Cell Culture
All cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). BT474 cells were maintained in improved minimal essential medium (IMEM, Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 μg/mL of insulin. SKBR3 cells were cultured in McCoy’s 5A medium (ATCC, Manassas, VA) supplemented with 10% FBS and 1% penicillin/streptomycin. MDA-MB-231 cells were maintained in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 5% FBS and 200 mg/mL of G418. All cells were grown at 37 °C with 5% CO2. Cells were cultured to 70-80% confluency and cell counts were determined with the Countess II FL automated cell counter (Thermo Fisher Scientific Inc., Waltham, MA).
Western Blot Evaluation of HER2 Expression and Quantification
BT474, SKBR3, and MDA-MB-231 breast cancer cells were washed with ice cold phosphate buffered saline and lysed for 10 minutes on ice with radioimmunoprecipitation assay buffer supplemented with protease inhibitor (Roche Applied Science, Indianapolis, IN). Lysates were centrifuged at 13,400 g for 20 minutes at 4° C and collected for bicinchoninic acid protein quantification assay via the Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA). For each cell line, 20 μg of protein was prepared in a solution of sodium dodecyl sulfate and β-mercaptoethanol and boiled for 5 minutes at 95° C. Samples were run on a NuPAGE Bis-Tris gel and transferred to a polyvinylidene fluoride membrane. The membrane was blocked in 5% dry milk in tris-buffered saline (TBST), probed with horseradish peroxidase (HRP) conjugated mouse anti-human β-actin overnight at 4° C and developed with the Amersham ECL western blot detection system (GE healthcare, Buckinghamshire, UK) for one minute at room temperature. Membranes were developed in the absence of light and visualized with an SRX-101A Medical Film Processor (Konica Minolta Medical and Garphic, Inc., Shanghai, China). The membrane was incubated in stripping buffer for 15 minutes, washed with TBST and probed with Rabbit anti-human HER2/ErbB2 primary antibody (Cell Signaling Technology, Danvers, MA) for 2 hours at room temperature. The membrane was washed, incubated with HRP conjugated goat anti-rabbit IgG secondary antibody (Cell Signaling Technology, Danvers, MA) for 1 hour at room temperature. After a final wash, the membrane was redeveloped and visualized for HER2 expression. Quantification of bands was conducted with the Image Studio Lite program, version 5.0 (LI-COR Biosciences, Lincoln, NE, USA). HER2 expression was normalized to β-actin expression for quantification.
Transfection of Breast Cancer Cell Lines
BT474, SKBR3, and MDA-MB-231 cells were transfected to express enhanced green fluorescent protein (EGFP) through the Sleeping Beauty Transposon System. An EGFP plasmid was obtained and cloned into a Sleeping Beauty compatible vector, psDBbi-Neo (Addgene, Cambridge, MA, plasmid #60525). Lipofectamine LTX (Thermo Fisher Scientific, Waltham, MA) was used to co-transfect the psDBbi-Neo vector and the pCMV (CAT) T7-SB100 (Addgene, Cambridge, MA, plasmid #34879) Sleeping Beauty transposase. Following transfection, cells were cultured in their respective media supplemented with 200 mg/mL G418 for four weeks to select for positively transfected cells. Fluorescence activated cell sorting was used to separate cells, and the 25% highest intensity EGFP expressing cells were used in the experiments. The pCMV (CAT)T7-SB100 plasmid was a gift from Dr. Zsuzsanna Izsvak [22] and the pSBbi-Neo was a gift from Dr. Eric Kowarz [23].
In Vitro Treatment Experiments
Experiment 1: Order of dosing
BT474-GFP cells were plated in 96-well plates at 5,000 cells/well. 24 hours later (Day 0), plates were placed in the IncuCyte S3 Live-Cell Analysis System (Essen BioScience, Ann Arbor, MI) and whole-well imaged every six hours with phase contrast and green channels (excitation 440-480, emission 504-544) for one week. Single agent trastuzumab treatment groups were treated with trastuzumab (0.1 ng/ml) from 0 to 48 hours (group 1), 24-72 hours (group 2), or 48-96 hours (group 3). For single agent radiation treatment groups, cells were treated with radiation (10 Gy) at 24 hours with the CellRad Dedicated Benchtop Cell Irradiator (Faxitron, Tucson, AZ). For combination treatment groups, cells received trastuzumab (0.1 ng/ml) before, with, or after radiation treatment (10 Gy) as detailed in Fig. 1a. Each treatment group has 24 replicates per experiment. The experiment was performed three times.
Experiment 2: Quantifying radiation + trastuzumab combination effects
BT474-GFP, SKBR3-GFP, or MDA-MB-231-GFP cells were plated in 96-well plates at 5,000 cells/well. 24 hours later (Day 0), cells were treated with either 0.1% saline, trastuzumab (0.1 ng/ml), or radiation (5 or 10 Gy) for control and single agent treatment groups. Combination groups received both trastuzumab (0.1 ng/ml) and radiation (5 or 10 Gy) on Day 0 (Fig. 1b). Plates were placed in the IncuCyte S3 Live-Cell Analysis System (Essen BioScience, Ann Arbor, MI) and whole-well imaged (4×, 2.82 um/pixel) every six hours with phase contrast and green channels (excitation 440-480, emission 504-544) for one week. Each treatment group has 30 replicates per experiment. The experiments were performed three times.
