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Results list Most used isolation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV170047	5/8	Homo sapiens	Cell culture supernatant	dUC (Differential) (ultra)centrifugation	Soekmadji C	2017	44%
Study summary Full title Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer. All authors Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC Journal Oncotarget Abstract Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + (Differential) (ultra)centrifugation Protein markers EV: Alix/ TSG101/ PSA/ CD9 non-EV: GAPDH Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 0.005 Western Blot Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected EV-associated proteins PSA Not detected contaminants GAPDH Characterization: Particle analysis TRPS Report type Size range/distribution,Mode Reported size (nm) 150 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170026	1/1	Mus musculus	Cell culture supernatant	DG dUC Filtration DC	Prakash Gangadaran	2017	44%
Study summary Full title Extracellular vesicles from mesenchymal stem cells activates VEGF receptors and accelerates recovery of hindlimb ischemia. All authors Gangadaran P, Rajendran RL, Lee HW, Kalimuthu S, Hong CM, Jeong SY, Lee SW, Lee J, Ahn BC Journal J Control Release Abstract Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are potential therapies for (show more...)Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are potential therapies for various diseases, but their angiogenic mechanisms of therapeutic efficacy remain unclear. Here, we describe how MSC-EVs, activates VEGF receptors and downstream angiogenesis pathways. Mouse MSC-EVs were isolated from cell culture medium and characterized using transmission electron microscopy, nanoparticle analysis, and western blotting. In vitro migration, proliferation, and tube formation assays using endothelial cells were used to assess the angiogenic potential of MSC-EVs, and revealed higher levels of cellular migration, proliferation, and tube formation after treatment. qRT-PCR and western blotting (WB) revealed higher protein and mRNA expression of the angiogenic genes VEGFR1 and VEGFR2 in mouse SVEC-4 endothelial cells after MSC-EVs treatment. Additionally, other vital pro-angiogenic pathways (SRC, AKT, and ERK) were activated by in vitro MSC-EV treatment. WB and qRT-PCR revealed enriched presence of VEGF protein and miR-210-3p in MSC-EV. The hindlimb ischemia mouse model was established and MSC-EVs with or without Matrigel (EV-MSC+Gel) were injected into the ischemic area and blood reperfusion was monitored using molecular imaging techniques. The in vivo administration of MSC-EVs increased both blood reperfusion and the formation of new blood vessels in the ischemic limb, with the addition of matrigel enhancing this effect further by releasing EVs slowly. MSC-EVs enhance angiogenesis in ischemic limbs, most likely via the overexpression of VEGFR1 and VEGFR2 in endothelial cells. These findings reveal a novel mechanism of activating receptors by MSC-EVs influence the angiogenesis. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC + Filtration + DC Adj. k-factor 253.9 (pelleting) / 253.9 (washing) Protein markers EV: Alix/ CD63 non-EV: calnexin/ cytochrome c/ GM130/ cytochromec Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells bone marrow-derived mesenchymal stem cells EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 60 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Wash: adjusted k-factor 253.9 Density cushion Density medium Iodixanol Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix, CD63 Not detected contaminants GM130, calnexin, cytochrome c Characterization: Particle analysis NTA Report type Mean Reported size (nm) 135 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170021	1/2	Homo sapiens	Cell culture supernatant	DG dUC	Liem, Michael	2017	44%
Study summary Full title Insulin mediated activation of PI3K/Akt signalling pathway modifies the proteomic cargo of extracellular vesicles. All authors Liem M, Ang CS, Mathivanan S Journal Proteomics Abstract Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (C (show more...)Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (CRC) and lowers the patient survival rate. An important attribute in diabetes and obesity is the presence of high levels of growth factors including insulin in blood which can activate the PI3K/Akt signalling pathway. Dysregulation of PI3K/Akt signalling pathway leads to sustained proliferative signals thereby allowing the cells susceptible to cancer. Extracellular vesicles (EVs), secreted nanovesicles of endocytic origin, are implicated in mediating the transfer of oncogenic cargo in the tumour microenvironment. In this study, CRC cells were treated with insulin to activate PI3K/Akt signaling pathway. Insulin treatment significantly increased the number of EVs secreted by CRC cells. Furthermore, pAkt was exclusively packaged in EVs secreted by PI3K/Akt activated cells. Quantitative proteomics analysis confirmed that the protein cargo of EVs are modified upon activation of PI3K/Akt signaling pathway. Bioinformatics analysis highlighted the enrichment of proteins implicated in cell proliferation in EVs secreted by PI3K/Akt activated cells. Furthermore, incubation of EVs secreted by PI3K/Akt activated cells induced proliferation in recipient CRC cells. These findings suggest that EVs can amplify the signal provided by the growth factors in the tumor microenvironment and hence aid in cancer progression. This article is protected by copyright. All rights reserved. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Insulin induced Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 253.9 (pelleting) / 89.2 (washing) Protein markers EV: TSG101/ TSG101,AKT,pAKT,beta-actin,FAT1,p-cadherin/ AKT/ Alix/ FAT1/ pAKT/ beta-actin/ p-cadherin non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Insulin induced EV-producing cells LIM1215 EV-harvesting Medium Serum free medium Cell viability 98 Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 60 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Densitometry (SYPRO Ruby) Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, TSG101,AKT,pAKT,beta-actin,FAT1,p-cadherin Proteomics database Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-150 EV concentration Yes Particle yield 3.08E+07 particles/million cells

