Cell lines, Cell Culture, and Reagents
Malignant human breast cancer cell lines (MDA-MB-231, SKBR3, MDA-MB-453, T47D, and ZR-75-1), a nonmalignant human breast epithelial cell line (MCF-10A), and human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (Rockville, MD). The MDA-MB-435EB1 human breast cancer cell line, which was established by transfecting c-erbB-2 cDNA into MDA-MB-435 and which overexpresses v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2), was a gift from Dr Michael Rosenblum at The University of Texas MD Anderson Cancer Center (Houston, TX). The IDC51N, IDC51T, and IDCm73T cells were cultured from breast cancer patient tissues. The breast cancer cells and HEK293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with penicillin (100 U/mL) (Invitrogen), streptomycin (100 μg/mL) (Invitrogen), and fetal bovine serum (FBS; 10%) (Atlanta Biologicals, Lawrenceville, GA), which is the complete DMEM. The v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS)–expressing, weakly tumorigenic MCF-10AT cells were purchased from The Barbara Ann Karmanos Cancer Institute (Detroit, MI). MCF-10A and MCF-10AT cells were maintained in DMEM: Nutrient Mixture F-12 (DMEM/F-12) (Invitrogen) supplemented with penicillin (100 U/mL) (Invitrogen), streptomycin (100 μg/mL) (Invitrogen), horse serum (5%) (Invitrogen), insulin (10 μg/mL) (Sigma-Aldrich, St Louis, MO), epidermal growth factor (20 ng/mL) (Invitrogen), and hydrocortisone (0.5 μg/mL) (Sigma-Aldrich). MCF-7, a malignant human breast cancer cell line, was a gift from Dr Melinda Hollingshead (Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis [DCTD], National Cancer Institute, Bethesda, MD). These cells were maintained in DMEM media supplemented with penicillin (100 U/mL) (Invitrogen), streptomycin (100 μg/mL) (Invitrogen), and FBS (10%) (Atlanta Biologicals). The identity of cell lines was confirmed by short tandem repeat genetic profiling.
Primary tumor and uninvolved normal human breast cells were isolated from surgery specimens obtained from breast cancer patients at The University of Texas MD Anderson Cancer Center according to an approved Institutional Review Board (IRB) protocol (LAB04-0083). Breast cancer cells (T) and matched uninvolved normal epithelial cells (N) were successfully cultured from two patients, Acc 51 and Acc 71, and used for this study. Patient Acc 51 was a 47-year-old woman of white race with invasive mammary carcinoma (nuclear grade 2, estrogen receptor positive [ER+
], progesterone receptor positive [PR+
], without metastasis), and patient Acc 73 was a 70-year-old woman of white race with invasive ductal carcinoma (IDC) (nuclear grade 2, ER+
, HER2 negative with lymph node metastasis). Paraffin-embedded tumor specimens (n = 223) were obtained from breast cancer patients who resided in the greater Baltimore area, as described previously (20
). Patients were recruited at the University of Maryland Medical Center (UMD), the Baltimore Veterans Affairs Medical Center, Union Memorial Hospital, Mercy Medical Center, and the Sinai Hospital in Baltimore between February 15, 1993, and August 27, 2003. All patients signed a consent form. Clinical and pathological information was obtained from medical records and pathology reports. Disease staging was performed according to the TNM staging system of the American Joint Committee on Cancer (AJCC) (21
) and the Union for International Cancer Control (UICC) (22
). Low stage was defined as stage I or II, and high stage was defined as stage III or IV. The Nottingham grading system (23
) was used to determine the tumor grade using the cumulative score of glandular differentiation, nuclear pleomorphism, and mitotic count. Low grade was defined as grade 1 or 2, and high grade was defined as grade 3.The collection of the tumor specimens and clinical and pathological information was reviewed and approved by the University of Maryland IRB for the participating institutions (UMD protocol number 0298229). IRB approval was then obtained at all institutions (Veterans Affairs Medical Center, Union Memorial Hospital, Mercy Medical Center, and Sinai Hospital). The research was also reviewed and approved by the Office of Human Subjects Research (OHSR) at the National Institutes of Health (OHSR number 2248).
