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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220035	1/3	Homo sapiens	PANC-1	(d)(U)C Filtration	Zhang Y	2018	33%
Study summary Full title Pancreatic cancer-derived exosomes suppress the production of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3. All authors Zhang Y, Huang S, Li P, Chen Q, Li Y, Zhou Y, Wang L, Kang M, Zhang B, Yang B, Dong X, Wu Y Journal Cancer Lett Abstract One hallmark of pancreatic cancer (PC) is the high prevalence of pancreatic cancer-associated diabet (show more...)One hallmark of pancreatic cancer (PC) is the high prevalence of pancreatic cancer-associated diabetes mellitus (PC-DM), but the mechanisms remain to be elucidated. Patients with PC who are diagnosed with new-onset diabetes/prediabetes have recently been shown to display significantly lower levels of glucose-dependent insulinotropic peptide (GIP) secreted mainly by enteroendocrine cells. We hypothesized that PC-derived exosomes are responsible for the decreased levels of incretins in patients with PC-DM. In this study, exosomes were successfully isolated from PANC-1, MIA PaCa-2 and SW620 cells and characterized. Only the exosomes from MIA PaCa-2 cells (Exo-Mia) reduce the production of GIP and glucagon-like peptide-1 (GLP-1) from STC-1 cells in vitro in a concentration- and time-dependent manner. Moreover, Exo-Mia increased the levels of the Gip and proglucagon mRNAs and decreased the expression of proprotein convertase subtilisin/kexin type 1/3 (PCSK1/3), which is responsible for the post-translational processing of Gip and proglucagon. Furthermore, differentially expressed exosomal miRNAs (miR-6796-3p, miR-6763-5p, miR-4750-3p and miR-197-3p) were identified and considered to be responsible for the inhibitory effects on GIP and GLP-1 production. To further determine the approach of cancer-derived exosomes reaching enteroendocrine cells, we analyzed the uptake and distribution of exosomes in animal model. It was observed that exosomes infused into the intestinal cavity were more easily internalized by the intestinal epithelium than exosomes injected into blood. In conclusion, pancreatic cancer-derived exosomes (Exo-Mia) suppress the synthesis of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3. Moreover, it may be the pancreatic juice that transport cancer-derived exosomes to target cells (K and L cells) in the gut. (hide) EV-METRIC 33% (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 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Alix/ CD63 non-EV: C-myc Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PANC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS 16h at 11,000g/ Other preparation 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: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ Alix Not detected contaminants C-myc Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Modus EM EM-type Transmission-EM Image type Close-up

	EV220035	2/3	Homo sapiens	MIA PaCa-2	(d)(U)C Filtration	Zhang Y	2018	33%
Study summary Full title Pancreatic cancer-derived exosomes suppress the production of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3. All authors Zhang Y, Huang S, Li P, Chen Q, Li Y, Zhou Y, Wang L, Kang M, Zhang B, Yang B, Dong X, Wu Y Journal Cancer Lett Abstract One hallmark of pancreatic cancer (PC) is the high prevalence of pancreatic cancer-associated diabet (show more...)One hallmark of pancreatic cancer (PC) is the high prevalence of pancreatic cancer-associated diabetes mellitus (PC-DM), but the mechanisms remain to be elucidated. Patients with PC who are diagnosed with new-onset diabetes/prediabetes have recently been shown to display significantly lower levels of glucose-dependent insulinotropic peptide (GIP) secreted mainly by enteroendocrine cells. We hypothesized that PC-derived exosomes are responsible for the decreased levels of incretins in patients with PC-DM. In this study, exosomes were successfully isolated from PANC-1, MIA PaCa-2 and SW620 cells and characterized. Only the exosomes from MIA PaCa-2 cells (Exo-Mia) reduce the production of GIP and glucagon-like peptide-1 (GLP-1) from STC-1 cells in vitro in a concentration- and time-dependent manner. Moreover, Exo-Mia increased the levels of the Gip and proglucagon mRNAs and decreased the expression of proprotein convertase subtilisin/kexin type 1/3 (PCSK1/3), which is responsible for the post-translational processing of Gip and