Proteomic and Lipidomic Profiling of Immune Cell-Derived Subpopulations of Extracellular Vesicles.

Background: Small vessel cerebral vascular disease (sCVD) is common to older individuals, often asymptomatic, but associated with incident stroke and future mortality. Multiple mechanisms for sCVD have been postulated, all of which include injury to the neurovascular unit. Analysis of extracellular vesicles (EVs), important constituents of the intercellular communication pathway, may offer new ways to evaluate pathological processes of sCVD as well as serve as novel biomarkers or identify new avenues for therapy. This abstract summarizes work by our group to subtype EVs and measure cargo proteins from the various cell types of the neurovascular unit from a group of cognitively normal individuals with a spectrum of sCVD. Method: Study participants consisted of 14 individuals, 50 % female, 76 + 7 years of age, having 17.6 + 1.9 years of education. White matter hyperintensities varied from 0.73 – 38.8 cc. EV isolation used an Exodus ultrafiltration system on platelet depleted plasma. EVs were further fractionated into EV subtypes with resin-conjugated antibodies against cell type-specific markers. Five EV subtypes were isolated by two rounds of immunoprecipitation with the following markers: NCAM1/ATP1A3 (excitatory neuron EV), CD49f/LRP1 (astrocyte EV), CD11b/LCP1 (activated microglia EV), LAMP2/FTH1 (oligodendrocyte EV), CD31/CD146 (activated endothelial EV). Following isolation of EV subtypes, samples were characterized for purity and yield by nanoparticle tracking analysis, imaged for protein expression by super resolution microscopy, and sequenced for biomarker identification by quantitative proteomics. Results: Nanoparticle tracking analysis confirmed high yields of all 5 EV subtypes, > 2E9 EVs/mL, along with identical size and surface charge profiles. Super resolution microscopy showed consistent canonical EV marker distributions on all EVs while EV subtypes expressed unique markers based off their cell type of origin. Quantitative proteomics identified 400 unique and differentially expressed proteins present amongst the various EV subtypes as compared to mean plasma EVs concentrations. Protein abundance varied widely across EV subpopulations, indicating distinct protein profiles. Conclusion: Preliminary results confirm the potential for biomarker discovery from novel EV subpopulations through identification of differentially expressed cargo proteins from the neurovascular unit. Future work will associate these findings with sCVD phenotypes.

Author response: Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—Enabling robust and non-invasive biomarker research

Author response: Role of cytoneme structures and extracellular vesicles in Trichomonas vaginalis parasite-parasite communication

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

Pancreatic tumors are characterized by poor vasculature and fibrous stromal tissue networksthat create an extensive hypoxic tumor microenvironment. Extracellular vesicles (EVs) play important roles in pancreatic tumor pathobiology by supporting inter-cellular communications. They arebroadly classified into three subtypes: exosomes (Exo; 30-150 nm), microvesicles (MVs; 100 nm-1 µm) and apoptotic bodies (Abs; 1-5 µm), according to their cellular origin. Here, we studied the effect of hypoxic stress on the release kinetics and size distribution of EVs in pancreatic cancer cells. Further, we also investigated the role of different EV subtypes in adaptive survival of pancreatic cancer cells under hypoxia.Our data demonstrated that under hypoxic conditions, pancreatic cancer cells (MiaPaCa and AsPC1) shed greater amount of EVs with most noticeable changes recorded for the small size EVs. Moreover, all EVs (small, moderate, large) showed a shift towards reduced size depending upon the extent of hypoxia. When examined for subtype-specific markers, we observed mixed profiles. Thrombospondin (marker for Abs) and Arf6 (marker for MVs) were exclusively detected in large and moderate size fractions, respectively, under both normoxia and hypoxia. However, CD9 (marker for Exo) was detected in both small and moderate size EVs under hypoxia. Furthermore, in release kinetics studies we observed cyclic increases in accumulation of EV subtypes under hypoxic conditions. In addition, our data demonstrated that EVs from hypoxic cancer cells promoted survival of cancer cells under hypoxia,with small EVs being the most active. Altogether, our findings establish that hypoxia alters shedding of EVs in supporting adaptive survival of pancreatic cancer cells; associated differences could possibly be exploited for effective cancer management. Citation Format: Mary C. Patton, Haseeb Zubair, Mohammad Aslam Khan, Seema Singh, Ajay P. Singh. Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5158.

Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.

