Cell lines and culture conditions
Two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and BT-549, and one hormone-receptor positive breast cancer (HRBC) cell lines, MCF-7, were from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, USA), 100 U/mL penicillin (Gibco, Grand Island, NE, USA), and 100 μg/mL streptomycin at 37°C in humidified air with 5% CO2. The culture medium was replaced every 3 days.
Deferoxamine (DFO), an iron-chelating agent, was purchased from Novartis (Basel, Switzerland). Deferasirox, an another iron-chelator, was purchased from Novartis Pharma (Basel, Switzerland). Eribulin was provided by Eisai Co. (Tokyo, Japan).
The sensitivity of the three human breast cancer cell lines to two iron chelators was evaluated using a cell proliferation (MTT) assay kit according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells (1 × 103 cells/well in 96-well plates) were cultured for 24 hours in 90 μL of DMEM with 10% FBS. A 10-μL aliquot of medium containing the indicated concentration of DFO or deferasirox was then added to each well. 72 hours after incubation, 10 μL of the MTT reagent was added to each well; 2 hours later, the medium was discarded and replaced by 100 μL of dimethyl sulfoxide. After shaking the plates for 10 min, the plate was measured as absorbance at 510 nm with a microplate reader (Perkin-Elmer, Waltham, MA, USA). Three independent experiments were performed. 
MDA-MB-231 cells were cultured in 96-well plates. After the cells reached 80–90% confluence, a wound was created in the cell monolayer using the WoundMaker tool (Essen Bioscience, Ann Arbor, MI, USA). The cells were cultured in DMEM with 1% FBS and 10 or 100 μM DFO for 30 h. Scratched fields were photographed every 2 hours using an Incucyte Live-Cell Imaging System (Essen Bioscience). The degree of cell migration was calculated as 100 × the wound closure area at each time point/the wound area at time 0 .
Analysis of cell cycle progression
Cells (1 × 106 cells/well) were plated into six-well tissue culture plates. After 24 hours, the cells were harvested and washed two times with phosphate-buffered saline and stained with the CycleTESTTM PLUS DNA Reagent kit according to the manufacturer’s instructions (Becton Dickinson Biosciences, CA-San Jose, USA). Staining intensity was quantified using BD LSR II flow cytometer with FACSDivaTM software (Becton Dickinson Biosciences) [17, 18].
Quantitative real-time-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). The RT step was performed at 37℃ for 15 min, 50℃ for 5 min, and then 98℃ for 5 min. cDNA was amplified via qRT-PCR with Taq DNA polymerase (Nippon Gene, Tokyo, Japan) and the StepOnePlus RT-PCR system (Applied Biosystems, Foster City, CA, USA). The following TaqMan gene expression assays for used: assay ID, Hs00154208 (CA9); assay ID, Hs00911700 (KDR); assay ID, Hs00983056 (CDH2); assay ID, Hs00232783 (ZEB1); assay ID, Hs01125301 (CD274); and assay ID, Hs02758991 (GAPHD) (Applied Biosystems). RT-PCR was performed at 95°C for 15 seconds, followed by 40 cycles at 60°C for 60 seconds .
In vivo tumor model
Invivo experiments were conducted on 4-week-old female athymic BALB/c nu/nu mice obtained from CLEA Japan (Tokyo, Japan). The animal experimental protocol was approved by Ethical Committee of the Osaka City University, Osaka, Japan. All studies on mice were conducted in accordance with the National Institute of Health (NIH) ‘Guide for the Care and Use of Laboratory Animals’. The mice were housed in a standard animal laboratory with ad libitum access to food and water. MDA-MB-231 cells (107 cells) were suspended in 100 μL of DMEM, and injected into the backs of the mice. After a week, the mice were randomized into two groups: (1) control (saline alone) and (2) DFO (20 mg/kg/day, 5 days/week). For evaluation of the combination therapy, the mice were randomized into four groups: (1) control (saline alone), (2) DFO alone, (3) eribulin alone (0.8 mg/kg/day, 5 days/week), and (4) DFO plus eribulin.
Both DFO and eribulin were dissolved in DMEM without FBS. DFO was directly injected into the tumor, and eribulin was intravenously administered. The resultant tumor volumes (length × width) were measured weekly. Animals were euthanized via isoflurane (Mylan, PA, USA) (1ml per one mouse) and cervical dislocation, and autopsied at 6 weeks after cell inoculation . In sacrifice, animals were unconscious.
Statistical analyses were performed with JMP13 software (SAS Institute, Cary, NC, USA). Student’s t-test was used to compare data between groups. A p value <0.05 was considered statistically significant.