proglucagon. Furthermore, differentially expressed exosomal miRNAs (miR-6796-3p, miR-6763-5p, miR-4750-3p and miR-197-3p) were identified and considered to be responsible for the inhibitory effects on GIP and GLP-1 production. To further determine the approach of cancer-derived exosomes reaching enteroendocrine cells, we analyzed the uptake and distribution of exosomes in animal model. It was observed that exosomes infused into the intestinal cavity were more easily internalized by the intestinal epithelium than exosomes injected into blood. In conclusion, pancreatic cancer-derived exosomes (Exo-Mia) suppress the synthesis of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3. Moreover, it may be the pancreatic juice that transport cancer-derived exosomes to target cells (K and L cells) in the gut. (hide) EV-METRIC 33% (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 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Alix/ CD63 non-EV: C-myc Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MIA PaCa-2 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation/ 16h at 11,000g 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: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ Alix Not detected contaminants C-myc Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Modus EM EM-type Transmission-EM Image type Close-up

	EV220035	3/3	Homo sapiens	SW620	(d)(U)C Filtration	Zhang Y	2018	33%
Study summary Full title Pancreatic cancer-derived exosomes suppress the production of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3. All authors Zhang Y, Huang S, Li P, Chen Q, Li Y, Zhou Y, Wang L, Kang M, Zhang B, Yang B, Dong X, Wu Y Journal Cancer Lett Abstract One hallmark of pancreatic cancer (PC) is the high prevalence of pancreatic cancer-associated diabet (show more...)One hallmark of pancreatic cancer (PC) is the high prevalence of pancreatic cancer-associated diabetes mellitus (PC-DM), but the mechanisms remain to be elucidated. Patients with PC who are diagnosed with new-onset diabetes/prediabetes have recently been shown to display significantly lower levels of glucose-dependent insulinotropic peptide (GIP) secreted mainly by enteroendocrine cells. We hypothesized that PC-derived exosomes are responsible for the decreased levels of incretins in patients with PC-DM. In this study, exosomes were successfully isolated from PANC-1, MIA PaCa-2 and SW620 cells and characterized. Only the exosomes from MIA PaCa-2 cells (Exo-Mia) reduce the production of GIP and glucagon-like peptide-1 (GLP-1) from STC-1 cells in vitro in a concentration- and time-dependent manner. Moreover, Exo-Mia increased the levels of the Gip and proglucagon mRNAs and decreased the expression of proprotein convertase subtilisin/kexin type 1/3 (PCSK1/3), which is responsible for the post-translational processing of Gip and proglucagon. Furthermore, differentially expressed exosomal miRNAs (miR-6796-3p, miR-6763-5p, miR-4750-3p and miR-197-3p) were identified and considered to be responsible for the inhibitory effects on GIP and GLP-1 production. To further determine the approach of cancer-derived exosomes reaching enteroendocrine cells, we analyzed the uptake and distribution of exosomes in animal model. It was observed that exosomes infused into the intestinal cavity were more easily internalized by the intestinal epithelium than exosomes injected into blood. In conclusion, pancreatic cancer-derived exosomes (Exo-Mia) suppress the synthesis of GIP and GLP-1 from STC-1 cells in vitro by down-regulating the PCSK1/3. Moreover, it may be the pancreatic juice that transport cancer-derived exosomes to target cells (K and L cells) in the gut. (hide) EV-METRIC 33% (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 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: Alix/ TSG101/ CD63 non-EV: C-myc Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW620 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation/ 16h at 11,000g 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: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ TSG101 Not detected contaminants C-myc Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Modus EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV220035
species	Homo sapiens
sample type	Cell culture
cell type	PANC-1	MIA PaCa-2	SW620
condition	Control condition	Control condition	Control condition
separation protocol	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration
Exp. nr.	1	2	3
EV-METRIC %	33	33	33