Improved Flow Cytometric Light Scatter Detection of Submicron-Sized Particles by Reduction of Optical Background Signals

Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of choice to rapidly assess heterogeneity of cell populations in suspension. With the research field of extracellular vesicles (EV) rapidly expanding, there is an increased demand to address heterogeneity of EV populations in biological samples. Although flow cytometry would be the ideal technique to do so, the available instruments are in general not equipped to optimally detect the dim light scatter signals generated by submicron-sized particles like EV. Although sideward scatter light and fluorescence are currently used as a threshold signal to identify EV within samples, the forward scatter light (FSC) parameter is often neglected due to the lack of resolution to distinguish EV-related signals from noise. However, after optimization of FSC detection by adjusting the size of the obscuration bar, we recently showed that certain EV-subsets could only be identified based on FSC. This observation made us to further study the possibilities to enhance FSC-detection of submicron-sized particles. By testing differently sized obscuration bars and differently sized pinholes in the focal plane behind the FSC detection lens, we generated a matrix that allowed us to determine which combination resulted in the lowest optical background in terms of numbers of events regarding FSC detection of submicron-sized particles. We found that a combination of an 8-mm obscuration bar and a 200-μm pinhole reduced optical background in a reproducible manner to such extent that it allowed a robust separation of 100-nm polystyrene beads from background signals within the FSC channel, and even allowed thresholding on FSC without the interference of massive background signals when both beads and EV were measured. These technical adaptations thus significantly improved FSC detection of submicron-sized particles and provide an important lead for the further development and design of flow cytometers that aid in detection of submicron-sized particles. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry

Acute joint inflammation induces a sharp increase in the number of synovial fluid EVs and modifies their phospholipid profile

Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis. LPS injection triggered a sharp increase of SF-EVs at 5-8 h post-injection, which started to decline at 24 h post-injection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0 h), SF-EVs collected at 5, 24, and 48 h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5 h and 24 h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation

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

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

BH3-only protein Noxa contributes to apoptotic control of stress-erythropoiesis

Apoptosis plays an essential role in the control of erythropoiesis under normal and pathological conditions. However, the contribution of individual proteins within cell death signalling pathways remains poorly defined. Here, we investigated the role of the pro-apoptotic Bcl-2 family member Noxa in the regulation of erythropoiesis. We found that expression of Noxa is induced during erythroid differentiation of human and murine precursor cells. Using in vitro model systems for erythroid progenitors, we observed rapid induction of Noxa upon cytokine deprivation. Knockdown or deletion of Noxa conferred significant protection against apoptosis upon cytokine withdrawal. In vivo, Noxa deficiency did not affect hematological blood parameters or erythroid progenitor composition of bone marrow and spleen under steady-state conditions. In contrast, in a model of acute haemolytic anemia, Noxa-deficiency enhanced hematocrit recovery. Moreover, in a model of chronic inflammation-induced anemia, Noxa-ablation resulted in a dramatic increase of erythroblast expansion. Our data indicate that induction of Noxa in erythroid progenitors sets a survival threshold that limits expansion beyond the number of cells that can be sustained by the available cytokines, which becomes apparent under conditions of induced anemi

Developing a novel cancer biomarker blood test by measuring mRNA levels of cell cycle genes in circulating extracellular vesicles

Background: Malignant cancer cells are highly proliferative. These cells actively release extracellular vesicles (EVs) in the tumor environment and into body fluids, such as blood. Cancer-derived EVs are enriched with cell cycle gene-related molecules, such as the target transcripts of the transcription factor family E2F. Quantification of cell cycle gene mRNA in blood-derived EVs of cancer patients can represent an indicator of this proliferation signature, which can be used to diagnose the presence of malignant tumors, predict the prognosis and efficacy of anti-cancer treatment and/or monitor disease progression, representing a potential new useful, minimally invasive, clinically applicable test. Methods: Six highly expressed cell cycle gene transcripts were selected based on the analysis of transcriptome data of cancer tissues available in public databases. The expression levels of these transcripts were then evaluated by qPCR in EVs isolated from cultured canine melanoma cells. To investigate whether the mRNA levels in tumor cells is mirrored into their released EVs, cell proliferation was blocked using a CDK4/6 inhibitor. As a first step to detect the cell cycle gene transcripts in plasmaderived EV, total RNA was extracted from the plasma of canine cancer patients and analyzed for the presence of the selected cell cycle transcripts. Results: The selected transcripts were detected in both large and small melanoma-derived EVs, but were most highly enriched in exosomes. CDK4/6 inhibitor treatment induced a decrease in cellular transcript levels, which were accompanied by a decrease of mRNA levels in EVs. In the plasma of canine cancer patients mRNA of some of the selected genes was detected with overall higher expression levels when compared to plasma of healthy controls. Conclusions: In vitro, cell cycle gene mRNAs are present within cancer cell-derived EVs and their levels reflect the expression levels and proliferation status of donor cells. Furthermore, cell cycle gene transcripts are detectable in the plasma of canine cancer patients. Further investigation will be applied to validate their association to circulating EVs and the correlation with tumor proliferation rate, patient response to therapy, and follow-up data

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery

Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis

Abstract Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co‐isolated. Furthermore, physical EV‐LPP complexes have been observed in purified EV preparations. Since co‐isolation or association of LPPs can impact EV‐based analysis and biomarker profiling, we investigated the presence and formation of EV‐LPP complexes in biological samples by using label‐free atomic force microscopy, cryo‐electron tomography and synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence‐based high sensitivity single particle flow cytometry. Furthermore, we evaluated the impact on flow cytometric analysis in the presence of LPPs using in vitro spike‐in experiments of purified tumour cell line‐derived EVs in different classes of purified human LPPs. Based on orthogonal single‐particle analysis techniques we demonstrate that EV‐LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence‐based flow cytometric EV analysis staining of LPPs, as well as EV‐LPP associations, can influence quantitative and qualitative EV analysis. Lastly, we demonstrate that the colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down‐stream EV analysis and EV biomarker profiling

Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

BACKGROUND. Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. METHODS. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. RESULTS. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. CONCLUSION. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. FUNDING. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID)

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery