is necessary to analyze and sort vesicle populations (3). One way to do this is by flow sorting such vesicles based on the presence of distinct vesicular surface proteins.

The ability to perform flow cytometric analysis and sorting of exosomes has been an ongoing area of controversy due to the small size of exosomes, which range in size from 40-130nm, near or below the diffraction limit of light. Nevertheless, a variety of groups have used this technique to analyze different subsets of small vesicles successfully (4-14), including proteomic analyses (4, 15-17). The efficacy of these flow-sorting experiments has been cross-validated by a variety of means, including western blots and co-localization of coincidently expressed factors. Fluorescence-Activated Vesicle Sorting (FAVS) uses light scattering properties of vesicles to analyze and sort individual exosomes using fluorescent labels. (See a previous blog on FAVS here.)

In the paper, “Identification and Characterization of EGF Receptor in Individual Exosomes by Fluorescence-Activated Vesicle Sorting (FAVS)”, published in the Journal of Extracellular Vesicles (JEV), Higginbotham and colleagues have used FAVS to analyze exosomal subsets that express varying amounts of EGFR in different cell-culture and in vivo contexts. This was done using DiFi cells, a human colorectal cancer (CRC) cell line, and A431, an epidermoid cancer cell line, which express approximately 5×10⁶ and 2.5×10⁶ EGFRs per cell, respectively (18, 19). The FAVS results showed that DiFi exosomes contain far more EGFR than do A431 exosomes, far exceeding the two-fold difference in EGFR levels present in these cell lines. Furthermore, using an antibody that recognizes an active form of EGFR, mAb806 (20-22), the amount of active EGFR was also found to be dramatically higher in DiFi exosomes than in A431 exosomes.

FAVS was also used to sort EGFR/CD9 double-positive and double-negative exosome populations, allowing enrichment of both subsets by post-sort analysis as well as western blot validation of the sorted exosomes (see Figure). Using human-specific reagents, FAVS was able to detect DiFi exosomes in the plasma of mice bearing DiFi xenografts. FAVS was also used to demonstrate that EGFR and one of its ligands, amphiregulin (AREG) are present in the plasma of normal individuals.

Results from the JEV paper derived from Fig 2. DiFi exosomes were flow sorted using antibodies to EGFR and CD9. Sorted purified double-negative vesicles (blue box/arrow) and double-positive vesicles (red box/arrow) were probed by western blot for markers as shown. These results validate the flow sorting enrichment of these different classes of vesicles. Also shown is a STORM image of an individual flow sorted double-positive vesicle.

This work joins flow-sorting work done by other labs using somewhat different techniques (6-14) and has implications for similar kinds of work done by other members of this consortium (23-25). Common to all these techniques was the use of lipid and/or specific extracellular vesicle markers to identify classes of secreted vesicles. Unlike FAVS, many sorting methods trigger vesicular events based on fluorescence rather than scatter. In all of these cases, analysis of secreted vesicle populations was performed. In some cases vesicle sorting was also achieved.

Thus, FAVS appears to be a promising technique to identify and purify distinct subsets of exosomes for discovery studies. It also holds promise for the detection of biomarkers in disease states including subsets of associated secreted RNAs.

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06.29.16 by Leonora Balaj

Protocols for studying extracellular vesicles and extracellular RNA

For researchers who have just begun studying extracellular vesicles (EVs) and their contents, including extracellular RNA, the Extracellular RNA Communication consortium (ERCC) published a protocols decision tree today, designed to help select a set of protocols for isolating EVs and exRNA from several biofluids of interest. Some of the protocols in the decision tree have been developed by ERCC researchers, but many relevant methods were published before the ERCC existed, and the versions on the ExRNA Portal include modifications and comments made by ERCC members in the course of their experiments, and are being periodically updated. In this blog, we introduce the ERCC protocols decision tree and discuss some of the nuanced differences between classes of EV isolation methods, highlighting methods that have existed in the field for some time.

