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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220326	1/4	Homo sapiens	Glioblastoma stem-like cells NCH421k	(d)(U)C DC	Lokumcu T	2024	78%
Study summary Full title Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. All authors Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V Journal ACS Nano Abstract Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide) EV-METRIC 78% (97th 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma stem-like cells NCH421k EV-harvesting Medium Serum free medium Cell viability (%) 91 - 93 Cell count 194200000 - 222312000 Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 100000 Density cushion Density medium Iodixanol Sample volume 7 Cushion volume 4 Density of the cushion 20% Centrifugation time 70 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 Detected contaminants 60S acidic ribosomal protein P0 (RPLP0) Not detected contaminants GM130/ Lamin B1/ Cytochrome c Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ Calnexin Not detected contaminants Argonaute 2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175.0 EV concentration Yes Particle yield particles per milliliter of starting sample: 4,58E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220326	2/4	Homo sapiens	Glioblastoma stem-like cells NCH644	(d)(U)C DC	Lokumcu T	2024	78%
Study summary Full title Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. All authors Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V Journal ACS Nano Abstract Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide) EV-METRIC 78% (97th 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma stem-like cells NCH644 EV-harvesting Medium Serum free medium Cell viability (%) 83 - 90 Cell count 225576000 - 231070000 Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 100000 Density cushion Density medium Iodixanol Sample volume 7 Cushion volume 4 Density of the cushion 20% Centrifugation time 70 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 Detected contaminants 60S acidic ribosomal protein P0 Not detected contaminants GM130/ Lamin B1/ Cytochrome c Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ Calnexin Not detected contaminants Argonaute 2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 169.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 1,87E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220326	3/4	Homo sapiens	Glioblastoma stem-like cells NCH705	(d)(U)C DC	Lokumcu T	2024	78%
Study summary Full title Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. All authors Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V Journal ACS Nano Abstract Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide) EV-METRIC 78% (97th 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma stem-like cells NCH705 EV-harvesting Medium Serum free medium Cell viability (%) 98 Cell count 204312000 - 225840000 Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 100000 Density cushion Density medium Iodixanol Sample volume 7 Cushion volume 4 Density of the cushion 20% Centrifugation time 70 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 Detected contaminants 60S acidic ribosomal protein P0 (RPLP0) Not detected contaminants GM130/ Lamin B1/ Cytochrome c Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ Calnexin Not detected contaminants Argonaute 2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 179.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 6,05E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220326	4/4	Homo sapiens	Glioblastoma stem-like cells NCH711d	(d)(U)C DC	Lokumcu T	2024	78%
Study summary Full title Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. All authors Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V Journal ACS Nano Abstract Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide) EV-METRIC 78% (97th 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma stem-like cells NCH711d EV-harvesting Medium Serum free medium Cell viability (%) 89 - 91 Cell count 102240000 - 116112000 Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 100000 Density cushion Density medium Iodixanol Sample volume 7 Cushion volume 4 Density of the cushion 20% Centrifugation time 70 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 Detected contaminants 60S acidic ribosomal protein P0 (RPLP0) Not detected contaminants GM130/ Lamin B1/ Cytochrome c Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ Calnexin Not detected contaminants Argonaute 2/ GM130/ PMP70/ Tamm-Horsfall protein/ Cytochrome c1 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 203.5 EV concentration Yes Particle yield particles per milliliter of starting sample: 2,87E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV220326
species	Homo sapiens
sample type	Cell culture
cell type	Glioblastoma stem-like cells NCH421k	Glioblastoma stem-like cells NCH644	Glioblastoma stem-like cells NCH705	Glioblastoma stem-like cells NCH711d
condition	Control condition	Control condition	Control condition	Control condition
separation protocol	dUC/ DC	dUC/ DC	dUC/ DC	dUC/ DC
Exp. nr.	1	2	3	4
EV-METRIC %	78	78	78	78