Image Analysis
Images were analyzed using the IncuCyte S3 Live-Cell Analysis System (Essen BioScience, Ann Arbor, MI) to segment discrete green fluorescent objects (cell counts). Background signal was estimated in 100 μm segments and subtracted from the image. Edges of clumped cells were determined to be the dimmest point between two objects. Cell growth was normalized to initial seeding density (0 hours).
Interaction Index Calculation
The interaction index [24] based on Bliss independence was used to determine effects of combination radiation and trastuzumab treatment in vitro. To calculate the effects of radiation (A), trastuzumab (B), and combination treatment (AB) compared to the control, let (f) represent the difference in normalized cell growth between control (NC) and treated (Ni) conditions:
Animal Procedures
Female nude athymic mice (NU/J) (N = 47) were purchased from The Jackson Laboratory (Bar Harbor, ME) at 3-4 weeks of age at an average weight of 12-15 g, and acclimated for one week in housing with a normal light cycle, sterile water and food, and microisolation cages. Mice were subcutaneously implanted with a 0.72 mg, 60-day release, 17β-estradiol pellet (Innovative Research of America, Sarasota, FL) in the nape of the neck. One day later, 107 BT474 cells were implanted subcutaneously in 100 μl of serum-free IMEM media with 30% growth factor-reduced Matrigel. Tumors were grown to approximately 250 mm3 (8-10 weeks) and entered into the study. Animals were randomly sorted into treatment groups: group 1) Control: 100 μl saline on Days 0, 3 (N = 6), group 2) Trastuzumab alone: 10 mg/kg on Days 0, 3 (N = 7), group 3) Radiation 5 Gy alone: Day 0 (N = 6) + 100 μl saline on Day 3, group 4) Radiation 10 Gy alone: Day 0 (N = 5) + 100 μl saline on Day 3, group 5) Radiation 5 Gy on Day 0 + Trastuzumab 10 mg/kg on Days 0, 3 (N = 5), group 6) Radiation 10 Gy on Day 0 + Trastuzumab 10 mg/kg on Days 0, 3 (N = 5). Trastuzumab and saline were administered via intraperitoneal (IP) injection. Radiation treatments (~4 Gy/min, 225 kV, 17.8 mA) were given with the MultiRad 350 Irradiation System (Faxitron, Tucson, AZ). Tumor measurements were taken three times per week using calipers for four weeks at which point mice were euthanized. Mice in the immunohistochemistry cohort were divided into three treatment groups: 1) Radiation 5 Gy on Day 0 (N = 4) + 100 μl saline on Day 3, 2) Trastuzumab 10 mg/kg on Days 0, 3 (N = 4), 3) Radiation 5 Gy on Day 0 + Trastuzumab 10 mg/kg on Days 0, 3 (N = 5). Tumor size was measured with calipers by measuring the longest axis of the tumor and the axis perpendicular to the longest axis. The minimum of these two measurements was assumed to be the height of the tumor to calculate the volume. Tumor size was measured three times for one week at which point mice were euthanized and tumors were extracted. One hour prior to sacrifice, mice were intravenously injected with 60 mg/kg of pimonidazole (Hypoxyprobe, Inc., Burlington, MA) in 100 μl of saline. Tumors were cut at the longest cross-section and half was fixed in 10% neutral buffered formalin (Fisher Scientific International Inc., Pittsburgh, PA) for 48 hours where it was then placed in 70% ethanol.
Immunohistochemistry
Formalin fixed tumor sections were embedded in paraffin and sliced into 4 μM sections. Sections were stained for hematoxylin and eosin (H&E), mouse anti-CD31, mouse anti-α-smooth muscle actin (α-SMA, Abcam, Cambridge, UK), mouse anti-CD45 (Invitrogen, Carlsbad, CA) and anti-pimonidazole (Hypoxyprobe, Inc., Burlington, MA). Immuno-stained slides were scanned (20×, 0.495 μm/pixel) with the Aperio ScanScope (Leica Microsystems, Wetzlar, Germany). Regions of necrosis were determined using manual segmentation. All other stains were quantitatively segmented based on color thresholds determined from positive and negative controls using in house MATLAB (MathWorks Inc., Natick, MA) routines. Images were converted to grayscale and registered to the corresponding tumor H&E via an intensity-based registration algorithm. A viable tissue mask was defined as total tumor area minus necrotic area. Inflammation (CD45), hypoxia (pimonidazole), and apoptosis (caspase3) were defined as the percent of positive stain per viable tissue area. Microvessel density (CD31) and vascular smooth muscle coverage (α-SMA) were calculated as the number of vessels per mm2 of tumor tissue. The vessel maturation index [40] was evaluated as the ratio of α-SMA coverage to microvessel density.
Statistical Analysis
Statistical analysis was conducted using MATLAB. A two-way analysis of variance (ANOVA), adjusting for multiple comparisons with Dunn’s, was used to determine longitudinal differences in order of dosing experiments. All in vitro data is presented as mean ± 95% confidence interval with P < 0.05 indicating significance. Statistical differences between in vivo tumor size at each time point as well as differences in ex vivo immunohistochemistry samples were determined using a nonparametric Wilcoxon rank sum test. All in vivo and ex vivo data is presented as mean ± standard error with P < 0.05 indicating significance. The Friedman’s test adjusting for multiple comparisons with Dunn’s test was used to assess longitudinal changes in tumor growth between treatment groups.