title: Nanoimmunoassay to Detect Responses in Head and Neck Cancer: Feasibility in a Mouse Model.
abstract: To demonstrate the feasibility of detecting and quantifying extracellular signal-related kinase (ERK) phosphorylation status using nanoimmunoassay (NIA).
  
  
  
  Analyses using Cal27, SCC25, and OSC19 head and neck squamous carcinoma cell lines in vitro and in a murine xenograft model.
  
  
  
  NIA and immunoblot were performed on whole-cell lysates, tumor lysates, and fine-needle aspirate biopsies to detect ERK phosphorylation states.
  
  
  
  Using NIA, all 6 isoforms of ERK1/2, including nonphosphorylated, monophosphorylated, and diphosphorylated species, could be reliably detected, distinguished, and quantified in a single assay using a single antibody. In vitro treatment of Cal27 cells with the epidermal growth factor receptor inhibitor gefitinib abolished phospho-ERK detection by immunoblot but resulted in residual detectable species by NIA. Residual phospho-ERK in gefitinib-treated cells could be further reduced by the addition of the insulin-like growth factor 1 receptor inhibitor OSI-906; this correlated with an additional decrease in proliferation over gefitinib alone. In a pilot study of 4 murine xenograft tumors, NIA performed on tumor lysates and fine-needle aspirate biopsies demonstrated altered ERK profiles after 2 days of gefitinib treatment compared with untreated mice.
  
  
  
  NIA offers a novel approach to quantitating the activation state of signaling molecules such as ERK in nanoscale in vitro and in vivo samples across a wide dynamic range. As such, it has potential to provide molecular diagnostic information before, during, and after treatment using a minimally invasive technique. Further study is warranted to determine its utility in assessing signaling proteins as biomolecular outcome predictors in clinical trials.
authors.affiliation: Division of Head and Neck Surgical Oncology, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, Virginia, USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Hubbard
authors.affiliation: Division of Head and Neck Surgical Oncology, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, Virginia, USA.|Department of Biochemistry, National Liver Institute, Menoufiya University, Al Minufya, Egypt.
authors.forename: Ashraf A
authors.initials: AA
authors.lastname: Khalil
authors.affiliation: Division of Head and Neck Surgical Oncology, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, Virginia, USA.
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Schoeff
authors.affiliation: Division of Head and Neck Surgical Oncology, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, Virginia, USA.
authors.forename: Amir
authors.initials: A
authors.lastname: Allak
authors.affiliation: Division of Head and Neck Surgical Oncology, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, Virginia, USA.
authors.forename: Kyle K
authors.initials: KK
authors.lastname: VanKoevering
authors.affiliation: Division of Head and Neck Surgical Oncology, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, Virginia, USA.
authors.forename: Anne K
authors.initials: AK
authors.lastname: Maxwell
authors.affiliation: Division of Head and Neck Surgical Oncology, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, Virginia, USA.
authors.forename: Oluwayemisi
authors.initials: O
authors.lastname: Adejumo
authors.affiliation: Division of Head and Neck Surgical Oncology, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, Virginia, USA.
authors.forename: Rolando E
authors.initials: RE
authors.lastname: Mendez
authors.affiliation: Division of Head and Neck Surgical Oncology, Department of Otolaryngology-Head and Neck Surgery, University of Virginia Health System, Charlottesville, Virginia, USA mark.jameson@virginia.edu.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Jameson
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000201:enzymology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004195:Disease Models, Animal; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005240:Feasibility Studies; D000077156:Gefitinib; D006258:Head and Neck Neoplasms / Q000201:enzymology*; D064593:Heterografts; D007118:Immunoassay / Q000379:methods; D066298:In Vitro Techniques; D051379:Mice; D036103:Nanotechnology; D011799:Quinazolines / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D011799:Quinazolines; D048049:Extracellular Signal-Regulated MAP Kinases; D000077156:Gefitinib
keywords: ERK; biomarker; head and neck cancer; nanoimmunoassay; targeted therapy