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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170065	1/1	Homo sapiens	Primary tumor-derived fibroblasts	(d)(U)C	Bhome R	2017	77%
Study summary Full title Exosomal microRNAs derived from colorectal cancer-associated fibroblasts: role in driving cancer progression. All authors Bhome R, Goh RW1, Bullock MD, Pillar N, Thirdborough SM, Mellone M, Mirnezami R, Galea D, Veselkov K, Gu Q, Underwood TJ, Primrose JN, De Wever O, Shomron N, Sayan AE, Mirnezami AH. Journal Aging (Albany NY) Abstract Colorectal cancer is a global disease with increasing incidence. Mortality is largely attributed to (show more...)Colorectal cancer is a global disease with increasing incidence. Mortality is largely attributed to metastatic spread and therefore, a mechanistic dissection of the signals which influence tumor progression is needed. Cancer stroma plays a critical role in tumor proliferation, invasion and chemoresistance. Here, we sought to identify and characterize exosomal microRNAs as mediators of stromal-tumor signaling. In vitro, we demonstrated that fibroblast exosomes are transferred to colorectal cancer cells, with a resultant increase in cellular microRNA levels, impacting proliferation and chemoresistance. To probe this further, exosomal microRNAs were profiled from paired patient-derived normal and cancer-associated fibroblasts, from an ongoing prospective biomarker study. An exosomal cancer-associated fibroblast signature consisting of microRNAs 329, 181a, 199b, 382, 215 and 21 was identified. Of these, miR-21 had highest abundance and was enriched in exosomes. Orthotopic xenografts established with miR-21-overexpressing fibroblasts and CRC cells led to increased liver metastases compared to those established with control fibroblasts. Our data provide a novel stromal exosome signature in colorectal cancer, which has potential for biomarker validation. Furthermore, we confirmed the importance of stromal miR-21 in colorectal cancer progression using an orthotopic model, and propose that exosomes are a vehicle for miR-21 transfer between stromal fibroblasts and cancer cells. (hide) EV-METRIC 77% (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 Colorectal cancer 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 Adj. k-factor 88.25 (pelleting) / 88.25 (washing) Protein markers EV: Alix/ CD81/ TSG101/ CD63 non-EV: cytochrome c/ GM130/ cytochromec Proteomics no Show all info Study aim Function, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary tumor-derived fibroblasts 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 50.3 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 88.25 Wash: time (min) 70 Wash: Rotor Type Type 50.3 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 88.25 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 100 Western Blot Detected EV-associated proteins Alix, CD63, CD81, TSG101 Not detected contaminants GM130, cytochrome c Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Electron microscopy Extra particle analysis NTA Report type Modus Reported size (nm) 113 EV concentration Yes Particle yield 1570000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV170065
species	Homo sapiens
sample type	Cell culture
cell type	Primary tumor-derived fibroblasts
condition	Colorectal cancer
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	77