The more the better – determining the optimal range when performing single-vesicle phenotyping

The characterization of extracellular vesicles (EVs) has evolved rapidly in recent years due to advances in straightforward technologies. Based on these more sensitive methods, it is now possible to describe EV populations in their entirety more precisely. However, these applications require an equivalently delicate experiment design and optimization steps to draw valid conclusions in the end. One of these methods is represented by the highly sensitive nanoflow cytometry (nFCM), by which particles can be analyzed not only on their size (< 40 nm) and concentration but also concerning surface markers. In this work, we addressed some of the potential caveats of this method, especially when characterizing particles with fluorescently labelled antibodies. In particular, we show, when using low particle concentrations, which are inevitably encountered when working with EVs, the characterization of surface markers is prone to significantly varying. We hypothesized that these technical limitations could respond to the stickiness of EVs and should be properly counteracted. As a reference, we strongly recommend performing particle number-based comparisons with at least 109 particles as staining input in nFCM analyses. Moreover, we provided representative particle-number based immunoblotting results, underlying the significance of this parameter as a normalizer in future EV research.

RNAs and extracellular vesicles - Keeping up the appearances

Since the advent of extracellular vesicle (EV) research in the last decade, these particles have been associated with RNAs. Traded as promising new biomarkers, RNA transport vehicles, or ultimately as potential therapeutic RNA delivery vehicles. However, this view is currently undergoing a change in which RNA may no longer be a major component of EVs. In this short opinion paper, we would like to encourage a reconsideration of our view on EVs and RNAs and open it up to new thoughts.

Establishment of a DFG-funded research group on the topic of plant-microbe communication through extracellular RNA

The exRNA consortium, a team of researchers funded by the Deutsche Forschungsgemeinschaft (DFG), addresses crucial aspects of cross-kingdom RNA interference (ckRNAi) and RNA application in plant protection, mainly focusing on mechanistic considerations and application efficiencies.

Extracellular vesicle therapeutics: the issue of one size fits all

Extracellular vesicles (EV) are mediators of intercellular communication locally in tissue microenvironments and enable distal across organ communication between cells of the same origin and those from different sources. EV surface proteins and lipids enable interaction with particular cells, whereas their internal payload of RNA, transcription factors, DNA, enzymes and metabolites functionally alters recipient cells. EV-interactions and uptake induce changes in cellular proliferation, differentiation, cell movement, as well as transcriptional and epigenetic regulation. These unique properties of EV poise them as attractive therapeutics in a broad range of pathologies, but questions remain in translating EV discoveries to effective therapies. Here, I briefly discuss the need for more stringent considerations for EV-therapeutic effects with a focus on EV biodistribution profiles in appropriate disease models and routes of EV administration with a particular focus on the vasculature.

Elucidating the role of extracellular vesicles in the Barley-Fusarium interaction

Fusarium graminearum (Fg) is a necrotrophic fungal pathogen that causes devastating diseases on its crop hosts barley and wheat. Recently, small RNAs (sRNAs) were identified as mobile communication signals between eukaryotes and their pathogens, symbionts or parasites. It has been shown that pathogens secrete sRNAs as effectors to suppress plant immunity and plants use endogenous sRNAs to resist infection, a phenomenon termed cross-kingdom RNAi; ckRNAi. However, little is known about the transport of fungus- or plant produced sRNAs to silence genes that contribute to immunity. Extracellular vesicles (EVs) are predicted playing a key role in the bidirectional transfer of sRNAs that mediate ckRNAi. To address this knowledge gap, we investigated the effects of EVs isolated from barley and Fg on their counterparts during plant-fungal interaction. Towards this, we developed a protocol for the isolation of EVs from Fg liquid cultures and assessed how Fg EVs contribute to fungal pathogenesis in barley using infiltration assays. To test the interdependence of EVs during Barley-Fg interaction, we treated Fg cultures with barley EVs. We found that infiltration of Fg EVs caused host specific phytotoxic effects in barley and barley EVs impaired Fg growth. Of note, Fg cultures showed an increase in purple pigmentation upon inoculation with barley EVs, suggesting a stress-induced premature formation of fruiting bodies. Together, our results demonstrate that EVs contribute to the Barley-Fg interaction, however, further studies are needed to unravel the nature of EV cargoes (e.g. protein and/or sRNA) responsible for affecting its plant/fungus counterpart.

Crossroads of the endosomal machinery: Multivesicular bodies, small extracellular vesicles and autophagy

The primary role of endosomal system is endocytic trafficking – to sort out internalized macromolecules and proteins to their destined cellular localizations. Incorporation of sorted cargos into multivesicular bodies (MVBs) confers specificities and determines their fates. This central point of the endosomal trafficking separates MVBs in two directions. The MVB populations fuse either with lysosomes to initiate autophagy or with plasma membrane to release small extracellular vesicles. Factors contributing to the selection of cargo and direction of trafficking incorporate the cells’ metabolic status and stress level. In this review, we discuss the molecular cues responsible for differential cargo sorting into MVBs and trafficking directions of MVBs in the endosomal network. Keywords: Exosomes; degradative MVB; secretory MVB; physiological stress; endocytic machinery; lysosome

Extracellular vesicles as biomarkers for diagnostic and prognostic application in cardiovascular disease

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide. Early diagnosis and the development of a prognosis is important for management or secondary prevention of the disease. In the past few decades, various biomarkers have been identified for improved risk assessment, more accurate diagnosis and prognosis, and a better understanding of the underlying pathophysiology in CVD. Extracellular vesicles (EVs) are thought to be important to cell-to-cell communication in the heart, and EV counts, cellular origin, and EV content have been related to CVD. This review examines current evidence for the potential application of EVs as a new class of biomarkers in CVD. Keywords: extracellular vesicles, biomarker, liquid biopsy, cardiovascular disease, myocardial infarction, heart failure, pulmonary arterial hypertension

Perspective – Escape from destruction: how cancer-derived EVs are protected from phagocytosis

There is evidence that cancer-derived extracellular vesicles (EVs) have nearby and distant effects in the body. In order to reach distant sites, EVs need to travel through the blood stream and organs where they encounter a hostile environment in the form or phagocytic cells. However, the stability and homeostasis in the blood circulation and in the tumor microenvironment are not well understood. Phagocytosis is an important mechanism for the clearance of apoptotic and necrotic cells. As exosomes (small EV) express “eat-me” signals such as phosphatidyl-serine, it is likely that they are cleared similar to dead cells. Here we discuss measures that cancer cells have developed to protect their EVs from rapid depletion. The expression of “don’t eat me” signals such as CD47 and CD24 on the tumor cell surface and in released exosomes is of vital importance. We will focus on the role of the CD24-Siglec-10 binding axis as a stop signal at the interface between tumor cells and phagocytic cells. Extending the lifetime of EVs is essential for the cancer to achieve systemic immune suppression and to prepare metastatic niches for spreading. Keywords: CD24, CD47, Extracellular vesicles, Siglecs, carbohydrates, phagocytosis