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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210302	1/2	Homo sapiens	MKN74	(d)(U)C Filtration qEV	Poças J	2023	78%
Study summary Full title Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival. All authors Poças J, Marques C, Gomes C, Otake AH, Pinto F, Ferreira M, Silva T, Faria-Ramos I, Matos R, Ribeiro AR, Senra E, Cavadas B, Batista S, Maia J, Macedo JA, Lima L, Afonso LP, Ferreira JA, Santos LL, Polónia A, Osório H, Belting M, Reis CA, Costa-Silva B, Magalhães A Journal Proc Natl Acad Sci U S A Abstract Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options (show more...)Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression. (hide) EV-METRIC 78% (97th 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 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 Filtration qEV Protein markers EV: Alix/ CD9/ CD63/ CD81/ HSP70/ SDCBP/ SDC4 non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1 Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN74 EV-harvesting Medium Serum free medium Cell viability (%) 88 Cell count 73000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 160 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 160 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ CD81/ HSP70/ SDCBP/ SDC4 Not detected contaminants CytochromeC Proteomics database PRIDE Proteomics Detected contaminants Calreticulin/ GM130/ PMP70/ Prohibitin Not detected contaminants Albumin/ Argonaute2/ CytochromeC/ Tubulin1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 102.1 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.90E+08 EM EM-type Transmission-EM/ Immuno-EM EM protein SDC4 Image type Close-up, Wide-field

	EV210302	2/2	Homo sapiens	MKN74	(d)(U)C Filtration qEV	Poças J	2023	78%
Study summary Full title Syndecan-4 is a maestro of gastric cancer cell invasion and communication that underscores poor survival. All authors Poças J, Marques C, Gomes C, Otake AH, Pinto F, Ferreira M, Silva T, Faria-Ramos I, Matos R, Ribeiro AR, Senra E, Cavadas B, Batista S, Maia J, Macedo JA, Lima L, Afonso LP, Ferreira JA, Santos LL, Polónia A, Osório H, Belting M, Reis CA, Costa-Silva B, Magalhães A Journal Proc Natl Acad Sci U S A Abstract Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options (show more...)Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression. (hide) EV-METRIC 78% (97th 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 SDC4 KO 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 Filtration qEV Protein markers EV: Alix/ CD9/ CD81/ HSP70/ SDCBP non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1 Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN74 EV-harvesting Medium Serum free medium Cell viability (%) 92 Cell count 74000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 160 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 160 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD81/ HSP70/ SDCBP Not detected contaminants CytochromeC/ Tubulin1 Proteomics database PRIDE Proteomics Detected contaminants Calreticulin/ GM130/ PMP70/ Prohibitin Not detected contaminants Albumin/ Argonaute2/ CytochromeC/ Tubulin1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 86 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.75E+08 EM EM-type Transmission-EM/ Immuno-EM EM protein SDC4 Image type Close-up, Wide-field

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EV-TRACK ID	EV210302
species	Homo sapiens
sample type	Cell culture
cell type	MKN74
condition	Control condition	SDC4 KO
separation protocol	dUC/ Filtration/ qEV	dUC/ Filtration/ qEV
Exp. nr.	1	2
EV-METRIC %	78	78