Serum-Free Media Supplements Carry miRNAs That Co-Purify With Extracellular Vesicles

Martin Auber, Dominik Fröhlich, Krämer-Albers Eva-Maria
Johannes Gutenberg University of Mainz, Germany

Numerous studies report the association of miRNAs with extracellular vesicles (EVs). In most cases, EVs were harvested from cell culture-conditioned media containing fetal bovine serum (FBS) or a defined media supplement as nutrient. Recently, Wei et al. (2016, PMID: 27503761) reported that miRNAs are co-isolating with EVs when harvested in media supplemented with FBS or vesicle-depleted FBS. To avoid serum-associated miRNA contamination, we performed RNA-Seq of EV-associated miRNAs derived from primary oligodendrocytes cultured under serum-free conditions and subsequently validated miRNAs by RT-qPCR including media and supplement controls. Intriguingly, several EV-associated miRNAs were robustly detected in un-conditioned media subjected to the EV-isolation protocol and the media supplements NS21 and B27, which are routinely used for neural cell culture. RNAse and detergent treatment of NS21 removed most but not all of the contaminating miRNAs. Detailed analysis of NS21-supplement by screening individual components excluded BSA as major source of the miRNA contamination and identified a single component as carrier of miRNAs. Media supplement lacking the miRNA-carrying components however appears to affect cell viability, indicating that deprival of this specific supplement is not a solution to get rid of contaminating miRNAs. Based on these findings, we designed a new RNA-Seq strategy including EV-samples collected under conditions of supplementation or supplement-deprivation as well as supplement only control.
Taken together, our study shows that a single component of defined media supplements may carry major contaminating miRNAs into EV-samples. Consequently, analysis of EV-RNA needs refined strategies including respective media controls.

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Normalizing Urinary Extracellular Messenger RNA Biomarkers: Theoretical Considerations and a Review of Experimental Findings

J. Brian Byrd1
Pradeep Moon Gunasekaran1, J. Matthew Luther2, J. Brian Byrd1
1) University of Michigan Medical School, USA;
2) Vanderbilt University School of Medicine, USA;

Messenger RNA (mRNA) has been extensively annotated, and its crucial role in the central dogma has made it a key target in many studies of biomarkers and drug targets. Extracellular vesicles shuttle mRNA, among other molecular cargo. mRNA in urinary extracellular vesicles has potential as a biomarker, particularly in diseases affecting cells of the urothelial tract. There is evidence that this mRNA could provide information about transcription in cells of urogenital tissues. However, the optimal means of normalizing these signals is unclear. In the more common cell lysate context, gene expression can be normalized to robustly expressed genes that have similar expression between cells. However, in the context of extracellular mRNA shuttled by extracellular vesicles, normalization strategies remain undefined. This talk will describe relevant first principles as well as research findings from our lab and other labs toward normalization of urine extracellular mRNA. The talk will focus on at least two hypothetical sources of confounding that might be important denominators when normalizing assays of urinary extracellular mRNA transcripts. The first is changes in the composition of the biofluid matrix broadly affecting analytes’ concentrations (i.e., intra-individual changes or inter-individual differences in urine composition due to having recently consumed water). The second is broad changes or differences in the expression of genes without direct relevance to the specific biology of interest. The extent to which each of these factors must be taken into account during normalization will depend on the magnitude of target gene’s expression signal in the disease relative to the magnitude of the noise created by these potentially confounding factors. The rationale for the use of urinary creatinine as the traditional reference molecule for many urinary assays will be discussed, as will advantages and disadvantages of urinary creatinine for normalization of urinary extracellular mRNA. In addition, the possibility of using reference genes to normalize assays of urinary extracellular mRNA will be discussed. Prior experiments bearing on the feasibility of these approaches will be reviewed. The talk will also discuss other potential strategies for normalization, such as normalizing to the concentration of extracellular vesicles. Finally, the talk will attempt to synthesize these ideas and the findings from the literature into suggestions for those developing urinary extracellular mRNA assays.