Production and Affinity Purification of Antigens and Antibodies
HERV-K SU gene was cloned into a pGEX-6P1-glutathione S-transferase (GST) expression vector (GE Healthcare Life Sciences, Piscataway, NJ) to obtain HERV-K env SU-GST recombinant fusion protein (K-GST). Protein expression was induced with isopropylthiogalactoside (IPTG) in Escherichia coli (E coli)
BL-21 (DE3), and fusion proteins were affinity purified using an ÄKTA Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare Life Sciences) equipped with a GSTrap column as described previously (18
). HERV-K SU was also cloned into a 6xHis-tagged pQE30 expression vector (Qiagen, Chatsworth, CA). The resultant construct (K-Q18), a recombinant fusion protein consisting of HERV-K env SU tagged with 6xHis, was expressed by induction with IPTG in M15 E coli
and affinity purified using the ÄKTA system. Purified K-Q18 fusion protein was used to immunize 6- to 8-week-old female BALB/c mice for production of hybridoma using standard technology. Previously, we developed mAbs against HERV-K, member 7 (18
). Mouse anti-HERV-K mAbs 6H5, 4D1, 4E11, 6E11, and 4E6 were selected for specific binding to K-GST antigen by enzyme-linked immunosorbent assay (ELISA) and immunoblot. These mAbs were produced from their respective hybridomas by the ascites method in female BALB/c mice and purified using Protein G HP resin (GE Healthcare Life Sciences) as described previously (18
). Briefly, hybridoma cells were produced by immunizing 6- to 8-week-old female BALB/c mice with K-Q18 fusion protein. The splenocytes of the immunized mice were fused with a myeloma cell line to derive hybridoma cells. Hybridoma cells that generated anti-HERV-K antibodies were selected by ELISA using K-GST protein. Sample endotoxin content was determined by limulus amebocyte lysate (LAL) assay (Lonza, Basel, Switzerland). Any endotoxin was removed from antibody preparations by affinity purification using Detoxi-Gel resin columns (Pierce, Rockford, IL) as per manufacturer's instructions.
A single-chain variable fragment (scFv) antibody was generated from 6H5 mAb as described previously (24
) using the Recombinant Phage Antibody System (GE Healthcare Life Sciences) to investigate possible advantages of using this antibody compared with HERV-K mAbs with respect to tumor uptake and immunogenicity. Briefly, the scFv clone G11D10 was selected using a colony lift assay followed by ELISA using K-GST protein as antigen. G11D10 scFv antibody was induced by IPTG in E. coli
HB2151, and the periplasmic extract was clarified using a 0.45-μm cellulose acetate filter (Corning Life Sciences, Corning, NY) and purified by ÄKTA FPLC using a HiTrap anti-Etag column (GE Healthcare Life Sciences). The purity of the antibodies and fusion proteins was evaluated by Coomassie blue staining or immunoblot.
Total protein lysates of cells (MCF-10A, MCF-10AT, SKBR3, T47D, MDA-MB-231, and MCF-7) were used for immunoblot analysis (50 μg protein per lane) as described previously (18
). The following primary antibodies were used: 6H5 mAb (1 μg/mL final concentration) and mouse anti-chicken β-actin (ACTB) mAb (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) (1:1000 dilution), followed by treatment with anti-mouse immunoglobulin (mIgG) HRP (1:5000 dilution) (Sigma-Aldrich). Rabbit anti-human caspase 3, mouse anti-human caspase 8, and mouse anti-human caspase 9 antibodies (Sigma-Aldrich; 1:1000 dilution) were used to detect cleavage of caspase proteins by immunoblot. Rabbit anti-human polyclonal antibody to active caspase 7 (Abcam, Cambridge, MA; 1 μg/mL final concentration) was used for detection of active caspase 7. Mouse anti-human cell death–inducing DFFA-like effector A (CIDEA) antibody (Abnova, Taipei, Taiwan; 1:1000 dilution) was used for detection of CIDEA expression in cells treated with 6H5 mAb or mIgG. Other primary antibodies used for immunoblot were mouse anti-human tumor protein p53 (TP53, 1 μg/mL final concentration), rabbit anti-human tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain (TNFRSF10D, 0.5 μg/mL final concentration), mouse anti-human cyclin-dependent kinase inhibitor 1A (CDKN1A, 1:500 dilution), mouse anti-human cyclin-dependent kinase 5 (CDK5) (all from Sigma-Aldrich); mouse anti-human myogenic differentiation 1 (MYOD1, 1 μg/mL final concentration), mouse anti-human glycosylphosphatidylinositol anchored molecule like protein (GML, 1 μg/mL final concentration), rabbit