Journal: PloS one

Article Title: Pre-clinical characterization of Dacomitinib (PF-00299804), an irreversible pan-ErbB inhibitor, combined with ionizing radiation for head and neck squamous cell carcinoma.

doi: 10.1371/journal.pone.0098557

Figure Lengend Snippet: Figure 1. EGFR is over-expressed in SCCHN cell lines. qRT-PCR was performed to measure ErbB transcript levels in the three SCCHN cell lines, normalized to that of the normal oral epithelial (NOE) cells. Expression fold change was determined by the 22DDCt method. ***p, 0.001; student’s t test. doi:10.1371/journal.pone.0098557.g001

Article Snippet: Phospho-EGFR immunohistochemistry was performed using microwave antigen retrieval in combination with the Level-2 Ultra Streptavidin System and antip-EGFR antibody (1:100 dilution; #3777, Cell Signalling), as previously described [28].

Techniques: Quantitative RT-PCR, Expressing

Journal: PloS one

Article Title: Pre-clinical characterization of Dacomitinib (PF-00299804), an irreversible pan-ErbB inhibitor, combined with ionizing radiation for head and neck squamous cell carcinoma.

doi: 10.1371/journal.pone.0098557

Figure Lengend Snippet: Figure 2. Dacomitinib demonstrated in vitro efficacy in EGFR over-expressing SCCHN cell lines. (A to D) Growth inhibition of three SCCHN and the NOE cell lines, treated with Dacomitinib, with or without IR. (B) FaDu; (C) UT-SCC-8; (D) UT-SCC-42a; and (E) NOE cells were treated with Dacomitinib (0, 10, 25, 50, 100, or 250 nM) alone, or in combination with IR (0, 2, or 4 Gy). MTS assays were conducted 72 hours post-treatment. The graphs represent data from 3 independent experiments, with the mean 6 SEM reported. *p,0.05, **p,0.01, ***p,0.001; one-way ANOVA. No statistically significant difference between curves for Figure 2A-D; two-way ANOVA. Combination of Dacomitinib plus RT did not result in a synergistic interaction for any of the dosing regimens in all three SCCHN cell lines (Chou-Talalay Method). doi:10.1371/journal.pone.0098557.g002

Article Snippet: Phospho-EGFR immunohistochemistry was performed using microwave antigen retrieval in combination with the Level-2 Ultra Streptavidin System and antip-EGFR antibody (1:100 dilution; #3777, Cell Signalling), as previously described [28].

Techniques: In Vitro, Expressing, Inhibition

Journal: PloS one

Article Title: Pre-clinical characterization of Dacomitinib (PF-00299804), an irreversible pan-ErbB inhibitor, combined with ionizing radiation for head and neck squamous cell carcinoma.

doi: 10.1371/journal.pone.0098557

Figure Lengend Snippet: Figure 4. Treatment of SCCHN cells with Dacomitinib inhibited EGFR signalling. FaDu, UT-SCC-8, and UT-SCC-42a cells were treated with Dacomitinib (D; 0, 10, 50, 100, 250, or 500 nM) in serum-free media for 24 hours. Thirty minutes prior to lysis, cells were stimulated with EGF (20 ng/ mL). Experiments were performed three independent times, with similar results; representative blots are shown. doi:10.1371/journal.pone.0098557.g004

Article Snippet: Phospho-EGFR immunohistochemistry was performed using microwave antigen retrieval in combination with the Level-2 Ultra Streptavidin System and antip-EGFR antibody (1:100 dilution; #3777, Cell Signalling), as previously described [28].

Techniques: Lysis

Journal: PloS one

Article Title: Pre-clinical characterization of Dacomitinib (PF-00299804), an irreversible pan-ErbB inhibitor, combined with ionizing radiation for head and neck squamous cell carcinoma.

doi: 10.1371/journal.pone.0098557

Figure Lengend Snippet: Figure 6. Dacomitinib delayed FaDu tumor growth, alone and in combination with IR. (A) FaDu-bearing mice were randomized to: (i) DMSO control; (ii) Dacomitinib (5 mg/kg/d) delivered orally on days 1 to 5; (iii) local tumor irradiation (IR; 2 Gy/treatment) delivered on days 2 and 5; or (iv) Dacomitinib plus IR. Mean tumor leg diameter (TLD) from three independent experiments (three mice per treatment group, per experiment) is reported 6 SEM. ***p,0.001, negative control vs. Dacomitinib only, or RT only vs. Dacomitinib plus RT; Student’s t test. (B) Mean mouse weight 6 SEM from experiment (A). (C) Ki-67 staining was performed on treated FaDu tumors: DMSO, radiation (IR), Dacomitinib (D), or Dacomitinib plus radiation (D+IR), as from (A). Tumors were extracted from mice 24 hours after the final treatment. Ki-67 positive cells stain dark brown. The right-hand panel represents the proportion of Ki-67-positive cells for each treatment group. Ki-67 scoring was conducted by counting the number of positive tumor cells in 3 representative sections for each tumor. Mean 6 SEM is reported. ***p,0.001; Student’s t test. (D) p-EGFR staining was performed on paraffin sections, as in (C). Positive Staining for p-EGFR is brown. doi:10.1371/journal.pone.0098557.g006

Article Snippet: Phospho-EGFR immunohistochemistry was performed using microwave antigen retrieval in combination with the Level-2 Ultra Streptavidin System and antip-EGFR antibody (1:100 dilution; #3777, Cell Signalling), as previously described [28].

Techniques: Control, Irradiation, Negative Control, Staining