Protein Composition Of Extracellular Vesicles Research Articles

Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer.

High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics.

Changes in proteomic cargo of circulating extracellular vesicles in response to lifestyle intervention in adolescents with hepatic steatosis

BackgroundRecent studies suggest that proteomic cargo of extracellular vesicles (EVs) may play a role in metabolic improvements following lifestyle interventions. However, the relationship between changes in liver fat and circulating EV-derived protein cargo following intervention remains unexplored. MethodsThe study cohort comprised 18 Latino adolescents with obesity and hepatic steatosis (12 males/6 females; average age 13.3 ±1.2 y) who underwent a six-month lifestyle intervention. EV size distribution and concentration were determined by light scattering intensity; EV protein composition was characterized by liquid chromatography tandem-mass spectrometry. ResultsAverage hepatic fat fraction (HFF) decreased 23% by the end of the intervention (12.5% [5.5] to 9.6% [4.9]; P = 0.0077). Mean EV size was smaller post-intervention compared to baseline (120.2 ± 16.4 nm to 128.4 ± 16.5 nm; P = 0.031), although the difference in mean EV concentration (1.1E+09 ± 4.1E+08 particles/mL to 1.1E+09 ± 1.8E+08 particles/mL; P = 0.656)) remained unchanged. A total of 462 proteins were identified by proteomic analysis of plasma-derived EVs from participants pre- and post- intervention, with 113 proteins showing differential abundance (56 higher and 57 lower) between the two timepoints (adj-p <0.05). Pathway analysis revealed enrichment in complement cascade, initial triggering of complement, creation of C4 and C2 activators, and regulation of complement cascade. Hepatocyte-specific EV affinity purification identified 40 proteins with suggestive (p < 0.05) differential abundance between pre- and post-intervention samples. ConclusionsCirculating EV-derived proteins, particularly those associated with the complement cascade, may contribute to improvements in liver fat in response to lifestyle intervention.

The Proteome of Extracellular Vesicles Released from Pulmonary Microvascular Endothelium Reveals Impact of Oxygen Conditions on Biotrauma.

The lung can experience different oxygen concentrations, low as in hypoxia, high as under supplemental oxygen therapy, or oscillating during intermittent hypoxia as in obstructive sleep apnea or intermittent hypoxia/hyperoxia due to cyclic atelectasis in the ventilated patient. This study aimed to characterize the oxygen-condition-specific protein composition of extracellular vesicles (EVs) released from human pulmonary microvascular endothelial cells in vitro to decipher their potential role in biotrauma using quantitative proteomics with bioinformatic evaluation, transmission electron microscopy, flow cytometry, and non-activated thromboelastometry (NATEM). The release of vesicles enriched in markers CD9/CD63/CD81 was enhanced under intermittent hypoxia, strong hyperoxia and intermittent hypoxia/hyperoxia. Particles with exposed phosphatidylserine were increased under intermittent hypoxia. A small portion of vesicles were tissue factor-positive, which was enhanced under intermittent hypoxia and intermittent hypoxia/hyperoxia. EVs from treatment with intermittent hypoxia induced a significant reduction of Clotting Time in NATEM analysis compared to EVs isolated after normoxic exposure, while after intermittent hypoxia/hyperoxia, tissue factor in EVs seems to be inactive. Gene set enrichment analysis of differentially expressed genes revealed that EVs from individual oxygen conditions potentially induce different biological processes such as an inflammatory response under strong hyperoxia and intermittent hypoxia/hyperoxia and enhancement of tumor invasiveness under intermittent hypoxia.

Herpes simplex virus type 1 modifies the protein composition of extracellular vesicles to promote neurite outgrowth and neuroinfection.

