Protein Content Of Extracellular Vesicles Research Articles

Role of Extracellular Vesicles (EVs) in Mediating Antioxidant Response to a Glucoraphanin Rich Meal

Aging is associated with increasing oxidative stress and declining antioxidant defences, making the body susceptible to various chronic conditions. Cruciferous vegetables contain glucoraphanin, which is metabolised into sulforaphane (SFN) in the gut(1). SFN activates a mechanism enhancing antioxidant defences that may contribute to a reduction in risk of age-related chronic diseases. Our hypothesis proposes that consuming cruciferous vegetables releases hepatic extracellular vesicles (EVs), which carry with a cargo of antioxidant proteins, into the systemic circulation(2) that are transported to ‘at risk’ tissues. We seek to characterise the antioxidant protein content of EVs from blood samples of healthy human volunteers from a prospective dietary intervention study trial called (GLOBE). The GLOBE study employs a randomised, single-blind, two-arm crossover design and involves 12 healthy male or female adults aged >55 years, at the time of enrolment, have a BMI in the range of 18.5-30 kg/m2, and have self-reported healthy (are not suffering from current illness like cancer, and gastrointestinal diseases including coeliac, Crohn’s, colitis, and irritable bowel syndrome) and not using any medications like antacids, laxatives and antibiotics which can interfere with normal digestive or metabolic processes. The dietary intervention consists of two commercially available treatments, one of which is a glucoraphanin-rich vegetable soup, while the other vegetable soup lacks glucoraphanin. Our initial focus lies in optimizing and standardizing a method for routinely characterizing EVs derived from healthy volunteers participating in dietary intervention studies. We specifically aim to extract EVs from a minimal plasma volume (2ml) using size exclusion chromatography (SEC). Subsequently, we intend to employ this method to analyse EVs obtained from 161 plasma samples collected from 12 participants during the GLOBE study for different time points like 0, 120, 240, 360, 480 min, 24 and 48 hr. Our goal is to gain insight into the role of EVs as part of the mechanisms by which consuming a moderate quantity of cruciferous vegetables may confer health benefits. Our research carries the potential to establish a standardised approach to the characterisation of EVs from healthy individuals which has several applications in nutrition research.

Abstract B041: Modeling the malignant transformation of fallopian tube epithelium driven by extracellular vesicles cargos in an organ-on-chip microphysiological system

Abstract Epithelial ovarian cancer (EOC) remains the leading cause of death from gynecological malignancy and the fifth leading cause of all cancer-related deaths among American women. The most common and lethal subtype, high-grade serous ovarian cancer (HGSOC) tends to originate from the fallopian tube. Precursor lesions of HGSOC such as secretory cell expansion (SCE), the loss of PAX2, PTEN, p53 mutation and serous tubal intraepithelial carcinoma (STIC) are often found at the fallopian tube epithelium (FTE). However, the mechanism of how neoplastic lesions disseminate in FTE and eventually colonize the ovary is unclear. We are interested in understanding if ovarian cancer initiation in the FTE is mediated by extracellular vesicles (EVs), which are nanosized biovesicles secreted by all living cells. EVs serve as vehicles for nucleic acids, proteins, and lipids. Cancer cells commonly secrete more EVs than healthy cells and shift their EV cargos to promote tumorigenesis. The alteration of molecular content of tumor-derived EV can potentially indicate disease states and serve as a new class of cancer biomarkers. Hence, our hypothesis is that EV cargos secreted by tumor cells contain factors promoting preneoplastic lesions in the FTE. Our goal is to interrogate the role of EVs in the initiation of ovarian cancer in the FTE at the early stage, for which there is no adequate in vitro model. We created a microfluidic model using a 3D dynamic culture system termed PREDICT-MOS to enable the direct interaction of hFTE with EVs and the long-term culture of the fallopian tube explant. We are interested to 1) test the role of EVs to drive preneoplastic transformation of hFTE progenitor cells and 2) to test whether tumor derived-EVs shift EV proteins cargos secreted by hFTE. The hFTE cultured on PREDICT-MOS is directly exposed to OVCAR3-derived EVs and embedded for transcriptomic profiling utilizing GeoMx Digital Spatial Profiler to identify differentially expressed genes in both ciliated and secretory cells within the epithelium. In parallel, we are characterizing the hFTE EV protein content with or without OVCAR3 EV treatment to detect the alteration in hFTE cargos using mass spectrometry proteomics. Our preliminary transcriptomic data from 8 patient fallopian tube samples demonstrates the deregulation of pathways associated with inflammation (TNF and NF-kappa B signaling), immune suppression (PD-1 checkpoint signaling) and epithelium morphogenesis. In addition, we have identified 295 proteins as the results of the first proteomic characterization of hFTE-derived EV proteins, which can serve as a “baseline” to evaluate whether the fallopian tube shifts its sEV cargo during ovarian cancer carcinogenesis. Further validation of transcriptomic findings on translational level using immunohistochemistry and identification of OVCAR3 EV protein cargos that induce the transcriptomic changes are needed to establish the role of EVs in the initiation of HGSOC in the fallopian tube. This project is supported by the Ovarian Cancer Research Fund Alliance Program Grant. Citation Format: Didi Zha, Jared Sipes, Sagar Rayamajhi, Leonidas E. Bantis, Angela Russo, Harsh Pathak, Andrew K. Godwin, Joanna E. Burdette. Modeling the malignant transformation of fallopian tube epithelium driven by extracellular vesicles cargos in an organ-on-chip microphysiological system [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B041.

Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles.

The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion.

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.

A Comparative Analysis of the Protein Cargo of Extracellular Vesicles from Helminth Parasites.

Helminth parasites cause debilitating-sometimes fatal-diseases in humans and animals. Despite their impact on global health, mechanisms underlying host-parasite interactions are still poorly understood. One such mechanism involves the exchange of extracellular vesicles (EVs), which are membrane-enclosed subcellular nanoparticles. To date, EV secretion has been studied in helminth parasites, including EV protein content. However, information is highly heterogeneous, since it was generated in multiple species, using varied protocols for EV isolation and data analysis. Here, we compared the protein cargo of helminth EVs to identify common markers for each taxon. For this, we integrated published proteomic data and performed a comparative analysis through an orthology approach. Overall, only three proteins were common in the EVs of the seven analyzed species. Additionally, varied repertoires of proteins with moonlighting activity, vaccine antigens, canonical and non-canonical proteins related to EV biogenesis, taxon-specific proteins of unknown function and RNA-binding proteins were observed in platyhelminth and nematode EVs. Despite the lack of consensus on EV isolation protocols and protein annotation, several proteins were shown to be consistently detected in EV preparations from organisms at different taxa levels, providing a starting point for a selective biochemical characterization.

The Proteomic Signature of Extracellular Vesicles from Sickle Cell Anemia Patients Provides Insights into Their Possible Role in the Pathophysiology of the Disease

