title: Effect of cetuximab and fractionated irradiation on tumour micro-environment.
abstract: OBJECTIVE
  Previous experiments have shown that application of the anti-EGFR monoclonal antibody C225 (cetuximab) improves local tumour control after irradiation in FaDu human squamous cell carcinoma (hSCC) due to the combined effect of decreased repopulation and improved reoxygenation. The present study investigates early changes of the pimonidazole hypoxic fraction of FaDu tumours and the expression and phosphorylation of the EGFR and its downstream signal transduction molecules after treatment with C225 alone or in combination with irradiation.
  
  
  METHODS
  FaDu tumour xenografts were irradiated with up to 3×3Gy with or without additional C225 treatment and excised at different time points. Tumour hypoxia was evaluated using pimonidazole. EGFR expression and phosphorylation and intratumoural distribution of C225 were assessed by immunofluorescence analysis. Western blots were performed to evaluate expression and phosphorylation of EGFR, ErbB2, AKT and MAPK (ERK1/2).
  
  
  RESULTS
  Hypoxia did not change during the 4days of treatment in the tumours treated with C225 alone or combined with irradiation. C225 treatment led to downregulation of the total EGFR in FaDu tumours, accompanied by a change of the spatial distribution of the receptor favouring the membranous expression. An induction of phosphorylation of the EGFR (tyr992, tyr1173) was observed with C225 alone or combined with irradiation. AKT phosphorylation was decreased, whereas MAPK phosphorylation remained unchanged. C225 membrane staining was homogeneously distributed over the whole tumour with no differences between hypoxic and non-hypoxic tumour cells.
  
  
  CONCLUSIONS
  Pimonidazole-hypoxia of FaDu tumours during the initial part of fractionated irradiation is not influenced by C225, indicating that external hypoxia markers may not be promising as biomarkers for tumour response to combined treatment. The downregulation of the total EGFR, but at the same time higher membrane staining, as well as the changes in downstream signal transduction molecules, warrants further investigation in other tumour models.
authors.affiliation: Department of Radiation Oncology, Medical Faculty and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany.
authors.forename: Alina
authors.initials: A
authors.lastname: Santiago
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eicheler
authors.forename: Jan
authors.initials: J
authors.lastname: Bussink
authors.forename: Paul
authors.initials: P
authors.lastname: Rijken
authors.forename: Ala
authors.initials: A
authors.lastname: Yaromina
authors.forename: Bettina
authors.initials: B
authors.lastname: Beuthien-Baumann
authors.forename: Albert J
authors.initials: AJ
authors.lastname: van der Kogel
authors.forename: Michael
authors.initials: M
authors.lastname: Baumann
authors.forename: Mechthild
authors.initials: M
authors.lastname: Krause
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology* / Q000627:therapeutic use; D061067:Antibodies, Monoclonal, Humanized; D002294:Carcinoma, Squamous Cell / Q000532:radiotherapy / Q000628:therapy; D045744:Cell Line, Tumor; D000068818:Cetuximab; D003131:Combined Modality Therapy; D019583:Dose Fractionation, Radiation; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D000860:Hypoxia; D008297:Male; D051379:Mice; D009369:Neoplasms / Q000532:radiotherapy / Q000601:surgery / Q000628:therapy; D009593:Nitroimidazoles / Q000494:pharmacology / Q000627:therapeutic use; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology / Q000627:therapeutic use; D015398:Signal Transduction / Q000187:drug effects; D014183:Transplantation, Heterologous; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D009593:Nitroimidazoles; D011838:Radiation-Sensitizing Agents; C033815:pimonidazole; D066246:ErbB Receptors; D000068818:Cetuximab