The highly prevalent herpes simplex virus type 1 (HSV-1) causes a range of diseases, including cold sores, blinding keratitis, and life-threatening encephalitis. HSV-1 initially replicates in epithelial cells, enters the peripheral nervous system via neurites, and establishes lifelong infection in the neuronal cell bodies. Neurites are highly dynamic structures that grow or retract in response to attractive or repulsive cues, respectively. Here, we show that infection with HSV-1, but not with a mutant virus lacking glycoprotein G (gG), reduced the repulsive effect of epithelial cells on neurite outgrowth and facilitated HSV-1 invasion of neurons. HSV-1 gG was required and sufficient to induce neurite outgrowth by modifying the protein composition of extracellular vesicles, increasing the amount of neurotrophic and neuroprotective proteins, including galectin-1. Antibodies directed against galectin-1 neutralized the capacity of extracellular vesicles released from HSV-1-infected cells to promote neurite outgrowth. Our study provides new insights into the neurotropism of HSV-1 and identifies a viral protein that modifies the protein composition of extracellular vesicles to stimulate neurite outgrowth and invasion of the nervous system.IMPORTANCEHerpes simplex virus type 1 (HSV-1) must infect neurites (or nerve endings) to establish a chronic infection in neurons. Neurites are highly dynamic structures that retract or grow in the presence of repulsive or attractive proteins. Some of these proteins are released by epithelial cells in extracellular vesicles and act upon interaction with their receptor present on neurites. We show here that HSV-1 infection of epithelial cells modulated their effect on neurites, increasing neurite growth. Mechanistically, HSV-1 glycoprotein G (gG) modifies the protein composition of extracellular vesicles released by epithelial cells, increasing the amount of attractive proteins that enhance neurite outgrowth and facilitate neuronal infection. These results could inform of therapeutic strategies to block HSV-1 induction of neurite outgrowth and, thereby, neuronal infection.

Isolation of Extracellular Vesicles Using Titanium Dioxide Microspheres.

Extracellular vesicles (EVs) are bilayer membrane particles released from several cell types to the extracellular environment. EVs have a crucial role in cell-cell communication, involving different biological processes in health and diseases. Due to the potential of biomarkers for several diseases as diagnostic and therapeutic tools, it is relevant to understand the biology of the EVs and their content. One of the current challenges involving EVs is regarding the purification method, which is a critical step for EV's functional and characterization studies. Ultracentrifugation is the most used method for EV isolation, where the nanoparticles are separated in sequential centrifugation to isolate the EVs based on their size. However, for viscous biofluids such as plasma, there is a co-isolation of the most abundant proteins, which can impair the EV's protein identification due to the low abundance of these proteins and signal suppression by the most abundant plasma proteins. Emerging techniques have gained attention in recent years. Titanium dioxide (TiO2) is one of the most promising techniques due to its property for selective isolation based on the interaction with phospholipids in the EV membrane. Using a small amount of TiO2 beads and a low volume of plasma, it is possible to isolate EVs with reduced plasma protein co-isolation. This study describes a comprehensive workflow for the isolation and characterization of plasma extracellular vesicles (EVs) using mass spectrometry-based proteomics techniques. The aim of this chapter is describe the EV isolation using TiO2 beads enrichment and high-throughput mass spectrometry techniques to efficiently identify the protein composition of EVs in a fast and straightforward manner.

Proteomics provides insights into the theranostic potential of extracellular vesicles.

Extracellular vesicles (EVs) encompass a diverse range of membranous structures derived from cells, including exosomes and microvesicles. These vesicles are present in biological fluids and play vital roles in various physiological and pathological processes. They facilitate intercellular communication by enabling the exchange of proteins, lipids, and genetic material between cells. Understanding the cellular processes that govern EV biology is essential for unraveling their physiological and pathological functions and their potential clinical applications. Despite significant advancements in EV research in recent years, there is still much to learn about these vesicles. The advent of improved mass spectrometry (MS)-based techniques has allowed for a deeper characterization of EV protein composition, providing valuable insights into their roles in different physiological and pathological conditions. In this chapter, we provide an overview of proteomics studies conducted to identify the protein contents of EVs, which contribute to their therapeutic and pathological features. We also provided evidence on the potential of EV proteome contents as biomarkers for early disease diagnosis, progression, and treatment response, as well as factors that influence their composition. Additionally, we discuss the available databases containing information on EV proteome contents, and finally, we highlight the need for further research to pave the way toward their utilization in clinical settings.