Sickle cell anemia (SCA) is marked by hemolysis and vaso-occlusion, which contribute to various other complications observed in the disease. Extracellular vesicles (EVs), released by activated cells, serve as carriers of various markers, proteins, and RNA derived from their parent cells. These structures have the ability to transfer their cargo to the recipient cells, modulating their activity. Despite EVs involvement in physiological processes, there is growing evidence of their role in certain blood diseases, making them a subject of extensive research in recent years. In sickle cell disease (SCD), EVs have been previously associated with hemolysis markers, leg ulcers, and endothelial cell activation and several other authors have emphasized the significant role of EVs in its pathophysiology. However, despite these advances, the precise protein content of these EVs, which may hold the key to unraveling their involvement in disease complications, remains largely unexplored. Here, we aimed to characterize the proteomic profile of EVs from patients with SCA. We conducted a comparative analysis, comparing the differentially abundant proteins in the SCA group to those in the control group. Additionally, we focused on analyzing the distinct protein profiles that were uniquely present in the SCA group studied. Nine healthy volunteers (HC) and nineteen patients with SCA were included in the study, with three receiving no treatment, twelve undergoing hydroxyurea (HU) therapy, and four on HU+Crizanlizumab (CRIZ) combination treatment. EVs were isolated from plasma using a combination of differential centrifugation and size exclusion chromatography techniques, and their quantification was performed using nano-tracking analysis. The protein profile of EVs was assessed through mass spectrometry after sample digestion and desalination in a developed protocol for EVs research. The obtained data were analyzed using MaxQuant and Perseus software. To determine differentially abundant proteins between the HC and SCA groups, statistical tests such as ANOVA followed by Benjamini-Hochberg correction were applied, considering a significance level of p<0.05. Additionally, gene ontology analysis was conducted to identify the relevant biological processes (FDR <0,05) associated with the differentially abundant proteins. Firstly, we identified 41 unique proteins in the SCA group. The relevant biological processes associated with these proteins are primarily related to erythroid differentiation, inflammation, cellular response to oxidative stress, and the upregulation of signaling pathways mediated by TNF and other cytokines. These findings align with the characteristic pathophysiology of SCA. The proteins found in higher concentrations in EVs of SCA patients compared to HC group (p<0,05) were mainly related to endocytosis and exocytosis, inflammation, immune response and coagulation process. Moreover, we observed higher levels of hemoglobin chains in SCA EVs. The delta chain was increased in the SCA group with no treatment in relation to the HC group and the SCA group with HU. Beta and alpha chains were also found in increased levels in the SCA group without HU compared to the HC group, SCA with HU and SCA with HU+CRIZ. Additionally, both transferrin (TF) and the transferrin receptor (TFRC-CD71) were increased in all three subgroups of SCA patients compared to the HC group. The identification of various types of hemoglobin in these EVs holds significant importance. This observation aligns with the potential role of these structures in promoting the generation of reactive oxygen species (ROS), activating endothelial cells, and reducing nitric oxide levels in individuals with SCA. Such findings may provide valuable insights into the underlying mechanisms contributing to the pathophysiology of SCA and its associated complications. Moreover, our data shows higher levels of Tenascin-C in the EVs of SCA patients (all subgroups) (p<0,05), a protein related to tissue damage and inflammation that has not yet been described in the pathophysiology of the disease. In summary, we access, for the first time, the protein content of EVs from patients with SCA. The data described here are novel and could help to elucidate the putative functions of EVs in the context of SCA providing valuable insights for future investigations and potential therapeutic strategies.

Extracellular vesicles in COVID-19 convalescence can regulate T cell metabolism and function

Long-term Tcell dysregulation has been reported following COVID-19 disease. Prolonged Tcell activation is associated with disease severity and may be implicated in producing long-covid symptoms. Here, we assess the role of extracellular vesicles (EV) in regulating Tcell function over several weeks post COVID-19 disease. We find that alterations in cellular origin and protein content of EV in COVID-19 convalescence are linked to initial disease severity. We demonstrate that convalescent donor-derived EV can alter the function and metabolic rewiring of CD4 and CD8 Tcells. Of note, EV following mild, but not severe disease, show distinctly immune-suppressive properties, reducing Tcell effector cytokine production and glucose metabolism. Mechanistically our data indicate the involvement of EV-surface ICAM-1 in facilitating EV-T cell interaction. Our data demonstrate that circulatory EV are phenotypically and functionally altered several weeks following acute infection, suggesting a role for EV as long-term immune modulators.

