A disulfide-linked homodimer of histidine-tagged extracellular domain of HER2 fused to human IgG1 Fc domain (HER2 ECD), a similar construct based on EGFR extracellular domain (EGFR ECD), and IgG1 isotype control antibody were from R&D Systems (MN, USA). Alkaline phosphatase (AP)- and horseradish peroxidase (HRP)-conjugated secondary antibodies to mouse and human IgGs, propidium iodide, trichloroacetic acid (TCA), Sulforhodamine B (SRB), Incomplete Freund’s Adjuvant, anti-calnexin antibody (#C4731), p-nitrophenyl phosphate, disodium salt (PNPP), 4', 6-diamidino-2-phenylindole (DAPI), insulin and Bromodeoxyuridine (BrdU) were from Sigma Aldrich (MO, USA). Anti-HER2 CB11antibody (#ab8054) was from Abcam (MA, USA). Anti-BrdU antibody (#M0744) was from Dako (Glostrup, Denmark). Anti-Akt (#9272), anti-phospho-Akt (#9271), anti-Erk 1/2 (#9107), anti-phospho-Erk 1/2 (#9106) antibodies were from Cell Signaling (MA, USA). Anti-phospho-HER2 (#sc-1011694) and anti-Cyclin D1 (#sc-246) antibodies were from Santa Cruz (California, USA). Alexa Fluor 488-conjugated secondary antibodies to mouse and human IgGs and Lipofectamine 2000 were from Invitrogen (CA, USA). HiTrap Protein G HP columns and ECL plus western blot detection kit were from GE-Healthcare (WI, USA). Recombinant human TNF-α was from PeproTech (NJ, USA). RNAse A and all restriction enzymes were from Fermentas (Vilnius, Lithuania). Trastuzumab (Herceptin), EDTA-free protease inhibitor cocktail tablets and phosSTOP (phosphatase inhibitor cocktail) tablets were from Roche Ltd (Basel, Switzerland). Antibody isotyping kit was from Thermo Fisher Scientific Inc (IL, USA). Protein G coupled Sepharose gel slurry was from East Coast Biologics (ME, USA).
SK-BR-3, MDA-MB-361, T47D, MCF-7 and BT-474 cell lines were obtained from ATCC. Huh-7 cell line is used in lab since 1995 and was last tested for authenticity in 2010 (originally from Jack Wands Lab at Massachusetts General Hospital, Boston, MA). All breast cancer cell lines have been tested and authenticated by short tandem repeat profiling in September 2009 and February 2012, as described previously
]. SK-BR-3 cells were grown in McCoy’s 5A medium. MDA-MB-361, T47D, MCF-7, BT-474 and Huh-7 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM), as described
]. Hybridomas were grown in RPMI 1640, DMEM or Serum Free Media (SFM) from Hyclone (IL, USA). Cell culture media were purchased from GIBCO (CA, USA), Sigma or Hyclone, and were supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (P/S), 1% non-essential amino acids and 1% L-glutamine, unless otherwise stated. BT-474 cell growth medium was supplemented additionally with 10 μg/ml insulin.
Fluorescent in situ hybridization analysis of ERBB2 copy numbers
MCF-7, T47D, MDA-MB-361, SK-BR-3 and BT-474 cells were harvested at confluence and fixed in Carnoy’s fixative (3:1 methanol/glacial acetic acid). A dual-color FISH was performed with Texas Red-labeled ERBB2
and FITC-labeled chromosome 17 α-satellite (D17Z1) DNA probes mix (LPS 001; Cytocell Ltd, Cambridge, UK), as described by supplier with minor modifications. The slides were treated at 72°C for 2 min in denaturation solution containing 70% formamide, 2XSSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0). The probe mixture was applied onto slides; the area was covered with a 22X22 mm glass coverslip, sealed with rubber cement. Slides were placed on a hot plate and heated at 75°C for 5 min. The hybridization was done overnight at 37°C in humidified chambers. Post-hybridization washes were done once with 0.4XSSC at 72°C for 2 min and once with 2XSSC containing 0.05% Tween 20, at room temperature for 30 s. After post-hybridization washes, the slides were dehydrated in a series of 70%, 85%, and 97% ethanol. Air-dried slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) in an anti-fade solution. Copy numbers in >100 nuclei of each cell line were counted under a Nikon Eclipse E600 epifluorescence microscope (Nikon Corp., Tokyo, Japan) equipped with filter sets appropriate for DAPI, FITC, and rhodamine. Individual single-color images of DAPI, FITC, and rhodamine were acquired through a high-sensitivity monochrome charge-coupled device (CCD) camera integrated to a Power Macintosh. Digitally acquired individual images were overlaid and operated with MacProbe image analysis software (PCI Scientific Systems League City, TX, USA). ERBB2
copy numbers and ploidy level based on average counts of centromere 17 in interphase and metaphase FISH were evaluated according to international standard cytogenetic nomenclature
Generation of anti-HER2 monoclonal antibodies
Animal experiments described here have been pre-approved by the Bilkent University Animal Experiments Ethical Review Panel, and conducted at Bilkent Animal House, which is certified by the Ministry of Agriculture of Turkey. All procedures complied with the guidelines of the Ministry of Agriculture (Official Gazette. No. 25464). BALB/c mice were immunized five times with live SK-BR-3 cells (5–10 × 106
cells in PBS) by intra-peritoneal injection at three-week intervals. As a final booster, each mouse received 20 μg recombinant HER2 ECD protein injected with incomplete Freund’s adjuvant. On the third day of last immunization, mice were sacrificed and their splenocytes were fused with SP2/O myeloma cells to produce hybridomas as previously described
HER2 ECD ELISA assay
96-well Polysorb (NUNC, Roskilde, Denmark) microtiter plates were coated overnight at 4°C with HER2-ECD (25–50 ng/well) in carbonate buffer. Following saturation with 1% BSA for one hour, plates were incubated with hybridoma supernatants or purified primary antibodies for 2–4 h, and bound antibodies were detected by further incubation with AP-conjugated anti-mouse IgG antibodies. Trastuzumab binding was tested using AP-conjugated anti-human IgG antibodies. Phosphatase activity was measured using PNPP substrate with absorbance (OD) reading at 405 nm using μQuant ELISA reader (BioTek, VT USA). The cross-reactivity tests with EGFR ECD were performed under the same conditions, except that ELISA plates were coated with EGFR ECD.
