title: Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors.
abstract: Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13- to 16-fold increased exosome yield and facilitated quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ proteomics. We identified several proteins linked to epithelial-mesenchymal transition, including increased abundance of vimentin and hepatoma-derived growth factor in the membrane, and casein kinase II α and annexin A2 in the lumen of exosomes, respectively, from metastatic cells. The change in exosome protein abundance correlated little, although significant for FL3 versus T24, with changes in cellular mRNA expression. Our proteomic approach may help identification of proteins in the membrane and lumen of exosomes potentially involved in the metastatic process.
authors.affiliation: Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Dennis Kjølhede
authors.initials: DK
authors.lastname: Jeppesen
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Nawrocki
authors.forename: Steffen Grann
authors.initials: SG
authors.lastname: Jensen
authors.forename: Kasper
authors.initials: K
authors.lastname: Thorsen
authors.forename: Bradley
authors.initials: B
authors.lastname: Whitehead
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Howard
authors.forename: Lars
authors.initials: L
authors.lastname: Dyrskjøt
authors.forename: Torben Falck
authors.initials: TF
authors.lastname: Ørntoft
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Marie Stampe
authors.initials: MS
authors.lastname: Ostenfeld
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition; D055354:Exosomes / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis / Q000378:metabolism; D008113:Liver Neoplasms / Q000556:secondary; D008175:Lung Neoplasms / Q000556:secondary; D013058:Mass Spectrometry; D051379:Mice; D009362:Neoplasm Metastasis / Q000473:pathology; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D001743:Urinary Bladder / Q000378:metabolism / Q000473:pathology*; D001749:Urinary Bladder Neoplasms / Q000378:metabolism / Q000473:pathology*; D014746:Vimentin / Q000032:analysis / Q000378:metabolism
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D020543:Proteome; D014746:Vimentin; C051610:hepatoma-derived growth factor
keywords: Bladder cancer; Cell biology; Epithelial-mesenchymal transition; Exosomes; Metastasis; Quantitative proteomics