	EV170021	2/2	Homo sapiens	Cell culture supernatant	dUC	Liem, Michael	2017	44%
Study summary Full title Insulin mediated activation of PI3K/Akt signalling pathway modifies the proteomic cargo of extracellular vesicles. All authors Liem M, Ang CS, Mathivanan S Journal Proteomics Abstract Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (C (show more...)Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (CRC) and lowers the patient survival rate. An important attribute in diabetes and obesity is the presence of high levels of growth factors including insulin in blood which can activate the PI3K/Akt signalling pathway. Dysregulation of PI3K/Akt signalling pathway leads to sustained proliferative signals thereby allowing the cells susceptible to cancer. Extracellular vesicles (EVs), secreted nanovesicles of endocytic origin, are implicated in mediating the transfer of oncogenic cargo in the tumour microenvironment. In this study, CRC cells were treated with insulin to activate PI3K/Akt signaling pathway. Insulin treatment significantly increased the number of EVs secreted by CRC cells. Furthermore, pAkt was exclusively packaged in EVs secreted by PI3K/Akt activated cells. Quantitative proteomics analysis confirmed that the protein cargo of EVs are modified upon activation of PI3K/Akt signaling pathway. Bioinformatics analysis highlighted the enrichment of proteins implicated in cell proliferation in EVs secreted by PI3K/Akt activated cells. Furthermore, incubation of EVs secreted by PI3K/Akt activated cells induced proliferation in recipient CRC cells. These findings suggest that EVs can amplify the signal provided by the growth factors in the tumor microenvironment and hence aid in cancer progression. This article is protected by copyright. All rights reserved. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 253.9 (pelleting) / 89.2 (washing) Protein markers EV: TSG101/ AKT/ Alix/ FAT1/ beta-actin/ p-cadherin/ TSG101,AKT,beta-actin,FAT1,p-cadherin non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells LIM1215 EV-harvesting Medium Serum free medium Cell viability 98 Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 60 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Densitometry (SYPRO Ruby) Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, TSG101,AKT,beta-actin,FAT1,p-cadherin Proteomics database Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-150 EV concentration Yes Particle yield 1.85E+07 particles/million cells

	EV170003	1/1	Mus musculus	Cell culture supernatant	dUC	Nager AR	2017	44%
Study summary Full title An Actin Network Dispatches Ciliary GPCRs into Extracellular Vesicles to Modulate Signaling. All authors Nager AR, Goldstein JS, Herranz-Pérez V, Portran D, Ye F, Garcia-Verdugo JM, Nachury MV Journal Cell Abstract Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. H (show more...)Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. Here, we find that when activated G protein-coupled receptors (GPCRs) fail to undergo BBSome-mediated retrieval from cilia back into the cell, these GPCRs concentrate into membranous buds at the tips of cilia before release into extracellular vesicles named ectosomes. Unexpectedly, actin and the actin regulators drebrin and myosin 6 mediate ectosome release from the tip of cilia. Mirroring signal-dependent retrieval, signal-dependent ectocytosis is a selective and effective process that removes activated signaling molecules from cilia. Congruently, ectocytosis compensates for BBSome defects as ectocytic removal of GPR161, a negative regulator of Hedgehog signaling, permits the appropriate transduction of Hedgehog signals in Bbs mutants. Finally, ciliary receptors that lack retrieval determinants such as the anorexigenic GPCR NPY2R undergo signal-dependent ectocytosis in wild-type cells. Our data show that signal-dependent ectocytosis regulates ciliary signaling in physiological and pathological contexts. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin GPCR signaling in BBS mutant Focus vesicles Ciliary Ectosome Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 253.9 (pelleting) / 99.86 (washing) Protein markers EV: CD81/ Arl13B/ BiotinylatedGPCRs non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition GPCR signaling in BBS mutant EV-producing cells mIMCD3 EV-harvesting Medium Serum-containing medium Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 90 Wash: Rotor Type TLS-55 Wash: speed (g) 100000 Wash: adjusted k-factor 99.86 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins CD81, Arl13B Characterization: Particle analysis EM EM-type Transmission-EM/ Immune-EM Image type Close-up, Wide-field Report size (nm) 70-120

	EV170067	4/6	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration Density gradient	Lässer C	2017	43%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide) EV-METRIC 43% (76th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Extracellular vesicle-like structures Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration + Density gradient Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Density gradient Density medium Sucrose Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Top-down Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes 1.24 - 1.31 g/mL Protein Concentration Method Not determined Characterization: Particle analysis EM EM-type Immuno-EM Image type Wide-field Report size (nm) 51

	EV170067	5/6	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Density gradient Filtration	Lässer C	2017	43%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide) EV-METRIC 43% (76th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Extracellular vesicle-like structures Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Density gradient + Filtration Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells TF-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Density gradient Density medium Sucrose Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Protein Concentration Method Not determined Characterization: Particle analysis