Extracellular vesicles of the Gram-positive gut symbiont Bifidobacterium longum induce immune-modulatory, anti-inflammatory effects

The gut microbiota is now well known to affect the host’s immune system. One way of bacterial communication with host cells is via the secretion of vesicles, small membrane structures containing various cargo. Research on vesicles secreted by Gram-positive gut bacteria, their mechanisms of interaction with the host and their immune-modulatory effects are still relatively scarce. Here we characterized the size, protein content, and immune-modulatory effects of extracellular vesicles (EVs) secreted by a newly sequenced Gram-positive human gut symbiont strain - Bifidobacterium longum AO44. We found that B. longum EVs exert anti-inflammatory effects, inducing IL-10 secretion from both splenocytes and dendritic cells (DC)-CD4+ T cells co-cultures. Furthermore, the EVs protein content showed enrichment in ABC transporters, quorum sensing proteins, and extracellular solute-binding proteins, which were previously shown to have a prominent function in the anti-inflammatory effect of other strains of B. longum. This study underlines the importance of bacterial vesicles in facilitating the gut bacterial immune-modulatory effects on the host and sheds light on bacterial vesicles as future therapeutics.

Rab11A Depletion in Microglia-Derived Extracellular Vesicle Proteome upon Beta-Amyloid Treatment

Microglia, the macrophage-like glial cells, behave as sentinels against exogenous pathogens invading the neural tissue. Their commitment is not only confined to the defensive function, but they also perform balancing trophic activities such as neuronal postnatal development, remodeling and pruning of synapses. Likewise, microglia-derived extracellular vesicles (EVs) can play strategic roles in maintaining a healthy brain by modulating neuronal activity and by controlling neurite outgrowth as well as innate immune response. Nevertheless, strong evidence also points to their role in the development of neurodegenerative pathologies such as Alzheimer’s disease (AD). Here, we explored EV protein content released by BV2 microglial cells in a resting state and after stimulation with beta-amyloid peptides (Aβ), mimicking conditions occurring in AD. In the resting BV2 cells, we extended the list of proteins present in mouse microglia EV cargo with respect to those reported in the Vesiclepedia exosome database while, in amyloid-triggered microglia, we highlighted a pronounced drop in EV protein content. Focusing on Rab11A, a key factor in the recycling routes of amyloid species, we observed a dramatic decrease of this protein in Aβ-treated microglia EV cargo with respect to the EVs from the untreated sample. This decrease might affect the delivery of Rab11A to neurons thus increasing the harmful amyloid burden in neuronal cells that eventually may lead to their death. We tentatively proposed that alterations observed in EVs derived from Aβ-treated microglia may represent molecular features that, among others, shape the disease-associated microglial phenotype, a recently proposed subset of microglial population, present in neurodegenerative pathologies.

Proteomic profile of extracellular vesicles in maternal plasma of women with fetal death

Objectives Fetal death is a complication of pregnancy caused by multiple etiologies rather than being the end-result of a single disease process. Many soluble analytes in the maternal circulation, such as hormones and cytokines, have been implicated in its pathophysiology. However, changes in the protein content of extracellular vesicles (EVs), which could provide additional insight into the disease pathways of this obstetrical syndrome, have not been examined. This study aimed to characterize the proteomic profile of EVs in the plasma of pregnant women who experienced fetal death and to evaluate whether such a profile reflected the pathophysiological mechanisms of this obstetrical complication. Moreover, the proteomic results were compared to and integrated with those obtained from the soluble fraction of maternal plasma. Methods This retrospective case-control study included 47 women who experienced fetal death and 94 matched, healthy, pregnant controls. Proteomic analysis of 82 proteins in the EVs and the soluble fractions of maternal plasma samples was conducted by using a bead-based, multiplexed immunoassay platform. Quantile regression analysis and random forest models were implemented to assess differences in the concentration of proteins in the EV and soluble fractions and to evaluate their combined discriminatory power between clinical groups. Hierarchical cluster analysis was applied to identify subgroups of fetal death cases with similar proteomic profiles. A p-value of <.05 was used to infer significance, unless multiple testing was involved, with the false discovery rate controlled at the 10% level (q < 0.1). All statistical analyses were performed by using the R statistical language and environment-and specialized packages. Results Nineteen proteins (placental growth factor, macrophage migration inhibitory factor, endoglin, regulated upon activation normal T cell expressed and presumably secreted (RANTES), interleukin (IL)-6, macrophage inflammatory protein 1-alpha, urokinase plasminogen activator surface receptor, tissue factor pathway inhibitor, IL-8, E-Selectin, vascular endothelial growth factor receptor 2, pentraxin 3, IL-16, galectin-1, monocyte chemotactic protein 1, disintegrin and metalloproteinase domain-containing protein 12, insulin-like growth factor-binding protein 1, matrix metalloproteinase-1(MMP1), and CD163) were found to have different plasma concentrations (of an EV or a soluble fraction) in women with fetal death compared to controls. There was a similar pattern of change for the dysregulated proteins in the EV and soluble fractions and a positive correlation between the log2-fold changes of proteins significant in either the EV or the soluble fraction (ρ = 0.89, p < .001). The combination of EV and soluble fraction proteins resulted in a good discriminatory model (area under the ROC curve, 82%; sensitivity, 57.5% at a 10% false-positive rate). Unsupervised clustering based on the proteins differentially expressed in either the EV or the soluble fraction of patients with fetal death relative to controls revealed three major clusters of patients. Conclusion Pregnant women with fetal death have different concentrations of 19 proteins in the EV and soluble fractions compared to controls, and the direction of changes in concentration was similar between fractions. The combination of EV and soluble protein concentrations revealed three different clusters of fetal death cases with distinct clinical and placental histopathological characteristics.

