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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180072	1/4	Homo sapiens	HCT116-TGFBR2	(d)(U)C Total Exosome Isolation UF	Fricke F	2019	57%
Study summary Full title TGFBR2‑dependent alterations of microRNA profiles in extracellular vesicles and parental colorectal cancer cells. All authors Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J. Journal Int J Oncol Abstract In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide) EV-METRIC 57% (92nd 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 extracellular vesicle 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 (d)(U)C Total Exosome Isolation UF Protein markers EV: non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116-TGFBR2 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type ClickSeal Biocontainment Rotor with Lid; 24 x 1.5/2.0 mL Tubes Pelleting: speed (g) 21100 Ultra filtration Cut-off size (kDa) 10000 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: RNA analysis RNA analysis Type RNA sequencing;(RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 128 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV180072	2/4	Homo sapiens	HCT116-TGFBR2	(d)(U)C Total Exosome Isolation UF	Fricke F	2019	57%
Study summary Full title TGFBR2‑dependent alterations of microRNA profiles in extracellular vesicles and parental colorectal cancer cells. All authors Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J. Journal Int J Oncol Abstract In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide) EV-METRIC 57% (92nd 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 genetically modified cell line Focus vesicles extracellular vesicle 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 (d)(U)C Total Exosome Isolation UF Protein markers EV: non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116-TGFBR2 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type ClickSeal Biocontainment Rotor with Lid Thermo Scientific Pelleting: speed (g) 21100 Ultra filtration Cut-off size (kDa) 10000 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing;(RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 125 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV180072	3/4	Homo sapiens	HCT116-AWE17	(d)(U)C Total Exosome Isolation UF	Fricke F	2019	29%
Study summary Full title TGFBR2‑dependent alterations of microRNA profiles in extracellular vesicles and parental colorectal cancer cells. All authors Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J. Journal Int J Oncol Abstract In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide) EV-METRIC 29% (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. 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 extracellular vesicle 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 (d)(U)C Total Exosome Isolation UF Protein markers EV: non-EV: Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116-AWE17 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Thermofisher Scientific, 75003435 Pelleting: speed (g) 21100 Ultra filtration Cut-off size (kDa) 10000 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis None Protein Concentration Method Bradford Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV180072	4/4	Homo sapiens	HCT116-AWE17	(d)(U)C Total Exosome Isolation UF	Fricke F	2019	29%
Study summary Full title TGFBR2‑dependent alterations of microRNA profiles in extracellular vesicles and parental colorectal cancer cells. All authors Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J. Journal Int J Oncol Abstract In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide) EV-METRIC 29% (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. 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 genetically modified cell line Focus vesicles extracellular vesicle 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 (d)(U)C Total Exosome Isolation UF Protein markers EV: non-EV: Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116-AWE17 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Thermo Scientific 75003435 Pelleting: speed (g) 21100 Ultra filtration Cut-off size (kDa) 10000 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis None Protein Concentration Method Bradford Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

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EV-TRACK ID	EV180072
species	Homo sapiens
sample type	Cell culture
cell type	HCT116-TGFBR2	HCT116-TGFBR2	HCT116-AWE17	HCT116-AWE17
condition	Control condition	genetically modified cell line	Control condition	genetically modified cell line
separation protocol	(d)(U)C Total Exosome Isolation UF	(d)(U)C Total Exosome Isolation UF	(d)(U)C Total Exosome Isolation UF	(d)(U)C Total Exosome Isolation UF
Exp. nr.	1	2	3	4
EV-METRIC %	57	57	29	29