	EV170067	6/6	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Density gradient	Lässer C	2017	43%
Study summary Full title Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. All authors Lässer C, Shelke GV, Yeri A, Kim DK, Crescitelli R, Raimondo S, Sjöstrand M, Gho YS, Van Keuren Jensen K, Lötvall J. Journal RNA Biol Abstract Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in (show more...)Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions. (hide) EV-METRIC 43% (76th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Extracellular vesicle-like structures Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Density gradient Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.09 - 1.31 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells TF-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Density gradient Density medium Sucrose Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 35 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 175000 Duration (min) 960 Fraction volume (mL) 3.9 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Protein Concentration Method Not determined Characterization: Particle analysis

	EV170054	1/5	Homo sapiens	Urine	dUC	Gheinani AH	2017	42%
Study summary Full title Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery. All authors Gheinani AH, Vögeli M, Baumgartner U, Vassella E, Draeger A, Burkhard FC, Monastyrskaya K Journal Sci Rep Abstract Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RN (show more...)Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50-150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies. (hide) EV-METRIC 42% (77th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 174.8 (pelleting) / 174.8 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 174.8 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 120000 Wash: adjusted k-factor 174.8 Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Protein Concentration 15.9 Characterization: Particle analysis NTA Report type Mode;mean;size range/distribution;D10;D50;D90 Reported size (nm) 174 EV concentration Yes Particle yield see Fig1A EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170006	1/8	Homo sapiens	Serum	dUC	Julich-Haertel H	2017	37%
Study summary Full title Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. All authors Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M Journal J Hepatol Abstract BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide) EV-METRIC 37% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles microparticle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 284.4 (pelleting) Protein markers EV: CD147/ EpCAM/ ASGPR1/ ANXA5/ CD133 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 60 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 284.4 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry MACSQuant® Analyzer 10 Calibration bead size 0.2,0.5 Extra information We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).

	EV170006	2/8	Homo sapiens	Serum	dUC	Julich-Haertel H	2017	37%
Study summary Full title Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. All authors Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M Journal J Hepatol Abstract BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide) EV-METRIC 37% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Inguinal hernia Focus vesicles microparticle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 284.4 (pelleting) Protein markers EV: CD147/ EpCAM/ ASGPR1/ ANXA5/ CD133 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Inguinal hernia Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 60 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 284.4 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry MACSQuant® Analyzer 10 Calibration bead size 0.2,0.5 Extra information We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).

	EV170006	3/8	Homo sapiens	Serum	dUC	Julich-Haertel H	2017	37%
Study summary Full title Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. All authors Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M Journal J Hepatol Abstract BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide) EV-METRIC 37% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Colon carcinoma Focus vesicles microparticle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 284.4 (pelleting) Protein markers EV: CD147/ EpCAM/ ASGPR1/ ANXA5/ CD133 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Colon carcinoma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 60 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 284.4 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry MACSQuant® Analyzer 10 Calibration bead size 0.2,0.5 Extra information We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).

	EV170006	4/8	Homo sapiens	Serum	dUC	Julich-Haertel H	2017	37%
Study summary Full title Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. All authors Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M Journal J Hepatol Abstract BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide) EV-METRIC 37% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Hepatocellular carcinoma Focus vesicles microparticle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 284.4 (pelleting) Protein markers EV: CD147/ EpCAM/ ASGPR1/ ANXA5/ CD133 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Hepatocellular carcinoma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 60 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 284.4 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry MACSQuant® Analyzer 10 Calibration bead size 0.2,0.5 Extra information We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).

	EV170006	5/8	Homo sapiens	Serum	dUC	Julich-Haertel H	2017	37%
Study summary Full title Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. All authors Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M Journal J Hepatol Abstract BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide) EV-METRIC 37% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Liver tumour Focus vesicles microparticle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 284.4 (pelleting) Protein markers EV: CD147/ EpCAM/ ASGPR1/ ANXA5/ CD133 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Liver tumour Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 60 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 284.4 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry MACSQuant® Analyzer 10 Calibration bead size 0.2,0.5 Extra information We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).

	EV170006	6/8	Homo sapiens	Serum	dUC	Julich-Haertel H	2017	37%
Study summary Full title Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. All authors Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M Journal J Hepatol Abstract BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide) EV-METRIC 37% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Cirrhosis Focus vesicles microparticle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 284.4 (pelleting) Protein markers EV: CD147/ EpCAM/ ASGPR1/ ANXA5/ CD133 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Cirrhosis Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 60 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 284.4 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry MACSQuant® Analyzer 10 Calibration bead size 0.2,0.5 Extra information We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).

	EV170006	7/8	Homo sapiens	Serum	dUC	Julich-Haertel H	2017	37%
Study summary Full title Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. All authors Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M Journal J Hepatol Abstract BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide) EV-METRIC 37% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Cholangiocarcinoma Focus vesicles microparticle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 284.4 (pelleting) Protein markers EV: ASGPR1/ EpCAM/ ANXA5/ CD133 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Cholangiocarcinoma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 60 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 284.4 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry MACSQuant® Analyzer 10 Calibration bead size 0.2,0.5 Extra information We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).

