We performed the pathological diagnosis and reviewed clinical data of 78 patients diagnosed with IDCs in the Department of Molecular Pathology, Nara Medical University from 2004 to 2009. We selected luminal subtype (47 cases), Her2 subtype (23 cases) and triple negative subtype (TNBC, 8 cases). Because no written informed consents were obtained, any identifying informational data were removed from the samples before analysis to protect privacy strictly (unlinkable anonymization). All procedures were done in accordance with the Ethical Guidelines for Human Genome/Gene Research which is issued by the Japanese Government. The procedures were approved by the Ethics Committee of Nara Medical University (approval number 937).
MCF-7 and MDA-468 human IDC cell lines were purchased from Dainihon Pharmaceutical Co. (Tokyo, Japan). Cells were maintained at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) which was supplemented with 10% fetal bovine serum (FBS). Cell growth was assessed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) dye assay (Wako Pure Chemical Corp. Ltd., Osaka, Japan), as previously described 27. A pH 7.0 DMEM was prepared from regular DMEM (pH 7.4) adjusted by the addition of HCl.
Antibody and reagents
The anti-human CLDN4 antibody 4D3 was developed by immunizing rats with a plasmid vector encoding human CLDN4, which recognized the extracellular domains of CLDN4 16. The anti-human CLDN1 extracellular domain antibody 2C1 was also developed by the same method 28. PTX (Wako) and estradiol (LKT Labs Inc., St. Paul, MN, USA) were purchased.
Apoptosis of cells was determined by staining with the Hoechst 33258 fluorescent dye (Wako). The number of apoptotic cells was counted by examining 1,000 stained cells 27.
BALB/c nude mice (4 weeks old, male) were purchased from SLC Japan (Shizuoka, Japan). All mice were housed 2 or 3 per wide cage. The holding room was maintained at 23°C, 50% humidity, with 12-hours light/12-hours dark cycle. The mice were euthanized at the end of experiments by cervical dislocation under anesthesia with isoflurane (3%). All experiments were performed according to the institutional guidelines and were approved by the Committee for Animal Experimentation of Nara Medical University (approval number 11725), which was accordant with the current regulations and standards of the Ministry of Health, Labor, and Welfare, Japan.
To establish a model for subcutaneous tumors, cells (1 × 107) were inoculated subcutaneously into the subscapular areas of nude mice. Then, with five mice in each group, PTX (10 mg/kg body weight BW) and/or 4D3 (1 mg/kg BW, diluted with saline) were injected concurrently into the peritoneal cavity on days 1, 3, and 7. Tumor size was observed each week. According to the institutional humane endpoint for animal experiments, moribund mice were euthanized. For anti-estrogen therapy, tamoxifen citrate (TAM, 500 mg per mouse in peanut oil, daily, s.c., LKT Labs) was administrated 29.
Lung metastasis model
IDC cell suspension (1×106 cells/50 μl of PBS) was injected into the caudal vein. PTX (10 mg/kg BW) and 4D3 (1 mg/kg BW) were also administrated (i.p.) on days 1, 3, and 7.
Bone metastasis model
Under inhalation anesthesia with 3% isoflurane (WAKO), percutaneous intraosseal injection was performed by drilling a 26-gauge needle into the tibia proximal to the tuberositas tibia, then IDC cell suspension (5×105 cells/20 μl of phosphate buffered saline PBS) was inserted 30. Mice were treated with bisphosphonate (BP, zoledronic acid, WAKO, 100 μg/kg in 100 μl PBS) 31, PTX (10 mg/kg BW) and 4D3 (1 mg/kg BW) administrated (i.p.) on days 1, 3, and 7.
In vivo imaging of tumor
IDC cell were labeled with VivoTrack 680 (ParkinElmer Inc., Waltham, MA, USA). A mouse was examined by Clairvivo OPT in vivo imager (Shimazu, Kyoto, Japan) under anesthesia 16. Fluorescence intensity was calculated with software equipped in the imager.
