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

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.

IntroductionOptical techniques are routinely used to size and count extracellular vesicles (EV). For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI) as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA) provides both size and relative light scattering intensity (rLSI) values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs.MethodsNTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively) and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm) and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values.ResultsThe following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364–1.375 and 1.398–1.414 for large placental EV (>200 nm). Large placental EV had a significantly higher RI than small placental EV (p<0.0001). The spread of RI values was narrower for small EV than for the more heterogeneous large EV.DiscussionUsing NTA and Mie scattering theory, we have demonstrated that it is possible to estimate the RI of sub-micron EV using NTA data. EV typically had a modal RI of 1.37–1.39, whereas values of >1.40 were observed for some large (>200 nm) microvesicles.ConclusionThis method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement.

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.

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.

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.

Extracellular vesicles (EVs) are heterogeneous and play important roles in intercellular communication, contributing to physiological and pathological processes. Since few markers currently exist to differentiate subtypes of EVs, this study aimed to determine proteomic and lipidomic differences among four EV subpopulations. Large and small EVs (L-EVs and S-EVs) were isolated from human mast cells (HMC-1) and monocytes (THP-1) by differential ultracentrifugation and then further separated by density cushions into two different densities [low-density (LD) and high-density (HD)]. L-EVs were pelleted at 16,500 × g, and S-EVs were pelleted at 118,000 × g. LD EVs were collected at 1.079-1.146g/mL, while HD EVs were collected at 1.146-1.185g/mL. The morphology, size and yield of EVs were determined by TEM and western blot. The proteome and lipidome of the EV subpopulations were determined with mass spectrometry. A total of 5364 proteins were quantified, and L-EVs LD were enriched in mitochondrial proteins such as TIMM/TOMM and MICOS proteins, while L-EVs HD were enriched in cytoskeleton- and cytokinesis-associated proteins, such as KIF proteins. S-EVs LD were enriched in tetraspanins, ADAM10 and ESCRT machinery proteins, while S-EVs HD were enriched in proteins commonly viewed as contaminants, such as histones, complement factors and collagen. Proteins involved in membrane trafficking between the plasma membrane and organelles, such as adaptor protein complexes, the conserved oligomeric Golgi complex, the trafficking protein particle complex, sortin-nexins, TBC1 domain proteins and coatomer subunits, were expressed at similar levels across all EV subtypes. Furthermore, 107 lipids were quantified, and phosphatidylethanolamine (PE) was less abundant in L-EVs LD as compared to the other EV subtypes, while ceramides were enriched in L-EVs as compared to S-EVs.This study demonstrates that there is a core proteome and lipidome that is similar across all four EV subtypes, but importantly, it also shows that a portion of the proteome and lipidome differs in EV subpopulations separated based on size and density. We suggest that these could be important markers in future EV studies and that they may reflect a different biogenesis and EV function.

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, &gt; 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.

Understanding communication mechanisms between unicellular parasites is crucial in the development of novel therapies, as they rely on diverse modes of communication (like extracellular vesicle release, cytoneme, and filopodia formation) to regulate their behavior and survival.

BackgroundDiabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking.MethodsFourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ.ResultsThe number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model.ConclusionsUrinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.

Background:Extracellular vesicles (EVs) are intercellular messengers with epigenetic potential since they can shuttle microRNA (miRNA). EVs and miRNA play a role in human immunodeficiency virus (HIV) infection immunopathogenesis. Chronic immune activation and systemic inflammation during HIV infection despite effective antiretroviral therapy (ART) are associated with non-acquired immunodeficiency syndrome (AIDS) comorbidities in people living with HIV (PLWH). Analysis of plasma EVs and their miRNA content may be useful as immune activation or inflammatory biomarkers in PLWH receiving ART. In this study, we hypothesized that the number, size, and miRNA of large and small EVs could reflect immune activation associated with an elevated CD8 T-cell count or a low CD4/CD8 ratio in PLWH.Methods:Plasma EVs subtype purified from PLWH and uninfected controls were sized using dynamic light scattering and quantified using flow cytometry and acetylcholine esterase (AChE) activity. Expression of mature miRNAs miR-92, miR-155, miR-223 was measured by quantitative reverse-transcriptase polymerase chain reaction in EVs and leucocytes.Results:HIV infection induces increased production of small EVs in plasma. EV subtypes were differentially enriched in miR-92, miR-155, and miR-223. Positive correlations between CD8 T-cell count and large EVs abundance and small EVs AChE activity were observed. CD4/CD8 ratio was negatively correlated with small EV AChE activity, and miRNA-155 level per small EV was negatively correlated with CD8 T-cell count.Conclusions:These findings suggest that quantifying large or small EVs and profiling miRNA content per EV might provide new functional biomarkers of immune activation and inflammation.

Tumor Microenvironment and Clonal Monocytes of Chronic Myelomonocytic Leukemia Induce a Procoagulant Climate within the Tumor Niche

Isolation of small extracellular vesicles by interacting with inorganic surface

There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag–liquid chromatography–tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers.

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.

Extracellular vesicles (EVs) are membranous structures containing bioactive molecules, secreted by most cells into the extracellular environment. EVs are classified by their biogenesis mechanisms into two major subtypes: ectosomes (enriched in large EVs; lEVs), budding directly from the plasma membrane, which is common in both prokaryotes and eukaryotes, and exosomes (enriched in small EVs; sEVs) generated through the multivesicular bodies via the endomembrane system, which is unique to eukaryotes. Even though recent proteomic analyses have identified key proteins associated with EV subtypes, there has been no systematic analysis, thus far, to support the general validity and utility of current EV subtype separation methods, still largely dependent on physical properties, such as vesicular size and sedimentation. Here, we classified human EV proteomic datasets into two main categories based on distinct centrifugation protocols commonly used for isolating sEV or lEV fractions. We found characteristic, evolutionarily conserved profiles of sEV and lEV proteins linked to their respective biogenetic origins. This may suggest that the evolutionary trajectory of vesicular proteins may result in a membership bias toward specific EV subtypes. Protein–protein interaction (PPI) network analysis showed that vesicular proteins formed distinct clusters with proteins in the same EV fraction, providing evidence for the existence of EV subtype-specific protein recruiters. Moreover, we identified functional modules enriched in each fraction, including multivesicular body sorting for sEV, and mitochondria cellular respiration for lEV proteins. Our analysis successfully captured novel features of EVs embedded in heterogeneous proteomics studies and suggests specific protein markers and signatures to be used as quality controllers in the isolation procedure for subtype-enriched EV fractions.