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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV170047	1/8	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 Enzalutamide 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 Enzalutamide 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 Not determined 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) 120 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170047	3/8	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 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: GAPDH 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 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 0.017 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 Proteomics database No 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

	EV170047	4/8	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 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: GAPDH 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 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 0.012 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 Proteomics database No Characterization: Particle analysis TRPS Report type Size range/distribution,Mode Reported size (nm) 120 EM EM-type Transmission-EM Image type Close-up, Wide-field

	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

	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

	EV170047	2/8	Homo sapiens	Cell culture supernatant	dUC (Differential) (ultra)centrifugation	Soekmadji C	2017	0%
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 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 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 Enzalutamide 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/ 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 Enzalutamide 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 Protein Concentration Method Not determined Characterization: Particle analysis TRPS EM

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EV-TRACK ID	EV170047
species	Homo sapiens
sample type	Cell culture
cell type	LNCaP	LNCaP	LNCaP	LNCaP	DUCaP	DUCaP	DUCaP	DUCaP
condition	Enzalutamide	DHT	Charcoal stripped serum	Control condition	DHT	Charcoal stripped serum	Control condition	Enzalutamide
isolation protocol	dUC dUC	dUC dUC	dUC dUC	dUC dUC	dUC dUC	dUC dUC	dUC dUC	dUC dUC
Exp. nr.	1	3	4	5	6	7	8	2
EV-METRIC %	44	44	44	44	33	33	33	0

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