P049 Proteomic characterization of serum extracellular vesicles from newly diagnosed patients with inflammatory bowel disease

Abstract Background The etiology and pathogenesis of inflammatory bowel disease (IBD), Crohn’s disease (CD), and ulcerative colitis (UC) are complex and the mechanisms that lead to the development of these diseases remain unclear. Extracellular vesicles (EVs) are small particles covered with a cell membrane, originating from the emitting cell, excreted into the extracellular medium, and subsequently captured by receptor cells. EVs have a role in multiple diseases and they could also have a function in IBD pathogenesis. Therefore, the analysis of EVs isolated from the serum of newly diagnosed IBD patients (before starting any treatment) may represent an appropriate experimental approach to elucidate their role in IBD pathogenesis. We aimed to evaluate EVs’ composition and potential effects in the pathogenesis of IBD. Methods The protein content of EVs isolated by size exclusion chromatography from the 500 µl of serum from 100 patients with IBD (50 patients with CD and 50 patients with UC) recently diagnosed and 50 healthy controls (HC) was characterized by proteomics and their size and concentration in serum by Nanoparticle tracking analysis (NTA). The biological function of EVs and alterations in signaling pathways related to IBD were determined using Ingenuity Pathways Analysis (IPA) to analyze the OMICs results. Results A total of 1,100 proteins have been identified, of which 105 proteins were differentially expressed by patients with CD versus HC, 111 proteins by patients with UC versus HC, and 32 proteins by patients with UC versus CD. IPA analysis revealed that proteins carried in EVs are involved in the dysregulation of immune pathways such as the acute phase response (Figure 1), LXR/RXR activation pathway (Figure 2), and the complement pathway. These pathways were regulated differentially in IBD patients compared with HC, but also when the CU group was compared with the CD group (Figure 3) being upregulated nuclear receptors signaling and cytotoxicity pathways. Conclusion EVs carry proteins that can be involved in the dysregulation of the immune system in IBD; this effect would be different in UC and CD since their EVs show a differential profile. Consequently, EVs may play role in IBD pathogenesis and source of strong biomarkers candidates for diagnosis in UC and CD. Further studies on their specific function during IBD are warranted.

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.

DNAJB6-Containing Extracellular Vesicles as Chaperone Delivery Systems: A Proteomic Analysis.