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Anesthesia and its impact on circulating extracellular miRNA profiles during cancer surgery

Dominik Buschmann
Dominik Buschmann1, Florian Brandes2/3, Anja Lindemann3, Melanie Maerte2, Petra Ganschow4, Alexander Chouker2, Gustav Schelling2, Michael W. Pfaffl1, Marlene Reithmair3
1) Division of Animal Physiology and Immunology, Technical University of Munich, Freising, Germany;
2) Department of Anesthesiology, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany;
3) Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany;
4) Department of General, Visceral, and Transplant Surgery, Ludwig-Maximilians-University Munich, Munich, Germany;

Even though tumor resection is crucial for survival of patients with solid tumors, surgery itself might promote tumor progression and metastasis. Data from both experimental and retrospective cohort studies suggest that using volatile anesthetic gases (VAG) during cancer surgery may enhance tumor growth and metastasis compared to surgery using total intravenous anesthesia (TIVA). For colorectal cancer surgery, overall and disease-free survival is significantly better in patients anesthetized with TIVA as opposed to VAG. Circulating cell-free miRNAs including those associated with extracellular vesicles are crucial mediators of malignant cell-to-cell communication and metastasis and might play a role in mediating these differential effects of anesthetics on tumor progression.
In this study, we characterized circulating extracellular miRNAs in a cohort of colorectal cancer surgery patients anesthetized using propofol (TIVA, n=8) or sevoflurane (VAG, n=9). Sera were sampled before anesthesia and after tumor resection at termination of surgery, and circulating miRNAs were profiled by Next-Generation Sequencing. Next, we assessed perioperative changes in miRNA expression induced by either anesthetic drug and compared their biological effects on tumor-relevant pathways.
Post-operative miRNA profiles were shifted in both anesthetic groups, resulting in clear separation of pre- and post-operative samples. Despite a significant overlap in miRNAs upregulated in both groups, we detected five (sevoflurane) and 36 (propofol) miRNAs to be specifically responsive to either anesthetic agent. In silico target analyses of miRNA expression patterns indicated an inhibitory effect of propofol on crucial carcinoma-related pathways such as proliferation and migration as well as enhanced apoptosis associated with propofol. Sevoflurane-associated changes in miRNA profiles impacted these pathways to a much smaller extent or not at all.
The differential effect of anesthetic agents on outcome of cancer surgery reported by retrospective studies might therefore be in part mediated by perioperative changes of circulating extracellular miRNA profiles.

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Characterization of Extracellular vesicles (EVs) Derived ExRNA in Cancer Using NGS

Hidetoshi Tahara1
Yukie Nishiyama1, Makoto Tahara2, Hidetoshi Tahara1
1) Department of Cellular and Molecular Biology,Institute of Biomedical & Health Sciences, Hiroshima university, Japan;
2) Department of Head and Neck Medical Oncology, National Cancer Center Hospital East, Kashiwa, Japan;

MicroRNAs (miRNAs) are small non-coding RNAs approximately 21 nucleotides in length that regulate posttranscriptional gene expression. miRNAs exist in exosomes, which are 50–100nm in size extracellular vesicles (EVs) and secrete from mammalian cells. Recent studies have demonstrated that miRNAs and other small RNAs stably exist in body fluids and their expression patterns in cancer patients are distinct from those in healthy individuals. In this study, we analyzed non-coding small RNAs including miRNAs that specifically exist in plasma/serum of patients with breast and tongue cancer by using NGS plat form. We identified a number of biomarker candidates such as mature miRNAs, isomiR, tRNA-derived fragments (tRFs), and other ncRNA, are known to regulate expression of genes involved in cell metabolism and are released into body fluid from various cells with extracellular vesicles. To test the possibilities that these candidate small RNAs are secreted from cancer cells, we purified EVs from serum in health volunteer and cancer patients, and analyzed small RNAs using NGS, and found that some of candidate small RNAs can be found in EVs. Interestingly, these small RNAs are also found in culture media in cancer cell lines. Based on our alogism, we identified significant biomarker secreted from cancer cells for early detection of cancer.

