Cell culture
MCF-7 cells were obtained from American Type Culture Collection and propagated in DMEM medium supplemented containing with 10% FBS and 1% penicillin/streptomycin (Sigma, St. Louis, MO). We established three different types of ERBC cell lines using MCF7 cells; Long-term tamoxifen treated cells in presence of estrogen (LTTE): MCF-7 cells were cultured in DMEM medium supplemented containing with 10% FBS and 1% penicillin/streptomycin with 1 uM 4-Hydroxytamoxifen (4-OHT), Long-term tamoxifen treated cells in absence of estrogen (LTT): MCF-7 cells were cultured in MEM (Richter's modification, no phenol red) medium supplemented containing with 5% CSS (charcoal stripped serum) and 1% penicillin/streptomycin with 1 uM 4-OHT, Long-term estrogen deprived cells (LTED); MCF-7 cells were cultured in MEM medium supplemented containing with 5% CSS and 1% penicillin/streptomycin. Letrozole resistant cells (LTLT-Ca) and AC1 as a parental cell line were obtained from Dr. Brodie at University of Maryland. Normal human lung fibroblasts (NHLF) were purchased from Lonza and grown in DMEM medium supplemented containing with 10% FBS and 1% penicillin/streptomycin (Sigma, St. Louis, MO). Cells were maintained under standard conditions of 37°C and 5% CO2. Cells were cultured for a maximum of 4 weeks before thawing fresh, early passage cells and confirmed to be Mycoplasma negative (Hoechst stain).
Conditioned media
TCM (tumor conditioned media): When ERBC cells were confluent in T175 tissue culture flasks, the normal cancer cell growth media was replaced with 8 ml serum-free media (SFM) containing with 2% serum. After 24 h incubation in a tissue culture incubator, the supernatant was centrifuged and filtered through 0.2 µm syringe filters (Corning, Corning, NY). The resulting tumor-conditioned media (TCM) was stored in aliquots at -80°C.
TCM-fibroblast CM (conditioned media from fibroblasts induced by TCM): When the fibroblast reached 30-40% confluence in T75 tissue culture flasks, the media was replaced with 30% of TCM of ERBC (TCM: appropriate media = 3:7) to allow the TCM to induce fibroblasts. The fibroblasts were allowed to grow in the media for 4 days at which point the media was replaced with 3 ml of SFM with 2% FBS (no supplements). After 24 h, the supernatant was centrifuged and filtered. The resulting tumor-induced fibroblast conditioned media (TCM-fibroblast) CM was stored in aliquots at -80°C to avoid multiple freeze thaws.
Cell Migration assay
ERBC cell migration was assessed by using the Oris™ cell migration kit (Platypus, Madison, WI). ERBC cells were pre-labeled with Cell Tracker Green (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. 50,000 labeled cells in complete media were added to each well of a 96-well plate containing stoppers to prevent the cells from settling in the center region of the wells. ERBC cells were allowed to adhere for 4 h, after which the stoppers were removed. Each (TCM-fibroblast) CM and SFM as a control (100 µl) was added, and the cells that migrated to the center of the well were quantified by measuring the fluorescence at 485/530 nm on a Victor V plate reader (PerkinElmer, Salem, MA). The migrated cells were also visualized by imaging on the Eclipse T-100 fluorescence microscope (Nikon, Brighton, MI).
Cell proliferation assay
The proliferation assay was measured by CyQUANT assay (Thermo fisher) according to the manufacturer’s protocol. Briefly, ERBC cells were plated at 2.5 x103 cells per well in 96-well plates, in triplicate, with 200µL (TCM-fibroblast) CM, with various inhibitors or vehicle for 3 days. 200 µL of the CyQUANT GR dye/cell-lysis buffer were added to each sample well. Mix gently and incubate the sample for 2–5 minutes at room temperature. Measure the sample fluorescence using a fluorescence microplate reader with filters appropriate for ~480 nm excitation and ~520 nm emission maxima. A percentage calculated of survival of drug-treated cells versus time-matched vehicle-treated cells.
Antibody array
For reverse western blot, human cytokine antibody array kits (R&D Systems) were used, according to the manufacturer’s instructions. The pixel densities were analyzed using ImageJ for the experimental analysis and the Array Quick Spots Tool from HLImage++ (Western Vision Software) for the spot intensity analysis.
UPlex Assay
U-Plex Assay (multiplex ELISA) was carried out as recommended by the manufacturer (Meso Scale Discoveries, Rockville, MD). Briefly, the conditioned media samples were generated and added to a pre-coated plate containing the capture antibody per well for the detection of the CXCL1. After 3 washes, sample, standards, or calibrator were added to the wells and incubated for 1 hour. Next, after 3 washes, detection antibody was added to the wells and incubated for 1 hour. After a final set of 3 washes, read buffer was added to the wells and the plate was read using the MSD SECTOR Imager 2400 (Meso Scale Discoveries, Rockville, MD) and the data was processed using the MSD Workbench 4.0 software (Meso Scale Discoveries, Rockville, MD).
Real-Time qRT-PCR
Total RNA was extracted with Trizol reagent (Invitrogen), and cDNA was synthesized from total RNA (2 µg) using an iScript™ cDNA Synthesis Kits (Bio-rad). Aliquots of cDNA were used as templates for real-time RT-qPCR procedure. Relative quantitation of mRNA expression was achieved using real-time PCR (CFX96 Touch™ Real-Time PCR Detection System, Bio-rad laboratory, Hercules, California). The SYBR® Green PCR Supermixes was used according to the manufacturer’s instruction.
Immuno blotting analysis
The western blotting analysis was performed with anti-ERK, anti-Phospho ERK (Santa cruz biotech), and anti-Actin antibody (Sigma, St. Louis, MO) as previously described (38). Briefly, ERBC cells were incubated with (TCM-fibroblast)CM for 1 hour with or without 1 uM reparixin, and fibroblasts were incubated with TCM of ERBC for 1 hour. The cells were lysed with RIPA buffer, 40 ug of protein separated using a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, and then transferred onto nitrocellulose membrane. Membranes were immunostained using the above antibodies.
Flow Cytometry
To examine the apoptosis, ERBC cells were harvested following 3 days treatment with various inhibitors, or a negative control (DMSO) and were analyzed using a Muse® Annexin V Dead Cell Kit (Millipore) and the Muse® Cell Analyzer (Luminex).
Statistical Analysis
Each experiment was repeated at least three times. The results of three independent experiments performed in triplicate were shown as mean ± SD or SEM compared with control. All statistical analyses were performed using a two-sided unpaired t-test using GraphPad Prism 6 (Graphpad, La Jolla CA). A p-value of 0.05 or less was considered significant.