Applications suite

Cell proliferation was monitored every 24h for 144 hours by manual cell counting using a 1/400² haemocytometer (Hawksley, Lancing, UK). Tumour cell invasion was assessed using 6mm Transwell plates with an 8.0μM pore size (Costar; Corning Incorporated, NY, USA) coated with basement membrane matrix (20% Matrigel; Invitrogen, Paisley, UK). Cells were seeded into the inner chamber at a density of 2.5×10⁵ for MDA-MB-231-IV and 5×10⁵ for MCF7 in RPMI without FCS and 5×10⁵ HS5 bone marrow cells in RPMI supplemented with 10% FCS were added to the outer chamber. Cells were removed from the top surface of the membrane 24 and 48 hours after seeding, and cells that have invaded through the pores were stained with haematoxylin and eosin before being imaged on Leica DM7900 light microscope and manually counted.
Migration of cells was investigated by analysing wound closure: Cells were seeded onto 0.2% gelatine in 6-well tissue culture plates (Costar; corning incorporated); once confluent, 10ug/ml mitomycin C was added to inhibit cell proliferation and a 50uM scratch made across the monolayer. The percentage of wound closure was measured 24 and 48 hours using a CTR7000 inverted microscope and LAS-AF v2.1.1 software (Leica Applications Suite; Leica Microsystems, Wetzlar, Germany).

Tissue Tek-embedded livers were cut with a cryostat CM3050S (Leica, Wetzlar, Germany) to obtain 14 µm sections which were mounted on SuperFrost Plus adhesion slides (Thermo Fisher Scientific, Waltham, MA, USA), air-dried overnight, and stored at room temperature until use. For hepatic neutral lipid staining, liver sections were fixed in 4% neutral buffered formalin, washed with tap water, and rinsed with 60% isopropanol. Hepatic lipids were stained with Oil Red O (Polysiences Inc., Warrington, FL, USA) for 15 min. Next, the liver sections were rinsed with 60% isopropanol, lightly stained with hematoxylin, and covered with a coverslip. Digital images of the sections were obtained using a Leica DMLB microscope (Leica Microsystems, Rijswijk, The Netherlands) equipped with software from the Leica Applications Suite (Leica Microsystems, Rijswijk, The Netherlands).

Calcium AS FS (DiaSys Diagnostic Systems, Holzheim, Germany) was used to quantify calcium content (normalized to the cell number per well) after cell lysis. OsteoImage^TM mineralization assay (Lonza, Basel, Switzerland) applied according to the manufacturer’s instructions, allowed for hydroxyapatite visualization and quantification via the high resolution fluorescence scanner. Cells were stained with Alexa Fluor 568 phalloidin and examined using a Leica SP5X laser scanning confocal microscope (Leica, Wetzlar, Germany) for qualitative assessment. Leica Applications Suite was used to obtain images.