anti-human TP53-regulated apoptosis–inducing protein-1 (TP53AIP1, 1 μg/mL final concentration) (all from Abcam); mouse anti-human FAS ligand (FASLG; TNF superfamily, member 6, 1:200 dilution), goat anti-human tumor necrosis factor receptor superfamily, member 8 (TNFSF8, 1:200 dilution) (both FASLG and TNFSF8 antibodies from Santa Cruz Biotechnology, Santa Cruz, CA), and antitumor necrosis factor receptor superfamily, member 25 (TNFRSF25, also known as DR3) (rabbit anti-human DR3, 1:1000 dilution) (Sigma-Aldrich). Samples treated with 1000 U of peptide-N-glycosidase F (PNGase F; New England Biolabs, Ipswich, MA) to remove all N-linked carbohydrates for glycoprotein analysis were incubated at 37°C overnight in reaction buffer provided by the manufacturer. These PNGase F-treated samples were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed using a glycoprotein detection kit (GLYCOPRO; Sigma) and an immunoblot assay with 6H5 mAb (1 μg/mL final concentration). All immunoblots were performed using 0.2 μm polyvinylidene fluoride (PVDF) membranes (Immun-blot; Bio-Rad, Hercules, CA) and blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (20 mM Tris [pH 7.5], 150 mM NaCl) with 0.2% Tween-20.This experiment was repeated at least two independent times.
Metabolic Turnover Assay
We examined the metabolic turnover of the target antigen HERV-K env protein in the absence of 6H5 mAb using a biotinylation pulse-chase assay described previously (25
). MDA-MB-231 and MCF-7 cell surface proteins were pulsed with biotin using a Sulfo-NHS-SS-biotin reagent (Pierce), as per manufacturer’s instructions, and incubated at 37°C for 0, 5, 15, 45, 90, and 180 minutes. At each time point the cells were lysed, biotinylated proteins were pulled down with streptavidin resin, and the pull-down proteins were analyzed by immunoblot for detection of HERV-K env expression (surface and full-length envelope protein) using 6H5 mAb (21
). This experiment was repeated at least three independent times.
Enzyme-Linked Immunosorbent Assay
Sensitivity and specificity of antibody–antigen interaction were analyzed by ELISA, as described previously (15
). For cell ELISA, breast cells (MDA-MB-231, MCF-7, SKBR3, MCF-10A) (2500 cells per well) were seeded in a 96-well plate and incubated at 37°C overnight. After washing the plate one time with 1× phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2
, 2 mM KH2
, pH 7.4), the liquid was removed, and the plate was incubated at 37°C overnight to dry. Cells were fixed with 3.7% formaldehyde (Sigma-Aldrich) for 8 minutes, washed 5 times with 1× PBS, and then blocked for 2 hours at room temperature with 3% BSA in 1× PBS containing 0.05% Tween-20. Serial dilutions of 6H5 mAb (1:1000, 1:3000, 1:9000, 1:27
000, and 1:2
000) or mIgG (using the same dilutions used for 6H5 mAb) were added, and the plate was incubated overnight at 4°C. After washing with 1× PBS, anti-mIgG–HRP (1:4000) was added, and the plate was incubated for 1 hour at room temperature. After washing with 1× PBS, the substrate 2,2’-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) (ABTS) (Sigma-Aldrich) was added for 10–30 minutes, and the plate was read on a Wallac Victor 2 V Microplate Reader (PerkinElmer, Waltham, MA). This experiment was repeated at least three independent times.
Immunofluorescence, Fluorescence-Activated Cell Sorting (FACS), and Antibody Conjugation Assays
For immunofluorescence assays, breast cells (MCF-10A, MCF-10AT, MCF-7, MDA-MB-231, MDA-MB-453) were cultured in six-well plates (2 × 105
cells per well) containing glass coverslips for 24 hours. Cells were fixed with 3.7% paraformaldehyde at room temperature for 8 minutes followed by washing six times with 1× PBS. Cells were incubated with 6H5 mAb (10 μg/mL) for 60 minutes followed by six washes with 1× PBS. Cells were incubated with goat anti-mIgG conjugated with AlexaFluor 488 (anti-mIgG-AF488) (Invitrogen) for 30 minutes followed by six washes with 1× PBS. Coverslips were mounted onto slides using 10% glycerol. Slides were examined with the use of a Zeiss LSM 510 confocal imaging system (Zeiss, Heidelberg, Germany). Cells were subjected to immunofluorescence staining with antibodies against caspases 3, 8, and 9, CDKN1A, and CDK5 (1 μg/100 μL per slide). To detect cytoplasmic expression of HERV-K env, cells were treated with 0.1% Triton X-100 as described previously (15
). This experiment was repeated at least three independent times.
