A rapid, automated surface protein profiling of single circulating exosomes in human blood.
ABSTRACT: Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30-150?nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids.
Project description:In this report, we use a proteomic strategy to identify glycoproteins on the surface of exosomes derived from myeloid-derived suppressor cells (MDSCs), and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. We report successful modification of a surface chemistry method for use with exosomes and identify 21 surface N-glycoproteins on exosomes released by mouse mammary carcinoma-induced MDSCs. These glycoprotein identities and functionalities are compared with 93 N-linked glycoproteins identified on the surface of the parental cells. As with the lysate proteomes examined previously, the exosome surface N-glycoproteins are primarily a subset of the glycoproteins on the surface of the suppressor cells that released them, with related functions and related potential as therapeutic targets. The "don't eat me" molecule CD47 and its binding partners thrombospondin-1 (TSP1) and signal regulatory protein ? (SIRP?) were among the surface N-glycoproteins detected. Functional bioassays using antibodies to these three molecules demonstrated that CD47, TSP1, and to a lesser extent SIRP? facilitate exosome-mediated MDSC chemotaxis and migration.
Project description:Exosomes are nanovesicles released by all cells that can be found in the blood. A key point for their use as potential biomarkers in cancer is to differentiate tumour-derived exosomes from other circulating nanovesicles. Heat shock protein-70 (HSP70) has been shown to be abundantly expressed by cancer cells and to be associated with bad prognosis. We previously showed that exosomes derived from cancer cells carried HSP70 in the membrane while those from non-cancerous cells did not. In this work, we opened a prospective clinical pilot study including breast and lung cancer patients to determine whether it was possible to detect and quantify HSP70 exosomes in the blood of patients with solid cancers. We found that circulating exosomal HSP70 levels, but not soluble HSP70, reflected HSP70 content within the tumour biopsies. Circulating HSP70 exosomes increased in metastatic patients compared to non-metastatic patients or healthy volunteers. Further, we demonstrated that HSP70-exosome levels correlated with the disease status and, when compared with circulating tumour cells, were more sensitive tumour dissemination predictors. Finally, our case studies indicated that HSP70-exosome levels inversely correlated with response to the therapy and that, therefore, monitoring changes in circulating exosomal HSP70 might be useful to predict tumour response and clinical outcome.
Project description:<b>Background:</b> Efficient and specific induction of cell death in liver cancer is urgently needed. In this study, we aimed to design an exosome-based platform to deliver ferroptosis inducer (Erastin, Er) and photosensitizer (Rose Bengal, RB) into tumor tissues with high specificity. <b>Methods:</b> Exosome donor cells (HEK293T) were transfected with control or CD47-overexpressing plasmid. Exosomes were isolated and loaded with Er and RB via sonication method. Hepa1-6 cell xenograft C57BL/6 model was injected with control and engineered exosomes via tail vein. <i>In vivo</i> distribution of the injected exosomes was analyzed via tracking the fluorescence labeled exosomes. Photodynamic therapy was conducted by 532 nm laser irradiation. The therapeutic effects on hepatocellular carcinoma and toxic side-effects were systemically analyzed. <b>Results:</b> CD47 was efficiently loaded on the exosomes from the donor cells when CD47 was forced expressed by transfection. CD47 surface functionalization (Exos<sup>CD47</sup>) made the exosomes effectively escape the phagocytosis of mononuclear phagocyte system (MPS), and thus increased the distribution in tumor tissues. Erastin and RB could be effectively encapsulated into exosomes after sonication, and the drug-loaded exosomes (Er/RB@Exos<sup>CD47</sup>) strongly induced ferroptosis both <i>in vitro</i> and <i>in vivo</i> in tumor cells after irradiation of 532 nm laser. Moreover, compared with the control exosomes (Er/RB@Exos<sup>Ctrl</sup>), Er/RB@Exos<sup>CD47</sup> displayed much lower toxicity in liver. <b>Conclusion</b>: The engineered exosomes composed of CD47, Erastin, and Rose Bengal, induce obvious ferroptosis in hepatocellular carcinoma (HCC) with minimized toxicity in liver and kidney. The proposed exosomes would provide a promising strategy to treat types of malignant tumors.
