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A microenvironment-inspired synthetic 3D model for pancreatic ductal adenocarcinoma organoids - CDM Sets

ABSTRACT: Cell-derived matrix (CDM) datasets from KPC-1 or HPDE cells after seven days of growth.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Pancreatic Ductal Carcinoma

SUBMITTER: Christopher Below

LAB HEAD: Claus Jorgensen

PROVIDER: PXD022509 | Pride | 2021-11-29

REPOSITORIES: Pride

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Project description:Extracellular vesicles (EVs) exert important functions in the extracellular space and in cell-to-cell communication. Ultracentrifugation, the most frequently used EV isolation technique, requires specialized equipment and provides relatively low purity and yield. A more recently applied and easily accessible EV isolation technique is size exclusion chromatography (SEC). Here, we hypothesized that EVs isolated from dilute cell culture media by concentration on a 10 kDa filter followed by SEC are suitable for proteomic and functional studies. We also hypothesized that the protein-rich SEC fractions are suitable controls to assess biological effects of the non-EV associated secretome. Cell-depleted medium from BEAS-2B bronchial epithelial cells (60 ml) was 0.22 µm filtered, concentrated to 0.5 ml by 10 kDa UF and run over a sepharose CL-4B column. Fractions were assessed for protein and EV content. EV-rich and protein-rich fractions were concentrated by 10 kDa UF. EV concentrates were characterized by tuneable resistive pulse sensing, cryo transmission electron microscopy and nanoLC-MS/MS. Finally, we assessed the effect of EVs or free secreted protein from unexposed cells or cells exposed to 1% (v/v) cigarette smoke extract (CSE) on IL-8 release by naïve BEAS-2B cells or on adhesion of THP-1 monocytes to endothelial cells. UF concentrated the EVs with approximately 100% recovery. Most of EVs eluted in SEC fractions 6-11, while protein became detectable at fraction 12. The final EV recovery was 40 ± 4%, as compared to 27 ± 8% by ultracentrifugation. Mass Spectrometry of the EV fractions identified 388 different proteins, including various EV markers. Only the protein, but not the EV fractions from media of CSE-exposed cells induced IL-8 release by naïve bronchial epithelial cells, whereas only EVs, but not the protein fractions induced THP-1 adhesion to endothelial cells. Thus, UF followed by SEC provides EV isolates with sufficient yield and purity for proteomic studies and allows directly comparing the functional effects of EVs and the non-EV associated secretome.

			Action	DRS
	20180216_JB_01.mgf	Mgf
	20180216_JB_01.mzML	Mzml
	20180216_JB_01.mzid.gz	Mzid
	20180216_JB_01.raw	Raw
	20180216_JB_01_Proteins.txt	Txt

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A microenvironment-inspired synthetic 3D model for pancreatic ductal adenocarcinoma organoids - CDM Sets

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