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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130168	1/2	Homo sapiens	NAY	(d)(U)C	Bijnsdorp IV	2013	11%
Study summary Full title Exosomal ITGA3 interferes with non-cancerous prostate cell functions and is increased in urine exosomes of metastatic prostate cancer patients All authors Bijnsdorp IV, Geldof AA, Lavaei M, Piersma SR, van Moorselaar RJ, Jimenez CR Journal J Extracell Vesicles Abstract BACKGROUND: Cancer cells are able to change the protein expression and behavior of non-cancerous sur (show more...)BACKGROUND: Cancer cells are able to change the protein expression and behavior of non-cancerous surrounding cells. Exosomes, secreted by prostate cancer (PCa) cells, may have a functional role in cancer metastasis and present a promising source for protein biomarkers. The aim of the present study was to identify which proteins in exosomes can influence non-cancerous cells, and to determine whether we can use urine exosomal proteins to identify high-risk PCa patients. METHOD: Exosomes were isolated by ultracentrifugation. Migration and invasion were studied by the transwell (invasion) assay. Proteomics was performed by LC-MS/MS and identified proteins were validated by Western blotting. Cellular uptake of fluorescent labeled PKH67-exosomes was measured by FACS. RESULTS: Based on comparative protein profiling by mass spectrometry-based proteomics of LNCaP- and PC3-exosomes, we selected ITGA3 and ITGB1, involved in migration/invasion, for further analyses. Inhibition of exosomal ITGA3 reduced the migration and invasion of non-cancerous prostate epithelial cells (prEC) almost completely. Cellular uptake of exosomes by prEC was higher with PC3-exosomes compared to LNCaP exosomes. Finally, ITGA3 and ITGB1 were more abundant in urine exosomes of metastatic patients (p<0.05), compared to benign prostate hyperplasia or PCa. CONCLUSION: These data indicate exosomal ITGA3 and ITGB1 may play a role in manipulating non-cancerous surrounding cells and that measurement of ITGA3 and ITGB1 in urine exosomes has the potential to identify patients with metastatic PCa in a non-invasive manner. (hide) EV-METRIC 11% (30th 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 NAY Focus vesicles exosomes 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 non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix

	EV130168	2/2	Homo sapiens	Urine	(d)(U)C	Bijnsdorp IV	2013	11%
Study summary Full title Exosomal ITGA3 interferes with non-cancerous prostate cell functions and is increased in urine exosomes of metastatic prostate cancer patients All authors Bijnsdorp IV, Geldof AA, Lavaei M, Piersma SR, van Moorselaar RJ, Jimenez CR Journal J Extracell Vesicles Abstract BACKGROUND: Cancer cells are able to change the protein expression and behavior of non-cancerous sur (show more...)BACKGROUND: Cancer cells are able to change the protein expression and behavior of non-cancerous surrounding cells. Exosomes, secreted by prostate cancer (PCa) cells, may have a functional role in cancer metastasis and present a promising source for protein biomarkers. The aim of the present study was to identify which proteins in exosomes can influence non-cancerous cells, and to determine whether we can use urine exosomal proteins to identify high-risk PCa patients. METHOD: Exosomes were isolated by ultracentrifugation. Migration and invasion were studied by the transwell (invasion) assay. Proteomics was performed by LC-MS/MS and identified proteins were validated by Western blotting. Cellular uptake of fluorescent labeled PKH67-exosomes was measured by FACS. RESULTS: Based on comparative protein profiling by mass spectrometry-based proteomics of LNCaP- and PC3-exosomes, we selected ITGA3 and ITGB1, involved in migration/invasion, for further analyses. Inhibition of exosomal ITGA3 reduced the migration and invasion of non-cancerous prostate epithelial cells (prEC) almost completely. Cellular uptake of exosomes by prEC was higher with PC3-exosomes compared to LNCaP exosomes. Finally, ITGA3 and ITGB1 were more abundant in urine exosomes of metastatic patients (p<0.05), compared to benign prostate hyperplasia or PCa. CONCLUSION: These data indicate exosomal ITGA3 and ITGB1 may play a role in manipulating non-cancerous surrounding cells and that measurement of ITGA3 and ITGB1 in urine exosomes has the potential to identify patients with metastatic PCa in a non-invasive manner. (hide) EV-METRIC 11% (33rd 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 Urine Sample origin NAY Focus vesicles exosomes 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 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine 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) 90 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix

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EV-TRACK ID	EV130168
species	Homo sapiens
sample type	Cell culture	Urine
cell type	NAY	NA
medium	serum free
condition	NAY	NAY
separation protocol	(d)(U)C	(d)(U)C
Exp. nr.	1	2
EV-METRIC %	11	11