Human breast cancer cell line MCF-7 was obtained from the Institute of Development, Aging and Cancer, Tohoku University. MDA-MB-231 was purchased from the American Type Culture Collection. Both cell lines were maintained in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS) and an antibiotic-antimycotic.
In vitro sequential selection of invasive populations from MCF-7 cells
BD BioCoat Matrigel Invasion Chambers (for a 6-well plate; BD Biosciences, San Jose, CA) were used to select invasive populations from MCF-7 cells. MCF-7 cell suspension (4 × 105 cells/well) in serum-free RPMI1640 was seeded into the upper chamber. After 60 hours' cultivation, cells that migrated through the membrane (named MCF-7-1) were harvested by trypsinization, proliferated on a dish, and then reseeded in a Matrigel Invasion Chamber. The cycle was repeated 14 times, and finally MCF-7-14 cells were obtained. In each cycle, invading cells on the underside of the chamber were fixed with 10% formalin-neutralized buffer, stained with 0.1% crystal violet, and imaged through a microscope (Axiovert 200M; Carl Zeiss, Jena, Germany) with a CCD camera (AxioCam; Carl Zeiss).
Cell proliferation assay
MCF-7 and MCF-7-14 cells (5 × 103 cells/well) were seeded into 96-well plates and cultured. The cells were counted at 24, 48, 72 and 96 h with a Cell Counting Kit-8 (MTT assay; Wako Pure Chemical Industries, Osaka, Japan).
Cell protein lysates were prepared from confluent cell cultures of MCF-7 and MCF-7-14 cells, subjected to SDS-PAGE, and transferred to PVDF membranes. The membranes were blocked (5% skim milk) and incubated with rat anti-human epidermal growth factor receptor 2, HER-2 (GeneTex, Irvine, CA) and mouse anti-estrogen receptor alpha, ER-α (Stressgen/Assay Designs, Ann Arbor, MI) monoclonal antibodies (mAbs), followed by the corresponding HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). Antibody complexes were detected with the ECL detection system (GE Healthcare).
In vitro wound healing assay
Migratory abilities of MCF-7 and MCF-7-14 cells were measured using the in vitro wound healing assay. Cells were plated on 10-cm culture dishes and grown to 100% confluence. Wounds were created by scraping monolayer cells with a sterile pipette tip. At 0, 24, 48, 72 and 96 h after the creation of wounds, three different areas were imaged through an Axiovert 200 M. Wound distances were measured at each time point and expressed as the average percent of wound closure by comparing the zero time.
Animal experiments were performed in accordance with the Guidelines of the Japanese Government for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Tokyo University of Science and the Ethics Committee of Bio Matrix Research Inc. MCF-7 and MCF-7-14 cells were transfected with pEGFP-C1 (encoding the enhanced green fluorescence protein, EGFP; Clontech Laboratories, Mountain View, CA) using Fugene 6 Transfection Reagent (Roche, Basel, Switzerland) in serum-free medium. MCF-7 and MCF-7-14 cells stably transfected with pEGFP-C1 (named MCF-7-EGFP and MCF-7-14-EGFP, respectively) were selected by G418 treatment (500 μg/ml) for 3 weeks. Suspensions of MCF-7-EGFP and MCF-7-14-EGFP cells (1 × 106 cells/50 μl) were mixed with the same volume of Matrigel, and injected into the fourth mammary fat pad of 10-week-old female BALB/cJ/nu/nu mice.
In vivo and ex vivo imaging of EGFP-labeled breast cancer cells
At 4, 8 and 12 weeks after implantation of MCF-7-EGFP and MCF-7-14-EGFP cells, mice were sacrificed and scanned by a luminescence/fluorescence imaging analyzer (LAS3000; Fujifilm, Tokyo, Japan) to detect metastatic lesions. Ex vivo fluorescence imaging was also performed on necropsy samples.
Histology and immunohistochemistry
Xenografts and metastatic lesions were rapidly frozen, and 6 μm thick sections were cut on a cryostat. The sections were analyzed by hematoxylin/eosin (HE) and methyl green pyronin (MGP) staining, or unstained sections were analyzed for EGFP fluorescence. For immunostaining epithelial and mesenchymal markers, mouse anti-cytokeratin (CK) 19 (A53-B/A2; 1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-E-cadherin (67A4; 1:25; Santa Cruz Biotechnology) and anti-vimentin (V9; 1:150; Dako, Glostrup, Denmark) mAbs were used. Briefly, frozen sections were treated with 3% H2O2 for 10 min and 5% skim milk in Tris-buffered saline with 0.2% Triton-X for 1 h. Primary antibodies were applied for 1 h at room temperature. Subsequently, the sections were incubated with Simple Stain Mouse MAX PO (Nichirei Biosciences, Tokyo, Japan) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200; Molecular Probes, Invitrogen, Carlsbad, CA) at room temperature. Diaminobenzidine (DAB) with 0.1% H2O2 (Dako) was used as the final chromogen and hematoxylin for nuclear counterstaining in immunohistochemistry. In immunofluorescence, cell nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI). Immunostaining of the endothelial marker, CD31, was performed with rat anti-mouse CD31 mAb (MEC 13.3; 1:50; BD Biosciences) and HRP-linked goat anti-rat IgG F(ab')2 fragment (1:200; Amersham ECL, GE Healthcare). Microscopic images were acquired using an Axiovert 200 M equipped with an AxioCam and analyzed by AxioVision software (Carl Zeiss).
