Extracellular vesicles in viral disease management

Extracellular vesicles (EVs) are heterogeneous biological nanoparticles secreted by all cell types. Identifying the proteins preferentially encapsulated in secreted EVs will help understand their heterogeneity. Src family kinases including Src and Fyn are a group of tyrosine kinases with fatty acylation modifications and/or multiple lysine residues (contributing charge interaction) at their N-terminus. Here, we demonstrate that Src and Fyn kinases were preferentially encapsulated in EVs and fatty acylation including myristoylation and palmitoylation facilitated their encapsulation. Genetic loss or pharmacological inhibition of myristoylation suppressed Src and/or Fyn kinase levels in EVs. Similarly, loss of palmitoylation reduced Fyn levels in EVs. Additionally, mutation of lysine at sites 5, 7, and 9 of Src kinase also inhibited the encapsulation of myristoylated Src into EVs. Knockdown of TSG101, which is a protein involved in the endosomal sorting complexes required for transport (ESCRT) protein complex mediated EVs biogenesis and led to a reduction of Src levels in EVs. In contrast, filipin III treatment, which disturbed the lipid raft structure, reduced Fyn kinase levels, but not Src kinase levels in EVs. Finally, elevated levels of Src protein were detected in the serum EVs of host mice carrying constitutively active Src-mediated prostate tumors in vivo. Collectively, the data suggest that different EVs biogenesis pathways exist and can regulate the encapsulation of specific proteins into EVs. This study provides an understanding of the EVs heterogeneity created by different EVs biogenesis pathways.

The Population of Circulating Extracellular Vesicles Dramatically Alters after Very Premature Delivery- a Previously Unrecognised Postnatal Adaptation Process?