ABSTRACTAntiretroviral therapy (ART) suppresses viral replication in most people living with HIV‐1 (PLWH). However, PLWH remain at risk of viral rebound. HIV‐1 infection modifies the content of extracellular vesicles (EVs). The changes in microRNA content in EVs are biomarkers of immune activation and viral replication in PLWH. Moreover, viral molecules are enclosed in EVs produced from infected cells. Our objective was to assess the value of EV‐associated HIV‐1 RNA as a biomarker of immune activation and viral replication in PLWH. Plasma samples were obtained from a cohort of 53 PLWH with a detectable viremia. Large and small EVs were respectively purified by plasma centrifugation at 17 000g and by precipitation with ExoQuick. HIV‐1 RNA and microRNAs were quantified in the EV subtypes by RT‐qPCR. HIV‐1 RNA content was higher in large EVs of ART‐naive PLWH. Small EVs HIV‐1 RNA was equivalent in ART‐naive and ART‐treated PLWH and positively correlated with the CD4/CD8 T cell ratio. In ART‐naive PLWH, HIV‐1 RNA content of large EVs correlated with small EV‐associated miR‐29a, miR‐146a, and miR‐155, biomarkers of viral replication and immune activation. A receiver operating characteristic analysis showed that HIV‐1 RNA in large EVs discriminated PLWH with a high CD8 T cell count. HIV‐1 RNA in large EVs was associated with viral replication and immune activation biomarkers. Inversely, HIV‐1 RNA in small EVs was related to immune restoration. Overall, these results suggest that HIV‐1 RNA quantification in purified EVs could be a useful parameter to monitor HIV‐1 infection.

In recent years, the cargo profiles of extracellular vesicles (EVs), which were inherited from their parent cells, have emerged as a reliable biomarker for liquid biopsy (LB) in disease diagnosis, prognosis, and treatment monitoring. EVs secreted by different cells exhibit distinct characteristics, particularly in terms of disease diagnosis and prediction. However, currently available techniques for the quantitative analysis of EV cargoes, including enzyme-linked immunosorbent assay (ELISA), cannot specifically identify the cellular origin of EVs, thus seriously affecting the accuracy of EV-based liquid biopsy. In light of this, we here developed ultrabright fluorescent nanosphere (FNs)-based test strips which have the unique capability to specifically assess the levels of PD-L1-positive EVs (PD-L1+ EVs) derived from both tumor cells and immune cells in bodily fluids. The levels of PD-L1+ EV subpopulations in human saliva were quantified using the ultrabright fluorescent nanosphere-based test strips with more convenience and higher efficiency (detection time <30 min). Results demonstrated that the fluorescence intensity of the test line exhibited a good linear relationship respectively with the PD-L1 levels of tumor cell- (R2 = 0.993) and immune cell-derived EVs (R2 = 0.982) in human saliva. By assessing the levels of PD-L1+ EV subpopulations, our test strips hold immense potential for advancing the application of PD-L1+ EV subpopulation-based predictions in tumor diagnosis and prognosis evaluation. In summary, by integrating the benefits of FNs and lateral flow chromatography, we here provide a strategy to accurately measure the cargo levels of EVs originating from diverse cell sources in bodily fluids.

The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery.

The therapeutic efficacy of mesenchymal stromal cells (MSCs), multipotent progenitor cells, is attributed to small (50-200 nm) extracellular vesicles (EVs). The presence of a lipid membrane differentiates exosomes and EVs from other macromolecules. Analysis of this lipid membrane revealed three distinct small MSC EV subtypes, each with a differential affinity for cholera toxin B chain (CTB), annexin V (AV), and Shiga toxin B chain (ST) that bind GM1 ganglioside, phosphatidylserine, and globotriaosylceramide, respectively. Similar EV subtypes are also found in biologic fluids and are independent sources of disease biomarkers. Here, we compare and contrast these three EV subtypes. All subtypes carry β-actin, but only CTB-binding EVs (CTB-EVs) are true exosomes, enriched with exosome proteins and derived from endosomes. No unique protein has been identified yet in AV-binding EVs (AV-EVs); ST-binding EVs (ST-EVs) carry RNA and a high level of extra domain A-containing fibronectin. Based on the CTB, AV, and ST subcellular binding sites, the origins of CTB-, AV-, and ST-EV biogenesis are the plasma membrane, cytoplasm, and nucleus, respectively. The differentiation of EV subtypes through membrane lipids underlies the importance of membrane lipids in defining EVs and implies an influence on EV biology and functions.