Project description:Exosomes are small membranous vesicles that contain proteins, lipids, genetic material, and metabolites with abundant information from parental cells. Exosomes carry and deliver bioactive contents that can reprogram the functions of recipient cells and modulate the tumor microenvironment to induce pathological events through cell-to-cell communication and signal transduction. Tumor-derived exosomes (TDEs) in head and neck squamous cell carcinoma (HNSCC) are involved in most aspects of cancer initiation, invasion, progression, immunoregulation, therapeutic applications, and treatment resistance. In addition, HNSCC-derived exosomes can be used to obtain information on diagnostic and therapeutic biomarkers in circulating blood and saliva. Currently, the biology, mechanisms, and applications of TDEs in HNSCC are still unclear, and further research is required. In this review, we discuss various aspects of exosome biology, including exosomal components, exosomal biomarkers, and molecular mechanisms involved in immunoregulation, cancer metastasis, and therapy resistance. We also describe recent applications to update our understanding of exosomes in HNSCC.
Project description:Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy is difficult, costly, and sometimes not even an option. Tumor-derived exosomes have attracted increasing interest in non-invasive cancer diagnosis and monitoring of treatment response. However, the biology and clinical value of exosomes remains largely unknown due in part to current technical challenges in rapid isolation, molecular classification and comprehensive analysis of exosomes. Here we developed a new microfluidic approach to streamline and expedite the exosome analysis pipeline by integrating specific immunoisolation and targeted protein analysis of circulating exosomes. Compared to the conventional methods, our approach enables selective subpopulation isolation and quantitative detection of surface and intravesicular biomarkers directly from a minimally invasive amount of plasma samples (30 ?L) within ~100 min with markedly improved detection sensitivity. Using this device, we demonstrated phenotyping of exosome subpopulations by targeting a panel of common exosomal and tumor-specific markers and multiparameter analyses of intravesicular biomarkers in the selected subpopulation. We were able to assess the total expression and phosphorylation levels of IGF-1R in non-small-cell lung cancer patients by probing plasma exosomes as a non-invasive alternative to conventional tissue biopsy. We foresee that the microfluidic exosome analysis platform will form the basis for critically needed infrastructures for advancing the biology and clinical utilization of exosomes.
Project description:<b>Purpose</b>: Exosomes, small extracellular vesicles (EVs) derived from the endocytic compartment of their parent cells, are present in plasma of cancer patients and may serve as non-invasive biomarkers of disease outcome. Here, we asked whether tumor-derived (TEX) and/or T-cell derived exosomes can predict outcome in head and neck squamous cell carcinoma (HNSCC) patients treated with oncological therapy. <b>Materials and Methods</b>: 18 HNSCC patients enrolled in phase I clinical trial and receiving a combination of cetuximab, ipilimumab and radiation therapy were serially monitored for TEX and T cell-derived exosomes. Exosomes isolated from plasma by size exclusion chromatography were fractionated into TEX and CD3 + T cell-derived exosomes by immunocapture. Exosome-associated proteins were quantified by on-bead flow cytometry. Exosome molecular cargos of patients whose tumors recurred within 2 years (N = 5) were compared to cargos of patients who remained disease free at 2 years (N = 13) after therapy. <b>Results</b>: The predictive value of the exosome molecular cargo for disease recurrence was evaluated pre-, during and post therapy. In patients whose disease recurred, total exosome proteins, TEX/total exosome ratios, total CD3+, CD3(-)PD-L1+ and CD3 + 15s+ (Treg-derived) exosomes increased from the baseline levels. In patients who remained disease free, total exosome protein and TEX levels decreased, CD3+ and CD3+ CD15s+ exosomes stabilized and CD3+ CTLA4+ exosomes declined after ipilimumab therapy. <b>Conclusion</b>: TEX and T cell-derived circulating exosomes instead of immune cells were used for monitoring of patients' responses to oncological therapy. The results support the potential role of exosomes as a non-invasive tumor and immune cell biomarkers in cancer.
