298 Farber Cancer Institute, Harvard Medical School, Boston, MA) [17] and used for B-RafV600E overexpression (transduc- tion) studies in NT cells (passage 0). For analysis, cells were pelleted then processed according to Nucera et al. [18]. More details are reported in the supplemental online data. WB Analysis WB assays and band densitometry were performed accord- ing to a standard procedure [19]. Cell Cycle Analysis, Bromodeoxyuridine Assay, and Apoptosis Assay 8505c, TPC-1, and NT cells were seeded at 3 105 cells/ 10-cm dish. After 72 hours of treatment with PLX4720 (1 M or 10 M) or vehicle, bromodeoxyuridine (BrdU, 10 M) (Upstate Cell Signaling Solutions, Temecula, CA) was added for 1 hour. The cell cycle analysis, BrdU assay, and apoptosis assay were performed in triplicate according to Nucera et al. [19]. Migration and Invasion Assays Migration and invasion assays were performed in triplicate using 8505c cells (8 103 cells/assay), TPC-1 cells (8 103 cells/assay), and NT cells (25 103 cells/assay) for 72 hours in culture. More details are reported in the supple- mental online data. Quantitative Multigene Profiling by Real-Time Reverse Transcriptase Polymerase Chain Reaction Analysis Quantitative multigene and absolute real-time reverse tran- scriptase polymerase chain reaction (RT-PCR) were per- formed according to Shih and Smith [20] (supplemental online data and supplemental online Table 1). Tumor Implantation Animal work was performed in the animal facility at Massachusetts General Hospital (Boston, MA) in accor- dance with federal, local, and institutional guidelines. Orthotopic thyroid tumor implantation was done as pre- viously described in severe combined immunodeficient (SCID) mice (Charles River Laboratories, Wilmington, MA) using 8505c cells (5 105) engineered with green fluorescent protein (GFP) [21] or TPC-1 cells (1 106). Given our early-intervention design, 1 week post–tumor implantation mice were treated with either PLX4720 (30 mg/kg per day, n 8) by oral gavage (Plexxikon, Berke- ley, CA) or vehicle (n 8) once daily for 21 days. Weight was recorded weekly. Tumor burden was evaluated weekly by palpation, and mice underwent necropsy 35 days after tumor implantation. PLX4720 and Preclinical Thyroid Cancer Models Tumor size was measured using an electronic caliper. Tu- mor volume was calculated as (1/2) length width height. Tumors, regional lymph nodes, and lungs were also analyzed by histology. In Vivo and Ex Vivo Bioimaging, Histological, and IHC Analysis of Orthotopic Thyroid Tumors The computed tomography, multispectral fluorescence scan- ner (CRi Maestro 500, CRi Inc., Woburn, MA), histopathol- ogy, and IHC are described in the supplemental online data. Scoring for Metastases Metastases were counted and averaged at 40 using hema- toxylin and eosin–stained, formalin-fixed sections of lymph nodes and lungs. Mass Spectrometric Genotyping Genomic DNA from thyroid cancer lines was purified and subjected to mass spectrometric genotyping as described previously [22]. The RET/PTC-1 analysis was performed according to Puxeddu et al. [23]. Statistical Analysis Statistical analyses were performed using Microsoft Excel with Student’s t-test and the 2 test. p-values .05 were considered significant (*p .05, **p .01, ***p .001). The data represent the average standard deviation or, in the case of real-time RT-PCR or mice experiments, mean standard error of the mean. RESULTS PLX4720 Downregulates ERK-1/ERK-2 Phosphorylation and Inhibits Migration and Invasion in 8505c Cells Harboring B-RafV600E For our in vitro studies, we used 8505c, an aggressive and authentic [24] human thyroid carcinoma cell line that we have shown to be tumorigenic and metastatic in vivo [21] and to harbor the V600E mutation and TP53 R248G muta- tion (by mass spectrometric genotyping) (Fig. 1A); 8505c cells were found to be negative for other mutations (e.g., H-, N-, and K-RAS mutations, etc.). Treatment with 1 M PLX4720 resulted in a 90% reduction in phospho- ERK-1/ERK-2 protein levels after 1 hour (Fig. 1B) with no significant difference in cell proliferation (BrdU up- take) even after 72 hours (control versus PLX4720 treat- ment, 22.6% 3.3% versus 18.4% 1.4%; p 0.1) (Fig. 2A, 2B, 2D), whereas treating 8505c cells with 10 M PLX4720 for 1 hour or 72 hours reduced phospho- ERK-1/ERK-2 (Fig. 1B, 1C), reduced BrdU uptake (19.8% 0.8% versus 3.7% 1.2% in control versus