For FACS assays, breast cells (MCF-10A, MCF-10AT, MCF-7, MDA-MB-231, SKBR3, T47D, IDC51N and IDC51T, and MDA-MB-453) were removed from culture flasks using 1× trypsin and added to a 96-well plate (1 × 106 cells per well). Cells were incubated with 6H5 mAb (1 μg/106 cells) for 1 hour at room temperature followed by six washes with 1× PBS. Cells were incubated with goat anti-mIgG-Alexa Fluor 647 (Invitrogen) (mIgG conjugated with AlexaFluor 647, 1 μg/106 cells) for 30 minutes, washed six times with 1× PBS, resuspended in 250 μL 1× PBS, and analyzed on a FACS Array (BD Biosciences, Franklin Lakes, NJ). For blocking experiments, 6H5 mAb (1 μg) was preincubated with K-GST (1 μg) for 30 minutes at room temperature before adding to cells. This experiment was repeated at least three independent times.
A quantitative indirect immunofluorescence (QIFI) assay was used for the quantitation of surface envelope levels. QIFI beads, which are microbeads coated with different known amounts of mIgG of IgG1
subclass to mimic cells coated with mAbs at saturating concentration, were stained with goat anti-mIgG-Alexa Fluor 647 (1 μg per 1 × 106
cells). The peak fluorescence position of each bead population vs the number of antibody molecules per calibration bead (1700, 11
000, and 561
000 molecules per bead) (determined by Dako, Glostrup, Denmark) was plotted, as described previously (27
). The number of surface molecules on the cells was determined with a calibration curve, so that arbitrary units of mean fluorescence intensity (MFI) can be transformed to molecules of equivalent fluorochrome values that can be compared from one assay to another.
For antibody conjugation to AlexaFluor dye, 20 μg of antibody in PBS (pH 8.0) were incubated with 1 μg of carboxylic acid succinimidyl ester–activated AlexaFluor dye (Invitrogen) at room temperature in the dark for 1 hour. The conjugate was purified from free dye using size exclusion chromatography (Bio-Rad P30 gel) in spin column format. Propidium iodide (Sigma-Aldrich) or 4’-6-diamidino-2-phenylindole (DAPI) (Invitrogen) was used for nuclear staining. To detect cytoplasmic expression of HERV-K env, cells were treated with 0.1% Triton X-100, as described previously (15
Kinetic Binding Assays
Surface plasmon resonance assays are used to evaluate kinetics of binding, primarily the rate of association (Ka) and dissociation (Kd). We used these assays to assess affinity or strength of binding of mAb or scFv antibody to its antigen by determining the affinity constant (KD) values of 6H5 mAb and G11D10 scFv antibody for the HERV-K env protein. Assays were performed on a Biacore X100 instrument at 10 μL/min flow rate. Two sensor surfaces (flow cell 1 and flow cell 2) of a Biacore CM5 sensor chip (BIACORE, Piscataway, NJ) were activated with 0.4M N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxysuccinimide (NHS). Two hundred resonance units (RU) of recombinant HERV-K SU env protein, that is, approximately 200 pg/mm2, were immobilized onto flow cell 2 in 20 mM sodium acetate (pH 4.5). One RU corresponds to 1 pg/mm2 of mass bound to the sensor surface. Flow cell 1 contained no ligand. Both flow cells were then quenched using 1 M ethanolamine. In separate experiments, a series of concentrations (3, 6, 12, 24, and 48 nM) of 6H5 mAb or G11D10 scFv antibody were prepared in HEPES-buffered saline (10 mM HEPES, 150 mM sodium chloride), with 3 mM EDTA and 0.005% Tween-20 (HBS-EP) running buffer (pH 7.4) and injected onto both the ligand (K-GST: flow cell 2) and control flow cells. Kinetic constants were determined from blank normalized curves using Biacore X100 evaluation software (BIACORE) by fitting to the 1:1 Langmuir binding model. The affinity of the antibody (6H5 mAb or G11D10 scFv antibody) for its ligand (K-GST) is determined by measuring the binding kinetics of the interaction and expressed as KD for the interaction of a single antibody combining site with a single epitope. All concentrations were injected in duplicate.