Bacteroides acidifaciens and its derived extracellular vesicles improve DSS-induced colitis

Introduction“Probiotic therapy” to regulate gut microbiota and intervene in intestinal diseases such as inflammatory bowel disease (IBD) has become a research hotspot. Bacteroides acidifaciens, as a new generation of probiotics, has shown beneficial effects on various diseases.MethodsIn this study, we utilized a mouse colitis model induced by dextran sodium sulfate (DSS) to investigate how B. acidifaciens positively affects IBD. We evaluated the effects ofB. acidifaciens, fecal microbiota transplantation, and bacterial extracellular vesicles (EVs) on DSS-induced colitis in mice. We monitored the phenotype of mouse colitis, detected serum inflammatory factors using ELISA, evaluated intestinal mucosal barrier function using Western blotting and tissue staining, evaluated gut microbiota using 16S rRNA sequencing, and analyzed differences in EVs protein composition derived from B. acidifaciens using proteomics to explore how B. acidifaciens has a positive impact on mouse colitis.ResultsWe confirmed that B. acidifaciens has a protective effect on colitis, including alleviating the colitis phenotype, reducing inflammatory response, and improving intestinal barrier function, accompanied by an increase in the relative abundance of B. acidifaciens and Ruminococcus callidus but a decrease in the relative abundance of B. fragilis. Further fecal bacterial transplantation or fecal filtrate transplantation confirmed the protective effect of eosinophil-regulated gut microbiota and metabolites on DSS-induced colitis. Finally, we validated that EVs derived from B. acidifaciens contain rich functional proteins that can contribute to the relief of colitis.ConclusionTherefore, B. acidifaciens and its derived EVs can alleviate DSS-induced colitis by reducing mucosal damage to colon tissue, reducing inflammatory response, promoting mucosal barrier repair, restoring gut microbiota diversity, and restoring gut microbiota balance in mice. The results of this study provide a theoretical basis for the preclinical application of the new generation of probiotics.

Circulating extracellular vesicles are associated with pathophysiological condition including metabolic syndrome-related dysmetabolism in children and adolescents with obesity.

Obesity of children and adolescents (OCA) is often accompanied by metabolic syndrome (MetS), which often leads to adult obesity and subsequent complications, yet the entire pathophysiological response is not fully understood. The number and composition of circulating extracellular vesicles (EV) reflect overall patient condition; therefore, we investigated the pathophysiological condition of OCA, including MetS-associated dysmetabolism, using circulating EVs. In total, 107 children and adolescents with or without obesity (boys, n = 69; girls, n = 38; median age, 10years) were enrolled. Circulating EV number and EV protein composition were assessed via flow cytometry and liquid chromatography tandem-mass spectrometry, respectively. In a multivariate analysis, relative body weight (standardized partial regression coefficient (SPRC) 0.469, P = 0.012) and serum triglyceride level (SPRC 0.548, P < 0.001) were detected as independent parameters correlating with circulating EV number. Proteomic analysis identified 31 upregulated and 45 downregulated EV proteins in OCA. Gene ontology analysis revealed upregulated proteins to be involved in various biological processes, including intracellular protein transport, protein folding, stress response, leukocyte activation, innate immune response, and platelet degranulation, which can modulate lipid and glucose metabolism, skeletal and cardiac muscle development, inflammation, immune response, carcinogenesis, and cancer progression. Notably, several identified EV proteins are involved in neuro-development, neurotransmitter release, and neuro-protective agents in OCA. Circulating EVs were derived from adipocytes, hepatocytes, B cell lymphocytes, and neurons. Circulating EV number is significantly associated with MetS-related dysmetabolism and the EV protein cargo carries a special "signature" that reflects the alteration of various biological processes under the pathophysiological condition of OCA. KEY MESSAGES: Circulating EV number correlates with physical and laboratory parameters for obesity in children and adolescents. Relative body weight and triglyceride are independent factors for increased circulating EVs. EV composition is significantly changed in obesity of children and adolescents. Identified EV composition changes associated with obesity and involves in metabolism, immune response, and cancer progression. Circulating EVs are partially derived from adipocyte, hepatocytes, B cells, and neurons.