Antibody isotyping and affinity purification
The isotypes of monoclonal antibodies were determined using a commercial kit. Selected hybridoma clones were expanded and the antibodies were purified from the culture supernatants by FPLC using HiTrap Protein G HP columns (GE Healthcare, WI, USA).
Cells were seeded on coverslips in 6-well plates. After 36 h of cell culture, they were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. Following saturation with 10% FCS, the fixed cells were incubated with anti-HER2 antibodies for 90 min. Primary antibody binding was detected using Alexa Fluor 488 conjugated goat anti-mouse IgG or Alexa Fluor 488 conjugated goat anti-human IgG antibodies, depending on the type of the primary antibody. Nuclei were counterstained with DAPI and cells were observed using Zeiss Axio Imager.A1 microscope (Jena, Germany).
Cells (3-5×105) were fixed with 2% paraformaldehyde and incubated 1 h with 5 μg/ml anti-HER2 antibodies or IgG1 isotype control antibody (in 1% BSA and 0.5 mg/ml NaN3 in PBS). Cells were then incubated 1 h with Alexa-488-conjugated anti-mouse IgG antibody and the percentage of positively stained cells were counted on FACSCalibur (BD Biosciences).
For detection of endogenous HER2 protein, cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris–HCl pH; 7.4, 1 mM EDTA, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS, 1X Protease Inhibitors)
], 2 mg protein were diluted with milder NP40 buffer (150 mM NaCl, 50 mM Tris–HCl pH: 8, 1% NP40, 1X Protease Inhibitors) and incubated with 20 μg antibody for 2 h on a rotator at 4°C. Then, 200 μl Protein G-coupled Sepharose gel slurry was added, and the mixture was incubated 4 h at 4°C.
Following immunoprecipitation experiments, antigen-antibody bound Protein G beads were boiled in SDS-loading buffer; solubilized proteins were run on 8% or 4-12% gradient SDS-PAGE. Proteins subjected to SDS-PAGE were transferred to PVDF membrane using XCell II Blot Module (Invitrogen, CA, USA). Unoccupied protein binding sites on the PVDF membrane were saturated by incubating with 5% dried milk (in TBS-T) for 1 h. Full length and truncated forms of HER2 protein were detected using CB11 antibody directed against intracellular domain. Briefly, membranes were incubated overnight with CB11 antibody (1/500 dilution), followed by 1 h incubation with HRP-conjugated secondary antibodies to mouse IgGs and bound antibody was detected using ECL plus western blot detection kit.
The experiment was based on testing of antibody binding to HER2 proteins that have been modified by deletion of different subdomains of its ECD. Mammalian expression plasmids were constructed and transiently transfected into Huh-7 cells. Antibody binding was tested by combined immunoprecipitation-western blot and indirect immunofluorescence assays. A pDEST26-derived plasmid vector expressing full-length human HER2 protein (OCABo5050G0219D; shortly called p219D here) was obtained from Source BioScience LifeSciences (Nottingham, UK). HER2 extracellular domain was extracted from p219D by Sac I/Eco RI digestion leaving the coding sequence for the N-terminal 22 amino acid signal peptide intact. pCC2001 was produced by inserting Not I and Xho I sites between this sequence and the sequence encoding the rest of HER2 protein starting from the transmembrane domain. DNA sequences allowing the inclusion of domains I, II, III and IV of ECD under different combinations were amplified by PCR and inserted into pCC2001 plasmid using the Not I and Xho I sites. The integrity of cloned sequences has been verified by DNA sequencing. Huh-7 cells (6×105) were transfected in 6-well plates with different HER2 plasmid constructs (5 μg each) using Lipofectamine 2000, following a protocol provided by the manufacturer. Following 8-h Lipofectamine incubation, cells were washed with PBS and incubated overnight with fresh complete media (without P/S). Next day, the cells were trypsinized and replated. On the second day of cell-expansion, the cells were collected, lysed in RIPA buffer and subjected to immunoprecipitation and western blot analyses as described above, except that 150 μg proteins were immunoprecipitated using 15 μg antibody.