Cycling of HERV-K env Protein and Net Antibody Internalization Assays
For qualitative and quantitative analysis of cycling of HERV-K env protein between the cell surface and intracellular stores, a cycling assay was used. MCF-7, MDA-MB-231, and T47D cells were incubated in 96-well U-bottom plates with unlabeled 6H5 mAb (10 μg/mL) at 4°C for 20 minutes, washed one time with 1× PBS, and incubated with unlabeled 6H5 mAb at 37°C for different time intervals (0, 1, 5, 15, and 45 minutes) as described previously (28
). At each time point, cells in one-half of the wells were labeled with 5 μg/mL of goat anti-mIgG-Alexa Fluor 647 at 4°C for 30 minutes to detect HERV-K env protein that remained on the cell surface. The percentage of internalization at each time point was 100 minus the percentage of label at time 0. Cells in the other half of the wells were treated with citrate phosphate buffer (0.131 M citric acid, 0.066 M sodium monohydrogen phosphate, pH 3) to strip 6H5 mAb from the surface and then reincubated with 4D1 mAb conjugated to AlexaFluor 555 (Invitrogen), to determine whether 6H5 mAb influenced the cell surface binding of HERV-K env protein.
Cell Growth and Cell Proliferation Assays
The viability of cell lines (MCF-10AT, MCF-7, MDA-MB-231, SKBR3, IDCm73T, and T47D) as a function of antibody dosage was tested in the presence or absence of 6H5 mAb (10 μg/mL per dose). Untreated and mIgG (10 μg/mL)-treated cells were used as controls in all assays. Cells were seeded in a 12-well plate (2 × 104 cells per well) and incubated overnight for attachment. One group of cells was treated with 6H5 mAb (10 μg/mL per dose) or control mIgG (10 μg/mL per dose) administered at 24, 48, and 72 hours after seeding and harvested at 96 hours after seeding (designated as dose 3). In a second protocol, two doses were administered at 48 and 72 hours after seeding and harvested at 96 hours after seeding (designated as dose 2). Finally, one dose was administered at 72 hours after seeding and harvested at 96 hours after seeding (designated as dose 1). Cells in each well treated with 6H5 mAb or mIgG were counted after trypan blue staining using a hemocytometer. Triplicate fields containing at least 100 cells per field were counted, and because the volume counted was known, the counts were converted to total cell numbers per well. The counts of cells treated with 6H5 mAb or mIgG under dose 1, dose 2, or dose 3 conditions were plotted.
To evaluate the effect of antibody concentration on cell proliferation, breast cell lines were plated in a 96-well plate (3000 cells per well) and incubated overnight for attachment. Cells were treated with a serial dilutions of 6H5 mAb (2.13 × 10−12
, 1.07 × 10−11
, 5.33 × 10−11
, 2.67 × 10−10
, 1.33 × 10−9
, 6.67 × 10−9
, 3.33 × 10−8
M) or mIgG (3.33 × 10−8
final concentration) for 72 hours, and cell proliferation was determined using the CellTiter 96 AQ Non-Radioactive Cell Proliferation Assay (MTS assay; Promega, Madison, WI). The absorbance produced by chromophore in all positively stained cells in each well was measured at 490 nm using a Wallac Victor 2 V Microplate Reader (Perkin Elmer, Waltham, MA). Cytotoxicity was determined by staining with crystal violet (26
). The absorbance was measured at 600 nm using a Wallac Victor 2 V Microplate Reader.
