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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180008	1/4	Homo sapiens	HCCLM3	DG (d)(U)C	Hao, Liu	2018	75%
Study summary Full title Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility All authors Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang Journal Oncogene Abstract Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (hide) EV-METRIC 75% (96th 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 exosome 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 DG (d)(U)C Protein markers EV: CD81/ TSG101/ CD63 non-EV: Grp94/ Argonaute-2 Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCCLM3 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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 Density gradient Only used for validation of main results Yes Density medium 255.8 Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 180 Pelleting-wash: rotor type 255.8 Pelleting-wash: speed (g) SW 41 Ti Pelleting-wash: adjusted k-factor 255.8 Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) 0.38 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81, TSG101 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV180008	4/4	Homo sapiens	HuH7	DG (d)(U)C	Hao, Liu	2018	75%
Study summary Full title Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility All authors Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang Journal Oncogene Abstract Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (hide) EV-METRIC 75% (96th 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 exosome 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 DG (d)(U)C Protein markers EV: CD81/ TSG101/ CD63 non-EV: Grp94/ Argonaute-2 Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HuH7 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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 Density gradient Only used for validation of main results Yes Density medium 255.8 Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 180 Pelleting-wash: rotor type 255.8 Pelleting-wash: speed (g) SW 41 Ti Pelleting-wash: adjusted k-factor 255.8 Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) 0.12 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81, TSG101 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV180008	2/4	Homo sapiens	HCCLM3	(d)(U)C Total Exosome Isolation	Hao, Liu	2018	50%
Study summary Full title Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility All authors Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang Journal Oncogene Abstract Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (hide) EV-METRIC 50% (87th 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 exosome 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 Total Exosome Isolation Protein markers EV: CD81/ TSG101 non-EV: Grp94 Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCCLM3 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) 0.76 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD81, TSG101 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 133 EM EM-type Transmission-EM Image type Wide-field

	EV180008	3/4	Homo sapiens	HuH7	(d)(U)C Total Exosome Isolation	Hao, Liu	2018	50%
Study summary Full title Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility All authors Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang Journal Oncogene Abstract Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (hide) EV-METRIC 50% (87th 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 exosome 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 Total Exosome Isolation Protein markers EV: CD81/ TSG101 non-EV: Grp94 Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HuH7 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) 0.2 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD81, TSG101 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 148 EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV180008
species	Homo sapiens
sample type	Cell culture
cell type	HCCLM3	HuH7	HCCLM3	HuH7
condition	Control condition	Control condition	Control condition	Control condition
separation protocol	DG (d)(U)C	DG (d)(U)C	(d)(U)C Total Exosome Isolation	(d)(U)C Total Exosome Isolation
Exp. nr.	1	4	2	3
EV-METRIC %	75	75	50	50