Mccoy medium

MIA PaCa2 pancreatic, MDA-MB-231 breast, A549 lung cancer cell lines were cultured in DMEM (Invitrogen, Germany) supplemented with 10% fetal calf serum (FCS: PAA, Germany), 1% penicillin/streptomycin. HCT-116 colorectal carcinoma cells were cultured in McCoy medium (Sigma, Germany). In our study we aimed at using cancer cell lines that simultaneously fulfill several criteria such as strong response to hypoxic environment, secretion of VEGF in culture supernatants and capacity to form tumors in vivo. Cancer cell lines were obtained from DSMZ-German Collection of Microorganisms and Culture Cells (Braunschweig, Germany) or authenticated at the Multiplexion (Heidelberg, Germany).

Human monocytes were isolated from a buffy coat. Cells were centrifugated on the Pancoll gradient (1.070 g/ml) and purified on the Percoll gradient (1.064 g/ml) to obtain the monocyte populations with a purity of >85%. Monocytes were cultured in McCoy medium (Sigma-Aldrich) with 15% FCS and 7% CO₂, in the absence or presence of 5 mg/ml recombinant human S100A9 (9254-S9-050, R&D). TNFSF13B expression level in the monocytes was detected using Human TNFSF13B Quantikine ELISA Kit (R&D).

For cell culture experiments, samples were sterilized under UV light, inserted into 12-well polystyrene cell culture plates (TPP, Switzerland; internal well diameter 21.4 mm) and seeded with: i) human osteoblast-like MG-63 cells (European Collection of Cell Cultures, Salisbury, UK; Cat. No. 86051601) suspended in Dulbecco's modified Eagle's Minimum Essential Medium (DMEM; Sigma, USA, Cat. No. D5648), ii) Saos-2 cells (European Collection of Cell Cultures, Salisbury, UK, Cat. No. 89050205) suspended in McCoy medium (Sigma, Cat. No. M4892), or iii) murine macrophage-like RAW 264.7 cells (ATCC, TIB-71, USA) suspended in RPMI-1640 medium (Sigma, Cat. No. R8758). All culture media were supplemented with 10% fetal bovine serum (Sebak GmbH, Aidenbach), and gentamicin (40 µg/mL, LEK). Each well contained i) 36,000 MG-63 cells (i.e., approximately 10,000 cells/cm²), ii) 20,000 Saos-2 cells (5,600 cells/cm²) or iii) 50,000 RAW 264.7 cells (14,000 cells/cm²), and 2 mL of the medium. The cells were cultured for 1 and 3 days (to evaluate cell numbers) or for 7 days (for PCR detection of markers of osteoblast differentiation, and to measure the potential immune activation of RAW cells) at 37°C in a humidified air atmosphere containing 5% CO₂. Two samples were used for each experimental group and time interval.