Bromodeoxyuridine (BrdU) Incorporation and Cell Cycle Assay
Cell cycle analysis was performed using BrdU, an analog of the DNA precursor thymidine, which incorporates into newly synthesized DNA. MCF-10A, MCF-10AT, MDA-MB-453, T47D, MCF-7, MDA-MB-231, and SKBR-3 cells (2.5 × 105 per well) were plated in a six-well plate (BD Biosciences) in their respective media. Culture media containing FBS was replaced with serum-free media 24 hours before analysis. Cells were pulsed with BrdU (30 μmol/mL) (APC BrdU Flow Kit; BD Biosciences) and cultured with 10 μg/mL 6H5 mAb or mIgG at 37°C for 24, 48, and 72 hours. Cells were resuspended in cytofix/cytoperm buffer (BD Biosciences) and placed on ice for 30 minutes. After washing with 1× PBS, cells were resuspended in cytoperm plus buffer (BD Biosciences) and incubated on ice for 10 minutes. After washing with 1× PBS, cells were refixed with cytofix/cytoperm buffer on ice for 5 minutes. After washing with 1× PBS, cells were resuspended in DNase (300 μg/mL, APC BrdU Flow Kit; BD Biosciences) in Dulbecco's phosphate-buffered saline (DPBS; Sigma-Aldrich) and incubated at 37°C for 1 hour. Cells were washed, stained with anti-BrdU conjugated with allophycocyanin (APC) at room temperature for 20 minutes, and then analyzed on a BD FACSArray. For total cellular DNA staining, 7-amino-actinomycin D (7-AAD) was used. This two-color flow cytometry analysis quantitates cells that are actively synthesizing DNA (BrdU incorporation) in terms of their cell cycle position, which is defined by 7-AAD staining intensities.
Ki-67 is a nuclear antigen and proliferation marker expressed only in cycling cells. Consequently, assessment of Ki-67 staining on paraffin-embedded tumor sections is an estimate of the proliferation index of individual tumors. Immunohistochemistry (IHC) was performed on paraffin-embedded tumor sections using mouse anti-human Ki-67 mAb (BD Bioscience) and a VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA). After deparaffinization, tumor sections were covered in 1 μg of anti-Ki-67 antibody diluted in 100 μL blocking buffer (1% BSA in 1× PBS) and incubated at room temperature for 60 minutes. Ten random fields were used for counting the Ki-67-positive cells under an Olympus 1X51 microscope (Olympus, Center Valley, PA) using the 20× objective.
Annexin V staining.
Annexin V staining precedes the loss of membrane integrity that characterizes the later stages of cell death, and therefore the vital dye 7-AAD, in conjunction with annexin V, was used to allow discrimination of early apoptotic cells. To measure the effects of 6H5 mAb on cellular apoptosis in vitro, breast cells MCF-10A, MCF-10AT, MCF-7, MDA-MB-231, MDA-MB-453, and T47D (5 × 105 cells/mL) were grown in their respective media and treated with 6H5 mAb at various concentrations (0, 1.25, 2.5, 5, and 10 μg/mL) at 37°C for 16 hours, or were treated with various mAbs (4D1, 4E11, 6H5, and 6E11) at a constant concentration of 10 μg/mL, or with an mIgG control antibody. Treated cells were harvested and resuspended in 200 μL 1× binding buffer (BD Pharmingen) at 1 × 106 cells per well. Cells were stained with annexin V-APC (red; BD Pharmingen) at room temperature for 15 minutes followed by one wash with 1× annexin V binding buffer. Cells were then incubated with 7-AAD-phycoerythrin-cyanide 7 (PE-Cy7) at room temperature for 10 minutes, and samples were analyzed on a BD FACSArray Bioanalyzer. The percentage of annexin V–positive cells for each cell line treated with 6H5 mAb was determined using FlowJo software (version 7.2.5).
Deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.
The ApopTag Peroxidase In Situ Apoptosis Detection kit (Chemicon International, Temecula, CA) was used for measuring apoptosis in paraffin-embedded tumor sections by labeling and detecting DNA strand breaks by the TUNEL method. Tumor sections (5 μM) were labeled at the free 3’OH DNA termini in situ with digoxigenin-labeled and unlabeled nucleotides added to the DNA by terminal deoxynucleotidyl transferase (TdT). The labeled DNA fragments then bind an anti-digoxigenin antibody conjugated to a peroxidase reporter molecule, which generates a strong stain from chromogenic substrates. Numbers of apoptotic cells were counted in 10 randomly selected high-power fields. Each slide was examined on at least two separate occasions by two individuals.