Differential proteomics argues against a general role for CD9, CD81 or CD63 in the sorting of proteins into extracellular vesicles.

The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock-out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P-1/EWI-F/PTGFRN and EWI-2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9- and CD81-deficient cells. In the case of EWI-2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P-1 and EWI-2 by CD9 and CD81.

Lactobacillus gasseri and Gardnerella vaginalis produce extracellular vesicles that contribute to the function of the vaginal microbiome and modulate host-Trichomonas vaginalis interactions.

Trichomonas vaginalis is an extracellular protozoan parasite of the human urogenital tract, responsible for a prevalent sexually transmitted infection. Trichomoniasis is accompanied by a dysbiotic microbiome that is characterised by the depletion of host-protective commensals such as Lactobacillus gasseri, and the flourishing of a bacterial consortium that is comparable to the one seen for bacterial vaginosis, including the founder species Gardnerella vaginalis. These two vaginal bacteria are known to have opposite effects on T. vaginalis pathogenicity. Studies on extracellular vesicles (EVs) have been focused on the direction of a microbial producer (commensal or pathogen) to a host recipient, and largely in the context of the gut microbiome. Here, taking advantage of the simplicity of the human cervicovaginal microbiome, we determined the molecular cargo of EVs produced by L. gasseri and G. vaginalis and examined how these vesicles modulate the interaction of T. vaginalis and host cells. We show that these EVs carry a specific cargo of proteins, which functions can be attributed to the opposite roles that these bacteria play in the vaginal biome. Furthermore, these bacterial EVs are delivered to host and protozoan cells, modulating host-pathogen interactions in a way that mimics the opposite effects that these bacteria have on T. vaginalis pathogenicity. This is the first study to describe side-by-side the protein composition of EVs produced by two bacteria belonging to the opposite spectrum of a microbiome and to demonstrate that these vesicles modulate the pathogenicity of a protozoan parasite. Such as in trichomoniasis, infections and dysbiosis co-occur frequently resulting in significant co-morbidities. Therefore, studies like this provide the knowledge for the development of antimicrobial therapies that aim to clear the infection while restoring a healthy microbiome.

DYT-TOR1A genotype alters extracellular vesicle composition in murine cell model and shows potential for biomarker discovery

Introduction: Biomarkers that can be used to identify patient subgroups with shared pathophysiology and/or that can be used as pharmacodynamic readouts of disease state are valuable assets for successful clinical trial design. In translational research for brain diseases, extracellular vesicles (EVs) have become a high-priority target for biomarker discovery because of their ubiquity in peripheral biofluids and potential to indicate brain state.Materials and methods: Here, we applied unbiased quantitative proteomics of EVs isolated from DYT-TOR1A knockin mouse embryonic fibroblasts and littermate controls to discover candidates for protein biomarkers. We further examined the response of genotype perturbations to drug treatment conditions to determine their pharmacodynamic properties.Results: We found that many DYT-TOR1A MEF EV differences were significantly corrected by ritonavir, a drug recently shown to correct DYT-TOR1A phenotypes in cell and mouse disease models. We also used tool compounds to explore the effect of the integrated stress response (ISR), which regulates protein synthesis and is implicated in dystonia pathogenesis. Integrated stress response inhibition in WT cells partially phenocopied the effects of DYT-TOR1A on EV proteome composition, and ISR potentiation in DYT-TOR1A caused changes that paralleled ritonavir treatment.Conclusion: These results collectively show that DYT-TOR1A genotype alters EV protein composition, and these changes can be dynamically modulated by a candidate therapeutic drug and ISR activity state. These mouse model findings provide proof-of-concept that EVs may be a useful source of biomarkers in human populations and further suggest specific homologs to evaluate in cross-species validation.