	EV170006	8/8	Homo sapiens	Serum	dUC	Julich-Haertel H	2017	37%
Study summary Full title Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. All authors Julich-Haertel H, Urban SK, Krawczyk M, Willms A, Jankowski K, Patkowski W, Kruk B, Krasnodębski M, Ligocka J, Schwab R, Richardsen I, Schaaf S, Klein A, Gehlert S, Sänger H, Casper M, Banales JM, Schuppan D, Milkiewicz P, Lammert F, Krawczyk M, Lukacs-Kornek V, Kornek M Journal J Hepatol Abstract BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV (show more...)BACKGROUND AND AIMS: we previously reported that large extracellular vesicles, specifically AnnexinV+EpCAM+CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, we aim to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+EpCAM+CD147+taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+EpCAM+ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, 202 control subjects were enrolled. RESULTS: our results indicate that AnnexinV+EpCAM+CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+EpCAM+ASGPR1+CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+EpCAM+ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest size of successfully detected cancers were ranging between 11-15 mm. AnnexinV+EpCAM+ASGPR1+ taMPs decreased at 7 days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+EpCAM+ASGPR1+ taMPs. CONCLUSION: we provide strong evidence that AnnexinV+EpCAM+ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possibly the extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis. (hide) EV-METRIC 37% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Non small cell lung carcinoma Focus vesicles microparticle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 284.4 (pelleting) Protein markers EV: ASGPR1/ EpCAM/ ANXA5/ CD133 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Non small cell lung carcinoma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 60 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 20000 Pelleting: adjusted k-factor 284.4 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry MACSQuant® Analyzer 10 Calibration bead size 0.2,0.5 Extra information We characterised various tumor-associated MPs (taMPs) in serum from various cancer patients aiming for the detection of liver cancer and differentiation from healthy subjects and other non-liver cancer entities. This led to several useful antigen combinations on taMPs that must be present simultaneously on the surface of the same MP in order to be accounted. That means, we reported several MP surface antigen combinations for the detection and differentiation of liver cancer (here: HCC and CCA).

	EV170070	1/1	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration	Tsang EK	2017	33%
Study summary Full title Small RNA Sequencing in Cells and Exosomes Identifies eQTLs and 14q32 as a Region of Active Export. All authors Tsang EK, Abell NS, Li X, Anaya V, Karczewski KJ, Knowles DA, Sierra RG, Smith KS, Montgomery SB. Journal G3 (Bethesda) Abstract Exosomes are small extracellular vesicles that carry heterogeneous cargo, including RNA, between cel (show more...)Exosomes are small extracellular vesicles that carry heterogeneous cargo, including RNA, between cells. Increasing evidence suggests that exosomes are important mediators of intercellular communication and biomarkers of disease. Despite this, the variability of exosomal RNA between individuals has not been well quantified. To assess this variability, we sequenced the small RNA of cells and exosomes from a 17-member family. Across individuals, we show that selective export of miRNAs occurs not only at the level of specific transcripts, but that a cluster of 74 mature miRNAs on chromosome 14q32 is massively exported in exosomes while mostly absent from cells. We also observe more interindividual variability between exosomal samples than between cellular ones and identify four miRNA expression quantitative trait loci shared between cells and exosomes. Our findings indicate that genomically colocated miRNAs can be exported together and highlight the variability in exosomal miRNA levels between individuals as relevant for exosome use as diagnostics. (hide) EV-METRIC 33% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Epstein-Barr virus-transformed Focus vesicles exosome Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration Protein markers EV: HSP70 non-EV: Calnexin Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Epstein-Barr virus-transformed EV-producing cells CEPH/UTAH family PEDIGREE 1463 peripheral blood B lymphocyte EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Wash: time (min) 70 Wash: Rotor Type TLA-100.3 Wash: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins HSP70 Not detected contaminants Calnexin Characterization: Particle analysis NTA Report type Modus Reported size (nm) 107 EM EM-type Transmission-EM Image type Close-up Report size (nm) 100