Analysis of Apoptosis and TP53 Signaling Pathways
Breast cancer cells (MCF-7) were treated with 6H5 mAb or mIgG (10 μg/mL) at 37°C for 24 hours, and total RNA was extracted using TRI Reagent Solution (Applied Biosystems, Foster, CA) and subjected to quantitative polymerase chain reaction (qPCR) analyses using Human Apoptosis and Human p53 Signaling Pathway PCR cDNA arrays, both from SA Biosciences (Frederick, MD). RNA extracts of cells treated with 6H5 mAb or mIgG were used for cDNA synthesis by reverse transcription (RT) using a RT2 First Strand Kit (SA Biosciences), per manufacturer's instructions. Briefly, RNA extracts (1 μL containing 0.5 μg) were mixed with 10 μL of the RT cocktail and incubated at 42°C for exactly 15 minutes. The reaction was immediately stopped by heating at 95°C for 5 minutes. Water (91 μL) was added, and the mixture was kept on ice until the PCR reaction. cDNA (102 μL reaction volume) obtained from cells treated with 6H5 mAb or mIgG was added to RT2 qPCR master mix (1350 μL), and 1248 μL water was added, for a total volume of 2700 μL; 25 μL of the mixture was aliquoted per well into the wells of a 96-well plate that contained 84 predispensed pathway gene–specific primer sets plus wells containing five housekeeping genes and three RNA and PCR quality controls (SA Biosciences). A model 7900HT real-time PCR system (Applied Biosystems) was used with a two-step cycling program for qPCR reactions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. The relative gene expression was determined by the difference in the Ct values (ΔΔCt) method; Ct is the PCR cycle at which the sample reaches the threshold, and ΔΔCt is ΔCt,sample − ΔCt,reference, where ΔCt,sample is the Ct value for any 6H5 mAb–treated sample normalized to the endogenous housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ΔCt,reference is the Ct value for the mIgG-treated sample also normalized to the endogenous housekeeping gene. Each array contained a panel of 96 primer sets for 84 relevant pathway- or disease-focused genes plus five housekeeping genes and three RNA and PCR quality controls. The relative expression levels for genes expressed after 6H5 mAb or mIgG treatment were determined.
In Vivo Studies
Mice were randomized into treatment groups, and researchers were blinded to the assignment of treatment groups in all in vivo studies. Anti-HERV-K sera were obtained from BALB/c mice immunized with K-GST as described previously(18
). Briefly, K-GST fusion proteins (100 μg in 100 μL 1× PBS with 100 μL Freund's complete adjuvant; Sigma-Aldrich) were used for subcutaneous immunization of 6- to 8-week-old female BALB/c mice (n = 3 per group), weighing between 21 and 25 g (NCI, Frederick, MD). Three weeks later, K-GST fusion proteins (100 μg in 100 μL 1× PBS with 100 μL Freund's incomplete adjuvant) were administered subcutaneously in weekly boosters for 3 weeks. Ten days after the final boost, the mice (n = 3 per group) were killed, and sera were collected by heart puncture. Anti-HERV-K serum antibody titers were determined by ELISA and immunoblot using K-GST fusion protein antigen.
Female immunodeficient athymic NCr-nu/nu mice (n = 15 per cell line), 6 to 8 weeks old and weighing between 21 and 25 g (NCI), were inoculated subcutaneously in the fourth mammary fat pad with 5 × 106 cells of breast cancer cell lines MCF-7 or MDA-MB-231 on day 1. The mice were randomly divided (n = 5 per group) and treated with 6H5mAb, or G11D10 scFvantibody, or mIgG, on days 4, 6, and 8. The 6H5 mAb or G11D10 scFv antibodies were administered intravenously at a dosage of 0.33 mg per injection, for a total dose of 1 mg per mouse over the 3-day dosing period. Immunodeficient mice were also treated with 6H5-r-Gel (0.33 mg per injection) or sera (200 μL per mouse) by intravenous injection. Mice (n = 5 per group) were injected intravenously with 0.33 mg of mIgG per injection as controls (n = 5 mice in all control groups). Additional control sera were obtained from unimmunized mice (n = 5). Tumor volumes (L × W × D; L = length; W = width; D = depth) for each group were compared. At least two independent experiments were done using the MCF-7 xenograft model, and at least three independent experiments were done using MDA-MB-231 xenografts. Mice were housed in a temperature-, humidity-, and light-controlled room and fed a stock regular diet. All mouse experiments were approved by the Institutional Animal Care and Use Committees (IACUC, protocol number 01-04-00733) of the University of Texas MD Anderson Cancer Center. The ventilated caging housing system consists of a polycarbonate or polysulfone cage, a polycarbonate or polysulfone filter lid with woven-fiber filter media, and a stainless steel wire-bar lid. The individual cages are maintained on a HEPA-filtered, ventilated, suspended cage-type rack. Per Keeling Center for Comparative Medicine and Research practice, the maximum mouse population per mouse cage is five adult mice of any size or age. Mice were placed in a chamber filled with CO2 and after breathing stopped and the mice were unconscious, euthanasia was completed by cervical dislocation. Tumors were excised on the final day of the study—day 31, day 39, or day 40. The tumor tissues were dissected, transferred to 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA), and embedded into paraffin blocks. Five-micrometer-thick formalin-fixed and paraffin-embedded biopsy sections were used for histological diagnosis, TUNEL, and Ki-67 assays.