Extracellular vesicle (EV) release in fungi was described for the first time in 2007 in the yeast-like pathogen Cryptococcus neoformans [1]. Since then, the phenomenon of EV production, which is present in all domains of life, has been observed in many different fungal species, including yeast cells and hyphae. Composition of EVs, the impact of their release on fungal pathogenesis, and their potential use as protective immunogens have been explored in a number of original studies and comprehensive reviews (see Fig 1 and [2] for a summary). However, many aspects related to the biological properties of fungal EVs remain obscure. In this manuscript, we will focus our discussion on three fundamental but still unanswered questions about fungal EVs. Fig 1 Overview of the functional aspects of fungal EVs. What Is the Role of Fungal EVs during Infection? It remains unknown whether fungal EVs are produced in vivo, which is likely linked to the lack of protocols and molecular markers for isolation of these membranous compartments from body fluids. Vesicle properties related to their stability in tissues are also obscure. C. neoformans EVs are rapidly disrupted by serum albumin at physiological concentrations [3]. This observation argues against the stability of EVs in vivo, but not against their potential functions. EV disruption might result in the release of internal and potentially immunomodulatory compounds into the extracellular space, possibly impacting the physiology of host cells. Different studies provided indirect evidence supporting the hypothesis that fungal EVs are produced during infection. Sera from patients with cryptococcosis or histoplasmosis reacted with EV components [4,5]. In addition, EVs were isolated from plasma of patients with Malassezia sympodialis-associated atopic eczema [6]. In vivo studies with C. neoformans suggested that EVs are produced during lung infection [1]. The immunobiological activity of fungal EVs and the mechanisms by which they modulate host cell physiology have been first explored in M. sympodialis, where allergen-containing EVs induced IL-4 and TNF-α responses [6]. In further studies with C. neoformans and Candida albicans [7,8], labeling of EVs with DiIC18, a lipophilic and fluorescent stain, allowed observation of vesicle internalization by murine phagocytes and consequent cytokine production. EVs co-localized with the lipid raft marker GM1, suggesting the participation of such domains during vesicle internalization [7,8]. Apparently, fungal EVs are internalized through phagocytosis, since DiIC18 labeling was restricted to the cytoplasm after 15 minutes of incubation with phagocytes. It remains unclear, however, whether fungal EVs also fuse with host cell membranes, as suggested after interaction of C. neoformans EVs with human brain microvascular endothelial cells (HBMEC) [9]. In this situation, vesicle cargo would be directly delivered into the cytoplasm of host cells. This mechanism appears to modulate HBMEC permeability during murine cryptococcosis, facilitating crossing of the blood-brain barrier and brain colonization by the fungus [9]. More recently, Wolf and colleagues investigated a strain of C. albicans lacking expression of a phosphatidylserine synthase [10]. EV cargo lacked characteristic virulence factors, including phospholipase Plb3 and adhesin Sim1. These EVs failed to induce NFκB activation in macrophages [10]. Thus, phospholipid biosynthesis appears to be required for EV cargo and functions. The molecules carried by EVs may impact antigen processing and, consequently, the immune response. C. albicans EVs stimulated dendritic cells (DCs) to produce IL-12p40, IL-10, and TNF-α, and induced upregulation of CD86 and MHC-II [7]. Treatment of murine macrophages with EVs from C. neoformans or C. albicans resulted in production of nitric oxide, IL-12, TGF-β, and IL-10 [7,8]. In addition, EVs from an acapsular strain of C. neoformans induced a high proinflammatory response [8]. The protective effect of EVs on the innate immune system has been suggested using the insect model Galleria mellonella. Treatment of larvae with EVs from C. albicans resulted in significant protection against subsequent challenges with this fungus [7]. These studies suggest that fungal EVs activate the innate immune response and may also promote, in other models, the development of adaptive responses (Table 1). A beneficial contribution of fungal EVs to humoral immunity is also expected. Enolase, HSP60, and GlcCer are examples of immunogens carried by EVs that can induce protective antibodies (reviewed in [11]). Table 1 Functional diversity of fungal EVs. EV Biogenesis: Where Do They Come From? Exosomes and ectosomes are major EVs produced by eukaryotic cells. Exosomes consist of small (40–100 nm) vesicles originated by invagination of the endosomal compartments membrane, which is driven by a protein complex named endosomal sorting complex required for transport (ESCRT) [12]. This complex regulates the release of small vesicles inside the lumen of the endosome, generating the so-called multivesicular bodies (MVBs). Upon fusion with the plasma membrane, MVBs release exosomes as EVs to the outer space [12]. Unlike exosomes, ectosomes are larger (up to 1 μm), ubiquitous vesicles that are assembled at and released from the plasma membrane [13]. In fungi, mechanisms of vesicle biogenesis and extracellular release are still obscure. Therefore, these extracellular membranous compartments are still collectively called EVs. MVB-like structures have been observed in C. neoformans [5]. Saccharomyces cerevisiae mutants lacking expression of ESCRT machinery proteins still produced EVs, but vesicle cargo was modified in the absence of ESCRT regulators [14]. Analysis of fungal EVs by electron microscopy revealed two kinds of populations in the cell wall periphery: large (up to 300 nm), individualized vesicles and small (up to 100 nm), grouped vesicles [15]. Groups of small EVs in the periplasm are consistent with exosome formation, as suggested in early studies with C. neoformans [16]. Observation of individualized and larger vesicles, however, is suggestive of membrane budding, likely resulting in ectosomes [17]. Membrane budding, in fact, has been observed more than a decade ago in C. neoformans [18]. EV formation can also include inverted macropinocytosis, a process by which fractions of the cytoplasm are sequestered by plasma membrane invaginations, resulting in individualized EV-like structures [19]. All the mechanisms described above would be consistent with the diversity in EV composition, which includes a number of cytoplasmic components (reviewed in [11]).