Label-free relative quantitative proteomics reveals extracellular vesicles as a vehicle for Salmonella effector protein delivery.

Extracellular vesicles are small vesicles with a diameter of 30-150 nm that are actively secreted by eukaryotic cells and play important roles in intercellular communication, immune responses, and tumorigenesis. Previous studies have shown that extracellular vesicles are involved in the process of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. However, changes in the protein content of extracellular vesicles elicited by S. Typhimurium infection have not been determined. Here, we extracted the extracellular vesicles with high purity from S. Typhimurium-infected Henle-407 cells, a kind of human intestinal epithelial cells, by ultracentrifugation combined with an extracellular vesicles purification kit, and analyzed their protein composition using label-free relative quantitative proteomics. The extracted extracellular vesicles exhibited an oval vesicular structure under electron microscopy, with a mean diameter of 140.4 ± 32.4 nm. The exosomal marker proteins CD9, CD63, and HSP70 were specifically detected. Compared with the uninfected group, nearly 1,234 specifically loaded proteins were uncovered in S. Typhimurium-infected Henle-407 cells. Among them were 409 S. Typhimurium-derived specific proteins, indicating a significant alteration in protein composition of extracellular vesicles by S. Typhimurium infection. Notably, these proteins included 75 secretory proteins and over 300 non-secretory proteins of S. Typhimurium, implicating novel pathways for bacterial protein delivery, although it remains unclear if their loading into extracellular vesicles is active or passive. To investigate the roles of these extracellular proteins, we exemplified the function of SopB, a well-known T3SS effector protein, and showed that the extracellular SopB could be taken up by RAW264.7 macrophages, activating the phosphorylation of Akt. This study provides new insights into the mechanism of Salmonella infection through extracellular vesicles that transport virulence proteins to uninfected neighboring cells to facilitate further infection.

Impact of Environmental Conditions on the Protein Content of Staphylococcus aureus and Its Derived Extracellular Vesicles.

Staphylococcus aureus, a major opportunistic pathogen in humans, produces extracellular vesicles (EVs) that are involved in cellular communication, the delivery of virulence factors, and modulation of the host immune system response. However, to date, the impact of culture conditions on the physicochemical and functional properties of S. aureus EVs is still largely unexplored. Here, we use a proteomic approach to provide a complete protein characterization of S. aureus HG003, a NCTC8325 derivative strain and its derived EVs under four growth conditions: early- and late-stationary growth phases, and in the absence and presence of a sub-inhibitory concentration of vancomycin. The HG003 EV protein composition in terms of subcellular localization, COG and KEGG categories, as well as their relative abundance are modulated by the environment and differs from that of whole-cell (WC). Moreover, the environmental conditions that were tested had a more pronounced impact on the EV protein composition when compared to the WC, supporting the existence of mechanisms for the selective packing of EV cargo. This study provides the first general picture of the impact of different growth conditions in the proteome of S. aureus EVs and its producing-cells and paves the way for future studies to understand better S. aureus EV production, composition, and roles.

Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies.

Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties.MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function.This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

Protein composition of extracellular vesicles from skin secretions of the amphibian Bombina maxima

Protein composition of extracellular vesicles from skin secretions of the amphibian Bombina maxima

Effect of caveolin-1 knockdown on the protein composition of extracellular vesicles secreted by non-small cell lung cancer cells

Background. Recent data show evidence that lipid rafts (LR) proteins could be involved in the formation of exosomes and the sorting of proteins that make up the exosomal cargo. Such data are available for flotillins, structural and functional components of flatted rafts. The presence of the main component of caveolar rafts, caveolin-1 (Cav-1), has been shown in exosomes produced by some cancer cells; however, its possible participation in the regulation of the protein composition of exosomes has not been studied previously.Materials and methods. Knockdown of Cav-1 by transduction of a lentiviral vector expressing precursors of short hairpin ribonucleic acid to Cav-1; isolation (by ultracentrifugation) and analysis (transmission electron microscopy, nanoparticle tracking analysis) of extracellular vesicles (EVs) from non-small cell lung cancer cells (NSCLC) H1299; analysis of proteins in cells and in EVs by immunoblotting.Results. Analysis of the effect of Cav-1 expression on the composition of EV proteins associated with exosome biogenesis revealed a decrease in the level of Alix and TSG101, an increase in the level of LR proteins and the absence of changes in the level of tetraspanin CD9. Conclusion. The obtained data demonstrate a Cav-1-dependent changes in the composition of EVs, indicating a change in the ratio of vesicles formed by the various molecular mechanisms.

Estimating Protein in Intact Extracellular Vesicles Based on Intrinsic Autofluorescence Spectroscopy

Background Extracellular vesicles (EVs) are submicron-sized particles that are secreted by cells within various organs and tissues with effects similar to endocrine hormones. Understanding how EVs affect cells and systems requires knowledge of EV function and composition of proteins, lipids and/or nucleic acids. Analyzing EV protein composition requires estimating total protein abundance. Conventional protein estimation methods consume sample, which can limit or eliminate the ability to perform downstream functional analysis. Since the hydrophobic amino acids tryptophan, tyrosine and phenylalanine possess intrinsic absorbance and autofluorescent properties, we hypothesized that protein can be estimated in intact EVs based on these intrinsic spectral properties and subsequently recovered for downstream analyses. Methods All mouse studies were approved by the Colorado State University Institutional Animal Care and Use Committee. Protein was estimated with bovine serum albumin (BSA) standards or EVs isolated from conditioned media of incubated mouse vastus lateralis skeletal muscle using Exoquick-TC for 24 hours in serum-free DMEM. EVs were either analyzed intact or after 10 minutes of sonication to disrupt morphology. Nanoparticle tracking analysis confirmed that our isolation protocol yielded particles with a median size of ~70 nm consistent with small EVs like exosomes. Absorbance and autofluorescence were measured simultaneously from 200-800 nm using a Horiba Aqualog. Bicinchoninic assay (BCA) was measured at 562 nm absorbance in parallel experiments as an orthogonal reference technique for estimating protein. Results We examined the absorbance spectra of BSA compared to EVs (A) and found that EVs lack a 280 nm absorbance peak due to UV absorbance below 280 nm. We next examined the fluorescence spectra for EVs and found a distinct protein peak at 280/340 ex/em (B). We generated standard curves by performing parallel analysis of BSA standards (1000, 500, 250, 100, 50 and 25, 10 and 5 µg/ml) combining BCA and autofluorescence measurements. To test whether our EV precipitation reagents directly interfere with protein estimation, the orthogonal BSA standard curves were repeated in the presence of 2% Exoquick-TC to mimic an EV isolation (C and D). The presence of 2% Exoquick-TC caused a shift in the slope in absorbance, but not fluorescence, compared to BSA alone. Plotting EVs on the orthogonal standard curve revealed a more dramatic overestimation of protein based on BCA than Exoquick-TC alone (E). Conclusions and Future Directions Our findings thus far indicate that protein autofluorescence may be a viable approach for estimating protein in intact, recoverable EVs if contaminating reagents can be removed. We are currently performing subsequent experiments to further optimize this method by utilizing complementary EV isolation methods (i.e. size exclusion chromatography) to: 1) reduce the presence of contaminating agents and 2) deplete residual non-EV proteins.

Secretion rates and protein composition of extracellular vesicles released by cancer-associated fibroblasts after radiation.