	EV170054	2/5	Homo sapiens	Urine	SEC dUC Filtration Ultrafiltration UF	Gheinani AH	2017	33%
Study summary Full title Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery. All authors Gheinani AH, Vögeli M, Baumgartner U, Vassella E, Draeger A, Burkhard FC, Monastyrskaya K Journal Sci Rep Abstract Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RN (show more...)Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50-150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography SEC + dUC + Filtration + Ultrafiltration + UF Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Polyethersulfone (PES) Size-exclusion chromatography Total column volume (mL) 23 Sample volume/column (mL) 0.4 Resin type Sepharose CL-2B Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Protein Concentration 5.7 Characterization: Particle analysis NTA Report type Mode;mean;size range/distribution;D10;D50;D90 Reported size (nm) 113 EV concentration Yes Particle yield see Fig1A EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170054	3/5	Homo sapiens	Urine	PEG precipitation dUC Other	Gheinani AH	2017	33%
Study summary Full title Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery. All authors Gheinani AH, Vögeli M, Baumgartner U, Vassella E, Draeger A, Burkhard FC, Monastyrskaya K Journal Sci Rep Abstract Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RN (show more...)Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50-150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography PEG precipitation + dUC + Other Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Other Name other isolation method PEG precipitation Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Protein Concentration 8.4 Characterization: Particle analysis NTA Report type Mode;mean;size range/distribution;D10;D50;D90 Reported size (nm) 138 EV concentration Yes Particle yield see Fig1A EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170054	4/5	Homo sapiens	Urine	PEG precipitation SEC dUC Other	Gheinani AH	2017	33%
Study summary Full title Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery. All authors Gheinani AH, Vögeli M, Baumgartner U, Vassella E, Draeger A, Burkhard FC, Monastyrskaya K Journal Sci Rep Abstract Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RN (show more...)Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50-150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography PEG precipitation + SEC + dUC + Other Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Size-exclusion chromatography Total column volume (mL) 23 Sample volume/column (mL) 0.4 Resin type Sepharose CL-2B Other Name other isolation method PEG precipitation Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Protein Concentration 2.3 Characterization: Particle analysis NTA Report type Mode;mean;size range/distribution;D10;D50;D90 Reported size (nm) 106 EV concentration Yes Particle yield see Fig1A EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170047	6/8	Homo sapiens	Cell culture supernatant	dUC (Differential) (ultra)centrifugation	Soekmadji C	2017	33%
Study summary Full title Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer. All authors Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC Journal Oncotarget Abstract Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide) EV-METRIC 33% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin DHT Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + (Differential) (ultra)centrifugation Protein markers EV: Alix/ TSG101/ PSA/ CD9 non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition DHT EV-producing cells DUCaP EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 1.6 Western Blot Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected EV-associated proteins PSA Proteomics database No Characterization: Particle analysis TRPS Report type Size range/distribution,Mode Reported size (nm) 120

	EV170047	7/8	Homo sapiens	Cell culture supernatant	dUC (Differential) (ultra)centrifugation	Soekmadji C	2017	33%
Study summary Full title Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer. All authors Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC Journal Oncotarget Abstract Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide) EV-METRIC 33% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Charcoal stripped serum Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + (Differential) (ultra)centrifugation Protein markers EV: Alix/ TSG101/ PSA/ CD9 non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Charcoal stripped serum EV-producing cells DUCaP EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 1.2 Western Blot Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected EV-associated proteins PSA Proteomics database No Characterization: Particle analysis TRPS Report type Size range/distribution,Mode Reported size (nm) 120

	EV170047	8/8	Homo sapiens	Cell culture supernatant	dUC (Differential) (ultra)centrifugation	Soekmadji C	2017	33%
Study summary Full title Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer. All authors Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC Journal Oncotarget Abstract Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide) EV-METRIC 33% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + (Differential) (ultra)centrifugation Protein markers EV: Alix/ TSG101/ PSA/ CD9 non-EV: Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells DUCaP EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 1.1 Western Blot Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected EV-associated proteins PSA Characterization: Particle analysis TRPS Report type Size range/distribution,Mode Reported size (nm) 140 EM EM-type Transmission-EM Image type Wide-field

	EV180003	2/3	Homo sapiens	Cell culture supernatant	dUC	André-Grégoire G	2017	33%
Study summary Full title Temozolomide affects Extracellular Vesicles Released by Glioblastoma Cells. All authors André-Grégoire G, Bidère N, Gavard J Journal Biochimie Abstract Glioblastoma multiforme (GBM) is the most aggressive primary tumour within the brain as well as the (show more...)Glioblastoma multiforme (GBM) is the most aggressive primary tumour within the brain as well as the most common and lethal cerebral cancer, mainly because of treatment failure. Indeed, tumour recurrence is inevitable and fatal in a short period of time. Glioblastoma stem-like cells (GSCs) are thought to participate in tumour initiation, expansion, resistance to treatments, including to the alkylating chemotherapeutic agent temozolomide, and relapse. Here, we assessed whether extracellular vesicles (EVs) released by GSCs could disseminate factors involved in resistance mechanisms. We first characterized EVs either circulating in peripheral blood from newly diagnosed patients or released by patient-derived temozolomide-resistant GSCs. We found that EVs from both sources were mainly composed of particles homogeneous in size (50-100 nm), while they were more abundant in liquid biopsies from GBM patients, as compared to healthy donors. Further, mass spectrometry analysis from GSC-derived EVs unveiled that particles from control and temozolomide-treated cells share core components of EVs, as well as ribosome- and proteasome-associated networks. More strikingly, temozolomide treatment led to the enrichment of EVs with cargoes dedicated to cell adhesion processes. Thus, while relatively inefficient in killing GSCs in vitro, temozolomide could instead increase the release of pro-tumoral information. (hide) EV-METRIC 33% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Temozolomide-treated Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 255.8 (pelleting) / 255.8 (washing) Protein markers EV: Alix/ HSP70 non-EV: TOM20 Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Temozolomide-treated EV-producing cells primary glioblastoma cells EV-harvesting Medium Serum free medium Cell viability 90 Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 255.8 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix, HSP70 Not detected contaminants TOM20 Proteomics database No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 90 EV concentration Yes

