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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120119	2/2	Homo sapiens	NAY	(d)(U)C Filtration	Ciravolo V	2012	22%
Study summary Full title Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy All authors Ciravolo V, Huber V, Ghedini GC, Venturelli E, Bianchi F, Campiglio M, Morelli D, Villa A, Della Mina P, Menard S, Filipazzi P, Rivoltini L, Tagliabue E, Pupa SM Journal J Cell Physiol Abstract Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and (show more...)Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness. (hide) EV-METRIC 22% (59th 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 NAY Focus vesicles exosomes 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 (d)(U)C Filtration Adj. k-factor 253.9 (pelleting) / 60.38 (washing) Protein markers EV: AChE/ Flotilin1/ CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted 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) 120 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 253.9 Wash: Rotor Type TLA100.3 Wash: adjusted k-factor 60.38 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotilin1/ AChE ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis EM EM-type immune EM EM protein CD63 Image type Close-up

	EV120119	1/2	Homo sapiens	Serum	(d)(U)C	Ciravolo V	2012	14%
Study summary Full title Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy All authors Ciravolo V, Huber V, Ghedini GC, Venturelli E, Bianchi F, Campiglio M, Morelli D, Villa A, Della Mina P, Menard S, Filipazzi P, Rivoltini L, Tagliabue E, Pupa SM Journal J Cell Physiol Abstract Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and (show more...)Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness. (hide) EV-METRIC 14% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin NAY Focus vesicles exosomes 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 (d)(U)C Adj. k-factor 253.9 (pelleting) / 60.38 (washing) Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum 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: time(min) 120 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 253.9 Wash: Rotor Type TLA100.3 Wash: adjusted k-factor 60.38 Characterization: Particle analysis None

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EV-TRACK ID	EV120119
species	Homo sapiens
sample type	Cell culture	Serum
cell type	NAY	NA
medium	EV Depleted
condition	NAY	NAY
separation protocol	(d)(U)C Filtration	(d)(U)C
Exp. nr.	2	1
EV-METRIC %	22	14