There are several methods and commercial kits available to isolate extracellular vesicles (EVs) and extracellular RNA (exRNA) from human biofluids. Multiple studies have reported on a variety of these methods, but to date there is not one method or kit that suits all studies. The type of biofluid, the sample volume, and the fraction of exRNAs of interest are some of the criteria used to determine what method should be used for a particular study. Here, we have compiled a list of methods and kits most widely reported in the literature for isolation of EVs, exRNA or other components of biofluids. Broadly speaking, all methods can be classified into five categories (see Table), the most widely used being ultracentrifugation.

Drawbacks of ultracentrifugation include the need for expensive instrumentation (ultracentrifuges and rotors) and the belief that the EV population after ultracentrifugation will be contaminated with cell-free DNA and proteins. Depending on the speed and time of ultracentrifugation, some ribonucleoprotein (RNP) and lipoprotein (LPP) complexes may also sediment. After ultracentrifugation, some researchers further purify the EV population using density gradients or size exclusion chromatography. Sucrose gradients have been used widely for several years but are being replaced more recently by iodixanol (OptiPrep) gradients because some groups have reported that sucrose may inhibit the biological effects of EVs, while EVs prepared with OptiPrep better retain their biological activity.

Size exclusion chromatography is also widely used and is suitable for fractionation of sedimented EVs, as well as unprocessed biofluids. Recently Izon introduced a commercial kit to speed up this method, with relatively good results. Size exclusion chromatography yields a very clean population of EVs with the drawback being loss of EVs during the multi-step purification process.

The first commercial EV isolation kit (ExoquickTM) was launched on the market about six years ago, and is based on the principle of polyethylene glycol/sodium chloride precipitation, which has long been used for concentration of viruses. Since that time, several other kits using a precipitation strategy were launched by other manufacturers. Each of these kits (SBI, Life Technologies, Norgen Biotek, and Exiqon) have slightly different proprietary approaches to EV precipitation. Drawbacks to EV precipitation kits include co-precipitation of other unwanted molecules found in the biofluids and the difficulty of isolating EVs from large volumes of starting material. Ultrafiltration (e.g. using Millipore Amicon filters) is often used by researchers to concentrate large volumes, either before or after EV purification.

Filtration based methods which isolate specific size ranges of EVs can also be performed using commercially available devices. In addition, several kits and protocols for affinity purification have been developed by biotechnology companies and academic research laboratories. Antibody-based affinity methods (ExoCap, Microfluids, µNMR), and heparin-coated agarose or magnetic beads have been shown to bind subpopulations of EVs from cell culture media, plasma and serum efficiently. Another affinity kit, the METE kit, includes a proprietary peptide that, according to the manufacturer, binds to heat shock proteins found on the surface of the plasma membrane, suggesting a possible method to enrich for EVs with high levels of heat shock proteins. All of these methods yield a pure sample of EVs and can be scaled up, although scale-up costs can be significant, particularly in the case of antibody-based methods. These methods are limited to isolating a subset of EVs that express a specific antigen. For researchers who are interested in targeting a specific population EVs that displays one of these antigens, this may be a good option. However, at this point, for most cases, the biology is unclear on the diversity of EVs released by cells. Therefore, one antibody, or a pool of three to four antibodies, may not isolate all relevant EVs present in the sample.

ExoRNeasy isolates EVs based on their affinity to a proprietary membrane. ERCC members using this kit have reported that it can efficiently separate EVs (they bind to the membrane with high affinity) from other exRNA-containing particles, such as RNP complexes, which can be collected in the flow through, so this kit offers the extra advantage of efficiently separating EVs from RNP complexes. The ExoRNeasy/exoEasy kit yields a pure EV population and can isolate EVs from a volume as low as 200 μL or as high as 4 mL of plasma or serum with one loading per column, and up to 100 mL of cultured media per column by loading the column 3-4 times. Larger volumes require loading multiple filters.