	EV170024	1/3	Homo sapiens	Cell culture supernatant	dUC Filtration	Volgers C	2017	33%
Study summary Full title Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells. All authors Volgers C, Benedikter BJ, Grauls GE, Savelkoul PHM, Stassen FRM Journal Aging (Albany NY) Abstract During infection, the release of nano-sized membrane vesicle is a process which is common both for b (show more...)During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release. (hide) EV-METRIC 33% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles membrane vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 156.9 (pelleting) Protein markers EV: CD81/ CD63,CD81/ CD63 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells THP1 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63,CD81 Flow cytometry specific beads Selected surface protein(s) CD63

	EV170024	2/3	Moraxella catarrhalis	Cell culture supernatant	dUC Filtration	Volgers C	2017	33%
Study summary Full title Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells. All authors Volgers C, Benedikter BJ, Grauls GE, Savelkoul PHM, Stassen FRM Journal Aging (Albany NY) Abstract During infection, the release of nano-sized membrane vesicle is a process which is common both for b (show more...)During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release. (hide) EV-METRIC 33% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles membrane vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 156.9 (pelleting) Protein markers EV: Moraxella catarrhalis antigen non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Moraxella catarrhalis Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Moraxella catarrhalis EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Moraxella catarrhalis antigen Flow cytometry specific beads Selected surface protein(s) Moraxella catarrhalis antigen Characterization: Particle analysis TRPS EV concentration Yes Particle yield 1.50E+09 particles/ml start sample

	EV170024	3/3	Pseudomonas aeruginosa	Cell culture supernatant	dUC Filtration	Volgers C	2017	33%
Study summary Full title Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells. All authors Volgers C, Benedikter BJ, Grauls GE, Savelkoul PHM, Stassen FRM Journal Aging (Albany NY) Abstract During infection, the release of nano-sized membrane vesicle is a process which is common both for b (show more...)During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release. (hide) EV-METRIC 33% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles membrane vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 156.9 (pelleting) Protein markers EV: Pseudomonas aeruginosa antigen non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Pseudomonas aeruginosa Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Pseudomonas aeruginosa EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Pseudomonas aeruginosa antigen Flow cytometry specific beads Selected surface protein(s) Pseudomonas aeruginosa antigen Characterization: Particle analysis TRPS EV concentration Yes Particle yield 1.00E+09 particles/ml start sample

	EV170000	1/2	Mus musculus	Serum	dUC Filtration	Thomou T	2017	29%
Study summary Full title Adipose-derived circulating miRNAs regulate gene expression in other tissues. All authors Thomou T, Mori MA, Dreyfuss JM, Konishi M, Sakaguchi M, Wolfrum C, Rao TN, Winnay JN, Garcia-Martin R, Grinspoon SK, Gorden P, Kahn Journal Nature Abstract Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism thro (show more...)Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism through the release of adipokines. Here we show that mice with an adipose-tissue-specific knockout of the microRNA (miRNA)-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, exhibit a substantial decrease in levels of circulating exosomal miRNAs. Transplantation of both white and brown adipose tissue-brown especially-into ADicerKO mice restores the level of numerous circulating miRNAs that are associated with an improvement in glucose tolerance and a reduction in hepatic Fgf21 mRNA and circulating FGF21. This gene regulation can be mimicked by the administration of normal, but not ADicerKO, serum exosomes. Expression of a human-specific miRNA in the brown adipose tissue of one mouse in vivo can also regulate its 3' UTR reporter in the liver of another mouse through serum exosomal transfer. Thus, adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues and thereby serve as a previously undescribed form of adipokine. (hide) EV-METRIC 29% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin NA Focus vesicles exosome Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Protein markers EV: CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Serum Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: speed (g) 100000 Wash: time (min) 60 Wash: speed (g) 100000 Characterization: Particle analysis TRPS Report type Size range/distribution EM EM-type Transmission-EM/ Immune-EM Image type Close-up, Wide-field Report size (nm) 80-200 Other particle analysis name(1) EXOCET ELISA assay EV-concentration Yes

	EV170036	8/12	Homo sapiens	Serum	dUC Filtration	Krafft C	2017	28%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 28% (77th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Benign prostate hyperplasia Focus vesicles EV12 Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 213.2 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Benign prostate hyperplasia Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes we proceeded with IR and RAMAN analysis of EVs isolated by 12000 x g (frequently designated as micro Protein Concentration Method microBCA Protein Concentration 4-6 for healty donors in EV12; 100 in cancer patients; Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-200 Particle yield 1.10E+11 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170036	10/12	Homo sapiens	Serum	dUC Filtration	Krafft C	2017	28%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 28% (77th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Prostate cancer Focus vesicles EV120 Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 213.2 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Prostate cancer Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes we proceeded with IR and RAMAN analysis of EVs isolated by 12000 x g (frequently designated as micro Protein Concentration Method microBCA Protein Concentration 4-6 for healty donors in EV12; 100 in cancer patients; Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-200 Particle yield 1.00E+11 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170005	1/4	Homo sapiens	Cell culture supernatant	dUC SEC UF	Suárez H	2017	28%
Study summary Full title A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles. All authors Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M Journal Sci Rep Abstract Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide) EV-METRIC 28% (58th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + SEC + UF Protein markers EV: CD81/ CD59/ CD63/ CD9/ MHC1 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells SK-MEL103 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Ultra filtration Cut-off size (kDa) Not spec Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 20 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 148.16 Characterization: Particle analysis NTA Report type Median