Cell-derived extracellular vesicles (EVs) are effectors of cell-to-cell communication that are in the spotlight as promising candidates for in vivo drug delivery because of their ability to enter cells and deliver cargo. For example, proteins of interest can be loaded into EVs to mediate protein transfer into target cells. To determine causality between EV content and function, which is also important to assess the clinical safety of EVs, it is crucial to comprehensively characterize their complete molecular composition. Here, we investigated EVs loaded with the chaperone protein DNAJB6. Chaperone proteins assist in protein folding and have been suggested to alleviate protein aggregation diseases, such as Alzheimer's disease and Huntington's disease. We analyzed and compared the proteome of EVs isolated from wildtype HEK293T cells with that of EVs from HEK 293T cells overexpressing DNAJB6-WT or loss-of-function mutant DNAJB6-M3. Comprehensive analysis of proteomics data showed enhanced levels of DNAJB6 as well as protein-folding-related proteins in EVs derived from DNAJB6-overexpression cells. Interestingly, upregulation of a chaperone and its protein-folding-related proteins resulted in downregulation of another chaperone plus its related proteins, and vice versa. This implies the presence of compensatory mechanisms in the cellular expression of chaperones. Collectively, we provide the proteomic EV signatures underlying EV mediated DNAJB6 transmission by HEK293T cells, with the aim of establishing a causal relationship between EV protein content and EV function.

Proteomic Profiling of Extracellular Vesicles Released by Leptin-Treated Breast Cancer Cells: A Potential Role in Cancer Metabolism.

Tumor extracellular vesicles (EVs), as endocytic vesicles able to transport nucleic acids, proteins, and metabolites in recipient cells, have been recognized fundamental mediators of cell-to-cell communication in breast cancer. The biogenesis and release of EVs are highly regulated processes and both the quantity of EVs and their molecular cargo might reflect the metabolic state of the producing cells. We recently demonstrated that the adipokine leptin, whose circulating levels correlate with adipose tissue expansion, is an inducer of EV release from breast cancer cells. Here, we show a specific proteomic signature of EVs released by MCF-7 breast cancer cells grown in the presence of leptin (Lep-EVs), in attempt to find additional molecular effectors linking obesity to breast cancer biology. An analysis of the proteomic profile of Lep-EVs by LC-MS/MS revealed a significant enrichment in biological processes, molecular functions, and cellular components mainly related to mitochondrial machineries and activity, compared to protein content of EVs from untreated breast cancer cells. Metabolic investigations, carried out to assess the autocrine effects of these vesicles on breast cancer cells, revealed that Lep-EVs were able to increase ATP levels in breast cancer cells. This result is associated with increased mitochondrial respiration evaluated by Seahorse analyzer, supporting the concept that Lep-EVs can modulate MCF-7 breast cancer cell oxidative metabolism. Moreover, taking into account the relevance of tumor immune cell crosstalk in the tumor microenvironment (TME), we analyzed the impact of these vesicles on macrophage polarization, the most abundant immune component in the breast TME. We found that tumor-derived Lep-EVs sustain the polarization of M0 macrophages, derived from the human THP-1 monocytic cells, into M2-like tumor-associated macrophages, in terms of metabolic features, phagocytic activity, and increased expression of CD206-positive population. Overall, our results indicate that leptin by inducing the release of EV-enriched in mitochondrial proteins may control the metabolism of MCF-7 breast cancer cells as well as that of macrophages. Characterization of tumor-derived EV protein cargo in an obesity-associated milieu, such as in the presence of elevated leptin levels, might allow identifying unique features and specific metabolic mechanisms useful to develop novel therapeutic approaches for treatment of breast cancer, especially in obese patients.

Multiple Myeloma-Derived Extracellular Vesicles Modulate the Bone Marrow Immune Microenvironment.

Multiple myeloma (MM), the third most frequent hematological cancer worldwide, is characterized by the proliferation of neoplastic plasma cells in the bone marrow (BM). One of the hallmarks of MM is a permissive BM microenvironment. Increasing evidence suggests that cell-to-cell communication between myeloma and immune cells via tumor cell-derived extracellular vesicles (EV) plays a key role in the pathogenesis of MM. Hence, we aimed to explore BM immune alterations induced by MM-derived EV. For this, we inoculated immunocompetent BALB/cByJ mice with a myeloma cell line, MOPC315.BM, inducing a MM phenotype. Upon tumor establishment, characterization of the BM microenvironment revealed the expression of both activation and suppressive markers by lymphocytes, such as granzyme B and PD-1, respectively. In addition, conditioning of the animals with MOPC315.BM-derived EV, before transplantation of the MOPC315.BM tumor cells, did not anticipate the disease phenotype. However, it induced features of suppression in the BM milieu, such as an increase in PD-1 expression by CD4+ T cells. Overall, our findings reveal the involvement of MOPC315.BM-derived EV protein content as promoters of immune niche remodeling, strengthening the importance of assessing the mechanisms by which MM may impact the immune microenvironment.