Tumour cells release diverse populations of extracellular vesicles (EVs) ranging in size, molecular cargo, and function. We sought to characterize mRNA and protein content of EV subpopulations released by human glioblastoma (GBM) cells expressing a mutant form of epidermal growth factor receptor (U87EGFRvIII) in vitro and in vivo with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87EGFRvIII cancer cells, non-cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87EGFRvIII glioma-bearing mice using differential centrifugation (EVs that sediment at 10,000 × g or 100,000 × g are termed large EVs and small EVs, respectively) were characterized using transmission electron microscopy (TEM), confocal microscopy, nanoparticle tracking analysis (NTA), flow cytometry, immunofluorescence (IF), quantitative-polymerase chain reaction (qPCR), droplet digital polymerase chain reaction (ddPCR) and micro-nuclear magnetic resonance (μNMR). We report that both U87EGFRvIII and HUVEC release a similar number of small EVs, but U87EGFRvIII glioma cells alone release a higher number of large EVs compared to non-cancer HUVEC. The EGFRvIII mRNA from the two EV subpopulations from U87EGFRvIII glioma cells was comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Similarly, EGFRvIII mRNA in large and small EVs isolated from the serum of U87EGFRvIII glioma-bearing mice is comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Here we report for the first time a direct comparison of large and small EVs released by glioma U87EGFRvIII cells and from serum of U87EGFRvIII glioma-bearing mice. Both large and small EVs contain tumour-specific EGFRvIII mRNA and proteins and combining these platforms may be beneficial in detecting rare mutant events in circulating biofluids.

Isolation methods of large and small extracellular vesicles derived from cardiovascular progenitors: A comparative study

The identification of surface markers that correlate with specific subpopulations of extracellular vesicles (EVs) is important for EV identification, classification, purification, sorting, and functional analysis. Tetraspanins, such as CD9, CD63, and CD81, were once considered to be universal markers of exosomes: small EVs released into the extracellular space when late endosomes/multivesicular bodies fuse with the plasma membrane. In contrast, plasma-membrane-derived ectosomes (also called microvesicles) have a different biogenesis, are often regarded as being larger than exosomes, and display a different surface proteome. However, recent studies have shown that tetraspanins, such as CD9 and CD81, are highly enriched on ectosomes derived from various sources. Thus, it is currently unclear how tetraspanin content correlates with specific EV subpopulations. Here, we present a modified immuno-TEM protocol that can be easily applied to heterogeneous EV populations comprising both small and large EVs (and presumably also a collection of exosomes and ectosomes). In EVs purified from U-2 OS cells by size-exclusion chromatography, we show that the percentage of particles positive for CD9 and CD81 is significantly higher in the subpopulation of EVs ≤ 100 nm (i.e., small EVs). These results also explain discrepancies in the size distribution profiles that we obtained using the same EV preparations by alternative single-vesicle characterization platforms, such as nano flow cytometry and SP-IRIS/ExoView. The latter only analyzes EVs that were previously captured based on the presence of tetraspanins, which introduces a bias in their size distribution.

Extracellular vesicles (EVs) are currently of tremendous interest in many research disciplines and EVs have potential for development of EV diagnostics or therapeutics. Most well-known single EV isolation methods have their particular advantages and disadvantages in terms of EV purity and EV yield. Combining EV isolation methods provides additional potential to improve the efficacy of both purity and yield.This review assesses the contribution and efficacy of using combined EV isolation methods by performing a two-step systematic literature analysis from all papers applying EV isolation in the year 2019. This resulted in an overview of the various methods being applied for EV isolations. A second database was generated for all studies within the first database that fairly compared multiple EV isolation methods by determining both EV purity and EV yield after isolation.From these databases it is shown that the most used EV isolation methods are not per definition the best methods based on EV purity or EV yield, indicating that more factors play a role in the choice which EV isolation method to choose than only the efficacy of the method. From the included studies it is shown that ~60% of all the included EV isolations were performed with combined EV isolation methods. The majority of EV isolations were performed with differential ultracentrifugation alone or in combination with differential ultrafiltration. When efficacy of EV isolation methods was determined in terms of EV purity and EV yield, combined EV isolation methods clearly outperformed single EV isolation methods, regardless of the type of starting material used. A recommended starting point would be the use of size-exclusion chromatography since this method, especially when combined with low-speed centrifugation, resulted in the highest EV purity, while still providing a reasonable EV yield.