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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220431	2/4	Homo sapiens	Serum	(d)(U)C ExoQuick IgG separation	Desai PP	2022	63%
Study summary Full title Combination of Small Extracellular Vesicle-Derived Annexin A2 Protein and mRNA as a Potential Predictive Biomarker for Chemotherapy Responsiveness in Aggressive Triple-Negative Breast Cancer. All authors Desai PP, Narra K, James JD, Jones HP, Tripathi AK, Vishwanatha JK Journal Cancers (Basel) Abstract Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane a (show more...)Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane as intraluminal vesicles of the multivesicular bodies, serve as vehicles that carry cargo influential in modulating the tumor microenvironment for the multi-step process of cancer metastasis. Annexin A2 (AnxA2), a calcium(Ca)-dependent phospholipid-binding protein, is among sEV cargoes. sEV-derived AnxA2 (sEV-AnxA2) protein is involved in the process of metastasis in triple-negative breast cancer (TNBC). The objective of the current study is to determine whether sEV-AnxA2 protein and/or mRNA could be a useful biomarkers to predict the responsiveness of chemotherapy in TNBC. Removal of Immunoglobulin G (IgG) from the serum as well as using the System Bioscience's ExoQuick Ultra kit resulted in efficient sEV isolation and detection of sEV-AnxA2 protein and mRNA compared to the ultracentrifugation method. The standardized method was applied to the twenty TNBC patient sera for sEV isolation. High levels of sEV-AnxA2 protein and/or mRNA were associated with stage 3 and above in TNBC. Four patients who responded to neoadjuvant chemotherapy had high expression of AnxA2 protein and/or mRNA in sEVs, while other four who did not respond to chemotherapy had low levels of AnxA2 protein and mRNA in sEVs. Our data suggest that the sEV-AnxA2 protein and mRNA could be a combined predictive biomarker for responsiveness to chemotherapy in aggressive TNBC. (hide) EV-METRIC 63% (94th 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 Serum Sample origin Triple negative breast cancer Focus vesicles small 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 ExoQuick IgG separation Protein markers EV: CD9/ CD81/ TSG101/ Annexin A2/ HSP70 non-EV: Calnexin/ Albumin/ IgG Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick Other Name other separation method ExoQuick Selected surface protein(s) Annexin A2 Other Name other separation method IgG separation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD81/ TSG101/ Annexin A2 Not detected EV-associated proteins HSP70 Detected contaminants Albumin/ IgG Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 77-106 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.89E+10 EM EM-type Cryo-EM Image type Close-up Report size (nm) 20-80

	EV220431	4/4	Mus musculus	Serum	(d)(U)C IgG separation Filtration	Desai PP	2022	56%
Study summary Full title Combination of Small Extracellular Vesicle-Derived Annexin A2 Protein and mRNA as a Potential Predictive Biomarker for Chemotherapy Responsiveness in Aggressive Triple-Negative Breast Cancer. All authors Desai PP, Narra K, James JD, Jones HP, Tripathi AK, Vishwanatha JK Journal Cancers (Basel) Abstract Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane a (show more...)Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane as intraluminal vesicles of the multivesicular bodies, serve as vehicles that carry cargo influential in modulating the tumor microenvironment for the multi-step process of cancer metastasis. Annexin A2 (AnxA2), a calcium(Ca)-dependent phospholipid-binding protein, is among sEV cargoes. sEV-derived AnxA2 (sEV-AnxA2) protein is involved in the process of metastasis in triple-negative breast cancer (TNBC). The objective of the current study is to determine whether sEV-AnxA2 protein and/or mRNA could be a useful biomarkers to predict the responsiveness of chemotherapy in TNBC. Removal of Immunoglobulin G (IgG) from the serum as well as using the System Bioscience's ExoQuick Ultra kit resulted in efficient sEV isolation and detection of sEV-AnxA2 protein and mRNA compared to the ultracentrifugation method. The standardized method was applied to the twenty TNBC patient sera for sEV isolation. High levels of sEV-AnxA2 protein and/or mRNA were associated with stage 3 and above in TNBC. Four patients who responded to neoadjuvant chemotherapy had high expression of AnxA2 protein and/or mRNA in sEVs, while other four who did not respond to chemotherapy had low levels of AnxA2 protein and mRNA in sEVs. Our data suggest that the sEV-AnxA2 protein and mRNA could be a combined predictive biomarker for responsiveness to chemotherapy in aggressive TNBC. (hide) EV-METRIC 56% (92nd 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 Serum Sample origin Control condition Focus vesicles small 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 IgG separation Filtration Protein markers EV: CD9/ TSG101/ Annexin A2 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 185000 Filtration steps 0.2 or 0.22 µm Other Name other separation method IgG separation Selected surface protein(s) Annexin A2 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101 Not detected EV-associated proteins Annexin A2/ CD9 Detected contaminants Calnexin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 97-126 EV concentration Yes Particle yield Not determined