Background: Extracellular vesicles (EVs) are released from cancer cells into the tumor microenvironment, there participating in intercellular communication by altering recipient cell function. Breast cancer (BC) derived EVs are hypothesized to have an impact on tumor growth, immunosuppression or metastatic development, indicating analytical significance of EV concentrations in BC patients. For this reason, we investigated plasmatic EV numbers from locally advanced, neoadjuvant treated (NACT) BC patients in association with clinical parameters and prognostic impact. Material and methods: Plasmatic extracellular vesicles were isolated using ExoQuick™ precipitation reagent (SBI Inc., Mountain View, CA, USA), according to manufacturer's instructions, from locally advanced BC patients before (n = 142) and after (n = 156) NACT as well as from healthy female controls (n = 16). Subsequently, number and EV particle size were analyzed using ZetaView Laser Scattering Video Microscope (Particle Metrix GmbH, Microtrac, Meerbusch, Germany). Samples were 1:50.000 in PBS pre-diluted to obtain particle concentrations of approx. 1 × 10⁁6 particles per ml. Circulating tumor cells (CTCs) and stem cell-like circulating tumor cells (slCTCs) were evaluated using the AdnaTests BreastCancer, StemCell and EMT, respectively (QIAGEN Hannover GmbH, Germany). Results: EV particle concentrations (mean ± SEM in 10⁁9/ml) were significantly (p < 0.0001) elevated in BC patients (n = 104, respectively) before (2370 ± 170) and after (3524 ± 523) NACT compared to healthy females (90 ± 19). Paired analysis before and after NACT revealed higher EV levels after NACT (p = 0.008). In association studies, high EV numbers before NACT were related to less differentiated carcinomas and to lymph node spread. ROC analysis showed optimal cut-off values of EV levels (i) before NACT: 3540 × 10⁁9/ml (sensitivity: 66.7%; specificity: 78.1%; AUC = 0.706) and (ii) after NACT: 2480 × 10⁁9/ml (sensitivity: 100%; specificity: 44,3%; AUC = 0.654). In Kaplan-Meier analysis, a decreased 3-year PFS was significantly associated with EV particle concentrations after NACT > 2480 × 10⁁9/ml (p = 0.005). A reduced OS of BC patients was significantly associated with (i) EV levels before NACT > 3540 × 10⁁9/ml) (p = 0.001) and (ii) EV levels after NACT > 2480 × 109/ml) (p = 0.003). Decreased EV levels were found in BC patients after NACT with CTCs expressing ERCC1 (p = 0.025) or the stem cell marker ALDH1 (p = 0.004). Conclusion: In conclusion, BC patients showed elevated plasmatic EV levels compared to healthy females, while increased EV levels after NACT might be due to therapeutic effects. The association between EV particle concentrations and clinical parameters as well as their prognostic impact on clinical outcome in BC indicate the importance of EVs as a mediator in the BC tumor microenvironment. Therefore, determination of plasmatic EV particle amount might serve as a biomarker for BC monitoring. Citation Format: Lisa König, Vera Rebmann, Oliver Hoffmann, Ann-Kathrin Bittner, Bettina Wagner, Luis Felipe Santos Manvailer, Sabine Schramm, Agnes Bankfalvi, Bernd Giebel, Rainer Kimmig, Peter A. Horn, Sabine Kasimir-Bauer. Breast cancer-derived extracellular vesicles: clinical and prognostic impact. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3152.

Association between Extracellular Vesicles (EVs) and Thrombosis in Antiphospholipid Syndrome

Extracellular vesicles (EVs) modulate inflammation, coagulation and vascular homeostasis in decompensated cirrhosis. To characterize the profile of plasmatic EVs in patients with decompensated cirrhosis and bacterial infections and evaluate the association between EVs and the development of hemostatic complications. We measured the levels of EVs using high-sensitivity flow cytometry and phospholipid-dependent clotting time (PPL) in a prospective cohort of hospitalized patients with acutely decompensated cirrhosis with versus without bacterial infections. A separate cohort of patients with bacterial infections without cirrhosis was also enrolled. We measured endothelium-, tissue factor (TF)-bearing, platelet- and leukocyte-derived EVs. In patients with infections, EVs were reassessed upon resolution of infection. Bleeding and thrombotic complications were recorded during 1-year follow-up. Eighty patients with decompensated cirrhosis were recruited (40 each with and without bacterial infections). Electron microscopy confirmed the presence of plasma EVs. Despite no difference in total EVs and PPL, patients with cirrhosis and infection had significantly higher TF+ EVs, P-Selectin+ EVs (activated platelet-derived), CD14+ EVs (monocyte/macrophages derived) and CD14+ TF+ EVs versus those with cirrhosis without infection. Upon infection resolution, levels of these EVs returned to those without infection. Patients with infections showed a significant association between reduced P-Selectin+ EVs and bleeding complications (HR 8.0 [95%CI 1.3-48.1]), whereas high levels of leukocyte-derived EVs (CD45+) and CD14+ EVs were significantly associated with thrombotic complications (HR 16.4 [95%CI 1.7-160] and 10.9 [95%CI 1.13-106], respectively). Results were confirmed in a validation cohort. Bacterial infections are associated with particular alterations of plasma EVs profile in decompensated cirrhosis. Bacterial infections trigger the release of EVs originating from various cell types, which may tip the precarious hemostatic balance of patients with acutely decompensated cirrhosis towards hyper- or hypocoagulability.

