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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140124	2/6	Homo sapiens	LNCaP	ExoQuick	Ramteke, Anand	2015	50%
Study summary Full title Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules All authors Anand Ramteke, Harold Ting, Chapla Agarwal, Samiha Mateen, Ranganathan Somasagara, Anowar Hussain, Michael Graner, Barbara Frederick, Rajesh Agarwal, Gagan Deep Journal Mol Carcinog Abstract Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mec (show more...)Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness. (hide) EV-METRIC 50% (87th 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 ExoQuick Protein markers EV: CD63/ MMP-9/ MMP-2/ TGF-beta2/ TNF1alpha/ IL6/ TSG101/ Akt/ ILK1/ beta-catenin/ PKM2/ HSP90/ Annexin II/ alpha-tubulin/ / Alix/ CD81/ HSP70 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD63/ HSP90/ Annexin II/ alpha-tubulin/ / MMP-9/ MMP-2/ TGF-beta2/ TNF1alpha/ IL6/ TSG101/ Akt/ ILK1/ beta-catenin/ PKM2/ HSP70/ Alix/ CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 187 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 0.26E8 EM EM-type Transmission-EM Image type Close-up

	EV140124	4/6	Homo sapiens	LNCaP	ExoQuick	Ramteke, Anand	2015	50%
Study summary Full title Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules All authors Anand Ramteke, Harold Ting, Chapla Agarwal, Samiha Mateen, Ranganathan Somasagara, Anowar Hussain, Michael Graner, Barbara Frederick, Rajesh Agarwal, Gagan Deep Journal Mol Carcinog Abstract Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mec (show more...)Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness. (hide) EV-METRIC 50% (87th 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 Hypoxia 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 ExoQuick Protein markers EV: CD63/ MMP-9/ MMP-2/ TGF-beta2/ TNF1alpha/ IL6/ TSG101/ Akt/ ILK1/ beta-catenin/ PKM2/ HSP90/ Annexin II/ alpha-tubulin/ / Alix/ CD81/ HSP70 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins Alix/ Annexin II/ alpha-tubulin/ / MMP-9/ MMP-2/ TGF-beta2/ TNF1alpha/ IL6/ TSG101/ Akt/ ILK1/ beta-catenin/ PKM2/ CD63/ HSP90/ HSP70/ CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 141 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 0.24E8 EM EM-type Transmission-EM Image type Close-up

	EV140124	1/6	Homo sapiens	LNCaP	(d)(U)C	Ramteke, Anand	2015	25%
Study summary Full title Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules All authors Anand Ramteke, Harold Ting, Chapla Agarwal, Samiha Mateen, Ranganathan Somasagara, Anowar Hussain, Michael Graner, Barbara Frederick, Rajesh Agarwal, Gagan Deep Journal Mol Carcinog Abstract Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mec (show more...)Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness. (hide) EV-METRIC 25% (63rd 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: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) Not specified Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 82656 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield NA NA

	EV140124	3/6	Homo sapiens	LNCaP	(d)(U)C	Ramteke, Anand	2015	25%
Study summary Full title Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules All authors Anand Ramteke, Harold Ting, Chapla Agarwal, Samiha Mateen, Ranganathan Somasagara, Anowar Hussain, Michael Graner, Barbara Frederick, Rajesh Agarwal, Gagan Deep Journal Mol Carcinog Abstract Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mec (show more...)Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness. (hide) EV-METRIC 25% (63rd 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 Hypoxia 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 yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) Not specified Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 82656 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield NA NA

	EV140124	5/6	Homo sapiens	PC3	ExoQuick	Ramteke, Anand	2015	0%
Study summary Full title Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules All authors Anand Ramteke, Harold Ting, Chapla Agarwal, Samiha Mateen, Ranganathan Somasagara, Anowar Hussain, Michael Graner, Barbara Frederick, Rajesh Agarwal, Gagan Deep Journal Mol Carcinog Abstract Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mec (show more...)Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness. (hide) EV-METRIC 0% (median: 14% 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 ExoQuick Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis None Protein Concentration Method Lowry-based assay Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield NA NA

	EV140124	6/6	Homo sapiens	PC3	ExoQuick	Ramteke, Anand	2015	0%
Study summary Full title Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules All authors Anand Ramteke, Harold Ting, Chapla Agarwal, Samiha Mateen, Ranganathan Somasagara, Anowar Hussain, Michael Graner, Barbara Frederick, Rajesh Agarwal, Gagan Deep Journal Mol Carcinog Abstract Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mec (show more...)Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness. (hide) EV-METRIC 0% (median: 14% 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 Hypoxia 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 ExoQuick Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis None Protein Concentration Method Lowry-based assay Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV140124
species	Homo sapiens
sample type	Cell culture
cell type	LNCaP	LNCaP	LNCaP	LNCaP	PC3	PC3
condition	Control condition	Hypoxia	Control condition	Hypoxia	Control condition	Hypoxia
separation protocol	ExoQuick	ExoQuick	(d)(U)C	(d)(U)C	ExoQuick	ExoQuick
Exp. nr.	2	4	1	3	5	6
EV-METRIC %	50	50	25	25	0	0