Project description:Objective:Peripheral artery disease (PAD) is a chronic condition characterized by inflammation. Emerging literature suggests that circulating exosomes and their microRNA (miRNA) contents may influence atherosclerosis and vascular remodeling. We hypothesize that circulating exosomes in patients with PAD directly modulate vascular cell phenotype and contain proinflammatory miRNAs. Methods:Exosomes (particle size, 30-150 nm) were isolated from plasma of healthy individuals (n = 6), patients with mild PAD (mPAD; median Rutherford class, 2.5; n = 6), and patients with severe PAD (sPAD; median Rutherford class, 4; n = 5). Exosome identity, size, and concentration were determined by Western blot and nanoparticle tracking analysis. Human vascular smooth muscle cell (VSMC) and endothelial cell (EC) migration was assessed by a standard wound closure assay after exposure to exosome preparations. Monocyte-derived macrophages isolated from healthy volunteers were exposed to exosome preparations, and targeted gene expression was analyzed using quantitative polymerase chain reaction. Exosome miRNA cargos were isolated, and a panel of defined, vascular-active miRNAs was assessed by quantitative polymerase chain reaction. Results:There was no difference in overall exosome particle concentration or size between the three groups (one-way analysis of variance [ANOVA], P > .05). Compared with exosomes from healthy individuals, exosomes from mPAD and sPAD patients increased VSMC migration (1.0 ± 0.09-fold vs 1.5 ± 0.09-fold vs 2.0 ± 0.12-fold wound closure; ANOVA, P < .0001) and inhibited EC migration (1.8 ± 0.07-fold vs 1.5 ± 0.04-fold vs 1.3 ± 0.02-fold wound closure; ANOVA, P < .01) in a stepwise fashion. Exosomes also induced changes in monocyte-derived macrophage gene expression that did not appear PAD specific. Hierarchical analysis of exosome miRNA revealed distinct clustering of vascular-active miRNAs between the three groups. Several miRNAs that promote inflammatory pathways in vascular cells were expressed at higher levels in exosomes from sPAD patients. Conclusions:Circulating exosomes from individuals with PAD exert in vitro functional effects on VSMCs and ECs that may promote adverse vessel remodeling. Exosomes from healthy individuals, mPAD patients, and sPAD patients contain distinct signatures of immune-regulatory miRNA. Together these data suggest that the proinflammatory cargo of circulating exosomes correlates with atherosclerosis severity in PAD patients and could influence vascular injury and repair. (JVS: Vascular Science 2020;1:28-41.). Clinical Relevance:Exosomes and their cargo have been implicated in several vascular remodeling processes including atherosclerosis, angiogenesis, and neointimal hyperplasia. In this study, we demonstrate that circulating exosomes from individuals with peripheral artery disease exert in vitro effects on vascular cells that may adversely affect vessel remodeling. Moreover, these exosomes contain elevated levels of vascular-active microRNA. Our results suggest that exosomes may serve as both biomarkers and effectors of vascular disease in patients with peripheral artery disease and motivate further investigation into the role of exosomes and their contents in aberrant remodeling in vascular diseases.
Project description:Whether circulating microvesicles convey bioactive signals in neurodegenerative diseases remains currently unknown. In this study, we investigated the biochemical composition and biological function of exosomes isolated from sera of patients with Parkinson's disease (PD).Proteomic analysis was performed on microvesicle preparations from grouped samples of patients with genetic and sporadic forms of PD, amyotrophic lateral sclerosis, and healthy subjects. Nanoparticle-tracking analysis was used to assess the number and size of exosomes between patient groups. To interrogate their biological effect, microvesicles were added to primary rat cortical neurons subjected to either nutrient deprivation or sodium arsenite.Among 1033 proteins identified, 23 exosome-associated proteins were differentially abundant in PD, including the regulator of exosome biogenesis syntenin 1. These protein changes were detected despite similar exosome numbers across groups suggesting that they may reflect exosome subpopulations with distinct functions. Accordingly, we showed in models of neuronal stress that Parkinson's-derived microvesicles have a protective effect.Collectively, these data suggest for the first time that immunophenotyping of circulating exosome subpopulations in PD may lead to a better understanding of the systemic response to neurodegeneration and the development of novel therapeutics.
Project description:Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.
Project description:Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential "developmental hemostatic mismatch risk" associated with transfusions containing plasma exosomes from adults.