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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210003	1/3	Homo sapiens	PC3	(d)(U)C DC	Qinyu, Ma	2021	44%
Study summary Full title Small extracellular vesicles deliver osteolytic effectors and mediate cancer‐induced osteolysis in bone metastatic niche All authors Qinyu Ma, Mengmeng Liang, Yutong Wu, Ce Dou, Jianzhong Xu, Shiwu Dong, Fei Luo Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment thro (show more...)Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment through mediating intercellular crosstalks. However, little is known about the contribution of EVs derived from cancer cells to the vicious cycle of bone metastasis. Here, we report a direct regulatory mode between tumour cells and osteoclasts in metastatic niche of prostate cancer via vesicular miRNAs transfer. Combined analysis of miRNAs profiles both in tumour‐derived small EVs (sEVs) and osteoclasts identified miR‐152‐3p as a potential osteolytic molecule. sEVs were enriched in miR‐152‐3p, which targets osteoclastogenic regulator MAFB. Blocking miR‐152‐3p in sEVs upregulated the expression of MAFB and impaired osteoclastogenesis in vitro. In vivo experiments of xenograft mouse model found that blocking of miR‐152‐3p in sEVs significantly slowed down the loss of trabecular architecture, while systemic inhibition of miR‐152‐3p using antagomir‐152‐3p reduced the osteolytic lesions of cortical bone while preserving basic trabecular architecture. Our findings suggest that miR‐152‐3p carried by prostate cancer‐derived sEVs deliver osteolytic signals from tumour cells to osteoclasts, facilitating osteolytic progression in bone metastasis. (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 prostate cancer 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 (Differential) (ultra)centrifugation Density cushion Protein markers EV: TSG101/ Alix/ CD63/ CD9/ CD81 non-EV: Argonaute2/ Histone 3/ LaminA/C Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Cell viability (%) 95 Cell count 1,00E+07 Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 39 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 Density cushion Density medium Sucrose Sample volume 30 Cushion volume 9 Density of the cushion 1,21 Centrifugation time 120 Centrifugation speed 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Not detected contaminants LaminA/C/ Histone 3/ Argonaute2 Characterization: RNA analysis RNA analysis Type 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) 70-140 EV concentration Yes Particle yield Yes, as number of particles per million cells 418000

	EV210003	2/3	Homo sapiens	C4	(d)(U)C DC	Qinyu, Ma	2021	44%
Study summary Full title Small extracellular vesicles deliver osteolytic effectors and mediate cancer‐induced osteolysis in bone metastatic niche All authors Qinyu Ma, Mengmeng Liang, Yutong Wu, Ce Dou, Jianzhong Xu, Shiwu Dong, Fei Luo Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment thro (show more...)Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment through mediating intercellular crosstalks. However, little is known about the contribution of EVs derived from cancer cells to the vicious cycle of bone metastasis. Here, we report a direct regulatory mode between tumour cells and osteoclasts in metastatic niche of prostate cancer via vesicular miRNAs transfer. Combined analysis of miRNAs profiles both in tumour‐derived small EVs (sEVs) and osteoclasts identified miR‐152‐3p as a potential osteolytic molecule. sEVs were enriched in miR‐152‐3p, which targets osteoclastogenic regulator MAFB. Blocking miR‐152‐3p in sEVs upregulated the expression of MAFB and impaired osteoclastogenesis in vitro. In vivo experiments of xenograft mouse model found that blocking of miR‐152‐3p in sEVs significantly slowed down the loss of trabecular architecture, while systemic inhibition of miR‐152‐3p using antagomir‐152‐3p reduced the osteolytic lesions of cortical bone while preserving basic trabecular architecture. Our findings suggest that miR‐152‐3p carried by prostate cancer‐derived sEVs deliver osteolytic signals from tumour cells to osteoclasts, facilitating osteolytic progression in bone metastasis. (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 prostate cancer 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 (Differential) (ultra)centrifugation Density cushion Protein markers EV: TSG101/ Alix/ CD63/ CD9/ CD81 non-EV: Argonaute2/ Histone 3/ LaminA/C Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells C4 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Cell viability (%) 96 Cell count 1,00E+07 Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 39 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 Density cushion Density medium Sucrose Sample volume 30 Cushion volume 9 Density of the cushion 1,21 Centrifugation time 120 Centrifugation speed 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Not detected contaminants LaminA/C/ Histone 3/ Argonaute2 Characterization: RNA analysis RNA analysis Type 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) 70-150 EV concentration Yes Particle yield Yes, as number of particles per million cells 461000

	EV210003	3/3	Homo sapiens	C4-2	(d)(U)C DC	Qinyu, Ma	2021	44%
Study summary Full title Small extracellular vesicles deliver osteolytic effectors and mediate cancer‐induced osteolysis in bone metastatic niche All authors Qinyu Ma, Mengmeng Liang, Yutong Wu, Ce Dou, Jianzhong Xu, Shiwu Dong, Fei Luo Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment thro (show more...)Extracellular vesicles (EVs) play critical roles in regulating bone metastatic microenvironment through mediating intercellular crosstalks. However, little is known about the contribution of EVs derived from cancer cells to the vicious cycle of bone metastasis. Here, we report a direct regulatory mode between tumour cells and osteoclasts in metastatic niche of prostate cancer via vesicular miRNAs transfer. Combined analysis of miRNAs profiles both in tumour‐derived small EVs (sEVs) and osteoclasts identified miR‐152‐3p as a potential osteolytic molecule. sEVs were enriched in miR‐152‐3p, which targets osteoclastogenic regulator MAFB. Blocking miR‐152‐3p in sEVs upregulated the expression of MAFB and impaired osteoclastogenesis in vitro. In vivo experiments of xenograft mouse model found that blocking of miR‐152‐3p in sEVs significantly slowed down the loss of trabecular architecture, while systemic inhibition of miR‐152‐3p using antagomir‐152‐3p reduced the osteolytic lesions of cortical bone while preserving basic trabecular architecture. Our findings suggest that miR‐152‐3p carried by prostate cancer‐derived sEVs deliver osteolytic signals from tumour cells to osteoclasts, facilitating osteolytic progression in bone metastasis. (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 prostate cancer 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 (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ TSG101/ CD63/ CD9/ CD81 non-EV: Argonaute2/ Histone 3/ LaminA/C Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells C4-2 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Cell viability (%) 96 Cell count 1,00E+07 Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 39 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 Density cushion Density medium Sucrose Sample volume 30 Cushion volume 9 Density of the cushion 1,21 Centrifugation time 120 Centrifugation speed 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101/ CD81 Not detected contaminants LaminA/C/ Histone 3/ Argonaute2 Characterization: RNA analysis RNA analysis Type 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) 70-150 EV concentration Yes Particle yield Yes, as number of particles per million cells 445000

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EV-TRACK ID	EV210003
species	Homo sapiens
sample type	Cell culture
cell type	PC3	C4	C4-2
condition	prostate cancer cell line	prostate cancer cell line	prostate cancer cell line
separation protocol	dUC DC	dUC DC	dUC DC
Exp. nr.	1	2	3
EV-METRIC %	44	44	44