title: Hepatocytes determine the hypoxic microenvironment and radiosensitivity of colorectal cancer cells through production of nitric oxide that targets mitochondrial respiration.
abstract: OBJECTIVE
  To determine whether host hepatocytes may reverse hypoxic radioresistance through nitric oxide (NO)-induced oxygen sparing, in a model relevant to colorectal cancer (CRC) liver metastases.
  
  
  METHODS
  Hepatocytes and a panel of CRC cells were incubated in a tissue-mimetic coculture system with diffusion-limited oxygenation, and oxygen levels were monitored by an oxygen-sensing fluorescence probe. To activate endogenous NO production, cocultures were exposed to a cytokine mixture, and the expression of inducible nitric oxide synthase was analyzed by reverse transcription-polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies.
  
  
  RESULTS
  Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide-producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance.
  
  
  CONCLUSIONS
  Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes.
authors.affiliation: Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium.
authors.forename: Heng
authors.initials: H
authors.lastname: Jiang
authors.forename: Valeri N
authors.initials: VN
authors.lastname: Verovski
authors.forename: Wim
authors.initials: W
authors.lastname: Leonard
authors.forename: Ka Lun
authors.initials: KL
authors.lastname: Law
authors.forename: Marieke
authors.initials: M
authors.lastname: Vermeersch
authors.forename: Guy
authors.initials: G
authors.lastname: Storme
authors.forename: Dirk
authors.initials: D
authors.lastname: Van den Berge
authors.forename: Thierry
authors.initials: T
authors.lastname: Gevaert
authors.forename: Alexandra
authors.initials: A
authors.lastname: Sermeus
authors.forename: Mark
authors.initials: M
authors.lastname: De Ridder
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D019069:Cell Respiration / Q000502:physiology*; D018920:Coculture Techniques; D015179:Colorectal Neoplasms; D004790:Enzyme Induction; D045325:HCT116 Cells; D019073:HT29 Cells; D022781:Hepatocytes / Q000378:metabolism* / Q000528:radiation effects; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000532:radiotherapy* / Q000556:secondary; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008930:Mitochondria, Liver / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010101:Oxygen Consumption / Q000502:physiology*; D011836:Radiation Tolerance / Q000502:physiology*; D059016:Tumor Microenvironment / Q000502:physiology
chemical_list: D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II