title: Tumour exosomes display differential mechanical and complement activation properties dependent on malignant state: implications in endothelial leakiness.
abstract: BACKGROUND
  Exosomes have been implicated in tumour progression and metastatic spread. Little is known of the effect of mechanical and innate immune interactions of malignant cell-derived exosomes on endothelial integrity, which may relate to increased extravasation of circulating tumour cells and, therefore, increased metastatic spread.
  
  
  METHODS
  Exosomes isolated from non-malignant immortalized HCV-29 and isogenic malignant non-metastatic T24 and malignant metastatic FL3 bladder cells were characterized by nanoparticle tracking analysis and quantitative nanomechanical mapping atomic force microscopy (QNM AFM) to determine size and nanomechanical properties. Effect of HCV-29, T24 and FL3 exosomes on human umbilical vein endothelial cell (HUVEC) monolayer integrity was determined by transendothelial electrical resistance (TEER) measurements and transport was determined by flow cytometry. Complement activation studies in human serum of malignant and non-malignant cell-derived exosomes were performed.
  
  
  RESULTS
  FL3, T24 and HCV-29 cells produced exosomes at similar concentration per cell (6.64, 6.61 and 6.46×10(4) exosomes per cell for FL3, T24 and HCV-29 cells, respectively) and of similar size (120.2 nm for FL3, 127.6 nm for T24 and 117.9 nm for HCV-29, respectively). T24 and FL3 cell-derived exosomes exhibited a markedly reduced stiffness, 95 MPa and 280 MPa, respectively, compared with 1,527 MPa with non-malignant HCV-29 cell-derived exosomes determined by QNM AFM. FL3 and T24 exosomes induced endothelial disruption as measured by a decrease in TEER in HUVEC monolayers, whereas no effect was observed for HCV-29 derived exosomes. FL3 and T24 exosomes traffic more readily (11.6 and 21.4% of applied exosomes, respectively) across HUVEC monolayers than HCV-29 derived exosomes (7.2% of applied exosomes). Malignant cell-derived exosomes activated complement through calcium-sensitive pathways in a concentration-dependent manner.
  
  
  CONCLUSIONS
  Malignant (metastatic and non-metastatic) cell line exosomes display a markedly reduced stiffness and adhesion but an increased complement activation compared to non-malignant cell line exosomes, which may explain the observed increased endothelial monolayer disruption and transendothelial transport of these vesicles.
authors.affiliation: The Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark.|Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Bradley
authors.initials: B
authors.lastname: Whitehead
authors.affiliation: Nanomedicine Laboratory, Centre for Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: LinPing
authors.initials: L
authors.lastname: Wu
authors.affiliation: The Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark.|Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Michael Lykke
authors.initials: ML
authors.lastname: Hvam
authors.affiliation: The Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark.
authors.forename: Husnu
authors.initials: H
authors.lastname: Aslan
authors.affiliation: The Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark.
authors.forename: Mingdong
authors.initials: M
authors.lastname: Dong
authors.affiliation: Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Dyrskjøt
authors.affiliation: Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Marie Stampe
authors.initials: MS
authors.lastname: Ostenfeld
authors.affiliation: Nanomedicine Laboratory, Centre for Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Seyed Moein
authors.initials: SM
authors.lastname: Moghimi
authors.affiliation: The Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark.|Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark; kenh@inano.au.dk.
authors.forename: Kenneth Alan
authors.initials: KA
authors.lastname: Howard
keywords: complement activation; endothelial disruption; extracellular vesicles; extravasation; metastatic cell-derived exosomes; nanomechanical properties