MCF-7, MCF-7-14 and MDA-MB-231 cells were plated on coverslips and fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 2 min, and then incubated in PBS containing 5% skim milk for 1 h at room temperature. Cells were incubated with anti-E-cadherin (1:100), anti-β-catenin (8E7; 1:100; Upstate Biotechnology/Millipore, Billerica, MA), anti-vimentin (1:300) and anti-fibronectin (clone 10; 1:200; BD Biosciences) mAbs for 1 h at room temperature, followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200-1:1000) and nuclear counterstaining with DAPI.
MCF-7, MCF-7-14 and MDA-MB-231 cells were synchronized in S-phase with the thymidine-hydroxyurea block method. Total RNA was isolated from cells arrested in S-phase using an RNeasy Mini kit (Qiagen, Hilden, Germany).
cDNA was synthesized from total RNA isolated from MCF-7, MCF-7-14 and MDA-MB-231 cells using a One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA). In vitro transcription reactions were performed using a GeneChip IVT Labeling Kit. Fifteen micrograms of the labeled cRNA was hybridized to a Human Genome U133 Plus 2.0 Array (Affymetrix). The array images were scanned and analyzed using Genechip operating software (GCOS; Affymetrix). The full microarray data set is available in the NCBI Gene Expression Omnibus public database under data series accession number GSE18903.
Microarray data analysis
Using GeneSpring GX 7.3.1 software (Agilent Technologies, Santa Clara, CA), microarray data of each chip were normalized to the 50th percentile of the measurements on that chip. For per-gene normalization, the measurements of each probe were normalized to the median of the measurements of that probe in MCF-7, MCF-7-14 and MDA-MB-231 cell lines. Average linkage hierarchical clustering was carried out on genes filtered on detection flags ("present" in two or more cell lines) and signal intensity (>50 in all cell lines). To identify potentially important differences in biological mechanisms regarding their invasive potential, genes up- or down-regulated >2-fold in both MCF-7-14 and MDA-MB-231 cells compared with MCF-7 cells were selected from the filtered genes. Ingenuity Pathway Analysis software (IPA 5.0; Ingenuity Systems, Redwood City, CA) was also utilized to identify the top significant canonical pathways for these selected genes and to functionally link the most differentially expressed genes.
cDNA was synthesized from 2 μg total RNA using SuperScript III reverse transcriptase (Invitrogen). Reverse transcription (RT) was run for 1 h at 50°C and stopped by heating for 5 min at 85°C. PCR was conducted in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). A 10 μl reaction containing 0.2 μl cDNA, 0.5 μM of each primer and 2.5 μl Power SYBR Green PCR Master Mix (Applied Biosystems) was used to monitor double-strand DNA synthesis. Primer sequences are provided in Additional file 1. Quantitative RT-PCR (qRT-PCR) was carried out following the recommended thermal profile: 95°C for 10 min (pre-incubation) followed by 40 cycles of 95°C for 15 sec (denaturation) and 60°C for 1 min (annealing and elongation). Fluorescence intensity of the amplified products was measured at the end of each PCR cycle. Two runs were performed with each data point run in triplicate. Results were normalized to internal control GAPDH mRNA and if necessary, represented relative to mRNA levels of MCF-7 cells.
MCF-7 and MCF-7-14 cells were plated on 96-well plates at a concentration of a single cell per well, which was confirmed visually. Wells containing either none or more than one cell were excluded from further analysis. Single-cell clones were cultured to allow the growth of individual colonies, which were picked and expanded in culture, and analyzed by Matrigel invasion assay and flow cytometry.
Three human breast cancer cell lines, MCF-7, MCF-7-14 and MDA-MB-231, and single-cell clones derived from the MCF-7 and MCF-7-14 cell lines were used in flow cytometry analysis. Cells were harvested with TrypLE Express (Invitrogen), and then suspended (2 × 106 cells/100 μl) with Stain Buffer containing 1% FBS (BD Biosciences). Phycoerythrin (PE)-conjugated mAbs against human CD44 (G44-26; BD Biosciences) or CD24 (ML5; BD Biosciences) were added to the cell suspension at the concentrations recommended by the manufacturer and incubated at 4°C in the dark for 60 min. The labeled cells were fixed in 100% methanol on ice for 5 min. Flow cytometry analysis were performed in triplicate using a FACSCalibur system (BD Biosciences).
Function-blocking antibody treatment
Cells (1 × 106 cells) were incubated with rat anti-human CD44 mAb (IM7; BD Biosciences) or normal rat IgG at 400 μg/ml for 20 min. After preincubation, a cell proliferation assay, cell migration assay and Matrigel invasion assay were performed. Cell proliferation was assessed by MTT assay. The cell migration assay was performed using an Oris Cell Migration Assay (Platypus Technologies, Madison, WI), which comprises a 96-well plate with silicone stoppers (2 mm diameter) in each well. Following cell seeding and cell attachment (2 × 104 cells), the stoppers were removed and migration proceeded for 72 h. Cells were then stained with crystal violet and imaged through a microscope. Matrigel Invasion Chambers (for a 24-well plate; BD Biosciences) were used to examine cell invasion. Pretreated cells (5 × 104 cells) in serum-free RPMI1640 were seeded into the upper chamber and invading cells were fixed and stained after 60 h cultivation.
Data are the means ± standard deviation (SD). Values were compared between MCF-7 and MCF-7-14 cells and between normal IgG and anti-CD44 mAb using Student's t test. Comparisons among cell lines were made using analysis of variance (ANOVA) and Tukey-Kramer multiple comparison, where appropriate. P < 0.05 was considered significant.