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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV250026	1/2	Homo sapiens	Blood plasma	(d)(U)C DG qEVoriginal 70	Richard M	2024	75%
Study summary Full title Monitoring concentration and lipid signature of plasma extracellular vesicles from HR metastatic breast cancer patients under CDK4/6 inhibitors treatment. All authors Richard M, Moreau R, Croyal M, Mathiot L, Frénel JS, Campone M, Dupont A, Gavard J, André-Grégoire G, Guével L Journal J Extracell Biol Abstract Extracellular vesicles (EVs) are cell-derived small membrane structures that transport various molec (show more...)Extracellular vesicles (EVs) are cell-derived small membrane structures that transport various molecules. They have emerged as potential circulating biomarkers for monitoring responses to cancer therapies. This study aimed to comprehensively characterize plasma-carried EVs in hormone receptor-positive (HR) metastatic breast cancer (MBC) patients treated with first-line CDK4/6 inhibitors (iCDK4/6) combined with endocrine therapy. MBC patients were classified into three groups based on their response to therapy: resistant, intermediate or sensitive. In a prospective cohort, we monitored the concentration of circulating EVs, analyzed their lipid signature and correlated these factors with treatment response. To facilitate the translation of EV research to clinical practice, we established a three-step procedure: (1) EVs were isolated from plasma using semi-automatized size exclusion chromatography (SEC)/ (2) EV concentration, termed vesiclemia, was determined by drop counting via interferometric light microscopy (ILM)/ and (3) EV lipid composition was analyzed by mass spectrometry. ILM-based vesiclemia values were highly fluctuating upon iCDK4/6 treatment, while early increase associated with accelerated progression. Of note, vesiclemia remained a steady parameter over a 1-year period in age-matched healthy women. Additionally, analysis of the EV cargo unveiled a distinct sphingolipid profile, characterized by increased levels of ceramides and sphingomyelins in resistant patients within the first 2 months of treatment. Based on 16 sphingolipid species, sensitive and resistant patients were correctly classified with an overall accuracy of 82%. This specific sphingolipid pattern was exclusively discernible within EVs, and not in plasma, highlighting the significance of EVs in the early prediction of individual responses to iCDK4/6 and disease progression. Overall, this study provides insights of the longitudinal characterization of plasma-borne EVs in both a healthy group and HR MBC patients under iCDK4/6 therapies. Combined vesiclemia and EV sphingolipid profile emphasize the promising potential of EVs as non-invasive biomarkers for monitoring early treatment response. (hide) EV-METRIC 75% (96th 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 Blood plasma Sample origin Control condition 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 gradient Commercial method Protein markers EV: CD9/ Syntenin non-EV: ApoA1 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type MLA-130 Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4% Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 100000 Pelleting-wash: duration (min) 180 Pelleting-wash: speed (g) SW 41 Ti Commercial kit qEVoriginal 70 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ Syntenin Detected contaminants ApoA1 Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS EV concentration Yes EM EM-type Cryo-EM Image type Close-up Report size (nm) 128 Other particle analysis name(1) Interferometry light microscropy EV-concentration Yes Particle yield No

	EV250026	2/2	Homo sapiens	Blood plasma	(d)(U)C DG qEVoriginal 70	Richard M	2024	63%
Study summary Full title Monitoring concentration and lipid signature of plasma extracellular vesicles from HR metastatic breast cancer patients under CDK4/6 inhibitors treatment. All authors Richard M, Moreau R, Croyal M, Mathiot L, Frénel JS, Campone M, Dupont A, Gavard J, André-Grégoire G, Guével L Journal J Extracell Biol Abstract Extracellular vesicles (EVs) are cell-derived small membrane structures that transport various molec (show more...)Extracellular vesicles (EVs) are cell-derived small membrane structures that transport various molecules. They have emerged as potential circulating biomarkers for monitoring responses to cancer therapies. This study aimed to comprehensively characterize plasma-carried EVs in hormone receptor-positive (HR) metastatic breast cancer (MBC) patients treated with first-line CDK4/6 inhibitors (iCDK4/6) combined with endocrine therapy. MBC patients were classified into three groups based on their response to therapy: resistant, intermediate or sensitive. In a prospective cohort, we monitored the concentration of circulating EVs, analyzed their lipid signature and correlated these factors with treatment response. To facilitate the translation of EV research to clinical practice, we established a three-step procedure: (1) EVs were isolated from plasma using semi-automatized size exclusion chromatography (SEC)/ (2) EV concentration, termed vesiclemia, was determined by drop counting via interferometric light microscopy (ILM)/ and (3) EV lipid composition was analyzed by mass spectrometry. ILM-based vesiclemia values were highly fluctuating upon iCDK4/6 treatment, while early increase associated with accelerated progression. Of note, vesiclemia remained a steady parameter over a 1-year period in age-matched healthy women. Additionally, analysis of the EV cargo unveiled a distinct sphingolipid profile, characterized by increased levels of ceramides and sphingomyelins in resistant patients within the first 2 months of treatment. Based on 16 sphingolipid species, sensitive and resistant patients were correctly classified with an overall accuracy of 82%. This specific sphingolipid pattern was exclusively discernible within EVs, and not in plasma, highlighting the significance of EVs in the early prediction of individual responses to iCDK4/6 and disease progression. Overall, this study provides insights of the longitudinal characterization of plasma-borne EVs in both a healthy group and HR MBC patients under iCDK4/6 therapies. Combined vesiclemia and EV sphingolipid profile emphasize the promising potential of EVs as non-invasive biomarkers for monitoring early treatment response. (hide) EV-METRIC 63% (90th 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 Blood plasma Sample origin metastatic hormone receptor-positive breast cancer 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 gradient Commercial method Protein markers EV: CD9/ Syntenin non-EV: APOA1 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type MLA-130 Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4% Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 100000 Pelleting-wash: duration (min) 180 Pelleting-wash: speed (g) SW 41 Ti Commercial kit qEVoriginal 70 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ Syntenin Detected contaminants APOA1 Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS EV concentration Yes Other particle analysis name(1) Interferometry light microscropy EV-concentration Yes Particle yield No

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EV-TRACK ID	EV250026
species	Homo sapiens
sample type	Blood plasma
condition	Control condition	metastatic hormone receptor-positive breast cancer
separation protocol	dUC/ Density gradient/ qEVoriginal 70	dUC/ Density gradient/ qEVoriginal 70
Exp. nr.	1	2
EV-METRIC %	75	63