	EV170005	4/4	Homo sapiens	Cell culture supernatant	dUC SEC UF	Suárez H	2017	28%
Study summary Full title A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles. All authors Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M Journal Sci Rep Abstract Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide) EV-METRIC 28% (58th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + SEC + UF Protein markers EV: CD81/ CD59/ CD63/ CD9/ MHC1 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Primary T-lymphoblasts EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Ultra filtration Cut-off size (kDa) Not spec Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 20 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 116.85 Characterization: Particle analysis NTA Report type Median

	EV170005	3/4	Homo sapiens	Cell culture supernatant	dUC	Suárez H	2017	25%
Study summary Full title A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles. All authors Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M Journal Sci Rep Abstract Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide) EV-METRIC 25% (57th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 264.9 (pelleting) / 264.9 (washing) Protein markers EV: CD59/ CD9 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells SK-MEL103 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: rotor type AH-627 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 264.9 Wash: time (min) 120 Wash: Rotor Type AH-627 Wash: speed (g) 100000 Wash: adjusted k-factor 264.9 Characterization: Protein analysis Protein Concentration Method BCA Characterization: Particle analysis NTA Report type Median

	EV170042	1/4	Homo sapiens	Urine	dUC	Silvers CR	2017	22%
Study summary Full title Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. All authors Silvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF Journal J Cell Sci Abstract The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are need (show more...)The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation. (hide) EV-METRIC 22% (48th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: Alix/ Annexin VII/ EHD4/ AnnexinVII non-EV: None Proteomics no Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting: time(min) 70 Pelleting: speed (g) 20000 Wash: time (min) 70 Wash: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Detected EV-associated proteins Alix, Annexin VII, EHD4

	EV170042	2/4	Homo sapiens	Cell culture supernatant	dUC	Silvers CR	2017	22%
Study summary Full title Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. All authors Silvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF Journal J Cell Sci Abstract The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are need (show more...)The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation. (hide) EV-METRIC 22% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: Alix/ TSG101/ CD9 non-EV: None Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells TCCSUP EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting: time(min) 70 Pelleting: speed (g) 20000 Wash: time (min) 70 Wash: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Detected EV-associated proteins Alix, CD9, TSG101 Proteomics database No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 35-300

	EV170042	3/4	Homo sapiens	Urine	dUC	Silvers CR	2017	22%
Study summary Full title Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. All authors Silvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF Journal J Cell Sci Abstract The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are need (show more...)The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation. (hide) EV-METRIC 22% (48th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Bladder cancer Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: SNB1/ EHD4/ Annexin VII/ HEXB/ Alix/ S100A4/ AnnexinVII non-EV: None Proteomics no Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Sample Condition Bladder cancer Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting: time(min) 70 Pelleting: speed (g) 20000 Wash: time (min) 70 Wash: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Detected EV-associated proteins Alix, Annexin VII, EHD4, HEXB, S100A4, SNB1 Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV170072	1/5	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation	Nabet BY	2017	14%
Study summary Full title Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer. All authors Nabet BY, Qiu Y, Shabason JE, Wu TJ, Yoon T, Kim BC, Benci JL, DeMichele AM, Tchou J, Marcotrigiano J, Minn AJ. Journal Cell Abstract Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, t (show more...)Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT. (hide) EV-METRIC 14% (39th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MRC5 EV-harvesting Medium EV-depleted medium Preparation of EDS 3h at 100,000g;Other preparation Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Protein Concentration Method Lowry-based assay Characterization: Particle analysis