Cell growth assay
This assay was performed as described previously
]. Briefly, cells (103
) were seeded in 96-well plates in triplicate and allowed to attach by 2 h of preincubation. Antibodies were diluted in cell culture medium and added to preincubated cells to obtain 0–20 μg/ml concentrations under a final volume of 200 μl medium/well. Cells were then incubated up to 6 days. TNF-α treatment was performed similarly except that cells were preincubated for 6 h. For antibody and TNF-α co-treatment experiments and checkerboard assays, cells were preincubated for 2 h for attachment, followed by 4-h incubation with antibodies prior to TNF-α addition. At the end of each time point, culture supernatants were discarded. Attached cells were washed twice with PBS, fixed with ice-cold 10% TCA for one hour, followed by Sulforhodamine B-based reading, as described
Drug interaction modeling
Drug interaction scores for pairwise combinations of TNF-α with individual antibodies at fixed concentrations were calculated as follows. Growth level in a drug condition was defined as the growth measurement in the drug condition normalized by growth measurement under no drug. To understand the interaction between an antibody and TNF-α at certain concentrations, growth levels under single antibody, TNF-α, antibody + TNF-α were compared. Expected growth level under no drug interaction was defined for each pairwise drug combination as the multiplication of the growth levels under each individual drug, following the Bliss Independence Model for drug interactions
]. The observed growth level for each combination was divided by the expected growth level to find an interaction score. According to this analysis, a score of 1 means no interaction, or independence, a score smaller than 1 means synergy, and a score larger than 1 means antagonism. For the drug interaction experiments where more than one concentration combination was experimentally tested, a single interaction score between two drugs was computed. For this, a modified version of the above algorithm and the data from all concentration combinations was used, as described previously
]. The scores and the scale of these scores are equivalent to the single combination drug interaction experiment and are interpreted in the same way (<1 = synergy; >1 = antagonism). Each interaction experiment was done in triplicate, and the average growth levels were used for interaction score calculation.
Bromodeoxyuridine incorporation assay
) were plated on coverslips in 12-well plates, and treated as described for cell growth assays. At 48th
hour, BrdU (10 μg/ml) was added into cell culture media and cells were further incubated for 24 h. The detection of BrdU incorporation was performed as described previously
], except that Alexa Fluor 488-conjugated anti-mouse IgG antibody was used for visualization. Cell nuclei were counterstained with DAPI, examined using Zeiss Axio Imager.A1 microscope and representative photographs (3–5 frames per coverslip) were acquired. Percent BrdU incorporation was calculated after manual counting of BrdU-positive (BrdU+
) and BrdU-negative (BrdU-
Cell cycle analysis
Analyses were performed as described previously
]. Briefly, 2.5×104
(for 6-day samples) or 105
(for 1 and 3-day samples) SK-BR-3 cells were seeded in 6-well dishes in McCoy’s 5A medium. After 36 h incubation, the medium was replaced with fresh McCoy’s 5A medium including BH1 or IgG1 isotype control antibody (final 5 μg/ml)
1000Unit/ml TNF-α. On days 1, 3 and 6 after the addition of antibodies, both suspended and adherent cells were collected, fixed and stained with propidium iodide (PI). PI-stained cells were measured and analysed by flow cytometry.
Analysis of the downstream protein expression
In order to analyze the effect of BH1 treatment on the downstream pathway, SK-BR-3 cells were treated for 1–24 h with 10 μg/ml BH1, 1000 Unit/ml TNF-α alone or their combination. Cells collected at each time point (1st, 2nd, 4th, 8th, 12th, 24th hour) were lysed with RIPA buffer (containing 1X PhosSTOP). Solubilized proteins were resolved by SDS-PAGE and analyzed by immunoblotting. PVDF membranes initially used for detection of phospho-Akt, phospho-Erk 1/2 and phospho-HER2 proteins were stripped once (62.5 mM Tris-Cl pH: 6.7, 2% SDS, 100 mM β-mercaptoethanol), washed thoroughly with TBS-T and re-blotted with anti-Akt, anti-Erk 1/2, anti-cyclin D1 and anti-calnexin antibodies. Following incubation with HRP-conjugated secondary antibodies to mouse or rabbit IgGs, bound antibodies were detected using ECL plus western blot detection kit.
Experimental data were presented as mean ± SD. Statistical analysis of experiments that have been performed at least in triplicate was done using Student’s t test.