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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV250046	1/2	Mus musculus	CT2A	UF qEV original	Schweiger MW	2024	38%
Study summary Full title Glioblastoma extracellular vesicles modulate immune PD-L1 expression in accessory macrophages upon radiotherapy. All authors Schweiger MW, Amoozgar Z, Repiton P, Morris R, Maksoud S, Hla M, Zaniewski E, Noske DP, Haas W, Breyne K, Tannous BA Journal iScience Abstract Glioblastoma (GBM) is the most aggressive brain tumor, presenting major challenges due to limited tr (show more...)Glioblastoma (GBM) is the most aggressive brain tumor, presenting major challenges due to limited treatment options. Standard care includes radiation therapy (RT) to curb tumor growth and alleviate symptoms, but its impact on GBM is limited. In this study, we investigated the effect of RT on immune suppression and whether extracellular vesicles (EVs) originating from GBM and taken up by the tumor microenvironment (TME) contribute to the induced therapeutic resistance. We observed that (1) ionizing radiation increases immune-suppressive markers on GBM cells, (2) macrophages exacerbate immune suppression in the TME by increasing PD-L1 in response to EVs derived from GBM cells which is further modulated by RT, and (3) RT increases CD206-positive macrophages which have the most potential in inducing a pro-oncogenic environment due to their increased uptake of tumor-derived EVs. In conclusion, RT affects GBM resistance by immuno-modulating EVs taken up by myeloid cells in the TME. (hide) EV-METRIC 38% (79th 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 Ultrafiltration qEV original Protein markers EV: Alix/ CD9/ CD81 non-EV: b-actin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells CT2A EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV original Other Name other separation method qEV original Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD9/ CD81 Not detected contaminants b-actin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution EV concentration Yes

	EV250046	2/2	Mus musculus	CT2A	UF qEV original	Schweiger MW	2024	38%
Study summary Full title Glioblastoma extracellular vesicles modulate immune PD-L1 expression in accessory macrophages upon radiotherapy. All authors Schweiger MW, Amoozgar Z, Repiton P, Morris R, Maksoud S, Hla M, Zaniewski E, Noske DP, Haas W, Breyne K, Tannous BA Journal iScience Abstract Glioblastoma (GBM) is the most aggressive brain tumor, presenting major challenges due to limited tr (show more...)Glioblastoma (GBM) is the most aggressive brain tumor, presenting major challenges due to limited treatment options. Standard care includes radiation therapy (RT) to curb tumor growth and alleviate symptoms, but its impact on GBM is limited. In this study, we investigated the effect of RT on immune suppression and whether extracellular vesicles (EVs) originating from GBM and taken up by the tumor microenvironment (TME) contribute to the induced therapeutic resistance. We observed that (1) ionizing radiation increases immune-suppressive markers on GBM cells, (2) macrophages exacerbate immune suppression in the TME by increasing PD-L1 in response to EVs derived from GBM cells which is further modulated by RT, and (3) RT increases CD206-positive macrophages which have the most potential in inducing a pro-oncogenic environment due to their increased uptake of tumor-derived EVs. In conclusion, RT affects GBM resistance by immuno-modulating EVs taken up by myeloid cells in the TME. (hide) EV-METRIC 38% (79th 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 5Gy irradiated 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 Ultrafiltration qEV original Protein markers EV: Alix/ CD9/ CD81 non-EV: b-actin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells CT2A EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEV original Other Name other separation method qEV original Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD9/ CD81 Not detected contaminants b-actin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution EV concentration Yes

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EV-TRACK ID	EV250046
species	Mus musculus
sample type	Cell culture
cell type	CT2A
condition	Control condition	5Gy irradiated
separation protocol	Ultrafiltration/ qEV original	Ultrafiltration/ qEV original
Exp. nr.	1	2
EV-METRIC %	38	38