Epidermal growth factor receptor as a novel molecular target for aggressive papillary tumors in the middle ear and temporal bone.

Adenomatous tumors in the middle ear and temporal bone are rare but highly morbid because they are difficult to detect prior to the development of audiovestibular dysfunction. Complete resection is often disfiguring and difficult because of location and the late stage at diagnosis, so identification of molecular targets and effective therapies is needed. Here, we describe a new mouse model of aggressive papillary ear tumor that was serendipitously discovered during the generation of a mouse model for mutant EGFR-driven lung cancer. Although these mice did not develop lung tumors, 43% developed head tilt and circling behavior. Magnetic resonance imaging (MRI) scans showed bilateral ear tumors located in the tympanic cavity. These tumors expressed mutant EGFR as well as active downstream targets such as Akt, mTOR and ERK1/2. EGFR-directed therapies were highly effective in eradicating the tumors and correcting the vestibular defects, suggesting these tumors are addicted to EGFR. EGFR activation was also observed in human ear neoplasms, which provides clinical relevance for this mouse model and rationale to test EGFR-targeted therapies in these rare neoplasms.

. A new mouse model of aggressive papillary ear tumor  Table S1. Ear tumor treatment References [28,31,32] Movies S1 (Before treatment) and S5 (After treatment). Mouse no. 9498 treated with the combination of afatinib and cetuximab Movies S2 (Before treatment) and S6 (After treatment). Mouse no. 8682 treated with WZ4002 Movies S3 (Coronal) and S4 (Horizontal). MRI scans of a head from mouse no. 9499

Immunoblotting analysis (IB)
Cells (5 x 10 5 cells per well) were plated in six-well plates. The following day, cells were treated with WZ4002 or equal volume of DMSO for 16 hours and lysed in 2 x lysis buffer as described previously [31]. For tumor-tissue homogenates in vivo, frozen tumors were allowed to thaw on ice, then homogenized in radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris (pH 8.0)] containing 2.5 mol/L h-glycerol phosphate, 0.2 mol/L sodium orthovanadate, 1.25 mol/L sodium fluoride, and 1 x protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) using a hand-held Tissue-Tearor homogenizer (Biospec Products, Bartlesville, OK, USA). Cell lysates or tumortissue homogenates with equal amounts of protein were separated by SDS-PAGE, and then transferred to nitrocellulose membranes. The membranes were blocked for 1 hour in blocking buffer (1 x TBS, 5% milk, 0.1% Tween 20), and placed in primary antibody diluted in 1 x TBS, 5% bovine serum albumin, 0.1% Tween 20, overnight at 4°C. The following day, membranes were washed thrice in wash buffer (0.1% Tween 20, 1 x TBS). Primary antibody was detected using a horseradish peroxidase-linked secondary antibody, and visualized with the enhanced chemiluminescent detection system (Amersham Biosciences, Pittsburgh, PA, USA). Immunoblot experiments were performed at least 3 times.

Immunohistochemical analysis (IHC)
Expression of mutant EGFR L858R+T790M (mEGFR L+T ) was confirmed by IB and IHC with an antibody of EGFR (L858R mutant specific, 43B2). A formalin-fixed with decalcification, paraffin-embedded (FFPE) mouse skull was sectioned, placed on slide, and stained with hematoxylin and eosin (H&E) (Histoserv Inc. Germantown, MD, USA). Antigen retrieval was carried out using preheated target retrieval solution (pH 6.0) from DakoCytomation (Carpinteria, CA, USA) for 30 minutes in a boiling rice cooker. Vectastain Elite ABC kits from Vector Laboratories (Burlingame, CA, USA) were used according to manufacturer's instructions for blocking, dilution of primary antibody, and labeling. Primary antibody was incubated with sections for 16 hours at 4°C. 3,3-Diaminobenzidine was prepared fresh from tablets (Sigma).
Specificity of staining was assessed by comparison with both samples stained in the absence of primary antibody and FFPE cell pellets from human non-small cell lung cancer (NSCLC) H1975 cells expressing mEGFR L+T protein.