Patient-Derived Extracellular Vesicles Proteins as New Biomarkers in Multiple Myeloma - A Real-World Study.

Multiple myeloma (MM) is a hematological malignancy of clonal antibody–secreting plasma cells (PCs). MM diagnosis and risk stratification rely on bone marrow (BM) biopsy, an invasive procedure prone to sample bias. Liquid biopsies, such as extracellular vesicles (EV) in peripheral blood (PB), hold promise as new minimally invasive tools. Real-world studies analyzing patient-derived EV proteome are rare. Here, we characterized a small EV protein content from PB and BM samples in a cohort of 102 monoclonal gammopathies patients routinely followed in the clinic and 223 PB and 111 BM samples were included. We investigated whether EV protein and particle concentration could predict an MM patient prognosis. We found that a high EV protein/particle ratio, or EV cargo >0.6 µg/108 particles, is related to poorer survival and immune dysfunction. These results were supported at the protein level by mass spectrometry. We report a set of PB EV-proteins (PDIA3, C4BPA, BTN1A1, and TNFSF13) with a new biomarker potential for myeloma patient outcomes. The high proteomic similarity between PB and BM matched pairs supports the use of circulating EV as a counterpart of the BM EV proteome. Overall, we found that the EV protein content is related to patient outcomes, such as survival, immune dysfunction, and possibly treatment response.

Extracellular Vesicle (EV) Dot Blotting for Multiplexed EV Protein Detection in Complex Biofluids.

Extracellular vesicles (EVs) are nanoscale vesicles secreted from cells, carrying biomolecular cargos similar to their cells of origin. Measuring the protein content of EVs in biofluids can offer a crucial insight into human health and disease. For example, detecting tumor-derived EVs' protein markers can aid in early diagnosis of cancer, which is life-saving. In order to use these EV proteins for diagnosis, sensitive and multiplexed methods are required. The current methods for EV protein detection typically require large sample consumption due to challenges with sensitivity and often need an EV isolation step for complex biofluid samples such as blood plasma. In this work, we have developed a simple and sensitive method for multiplexed detection of protein markers on EV membrane surfaces, which we call "EV dot blotting", inspired by conventional dot blotting techniques. After optimization of multiple factors such as antibody concentration, blocking reagent, type of 3D membranes, and use of gold nanoparticles for signal enhancement, cancer-cell-derived EVs were spiked in pooled normal human plasma for conducting a multiplexed assay in a microarray format. Without the need of isolating EVs from blood plasma, a limit of detection of 3.1 × 105 EVs/mL or 1863 EVs/sample was achieved for CD9 protein, 4.7 × 104 EVs/mL or 281 EVs/sample for CD24, and 9.0 × 104 EVs/mL or 538 EVs/sample for EpCAM, up to 4 orders of magnitude lower than those of conventional ELISA. This platform offers sensitive, multiplexed, simple, and low-cost EV protein detection directly from complex biofluids with minimal sample consumption, providing a useful tool for multiplexed EV protein quantification for a variety of applications.

