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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170040	1/2	Rattus norvegicus	MTPa, MTPa-Tspan8	(d)(U)C Filtration UF	Voglstaetter, Maren	2019	62%
Study summary Full title Tspan8 is expressed in breast cancer and regulates E‐cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles All authors Maren Voglstaetter, Andreas R Thomsen, Jerome Nouvel, Arend Koch, Paul Jank, Elena Grueso Navarro, Tanja Gainey‐Schleicher, Richa Khanduri, Andrea Groß, Florian Rossner, Carina Blaue, Clemens M Franz, Marina Veil, Gerhard Puetz, Andreas Hippe, Jochen Dindorf, Jubin Kashef, Wilko Thiele, Bernhard Homey, Celine Greco, Claude Boucheix, Andreas Baur, Thalia Erbes, Cornelius F Waller, Marie Follo, Ghamartaj Hossein, Christine Sers, Jonathan Sleeman, Irina Nazarenko Journal J Pathol Abstract Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus (show more...)Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. (hide) EV-METRIC 62% (92nd 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 (d)(U)C Filtration UF Protein markers EV: TSG101/ E-Cadherin/ Tspan8/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells MTPa, MTPa-Tspan8 EV-harvesting Medium Serum free medium Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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 No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.1 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101 Other 1 Co-immunoprecipitation Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type particle size distribution by intensity and mass are reported Reported size (nm) 100 NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield 6.00E+10 particles/million cells EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170040	2/2	Rattus norvegicus	Serum	Filtration PEG precipitation	Voglstaetter, Maren	2019	37%
Study summary Full title Tspan8 is expressed in breast cancer and regulates E‐cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles All authors Maren Voglstaetter, Andreas R Thomsen, Jerome Nouvel, Arend Koch, Paul Jank, Elena Grueso Navarro, Tanja Gainey‐Schleicher, Richa Khanduri, Andrea Groß, Florian Rossner, Carina Blaue, Clemens M Franz, Marina Veil, Gerhard Puetz, Andreas Hippe, Jochen Dindorf, Jubin Kashef, Wilko Thiele, Bernhard Homey, Celine Greco, Claude Boucheix, Andreas Baur, Thalia Erbes, Cornelius F Waller, Marie Follo, Ghamartaj Hossein, Christine Sers, Jonathan Sleeman, Irina Nazarenko Journal J Pathol Abstract Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus (show more...)Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. (hide) EV-METRIC 37% (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 Serum Sample origin Breast cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration PEG precipitation Protein markers EV: TSG101/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Serum Separation Method Filtration steps 0.22µm or 0.2µm Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 10 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type size distribution by intensty and by mass are reported Reported size (nm) 10-100 nm; 1000-9000 nm NTA Report type Size range/distribution Reported size (nm) 100 EV concentration Yes Particle yield 2.00E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV170040
species	Rattus norvegicus
sample type	Cell culture	Serum
cell type	MTPa MTPa-Tspan8	NA
medium	Serum free medium	NA
condition	Control condition	Breast cancer
separation protocol	(d)(U)C Filtration UF	Filtration PEG precipitation
Exp. nr.	1	2
EV-METRIC %	62	37