Activation of TLR3 and its adaptor TICAM-1 increases miR-21 levels in extracellular vesicles released from human cells

Although effective antiretroviral therapy (ART) has improved the life expectancy of people with HIV (PWH), the prevalence of milder forms of HIV-associated neurocognitive disorders (HAND) persist, and it is associated with systemic and neuro-inflammatory processes that could impact other organ systems. However, the complex signaling mechanisms between the bone-kidney systems and the brain in HAND remain unknown. Extracellular vesicles (EVs) play a potential role in inter-organ communication and are involved in regulating cell activity in distant tissues. In this study, we examined whether levels of EVs from bone-and kidney-related cells associate with cognitive dysfunction and explored the relationship between kidney-bone EV axis in PWH experiencing cognitive deficits. EV subtypes were characterized in plasma from 61 PWH with either cognitive impairment (CI, n = 53) or normal cognition (NC, n = 8) based on the American Academy of Neurology criteria for HIV-associated dementia (HAD, n = 11), minor cognitive motor disorder (MCMD, n = 25) or asymptomatic neurocognitive impairment (ANI, n = 17) by spectral flow cytometry. EVs were profiled with markers reflecting bone and kidney cell origin. A support vector machine learning-based model was employed for analyses of EV phenotypes to predict the cognitive dysfunction. Plasma-EVs expressing osteocalcin, sclerostin, and nephrin were significantly higher in the cognitive impairment group compared to the normal cognition group. EVs bearing kidney cell markers correlated significantly with bone-derived EVs. A machine learning-based model, comprised of osteocalcin+, nephrin+, and CD24+ EVs predicted cognitive impairment in PWH on ART. Our study reveals that neurocognitive impairment in PWH is associated with increased levels of plasma EVs enriched with the bone markers osteocalcin and sclerostin and the kidney marker nephrin, suggesting that these EV subtypes may be novel candidate biomarkers for disease-spanning neurocognitive dysfunction. Moreover, the relationship between bone-derived EVs with kidney-derived EVs may suggest their role in mediating inter-organ crosstalk in the pathogenesis of HIV-associated cognitive impairment.

Stereotactic ablative radiotherapy (SABR) has shown durable response rates in a subset of prostate cancer patients presenting with low metastatic burden (oligometastasis). By delivering a highly focused dose of radiation, SABR optimizes local control and induces a systemic antitumor immune response which causes tumor regression in non-irradiated metastases at distant sites (called abscopal response). However, cases of abscopal response remain scarce and the disease in majority of patients progresses by developing wide-spread metastasis. Therefore, it is crucial to elucidate the underlying mechanisms of resistance to SABR in non-responder patients and develop more effective combination therapies. By analyzing plasma samples from metastatic castration-resistant prostate cancer (mCRPC) patients, we have observed that radiotherapy induces release of tumor fragments, called extracellular vesicles (EVs). Patients with high levels of tumor-derived EVs were more at risk of developing metastases following radiotherapy. We hypothesize that radiotherapy can inhibit a systemic antitumor immune response by inducing the release of immunosuppressive tumor-derived EVs in prostate cancer. Prostate cancer cell lines (PC3 and DU145) were treated with single dose or fractionated radiotherapy. Levels of EVs were measured with Apogee Micro60-Plus nanoscale flow cytometer and the molecular composition of EVs was analyzed by proteomics and western-blot. Co-culture of prostate cancer cells with CD8+-T cells isolated from healthy donors was used to evaluate the impact of EVs on T-cell activity.For the first time, we identified irradiated PC3 and DU145 prostate cancer cells with both single dose and fractionated radiotherapy exhibiting significant increases in B7H3 protein expression. In accordance with total B7H3 protein level changes, the levels of surface B7H3 protein expression in irradiated PC3 and DU145 cells also significantly increased. Lastly, not only did radiation cause increases in the levels of PC3 and DU145 cell EV release, but also increased B7H3 protein enrichment in those EVs. Given the increases in the B7H3 protein levels in irradiated PC3 and DU145 cell surface and EVs, B7H3 enriched- PC3 and DU145 cell EV treatment to CD8+ T-cells caused significant decreases in the proliferation rates of CD8+ T-cells compared to the proliferation rates of CD8+ T-cells without EV treatment or with non-irradiated PC3 and DU145 cell-derived EV treatment.We anticipate that this proposed study can unveil novel cellular mechanisms underscoring the role of B7H3-enriched prostate cancer cell EVs in halting the radiation-induced anti-tumor immune response. This study warrants investigation into potential therapeutic strategies paired with SABR, making more efficacious anti-metastatic prostate cancer treatments. Citation Format: Yohan Kim, Roxane R. Lavoie, Haidong dong, Sean Park, Fabrice Lucien-Matteoni. Radiotherapy inhibits the antitumor immune response through release of immunosuppressive tumor-derived extracellular vesicles in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 675.