All animal facilities of The University of Texas MD Anderson Cancer Center are under the direction of full-time veterinarians and are fully accredited by the American Association of Accreditation of Laboratory Animal Care. The University of Texas MD Anderson Cancer Center complies with the National Institutes of Health's policy on animal welfare, the Animal Welfare Act and all applicable federal, state, and local laws.
Analysis of HERV-K env Expression by IHC
IHC was performed on 5-μm formalin-fixed paraffin-embedded tissue sections using standard protocols and the VECTASTAIN Elite ABC Kit as described previously (18
). The expression of antigens was evaluated with the following primary antibodies: 6H5 mAb (1:200 dilution) for HERV-K env and mouse anti-mouse inducible nitric oxide synthase (NOS2; amino acids 961-1144) mAb (1:250 dilution) (DakoCytomation Envision System). Briefly, the slides were baked in an oven at 60°C for 1 hour, then deparaffinized with 100% xylene at room temperature for 1 minute, then hydrated in a graded alcohol series consisting of two 30-second dips each in 100% and 95% ethyl alcohol diluted in water (total volume is 5 mL) at room temperature, and then hydrated in water. Sections were incubated in 3% hydrogen peroxidase in water at room temperature for 10 minutes to block endogenous peroxidase activity. After washing the slides for 5 minutes in water, blocking solution (four drops of stock horse serum in 10 mL of 1× PBS; horse serum was provided in the VECTASTAIN Elite ABC Kit) was added, and the slides were incubated at room temperature for 30 minutes. Slides were then incubated with 6H5 mAb (1 μg in 150 μL normal serum buffer per slide) at 4°C for 16 hours. After three washes with 1× PBS, slides were incubated for 30 minutes with anti-mIgG-HRP secondary antibody (1:600 dilution) in blocking buffer. After three washes with 1× PBS, slides were incubated in 3,3-diaminobenzidine for 5 minutes and counterstained with hematoxylin. Envelope expression was categorized by intensity (0 = absent; 1 = weak; 2 = moderate; 3 = strong) and distribution (percent tumor positive for envelope) (18
). Intensity and distribution scores were multiplied to obtain the final score (0–300) for envelope expression in a tumor. The expression level of NOS2 was categorized as low and high based on a combined score of intensity and distribution, as described previously (20
For cell culture studies, differences between treatment groups were analyzed by Student's t
test, and means, standard deviations, and 95% confidence intervals (CIs) were calculated using GraphPad Prism 5 (GraphPad Software Inc, San Diego, CA). The χ2
and Fisher exact tests were used to assess associations between HERV-K env expression and clinicopathologic features of breast cancer, such as grade (grade 1 or 2 vs grade 3), disease stage (TNM stage I or II vs III or IV), node status (negative vs positive), or tumor hormone receptor status (ER α negative vs ER α positive), as described previously (29
). Adjusted logistic regression was used to analyze dichotomized data for independent association with clinicopathologic features of breast cancer and to calculate odds ratios (ORs), 95% confidence intervals, and P
A repeated measures linear mixed model was applied to analyze the difference in tumor growth in vivo, allowing for one level of variation between mice and another level of variation within mice (repeated tumor size measures over time). Observations were recorded longitudinally for individual mice. Using a human breast cancer xenograft model, we observed previously in our laboratory (F. Wang-Johanning, unpublished data) that the control group had a mean tumor volume of 2000 mm3
(SD = 856 mm3
) by day 36, and the group treated with an anti-HERV-K mAb had a mean tumor volume of 170 mm3
(SD = 24.5 mm3
). Using these numbers as a guide, five mice per group were needed to ensure a power of 80% to detect a difference at alpha level of 0.05. All statistical tests were two-sided, and all P
values less than .05 were considered statistically significant. Data analysis was performed using R statistical free software (http://www.r-project.org/