Mammocult media

For tumor sphere formation assays, 1000 cells per ml were plated in 2ml in 6-well ultra-low attachment plate (Corning) in MammoCult medium (Stem cell Technologies) and after the indicated timepoint, 10×10 stitch imaging was done at 10x (100 random images acquired) using an Retiga Aqua Blue camera (Q Imaging, Vancouver, BC) connected to a Leica DMI6000 inverted microscope. Individual images were taken and then a composite image was generated using the scan slide function in Metamorph Imaging Software (Molecular Devices, Downington, PA). Subsequent integrated analysis also used Metamorph software. For limiting dilution sphere assays, BD FACS Aria II sorter was used to sort cells directly into 96-well ultra-low attachment plates in 200μl mammocult media per well (Corning). After 7 days, the number of wells with tumor spheres was counted and the data was analyzed by Extreme Limited Dilution Analysis [ELDA] platform to determine the stem cell frequency.

MDA-MB-231 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, CA, USA) and HCC1143 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Carlsbad, CA, USA). Media was supplemented with 10% foetal bovine serum (FBS; Corning, Corning, NY, USA) and 1% penicillin-streptomycin solution (Life Technologies). MCF-12A cells were grown in MammoCult™ media supplemented with 5% FBS (Corning, NY, USA), 10% MammoCult™ proliferation supplement, 0.5% hydrocortisone and 0.2% heparin (all from Stem Cell Technologies, Vancouver, BC, Canada). MDA-MB-231-Luc cells were cultured in L-15 media supplemented with 15% FBS and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA). A week before the animal experiment, the cells were harvested, washed in Dulbecco’s phosphate-buffered saline (DPBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and grown as mammospheres in MammoCult™ media (described below).

Cells were pre-treated with IgG3, anti-APO-1, LzCD95L, IFNβ, or received no treatment for 6 days. Cell suspensions were passed through a 40 μm sterile cell strainer (Fisher Scientific) to obtain single cell suspension. The strained cell suspension was diluted in Mammocult media and seeded at 5000 cell/well in triplicate in ultra-low adherence 24-well plates (Corning). The cells were cultured in Mammocult media (Cell Stem Technology; prepared according to manufacturer’s instructions) supplemented with 4 μg/ml Heparin, 0.5 μg/ml hydrocortisone and 10% Mammocult Proliferation Supplement. Spheres were counted using a light microscope 6 days after seeding.