	EV170072	2/5	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation	Nabet BY	2017	14%
Study summary Full title Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer. All authors Nabet BY, Qiu Y, Shabason JE, Wu TJ, Yoon T, Kim BC, Benci JL, DeMichele AM, Tchou J, Marcotrigiano J, Minn AJ. Journal Cell Abstract Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, t (show more...)Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT. (hide) EV-METRIC 14% (39th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS 3h at 100,000g;Other preparation Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Protein Concentration Method Lowry-based assay Characterization: Particle analysis NTA Report type Mean Reported size (nm) 133.4 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV180054	1/2	Homo sapiens	Urine	dUC SEC Ultrafiltration UF	Lozano-Ramos SI	2017	14%
Study summary Full title Molecular profile of urine extracellular vesicles from normo-functional kidneys reveal minimal differences between living and deceased donors. All authors Lozano-Ramos SI, Bancu I, Carreras-Planella L, Monguió-Tortajada M, Cañas L, Juega J, Bonet J, Armengol MP, Lauzurica R, Borràs FE Journal BMC Nephrol Abstract BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney disease (show more...)BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney diseases leading to irreversible kidney failure. Considering the origin of the graft, several studies have reported differences between living (LD) and deceased donors (DD) in graft and patient survival. These differences seem to be related to multiple factors including, donor age and time of cold ischemia among others. Many of transplanted organs come from old-aged DDs, in which pre-transplant biopsy is recommended. However, kidney biopsy has several limitations, and there is a need to develop alternatives to assess the status of a kidney before transplantation. As the analysis of urinary extracellular vesicles (uEVs) rendered promising results as non-invasive biomarkers of kidney-related pathologies, this pilot study aimed to investigate whether profiling uEVs of LDs and DDs may be of help to assess the quality of the kidney before nephrectomy. METHODS: uEVs from 5 living donors and 7 deceased donors were isolated by size-exclusion chromatography, and their protein and miRNA content were analysed by liquid chromatography followed by mass spectrometry and next generation sequencing, respectively. Then, hierarchical clustering and venn diagrams were done with Perseus software and InteractiVenn tool. Specific EVs data bases were also used for Gene Ontology analysis. RESULTS: Next generation sequencing revealed that uEVs from DDs contained less miRNAs than LDs, but most of the DD-expressed miRNAs were shared with LDs (96%). Only miR-326 (targeting the apoptotic-related Bcl2) was found significantly over-represented in LD. Focusing on the protein content, we detected a low intra-group correlation in both types of donors. Despite these differences, hierarchical clustering of either miRNA or protein data could not identify a differential profile between LDs and DDs. Of note, 90% of transplanted patients had a functional graft after a year from KTx. CONCLUSIONS: In this pilot study we found that, in normo-functional grafts, minor differences in uEVs profile could not discriminate between LDs and DDs. (hide) EV-METRIC 14% (39th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Deceased Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + SEC + Ultrafiltration + UF Protein markers EV: CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Sample Condition Deceased Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 12 Sample volume/column (mL) 0.3 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Nanodrop Proteomics database No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 124-250 EV concentration Yes

	EV180054	2/2	Homo sapiens	Urine	dUC SEC Ultrafiltration UF	Lozano-Ramos SI	2017	14%
Study summary Full title Molecular profile of urine extracellular vesicles from normo-functional kidneys reveal minimal differences between living and deceased donors. All authors Lozano-Ramos SI, Bancu I, Carreras-Planella L, Monguió-Tortajada M, Cañas L, Juega J, Bonet J, Armengol MP, Lauzurica R, Borràs FE Journal BMC Nephrol Abstract BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney disease (show more...)BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney diseases leading to irreversible kidney failure. Considering the origin of the graft, several studies have reported differences between living (LD) and deceased donors (DD) in graft and patient survival. These differences seem to be related to multiple factors including, donor age and time of cold ischemia among others. Many of transplanted organs come from old-aged DDs, in which pre-transplant biopsy is recommended. However, kidney biopsy has several limitations, and there is a need to develop alternatives to assess the status of a kidney before transplantation. As the analysis of urinary extracellular vesicles (uEVs) rendered promising results as non-invasive biomarkers of kidney-related pathologies, this pilot study aimed to investigate whether profiling uEVs of LDs and DDs may be of help to assess the quality of the kidney before nephrectomy. METHODS: uEVs from 5 living donors and 7 deceased donors were isolated by size-exclusion chromatography, and their protein and miRNA content were analysed by liquid chromatography followed by mass spectrometry and next generation sequencing, respectively. Then, hierarchical clustering and venn diagrams were done with Perseus software and InteractiVenn tool. Specific EVs data bases were also used for Gene Ontology analysis. RESULTS: Next generation sequencing revealed that uEVs from DDs contained less miRNAs than LDs, but most of the DD-expressed miRNAs were shared with LDs (96%). Only miR-326 (targeting the apoptotic-related Bcl2) was found significantly over-represented in LD. Focusing on the protein content, we detected a low intra-group correlation in both types of donors. Despite these differences, hierarchical clustering of either miRNA or protein data could not identify a differential profile between LDs and DDs. Of note, 90% of transplanted patients had a functional graft after a year from KTx. CONCLUSIONS: In this pilot study we found that, in normo-functional grafts, minor differences in uEVs profile could not discriminate between LDs and DDs. (hide) EV-METRIC 14% (39th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + SEC + Ultrafiltration + UF Protein markers EV: CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 12 Sample volume/column (mL) 0.3 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Nanodrop Proteomics database No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 124-250 EV concentration Yes

	EV170042	4/4	Homo sapiens	Cell culture supernatant	dUC	Silvers CR	2017	14%
Study summary Full title Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. All authors Silvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF Journal J Cell Sci Abstract The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are need (show more...)The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation. (hide) EV-METRIC 14% (39th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells SVHUC EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting: time(min) 70 Pelleting: speed (g) 20000 Wash: time (min) 70 Wash: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 35-300

	EV170036	1/12	Homo sapiens	Blood plasma	dUC	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (46th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Prostate cancer Focus vesicles EV12 Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Prostate cancer Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 90 Pelleting: speed (g) 12000 Protein Concentration Method microBCA Protein Concentration 311 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EV concentration Yes Particle yield 1.55E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1)

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