CD14 and Tissue Factor Positive Extracellular Vesicles Predict Response to Dovitinib in Patients with GBM: A Pilot Study

Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.

BackgroundAntiphospholipid syndrome (APS) is characterized by thrombosis or pregnancy complications associated with the presence of antiphospholipid antibodies (aPLs). Although the exact mechanisms are unclear, aPLs can increase the expression of tissue factor on platelets, leukocytes, and endothelial cells, leading to hypercoagulability. Extracellular vesicles (EVs) can also be released during this process and play a key role in immune regulation and thrombosis related to APS.AimsTo evaluate the association between circulating levels of EVs and thrombosis related to APS, as well as inflammatory markers.MethodsCase-control study including patients with thrombotic APS (t-APS) and healthy controls (HC). EVs expressing the following antigens were quantified by flow cytometry: CD41 (platelet integrin alpha IIb), CD162 (P-selectin glycoprotein ligand 1), CD31 (platelet and endothelial cell adhesion molecule 1), CD142 (tissue factor), and CD62 (P-selectin). EV levels were compared between groups and correlated with APS clinical and inflammatory parameters.ResultsA total of 69 t-APS patients and 46 HC were included. CD162+EV, CD31+EV, and CD41+EV levels were higher in t-APS patients compared to controls. CD41+EV levels were associated with venous thrombosis (p = .04) and multiple thrombosis (p = .07). Levels of CD162+EV, CD31+EV, CD142+EV and CD62P + EV were positively correlated with levels of interleukin-1 beta (IL-1β).ConclusionEVs expressing antigens related to platelet and endothelial cell activation and adhesion, as well as platelet-leukocyte interaction, were associated with thrombosis related to APS. The correlation between EV levels and IL-1β levels further underscore the association between EV release and thromboinflammatory responses in APS. Our results demonstrate the involvement of EVs in the interaction between inflammation and thrombosis in APS.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Neoadjuvant chemotherapy (NAC) is widely used as a standard treatment for early-stage triple-negative breast cancer (TNBC). While patients who achieve pathologic complete response (pCR) have a highly favorable outcome, patients who do not achieve pCR have variable prognoses. It is important to identify patients who are most likely to have poor survival outcomes to identify candidates for more aggressive therapeutic approaches after NAC. Many studies have demonstrated that cytokines and growth factors packaged into extracellular vesicles (EVs) have an essential role in tumor progression and drug resistance. In this study, we examined the role of serum-derived EV-associated cytokines as prognostic biomarkers for long-term outcomes in patients who underwent anthracycline-taxane-based NAC. We isolated extracellular vesicles from the serum of 190 TNBC patients who underwent NAC between 2015 and 2018 at Samsung Medical Center. EV-associated cytokine concentrations were measured with ProcartaPlex Immune Monitoring 65-plex panels. The prognostic value of EV-associated cytokines was studied. We found that patients with high EV_APRIL, EV_CXCL13, and EV_VEGF-A levels had shorter overall survival (OS). We further evaluated the role of these selected biomarkers as prognostic factors in patients with residual disease (RD) after NAC. Even in patients with RD, high levels of EV_APRIL, EV_CXCL13, and EV_VEGF-A were correlated with poor OS. In all subgroup analyses, EV_CXCL13 overexpression was significantly associated with poor overall survival. Moreover, multivariate analysis indicated that a high level of EV_CXCL13 was an independent predictor of poor OS. Correlation analysis between biomarker levels in EVs and serum showed that EV_VEGF-A positively correlated with soluble VEGF-A but not CXCL13. An elevated level of soluble VEGF-A was also associated with poor OS. These findings suggest that EV_APRIL, EV_CXCL13, and EV_VEGF-A may be useful in identifying TNBC patients at risk of poor survival outcomes after NAC.

ACE2-enriched extracellular vesicles enhance infectivity of live SARS-CoV-2 virus.