Anti-Trop2 blockade enhances the therapeutic efficacy of ErbB3 inhibition in head and neck squamous cell carcinoma

ErbB3 has been widely implicated in treatment resistance, but its role as a primary treatment target is less clear. Canonically ErbB3 requires EGFR or ErbB2 for activation, whereas these two established treatment targets are thought to signal independently of ErbB3. In this study, we show that ErbB3 is essential for tumor growth of treatment-naive HNSCC patient-derived xenografts. This ErbB3 dependency occurs via ErbB3-mediated control of EGFR activation and HIF1α stabilization, which require ErbB3 and its ligand neuregulin-1. Here, we show that ErbB3 antibody treatment selects for a population of ErbB3-persister cells that express high levels of the transmembrane protein Trop2 that we previously identified as an inhibitor of ErbB3. Co-treatment with anti-ErbB3 and anti-Trop2 antibodies is synergistic and produces a greater anti-tumor response than either antibody alone. Collectively, these data both compel a revision of ErbB-family signaling and delineate a strategy for its effective inhibition in HNSCC.

generated by separating off Swiss 3T3 feeder cells with 0.05% trypsin and washing with Dulbecco's phosphate buffered saline, followed by 0.25% trypsin to produce a suspension of CRC cells in media. 2 X 10 6 cells in media and 30% Matrigel (BD Biosciences) were injected subcutaneously into both flanks of each mouse. Tumors were measured in length and width with calipers several times per week and volumes were calculated using the formula (length X width 2 )/2. Tumors were allowed to reach an approximate volume of 150 mm 3 before being round-robined into treatment groups. Mice were treated with intraperitoneal injections of anti-ErbB3 antibody (Merrimack) at 25 mg/kg every three days, or with PBS for control. Mice were sacrificed according to IACUC approved protocol upon reaching two centimeters diameter in one dimension or two days after last treatment. Tumor material was harvested for both protein analyses by western by snap freezing in liquid nitrogen and histological studies by formalin fixation.

Histological Studies
Tumor samples were fixed in ten percent neutral buffered formalin for 24 to 48 hours and embedded in paraffin wax at the end of experiments or upon reaching two cm diameter in one direction. Sequential slices of each tumor were transferred to charged microscope slides and samples were processed for Hematoxylin & Eosin and protein expression (anti-EGFR Cell Signaling Technology #4267 1:50, anti-ErbB3 Cell Signaling Technology #12708 1:100, anti-ErbB2 Cell Signaling Technology #4290 1:200, HIF1α Abcam ab51608 1:100, and anti-Trop2 R & D Systems AF650 1:100). Samples were deparaffinized with two, five minute washes in xylenes. Samples were then rehydrated through ethanol series. Heat Induced Antigen Retrieval was then performed using either citric acid solution (H3300) or basic EDTA solution (CTS013) according to primary antibody recommendation utilizing a microwave. Primary antibodies were diluted in antibody solution: PBS, 1% Normal Donkey Serum, 0.3% Triton X-100, and NaN 3 0.0003% pH 7.4. Samples were incubated overnight with primary antibody at 4⁰C in moist chamber. The following day, samples were washed three times with PBS for five minutes each and incubated with species specific biotinylated secondary antibody for an hour at room temperature. Samples were again washed three times with PBS and then incubated with avidinbiotin HRP complex solution (Vector) prepared thirty minutes before use for thirty minutes.
Following three, five minute washes, samples were developed using diaminobenzidine substrate and counterstained with hematoxylin. Slides were then dehydrated through ethanol series and sealed using xylene based solution. Histology images were collected with Olympus BX51 microscope with Olympus DP70 camera using 4x, 20x, and 40x objective lenses. 3

Western Blots analysis
For CRC cell line protein expression, Swiss 3T3 fibroblasts were first trypsinized off with 0.05% trypsin. Culture plates were washed with buffer and cell pellets were collected with cell scrapers and lysed with standard RIPA buffer supplemented with 1 milliMolar (mM) sodium orthovanidate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail P8340 (Sigma-Aldrich). Cell pellets were incubated on ice for twenty min with constant agitation, centrifuged for two min and supernatants were collected. For xenograft tumor protein expression, tumor chunks were homogenized using pestles and lysed in RIPA buffer for twenty min before sonication, centrifugation for 10 min at 4°C, and supernatant collection. Total protein concentrations were determined by Quick Start™ Bradford Assay (BioRad). Membranes were blocked with 5% milk in 1x PBS-0.1% Tween20 for 30 min at room temperature. Primary antibodies diluted in PBS-0.1% Tween20, 3% Bovine Serum Albumen (BSA), 0.02% sodium azide (NaN 3) solution were incubated overnight on a rocker at 4°C. After primary antibody incubation, membranes were washed three times in 1x PBS-0.1% Tween20 for 8 min each. Species-specific HRP conjugated secondary monoclonal antibodies were diluted in 5% milk in 1x PBS-0.1% Tween20 at a concentration of 1:5000 and incubated for 1 hour at room temperature. After secondary antibody incubation, membranes were washed three times in 1x PBS-0.1% Tween20 for 8 min each. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) was used for visualization. Images were acquired using Chemidoc XRS+ imaging system and Image Lab Software. Primary antibodies used are as follows: anti-Phospho-ErbB2 Cell Signaling Technology #2243, anti-ErbB2 Cell Signaling Technology #4290, anti-Carbonic anhydrase IX Novus Biologicals NB100-417, Hypoxia Pathway Antibody Panel GeneTex GTX3000061, and anti-βActin Sigma-Aldrich A1978.

BRDU
Irradiated Swiss 3T3 fibroblasts were plated in 4 well chamber slides at 50,000 cells in 0.5 mL media. CRC lines were plated on irradiated fibroblasts 3-5 hours later at 10,000 cells per well in F media. 24 hours following CRC culture splitting procedure, regular F media (control samples) or Dl3.6b (100µg/mL) supplemented F media (experimental samples) was added. Following incubation at 37°C for 72 hours, fresh F media with and without Dl3.6b was added. Samples were incubated for another 72 hours. Samples were washed 3 times with PBS before each of the following steps: incubation at 37°C for 1 hour in BRDU labeling medium (1:1000), incubation at 4°C for 20 minutes in ethanol fixative, incubation at 37°C in mouse anti-BRDU antibody solution, incubation at 37°C in anti-mouse fluorescein conjugated solution, mounting with solution containing DAPI. Samples were cured in the dark for 24 hours before imaging. Images were collected with Olympus IX70 microscope with Olympus DP72 camera using 20x objective lens. CellSens Entry software was used for capturing images. Swiss 3T3 fibroblasts were excluded from counts visually.

CoCl 2
Irradiated Swiss 3T3 fibroblasts were plated in 60 mm plates at 500,000 cells in 2 ml media. CRC lines were plated on irradiated fibroblasts 3-5 hours later at 50000 cells per well in CRC culture media. Cells were grown to 50% confluence, and then CoCl 2 was added to a final concentration of 200µM. Samples were collected following three hours incubation. Irradiated fibroblasts were trypsinized off, culture plates were washed with buffer, and cell pellets were collected with cell scrapers and lysed with RIPA buffer.

Supplemental Figures
Supplemental Figure 1. Heterogeneity of Trop2 expression is in HNSCC and PDX models.