	EV220431	1/4	Homo sapiens	Serum	(d)(U)C ExoQuick IgG separation	Desai PP	2022	50%
Study summary Full title Combination of Small Extracellular Vesicle-Derived Annexin A2 Protein and mRNA as a Potential Predictive Biomarker for Chemotherapy Responsiveness in Aggressive Triple-Negative Breast Cancer. All authors Desai PP, Narra K, James JD, Jones HP, Tripathi AK, Vishwanatha JK Journal Cancers (Basel) Abstract Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane a (show more...)Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane as intraluminal vesicles of the multivesicular bodies, serve as vehicles that carry cargo influential in modulating the tumor microenvironment for the multi-step process of cancer metastasis. Annexin A2 (AnxA2), a calcium(Ca)-dependent phospholipid-binding protein, is among sEV cargoes. sEV-derived AnxA2 (sEV-AnxA2) protein is involved in the process of metastasis in triple-negative breast cancer (TNBC). The objective of the current study is to determine whether sEV-AnxA2 protein and/or mRNA could be a useful biomarkers to predict the responsiveness of chemotherapy in TNBC. Removal of Immunoglobulin G (IgG) from the serum as well as using the System Bioscience's ExoQuick Ultra kit resulted in efficient sEV isolation and detection of sEV-AnxA2 protein and mRNA compared to the ultracentrifugation method. The standardized method was applied to the twenty TNBC patient sera for sEV isolation. High levels of sEV-AnxA2 protein and/or mRNA were associated with stage 3 and above in TNBC. Four patients who responded to neoadjuvant chemotherapy had high expression of AnxA2 protein and/or mRNA in sEVs, while other four who did not respond to chemotherapy had low levels of AnxA2 protein and mRNA in sEVs. Our data suggest that the sEV-AnxA2 protein and mRNA could be a combined predictive biomarker for responsiveness to chemotherapy in aggressive TNBC. (hide) EV-METRIC 50% (89th 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 Serum Sample origin Control condition Focus vesicles small 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 ExoQuick IgG separation Protein markers EV: CD9/ CD81/ TSG101/ Annexin A2/ HSP70 non-EV: Calnexin/ Albumin/ IgG/ Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick Other Name other separation method ExoQuick Selected surface protein(s) Annexin A2 Other Name other separation method IgG separation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD81/ TSG101/ Annexin A2 Not detected EV-associated proteins HSP70 Detected contaminants Albumin/ IgG Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 77-106 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.89E+10

	EV220431	3/4	Mus musculus	Serum	(d)(U)C ExoQuick IgG separation	Desai PP	2022	50%
Study summary Full title Combination of Small Extracellular Vesicle-Derived Annexin A2 Protein and mRNA as a Potential Predictive Biomarker for Chemotherapy Responsiveness in Aggressive Triple-Negative Breast Cancer. All authors Desai PP, Narra K, James JD, Jones HP, Tripathi AK, Vishwanatha JK Journal Cancers (Basel) Abstract Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane a (show more...)Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane as intraluminal vesicles of the multivesicular bodies, serve as vehicles that carry cargo influential in modulating the tumor microenvironment for the multi-step process of cancer metastasis. Annexin A2 (AnxA2), a calcium(Ca)-dependent phospholipid-binding protein, is among sEV cargoes. sEV-derived AnxA2 (sEV-AnxA2) protein is involved in the process of metastasis in triple-negative breast cancer (TNBC). The objective of the current study is to determine whether sEV-AnxA2 protein and/or mRNA could be a useful biomarkers to predict the responsiveness of chemotherapy in TNBC. Removal of Immunoglobulin G (IgG) from the serum as well as using the System Bioscience's ExoQuick Ultra kit resulted in efficient sEV isolation and detection of sEV-AnxA2 protein and mRNA compared to the ultracentrifugation method. The standardized method was applied to the twenty TNBC patient sera for sEV isolation. High levels of sEV-AnxA2 protein and/or mRNA were associated with stage 3 and above in TNBC. Four patients who responded to neoadjuvant chemotherapy had high expression of AnxA2 protein and/or mRNA in sEVs, while other four who did not respond to chemotherapy had low levels of AnxA2 protein and mRNA in sEVs. Our data suggest that the sEV-AnxA2 protein and mRNA could be a combined predictive biomarker for responsiveness to chemotherapy in aggressive TNBC. (hide) EV-METRIC 50% (89th 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 Serum Sample origin Control condition Focus vesicles small 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 ExoQuick IgG separation Protein markers EV: CD9/ CD81/ TSG101/ Annexin A2/ HSP70 non-EV: Calnexin/ Albumin/ IgG Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick Other Name other separation method ExoQuick Selected surface protein(s) Annexin A2 Other Name other separation method IgG separation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101 Not detected EV-associated proteins Annexin A2/ CD9 Detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 77-106 EV concentration Yes Particle yield Not determined

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EV-TRACK ID	EV220431
species	Homo sapiens	Mus musculus	Homo sapiens	Mus musculus
sample type	Serum	Serum	Serum	Serum
condition	Triple negative breast cancer	Control condition	Control condition	Control condition
separation protocol	dUC/ ExoQuick/ IgG separation	dUC/ IgG separation/ Filtration	dUC/ ExoQuick/ IgG separation	dUC/ ExoQuick/ IgG separation
Exp. nr.	2	4	1	3
EV-METRIC %	63	56	50	50