Extracellular Vesicles From LPS-Treated Macrophages Aggravate Smooth Muscle Cell Calcification by Propagating Inflammation and Oxidative Stress

Background: Vascular calcification (VC) is a cardiovascular complication associated with a high mortality rate among patients with diseases such as atherosclerosis and chronic kidney disease. During VC, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete a heterogeneous population of extracellular vesicles (EVs). Recent studies have shown involvement of EVs in the inflammation and oxidative stress observed in VC. We aimed to decipher the role and mechanism of action of macrophage-derived EVs in the propagation of inflammation and oxidative stress on VSMCs during VC. Methods: The macrophage murine cell line RAW 264.7 treated with lipopolysaccharide (LPS-EK) was used as a cellular model for inflammatory and oxidative stress. EVs secreted by these macrophages were collected by ultracentrifugation and characterized by transmission electron microscopy, cryo-electron microscopy, nanoparticle tracking analysis, and the analysis of acetylcholinesterase activity, as well as that of CD9 and CD81 protein expression by western blotting. These EVs were added to a murine VSMC cell line (MOVAS-1) under calcifying conditions (4 mM Pi—7 or 14 days) and calcification assessed by the o-cresolphthalein calcium assay. EV protein content was analyzed in a proteomic study and EV cytokine content assessed using an MSD multiplex immunoassay. Results: LPS-EK significantly decreased macrophage EV biogenesis. A 24-h treatment of VSMCs with these EVs induced both inflammatory and oxidative responses. LPS-EK-treated macrophage-derived EVs were enriched for pro-inflammatory cytokines and CAD, PAI-1, and Saa3 proteins, three molecules involved in inflammation, oxidative stress, and VC. Under calcifying conditions, these EVs significantly increase the calcification of VSMCs by increasing osteogenic markers and decreasing contractile marker expression. Conclusion: Our results show that EVs derived from LPS-EK–treated-macrophages are able to induce pro-inflammatory and pro-oxidative responses in surrounding cells, such as VSMCs, thus aggravating the VC process.

Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular Vesicles.

Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses in vitro. In the present study, we evaluated the impact of in vitro infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s).

The Extracellular Vesicles from the Commensal Staphylococcus Epidermidis ATCC12228 Strain Regulate Skin Inflammation in the Imiquimod-Induced Psoriasis Murine Model.

Extracellular vesicles (EVs) are evaginations of the cytoplasmic membrane, containing nucleic acids, proteins, lipids, enzymes, and toxins. EVs participate in various bacterial physiological processes. Staphylococcus epidermidis interacts and communicates with the host skin. S. epidermidis’ EVs may have an essential role in this communication mechanism, modulating the immunological environment. This work aimed to evaluate if S. epidermidis’ EVs can modulate cytokine production by keratinocytes in vitro and in vivo using the imiquimod-induced psoriasis murine model. S. epidermidis’ EVs were obtained from a commensal strain (ATC12228EVs) and a clinical isolated strain (983EVs). EVs from both origins induced IL-6 expression in HaCaT keratinocyte cultures; nevertheless, 983EVs promoted a higher expression of the pro-inflammatory cytokines VEGF-A, LL37, IL-8, and IL-17F than ATCC12228EVs. Moreover, in vivo imiquimod-induced psoriatic skin treated with ATCC12228EVs reduced the characteristic psoriatic skin features, such as acanthosis and cellular infiltrate, as well as VEGF-A, IL-6, KC, IL-23, IL-17F, IL-36γ, and IL-36R expression in a more efficient manner than 983EVs; however, in contrast, Foxp3 expression did not significantly change, and IL-36 receptor antagonist (IL-36Ra) was found to be increased. Our findings showed a distinctive immunological profile induction that is dependent on the clinical or commensal EV origin in a mice model of skin-like psoriasis. Characteristically, proteomics analysis showed differences in the EVs protein content, dependent on origin of the isolated EVs. Specifically, in ATCC12228EVs, we found the proteins glutamate dehydrogenase, ornithine carbamoyltransferase, arginine deiminase, carbamate kinase, catalase, superoxide dismutase, phenol-soluble β1/β2 modulin, and polyglycerol phosphate α-glucosyltransferase, which could be involved in the reduction of lesions in the murine imiquimod-induced psoriasis skin. Our results show that the commensal ATCC12228EVs have a greater protective/attenuating effect on the murine imiquimod-induced psoriasis by inducing IL-36Ra expression in comparison with EVs from a clinical isolate of S. epidermidis.