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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230372	7/14	Homo sapiens	H2228	(d)(U)C DG	Schöne N	2024	100%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 100% (99th 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 large EVs 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 Protein markers EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6 non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H2228 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ Mitofilin/ Arf6/ CD81 Not detected EV-associated proteins Syntenin Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 196.8 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.83E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230372	9/14	Homo sapiens	H596	(d)(U)C DG	Schöne N	2024	100%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 100% (99th 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 large EVs 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 Protein markers EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6 non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H596 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ Mitofilin/ Arf6 Not detected EV-associated proteins Syntenin/ CD81 Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 188.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.33E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230372	13/14	Homo sapiens	Blood plasma	(d)(U)C	Schöne N	2024	100%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 100% (99th 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 NSCLC Focus vesicles large EVs 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 Protein markers EV: Actinin-4/ Rgap1/ EMMPRIN/ EpCAM/ EGFR non-EV: ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ EMMPRIN Detected contaminants ApoB Not detected contaminants ApoA1 Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 187 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.05E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230372	3/14	Homo sapiens	HCC-78	(d)(U)C DG	Schöne N	2024	78%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (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 large EVs 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 Protein markers EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6 non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCC-78 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ Syntenin-1/ Arf6/ Mitofilin Not detected EV-associated proteins CD81 Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 200.13 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.63E+09

	EV230372	8/14	Homo sapiens	H2228	(d)(U)C	Schöne N	2024	78%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (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 small EVs 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 Protein markers EV: CD81/ Syntenin/ Actinin-4/ Arf6/ Rgap1/ Mitofilin non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H2228 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Syntenin/ Arf6/ CD81 Not detected EV-associated proteins Actinin-4/ Rgap1/ Mitofilin Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 163.8 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.10E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230372	10/14	Homo sapiens	H596	(d)(U)C DG	Schöne N	2024	78%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (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 small EVs 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 Protein markers EV: CD81/ Syntenin/ Actinin-4/ Arf6/ Rgap1/ Mitofilin non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H596 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Syntenin/ Actinin-4/ Arf6/ CD81 Not detected EV-associated proteins Rgap1/ Mitofilin Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 141.2 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.09E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230372	1/14	Homo sapiens	A549	(d)(U)C	Schöne N	2024	56%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 56% (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 Cell culture supernatant Sample origin Control condition Focus vesicles large EVs 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 Protein markers EV: Actinin-4/ CD81/ Rgap1/ Syntenin/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin/ Arf6/ HDAC1 non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A549 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Mitofilin/ Arf6 Not detected EV-associated proteins CD81/ Rgap1/ Syntenin Detected contaminants ApoB Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1 Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 163.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.71E+08

	EV230372	2/14	Homo sapiens	A549	(d)(U)C	Schöne N	2024	56%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 56% (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 Cell culture supernatant Sample origin Control condition Focus vesicles small EVs 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 Protein markers EV: CD81/ Syntenin/ Actinin-4/ Arf6/ Rgap1/ Mitofilin non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A549 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD81/ Syntenin/ Actinin-4/ Arf6 Not detected EV-associated proteins Rgap1/ Mitofilin Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 147.13 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.95E+09

	EV230372	4/14	Homo sapiens	HCC-78	(d)(U)C	Schöne N	2024	56%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 56% (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 Cell culture supernatant Sample origin Control condition Focus vesicles small EVs 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 Protein markers EV: CD81/ Syntenin/ Actinin-4/ Arf6/ Rgap1/ Mitofilin non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCC-78 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD81/ Syntenin/ Arf6 Not detected EV-associated proteins Rgap1/ Actinin-4/ Mitofilin Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 163.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.26E+10

	EV230372	5/14	Homo sapiens	H1975	(d)(U)C	Schöne N	2024	56%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 56% (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 Cell culture supernatant Sample origin Control condition Focus vesicles large EVs 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 Protein markers EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6 non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H1975 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ Mitofilin/ Arf6 Not detected EV-associated proteins CD81/ Syntenin Detected contaminants GM130 Not detected contaminants HDAC1/ Albumin/ ApoA1/ ApoB Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 180.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.37E+10

	EV230372	6/14	Homo sapiens	H1975	(d)(U)C	Schöne N	2024	56%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 56% (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 Cell culture supernatant Sample origin Control condition Focus vesicles small EVs 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 Protein markers EV: CD81/ Syntenin/ Actinin-4/ Arf6/ Rgap1/ Mitofilin non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H1975 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD81/ Syntenin/ Actinin-4 Not detected EV-associated proteins Rgap1/ Mitofilin/ Arf6 Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 163.9 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.43E+10

	EV230372	11/14	Homo sapiens	Blood plasma	(d)(U)C	Schöne N	2024	56%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 56% (88th 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 large EVs 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 Protein markers EV: Actinin-4/ Rgap1/ EMMPRIN/ EpCAM non-EV: ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ EMMPRIN Detected contaminants ApoB Not detected contaminants ApoA1/ ApoA1 Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 185.2 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.01E+08

	EV230372	14/14	Homo sapiens	Blood plasma	(d)(U)C	Schöne N	2024	43%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 43% (75th 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 NSCLC Focus vesicles small EVs 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Characterization: Protein analysis None Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 143.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.85E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230372	12/14	Homo sapiens	Blood plasma	(d)(U)C	Schöne N	2024	14%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 14% (38th 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 small EVs 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Characterization: Protein analysis None Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 137.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.41E+08

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EV-TRACK ID	EV230372
species	Homo sapiens
sample type	Cell culture	Cell culture	Blood plasma	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Cell culture	Blood plasma	Blood plasma	Blood plasma
cell type	H2228	H596	NA	HCC-78	H2228	H596	A549	A549	HCC-78	H1975	H1975	NA	NA	NA
medium	EV-depleted medium	EV-depleted medium	NA	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	EV-depleted medium	NA	NA	NA
condition	Control condition	Control condition	NSCLC	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	NSCLC	Control condition
separation protocol	dUC/ Density gradient	dUC/ Density gradient	dUC	dUC/ Density gradient	dUC	dUC/ Density gradient	dUC	dUC	dUC	dUC	dUC	dUC	dUC	dUC
vesicle related term	large EVs	large EVs	large EVs	large EVs	small EVs	small EVs	large EVs	small EVs	small EVs	large EVs	small EVs	large EVs	small EVs	small EVs
Exp. nr.	7	9	13	3	8	10	1	2	4	5	6	11	14	12
EV-METRIC %	100	100	100	78	78	78	56	56	56	56	56	56	43	14

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