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Circular RNAs In EVs – A Novel Class Of Released Non-coding RNAs

Christian Preußer
Justus Liebig University of Giessen, Institute of Biochemistry

Non-coding RNAs (ncRNAs) were established in the last decade as a new valuable biomarker class for human diseases. Specifically, circular RNAs (circRNAs), which represent the newest class of ncRNAs, turned into research focus within the last decade. Although single examples of these particular RNA class had been known for more than forty years, circRNAs were established as a large RNA class only a few years ago, based on the advent of tailored RNA-Seq technologies and Bioinformatics. CircRNAs were identified in all eukaryotic cells investigated so far, are cell-type specifically expressed, and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form, resulting in a metabolically more stable form of RNA compared to their linear counterparts. These biological properties and the correlation with various human diseases, such as cardiovascular diseases and cancer, immediately suggesting their potential use as novel attractive RNA-based biomarkers. We have focused on the analysis of circRNAs and the corresponding linear splice isoforms from human blood cells, in particular platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles (EVs) from purified and in vitro activated human platelets, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. Furthermore, to unravel mechanisms that contribute to the specific packaging of RNAs into EVs, we developed a novel approach to determine specific sequence motifs required for selective loading of (circ)RNA into EVs. This unbiased method should contribute to our understanding of how RNAs are specifically packaged into EVs. In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and harbor great biomarker potential. In addition, we would also like to stress the strong need for standardization and for setting up minimal requirements for validating this class of ncRNA.

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Standardization in EV and exRNA Research: MISEV and Beyond

Kenneth Witwer
The Johns Hopkins University School of Medicine, United States of America

Rapidly growing interests in physiologic and pathologic processes associated with extracellular vesicles (EVs), including presumed functions of various EV RNA molecules, have created a need for standardization initiatives to shape and guide the field. As an example, the International Society for Extracellular Vesicles (ISEV) first published the Minimal Information for Studies of Extracellular Vesicles (“MISEV”) in 2014 to call attention to rigorous EV characterization approaches and experimental controls. In this presentation, I review recent developments in EV and exRNA standardization, focusing especially on the “MISEV2018” guidelines in the Journal of Extracellular Vesicles. These guidelines were prepared with methods for broad community input and buy-in that may serve as a model for ongoing and future initiatives. We will discuss outstanding needs in the field and what might be done to address them.

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Mesenchymal Stem Cell-derived Extracellular Vesicles: a Potential New Tool in Regenerative Medicine

Bernd Giebel
Institute for Transfusion Medicine, Germany

Human mesenchymal stem/stromal cells (MSCs) represent a promising tool in regenerative medicine. Up to now, more than 800 NIH-registered clinical trials investigated their immunomodulatory and pro-regenerative therapeutic potential in various diseases, including graft-versus-host disease (GvHD) and ischemic stroke. Despite controversial reports regarding the efficacy of MSC-treatments, MSCs seem to exert their beneficial effects rather in a paracrine manner than by cell replacement. In this context, extracellular vesicles (EVs), such as exosomes and microvesicles, are discussed to execute the MSCs’ therapeutic effects. Indeed, we observed beneficial therapeutic impacts of MSC-EVs in a patient, who suffered from steroid-refractory acute GvHD. Furthermore, beneficial effects were observed in animal models for several different diseases. According to controversial reports in the MSC field, especially since a phase III clinical trial failed to show clinical efficacy in MSC treated GvHD patients, we have started to compare immunomodulatory effects of independent MSC-EV preparations. Indeed, in our in vitro assays independent MSC-EV fractions reveal different immunomodulatory capabilities. To unravel the basis for these differences we are currently using several methods to dissect the heterogeneity between and within given MSC-EV samples.

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