title: Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer.
abstract: OBJECTIVE
  Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable.
  
  
  METHODS
  Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells.
  
  
  RESULTS
  Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03).
  
  
  CONCLUSIONS
  Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications.
authors.affiliation: Department of Pathology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA.
authors.forename: Chang-Sook
authors.initials: CS
authors.lastname: Hong
authors.affiliation: Department of Pathology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA.|Department of Otolaryngology, University of Duisburg-Essen, Essen, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Funk
authors.affiliation: Department of Pathology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA.|Department of Head & Neck Surgery, University Hospital, Basel, Switzerland.
authors.forename: Laurent
authors.initials: L
authors.lastname: Muller
authors.affiliation: Department of Medicine, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Boyiadzis
authors.affiliation: Department of Pathology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; whitesidetl@upmc.edu.
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Whiteside
keywords: exosome functions; exosome isolation; exosome morphology; human cancer; human plasma; mini size-exclusion column chromatography