title: Hypoxic enhancement of exosome release by breast cancer cells.
abstract: BACKGROUND
  Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling.
  
  
  METHODS
  Breast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant.
  
  
  RESULTS
  Exposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions.
  
  
  CONCLUSIONS
  These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion.
authors.affiliation: Renal Department, Flinders Medical Centre, Flinders University School of Medicine, Bedford Park, South Australia, 5042, Australia.
authors.forename: Hamish W
authors.initials: HW
authors.lastname: King
authors.forename: Michael Z
authors.initials: MZ
authors.lastname: Michael
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Gleadle
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D017077:Culture Media, Conditioned / Q000737:chemistry; D055354:Exosomes / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D051793:Hypoxia-Inducible Factor 1; C543800:MIRN210 microRNA, human; D035683:MicroRNAs