Tumors were penetrated by 18G needle, through which lumen a fine needle probe of pH meter (Chemical Instruments, Co. LTD., Hachioji, Japan) was inserted into tumors under anesthesia. pH was monitored for 5 min to calculate the mean value in one site. The measurement was performed at five sites for each tumor. Representative pH was a mean of values in the five sites.
Thin sections (4-μm) of paraffin-embedded specimens were immunohistochemically stained with 0.2 µg/mL of 4D3 or 2C1 by a previously described immunoperoxidase technique 32. Secondary antibodies conjugated with peroxidase (Medical and Biological Laboratories, Nagoya, Japan) were used (0.2 µg/mL). Color development were performed with diamine benzidine hydrochloride (DAKO, Glastrup, Denmark). A counterstain was done with Meyer’s hematoxylin (Sigma). Immunopositive cells at the cytoplasmic membrane were counted. Staining strength was scored from 0 to 3 (a score of 1 was used to describe the expression level in normal pancreatic duct epithelium). The staining index was calculated as the staining strength score multiplied by the staining area (%) 14-16. For a negative control, non-immunized rat IgG (Santa-Cruz Biotechnology, Santa-Cruz, CA, USA) was used as the primary antibody.
Whole-cell lysates were prepared as previously described 33. Lysates (20 μg) were used to immunoblot analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12.5%), and then transferred electronically onto nitrocellulose filters. The filters were incubated with primary antibodies and peroxidase-conjugated secondary antibodies (Medical and Biological Laboratories). Anti-tubulin antibody was used as a loading standard (Oncogene Research Products, Cambridge, MA, USA). The signals were visualized with an Enhanced Chemiluminescence Western-blot detection system (Amersham, Aylesbury, UK). Antibodies for CLDN4 (4D3), CLDN1 (2C1), and ER (Proteintech Group Inc., Rosemont, IL, USA) were used as primary antibodies.
Reverse transcription-polymerase chain reaction (RT-PCR)
To assess human CLDN4 mRNA expression, RT-PCR was performed with total RNA (0.5 µg ) extracted using an RNeasy kit (Qiagen, Germantown, MD, USA). The primer sets were as follows; mouse Batf2 (M1 macrophage), forward, 5'-AGC ACG AAT CCT TGG AGA AA AA-3′ and reverse, 5'-GTT CCT GGC AGC CAT TGT AT-3′ (National Center for Biotechnology Information NCBI Reference Sequence: BC024521.1); mouse Fizz1 (M2 macrophage), forward, 5'-CCC TTC TCA TCT GCA TCT CC-3′ and reverse, 5'-CAG TAG CAG TCA TCC CAG CA-3′ (NCBI Reference Sequence: AF316397.2); mouse CD73 (mesenchymal stem cell), forward, 5'-CCT CTC AAA TCC AGG GAC AA-3′ and reverse, 5'-TTT GGA AGG TGG ATT TCC TG-3′ (NCBI Reference Sequence: L12059.1); which were synthesized by Sigma Genosys, Ishikari, Japan. PCR products were electrophoresed with a 2% agarose gel and stained with ethidium bromide. The β-actin (ACTB) mRNA was also amplified as an internal control (GenBank Accession No. NM_001101).
Enzyme-linked immunosorbent assay (ELISA)
ELISA kits were used to measure the concentrations of PTX (anti-5-PTX antibody-derived ELISA, Absolute Antibody, UK), according to the manufacturers’ instructions.
Statistical significance was calculated using chi-square test and Kruskal-Wallis test with InStat software (GraphPad, Los Angeles, CA, USA). Survival analysis was performed using the Kaplan-Meier method along with the log-rank test (SPSS Statistics, IBM Japan, Tokyo, Japan). Statistical significance was defined as a two-sided p-value of < 0.05.