Magnetic resonance imaging (MRI) Scanning and total tumor volume measurement
Anesthesia was induced then maintained with 1%-2% isoflurane administered via nosecone in an imaging chamber, and mice with vitals and body core temperature maintained were placed in a 72/25 mm volume/surface coil ensemble and positioned in a 7T horizontal MRI scanner. T 2 weighted anatomical images, encompassing the whole brain, were acquired in the coronal and horizontal planes (resolution= 75x75x750 µm, TR/TE= 2500/24 ms). For high resolution MRI, 3-Dimensional gradient-echo (GE) images (3D-MRI) of 0.2% gadoliniumperfused head were obtained at 14 T vertical scanner (TR/TE = 75/6 ms, isotropic resolution = 30x25x24 µm). The acquired images were viewed and analyzed using ImageJ version 1.43 software (http://rsbweb.nih.gov/ij/index.html). Ear tumor areas on the image sections were traced as regions of interest (ROIs). The areas of ROIs segmented with ImageJ were multiplied by the section thickness (750 μm), and then summed to obtain an estimate of the total tumor volume.
Criteria to classify tumor responses to drug treatment were previously described [28] as the following: 1) complete response (CR): the disappearance of all target lesions; 2) partial response (PR): at least a 30% decrease in the volume of target lesions, taking as reference the baseline tumor volume; 3) progressive disease (PD): at least a 20% increase in the volume of target lesions, taking as reference the baseline tumor volume, and 4) stable disease (SD): neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the baseline tumor volume.

Micro-computed tomography (CT) scanning
Anesthesia was induced then maintained with 1%-2% isoflurane administered via nosecone in an imaging chamber. Micro-CT scan was performed with an Inveon CT (Siemens Preclinical Solutions, Knoxville, TN, USA) using a 0.5 mm Al filter and 80 kVp X-rays with 380-uA current. The image acquisition was respiratory-gated. The isotropic resolution obtained was 43 μm. The resulting raw data were reconstructed to a final image volume of 480 X 480 X 480 slices at (87 μm) 3 voxel dimensions using Siemens Inveon Acquisition Workplace version 1.4. The reconstructed images were viewed and analyzed using the ImageJ version 1.43 software. Formalin-fixed cells were sent to Histoserv Inc. and processed for FFPE pellets.

Sequencing of von Hippel-Lindau (Vhl) gene
For DNA preparation, the entire ear tumor pieces from SP-C/mEGFR L+T mice were dissected and flash-frozen followed by stored at -70 ○ C until being used. Identical weight of lung tissues from above mice were procured under identical procedures as internal controls for individual tumors. Tissues were chopped into <0.5 mm 3 pieces on dry ice and DNA was extracted with Qiagen DNAeasy Blood & Tissue kit (QIAGEN Inc., Valencia, CA, USA). In PCR process, 6 pairs of primers were used to amplify the 2.7 kb fragment of Vhl gene. In brief, one pair was used for exon 1 and exon 2 amplification, respectively. Four pairs were used for exon 3 amplification; the overlapped 4 fragments (3a, 3b, 3c, 3d) cover the entire exon 3. Taq high-fidelity polymerase (Life Technologies, Grand Island, NY, USA) was used for PCR amplification, following below conditions: 95 ○ C denaturation for 2 min, followed by 35 cycles of 95 ○ C for 45 sec, 57 ○ C for 45 sec, 72 ○ C for 45 sec, followed by a 5min extension at 72 ○ C. PCR products were visualized as single bands on agarose-gels and the bands were resected from the gel and DNAs were purified with Qiagen's Gel Extraction kit. PCR products were then cloned into Invitrogen's Topo10 vectors (Life Technologies) using the Topo-TA Cloning kit. After blue/white screening, 6 clones of each PCR product were prepared using Qiagen's DNA miniprep kit. Cloning products were sequenced with Sanger's approach at W.M. Keck DNA Sequencing Facility at Yale University School of Medicine. Gene-specific primers (listed below) and vector's M13f/r primers were used for sequencing.