Two kits available on the market are based on a two-step procedure where the sample is first either filtered (e.g. PureExo) or concentrated (e.g. Exo-Spin) and then resuspended and allowed to bind to proprietary beads. Intact EVs are then eluted in PBS and can be used for a variety of downstream assays. These kits do not offer feasible approaches to scale up to larger volumes.

Many other combinations and variations on these methods have also been reported in the literature, and this list is not meant to comprehensively encompass all reports in the field. It is a simple overview of the major classes of EV and RNA isolation methods present in the EV isolation field.

06.15.16 by Chiara Gai, Alessandra Iavello and Giovanni Camussi

Role of Alix in miRNA packaging during extracellular vesicle biogenesis

Extracellular vesicles (EVs) play an important role in cell-to-cell communication. Recently, EVs have been shown to be involved in immune modulation, tumor biology, and tissue regeneration. The mechanisms of action of EVs are associated with their ability to stimulate target cells directly and to transfer proteins, biologically active lipids, and nucleic acids to the target cells. In fact, mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) can be compartmentalized into EVs, escape enzymatic degradation, and be delivered to target cells. This horizontal transfer of extracellular RNAs carried by EVs can induce epigenetic alterations in recipient cells. The result is a change of phenotype or even a genetic and functional reprogramming of the recipient cells. Furthermore, EVs carry a selection of miRNAs different from the miRNAs most expressed in the cells of origin. However, little is known about the mechanisms of miRNA enrichment in EVs.

Argonaute2

Alix

We hypothesized a possible interaction between the Alix and Argonaute 2 (Ago2) proteins. The resulting complex may have a role in miRNA transport into EVs. Ago2 is a reasonable candidate to play a role in miRNA packaging within multivesicular bodies during EV biogenesis because of its central role in miRNA maturation. We observed that Ago2, as well as several other ribonucloproteins involved in RNA storage and stability, is expressed in EVs derived from adult human liver stem-like cells (HLSCs). Cells which express mesenchymal and embryonic markers.

Alix Figure 1

Alix is a multifunctional protein commonly used as a marker of EVs. It is an accessory protein of the Endosomal Sorting Complex Required for Transport (ESCRT), and several studies indicate that ESCRT is involved in the biogenesis of EVs.

We observed that HLSC-derived EVs express both Alix and Ago2. Co-immunoprecipitation (Co-IP) experiments with Alix or Ago2 antibody showed that the two proteins are associated. We also found that the miRNAs enriched in HLSC-EVs precipitate with the Alix – Ago2 complex. After the incubation of HLSC-EVs with human endothelial cells, we observed that miRNAs from HLSC-EVs are transferred to these cells.

Alix Figure 4

After the silencing of Alix expression in HLSCs, we observed the absence of both Alix and Ago2 proteins in EVs derived from the knockdown HLSCs and a strong reduction in the number of miRNAs normally enriched in HLSC-EVs. On the other hand, EV size, surface expression of CD63 and Tsg101, and the number of released EVs were not affected. After incubation with endothelial cells, EVs derived from Alix-knockdown HLSC do not transfer miRNAs to cells.

Alix is known to be involved in endocytic membrane trafficking and cytoskeletal remodeling. It is also associated with the ESCRT machinery, which participates in processes of vesiculation and cargo sorting, including multivesicular body biogenesis. Our data suggest that Alix binds Ago2 and drives it into EVs together with the associated miRNAs.

Alix Figure 5

This might be a general mechanism of miRNA transport into EVs, common to other cell types. Enrichment of a selected set of miRNAs might also depend on the affinity of miRNAs for carrier proteins such as Ago2.

Source: Iavello A, Frech VS, Gai C, Deregibus MC, Quesenberry PJ, Camussi G. Role of Alix in miRNA packaging during extracellular vesicle biogenesis. Int J Mol Med. 2016 37:958-966. doi: 10.3892/ijmm.2016.2488. PMID: 26935291.