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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210072	1/5	Homo sapiens	HOS	(d)(U)C	Gong, Liangzhi	2018	44%
Study summary Full title Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1 All authors Liangzhi Gong, Qiyuan Bao, Chuanzhen Hu, Jun Wang, Qi Zhou, Li Wei, Lei Tong, Weibin Zhang, Yuhui Shen Journal Biochem Biophys Res Commun Abstract Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer (show more...)Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS. (hide) EV-METRIC 44% (84th 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 exosome 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 Protein markers EV: Alix/ CD81/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HOS EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Alix/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-250 EM EM-type Transmission-EM Image type Wide-field

	EV210072	2/5	Homo sapiens	Well5	(d)(U)C	Gong, Liangzhi	2018	33%
Study summary Full title Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1 All authors Liangzhi Gong, Qiyuan Bao, Chuanzhen Hu, Jun Wang, Qi Zhou, Li Wei, Lei Tong, Weibin Zhang, Yuhui Shen Journal Biochem Biophys Res Commun Abstract Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer (show more...)Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS. (hide) EV-METRIC 33% (75th 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 exosome 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 Protein markers EV: Alix/ CD81/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Well5 EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Alix/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-300

	EV210072	3/5	Homo sapiens	143B	(d)(U)C	Gong, Liangzhi	2018	33%
Study summary Full title Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1 All authors Liangzhi Gong, Qiyuan Bao, Chuanzhen Hu, Jun Wang, Qi Zhou, Li Wei, Lei Tong, Weibin Zhang, Yuhui Shen Journal Biochem Biophys Res Commun Abstract Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer (show more...)Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS. (hide) EV-METRIC 33% (75th 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 exosome 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 Protein markers EV: Alix/ CD81/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 143B EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-250

	EV210072	4/5	Homo sapiens	MG63	(d)(U)C	Gong, Liangzhi	2018	33%
Study summary Full title Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1 All authors Liangzhi Gong, Qiyuan Bao, Chuanzhen Hu, Jun Wang, Qi Zhou, Li Wei, Lei Tong, Weibin Zhang, Yuhui Shen Journal Biochem Biophys Res Commun Abstract Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer (show more...)Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS. (hide) EV-METRIC 33% (75th 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 exosome 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 Protein markers EV: Alix/ CD81/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MG63 EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Alix/ CD81 Detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-200

	EV210072	5/5	Homo sapiens	Blood plasma	(d)(U)C	Gong, Liangzhi	2018	0%
Study summary Full title Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1 All authors Liangzhi Gong, Qiyuan Bao, Chuanzhen Hu, Jun Wang, Qi Zhou, Li Wei, Lei Tong, Weibin Zhang, Yuhui Shen Journal Biochem Biophys Res Commun Abstract Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer (show more...)Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS. (hide) EV-METRIC 0% (median: 22% 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 Blood plasma Sample origin Osteosarcoma Focus vesicles exosome 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

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EV-TRACK ID	EV210072
species	Homo sapiens
sample type	Cell culture	Cell culture	Cell culture	Cell culture	Blood plasma
cell type	HOS	Well5	143B	MG63	NA
medium	Not specified	Not specified	Not specified	Not specified	NA
condition	Control condition	Control condition	Control condition	Control condition	Osteosarcoma
separation protocol	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C
Exp. nr.	1	2	3	4	5
EV-METRIC %	44	33	33	33	0