Reciprocal communication between the malignant and non-malignant cellular elements in tumors is essential for cancer sustainability and plays an important role in the response of cancers to treatments. Some of this cellular crosstalk takes place via secretion of vesicles that are actively released into the extracellular space by most cell types in tumors. Recent studies have demonstrated radiation-induced changes in the secretion rate and composition of extracellular vesicles (EVs), with impact on radiation-related cellular communication. However, little is known about the effects of different radiation regimens on the release of EVs by cells of the tumor microenvironment. In this study, we provide a comprehensive molecular characterization of EVs released by cultured primary lung tumor fibroblasts. We explore the quantitative and morphological changes triggered by ionizing radiation (IR), delivered as a single dose of 18 Gy or three consecutive daily medium-doses of 6 Gy. Cancer-associated fibroblasts (CAFs) secrete EVs with sizes ranging from 80 to 200 nm, expressing some of the classical exosome markers. Exposing CAFs to a single-high radiation dose (1 × 18 Gy) or fractionated medium-dose did not alter the release of CAF-EVs. The protein composition of CAF-EVs was analyzed by LC-MS/MS proteomics and revealed that CAF-EVs are enriched with heat shock proteins, integrins, tetraspanins, proteinases, collagens, growth factors and an array of molecules involved in the regulation of cell migration and the immune system. Quantitative proteomic analyses revealed minor changes in the protein composition of CAF-EVs after radiation exposure. Taken together, this study presents original data on lung tumor CAF-EV composition and reveals that release and protein cargo of CAF-EVs are largely unaltered after exposing CAFs to IR.

Sequencing-Based Protein Analysis of Single Extracellular Vesicles.

Circulating extracellular vesicles (EVs)-biological nanomaterials shed from most mammalian cells-have emerged as promising biomarkers, drug delivery vesicles, and treatment modulators. While different types of vesicles are being explored for these applications, it is becoming clear that human EVs are quite heterogeneous even in homogeneous or monoclonal cell populations. Since it is the surface EV protein composition that will largely dictate their biological behavior, high-throughput single EV profiling methods are needed to better define EV subpopulations. Here, we present an antibody-based immunosequencing method that allows multiplexed measurement of protein molecules from individual nanometer-sized EVs. We use droplet microfluidics to compartmentalize and barcode individual EVs. The barcodes/antibody-DNA are then sequenced to determine protein composition. Using this highly sensitive technology, we detected specific proteins at the single EV level. We expect that this technology can be further adapted for multiplexed protein analysis of any nanoparticle.

Characterization and proteome analysis of the extracellular vesicles of Phytophthora capsici

Extracellular vesicles (EVs) are important for the transport of biomolecular materials and intercellular communication in eukaryotes. Recent research has revealed that they are involved in plant–pathogen interaction and pathogenesis of infected cells. Phytophthora capsici is a highly devastating oomycete pathogen with a broad host range. To increase infection and facilitate colonization, it secretes effector proteins during interaction with plants. In this study, we characterize for the first time the EVs from pathogen P. capsici through transmission electron microscopy. For the biological study of EVs, results showed that mixing high concentrations of EVs with zoospores could enhance the virulence of P. capsici. By sequencing the protein composition of EVs by liquid chromatography in tandem with mass spectrometry we found that there are many proteins related to metabolism, oxidation/reduction, and transport in EVs, indicating that they have important roles in pathogenesis and immunological processes within the host. SignificanceExtracellular vesicles (EVs) are important both at normal physiological processes as well as pathological progression during pathogen and host interaction. In this paper we first establish the extraction method of EVs from the important oomycete pathogen Phytophthora capsici. Bioinformatics analysis of EV proteomics revealed a variety of pathogenic-related proteins, like oxidation/reduction-related proteins, stress response proteins as well as elicitors. Our results will help better understanding the biological function of the EVs during plant and P. capsici interaction and providing the evidence for the role of EVs in pathogenesis of the P. capsici.