title: Efficacy of Beta1 Integrin and EGFR Targeting in Sphere-Forming Human Head and Neck Cancer Cells.
abstract: BACKGROUND
  Resistance to radiotherapy continues to be a limiting factor in the treatment of cancer including head and neck squamous cell carcinoma (HNSCC). Simultaneous targeting of β1 integrin and EGFR was shown to have a higher radiosensitizing potential than mono-targeting in the majority of tested HNSCC cancer models. As tumor-initiating cells (TIC) are thought to play a key role for therapy resistance and recurrence and can be enriched in sphere forming conditions, this study investigated the efficacy of β1 integrin/EGFR targeting without and in combination with X-ray irradiation on the behavior of sphere-forming cells (SFC).
  
  
  METHODS
  HNSCC cell lines (UTSCC15, UTSCC5, Cal33, SAS) were injected subcutaneously into nude mice for tumor up-take and plated for primary and secondary sphere formation under non-adhesive conditions which is thought to reflect the enrichment of SFC and their self-renewal capacity, respectively. Treatment was accomplished by inhibitory antibodies for β1 integrin (AIIB2) and EGFR (Cetuximab) as well as X-ray irradiation (2 - 6 Gy single doses). Further, flow cytometry for TIC marker expression and cell cycling as well as Western blotting for DNA repair protein expression and phosphorylation were employed.
  
  
  RESULTS
  We found higher primary and secondary sphere forming capacity of SAS cells relative to other HNSCC cell lines, which was in line with the tumor up-take rates of SAS versus UTSCC15 cells. AIIB2 and Cetuximab administration had minor cytotoxic and no radiosensitizing effects on SFC. Intriguingly, secondary SAS spheres, representing the fraction of surviving SFC upon passaging, showed greatly enhanced radiosensitivity compared to primary spheres. Intriguingly, neither AIIB2 nor Cetuximab significantly altered basal sphere forming capacity and radiosensitivity. While an increased accumulation of G0/G1 phase cells was observable in secondary SAS spheres, DNA double strand break repair indicated no difference on the basis of significantly enhanced ATM and Chk2 dephosphorylation upon irradiation.
  
  
  CONCLUSIONS
  In the HNSCC model, sphere-forming conditions select for cells, which are unsusceptible to both anti-β1 integrin and anti-EGFR inhibitory antibodies. With regard to primary and secondary sphere formation, our data suggest that both of these SFC fractions express distinct survival strategies independent from β1 integrin and EGFR and that future work is warranted to better understand SFC survival and enrichment before and after treatment to untangle the underlying mechanisms for identifying novel, druggable cancer targets in SFC.
authors.affiliation: 1. OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, and Helmholtz-Zentrum Dresden - Rossendorf, Germany;; 2. Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden - Rossendorf, Germany;
authors.forename: Katja
authors.initials: K
authors.lastname: Zscheppang
authors.affiliation: 1. OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, and Helmholtz-Zentrum Dresden - Rossendorf, Germany;; 3. National Center for Tumor Diseases (NCT), Partner Site Dresden, Germany;
authors.forename: Ina
authors.initials: I
authors.lastname: Kurth
authors.affiliation: 1. OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, and Helmholtz-Zentrum Dresden - Rossendorf, Germany;
authors.forename: Nicole
authors.initials: N
authors.lastname: Wachtel
authors.affiliation: 1. OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, and Helmholtz-Zentrum Dresden - Rossendorf, Germany;; 5. Institute of Radiooncology, Helmholtz-Zentrum Dresden - Rossendorf, Dresden, Germany;; 6. German Cancer Consortium (DKTK), Dresden, Germany;; 7. German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Dubrovska
authors.affiliation: 1. OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, and Helmholtz-Zentrum Dresden - Rossendorf, Germany;; 5. Institute of Radiooncology, Helmholtz-Zentrum Dresden - Rossendorf, Dresden, Germany;
authors.forename: Leoni A
authors.initials: LA
authors.lastname: Kunz-Schughart
authors.affiliation: 1. OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, and Helmholtz-Zentrum Dresden - Rossendorf, Germany;; 4. Department of Radiation Oncology, University Hospital and Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Germany;; 5. Institute of Radiooncology, Helmholtz-Zentrum Dresden - Rossendorf, Dresden, Germany;; 6. German Cancer Consortium (DKTK), Dresden, Germany;; 7. German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Cordes
keywords: EGFR.; Sphere formation; beta1 integrin; head and neck squamous cell carcinoma; radiosensitivity