Foetal bovine serum (fbs)

Caco‐2 and HT‐29 MTX P8 colorectal adenocarcinoma cells were maintained at 37°C in complete Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with penicillin/streptomycin, non‐essential amino acids (Sigma‐Aldrich) and 10% foetal bovine serum (Biowest) in a 5% CO₂ humidified incubator (Panasonic). For FUdR pre‐treatment, 50 μM of FUdR was added for 24 h prior to the start of the assay and removed by washing the cells with PBS. Exposure to sulphide was achieved by adding 3 mM sodium sulphide (Na₂S) as a sulphide donor in the solution. Samples were treated with sulphide two times 2 h apart for a total duration of 4 h and then washed with PBS before the addition of fresh DMEM media for XTT viability assays. XTT assays were carried out according to the manufacturer’s instructions (Biotium) and samples were read using a Synergy H1 microplate reader (Biotek) at 475 nm and subtracting background at 660 nm.

Adipose-Derived human Mesenchymal Stem Cells (AD-hMSCs, Lifeline Cell Technology, Frederick, MD, USA) were expanded at 37°C and 5% CO2 in Mesenchimal Stem Cell Growth Medium 2, supplemented with Mesenchymal Stem Cell Growth Medium 2 Supplement Mix (PromoCell, Heidelberg, Germany). For differentiation experiments, confluent cells were switched to Mesenchymal Stem Cell Growth Medium 2 supplemented with 10% Foetal Bovine Serum (Biowest #S1810-500, Nuaillé, France), 0.2 μΜ Indomethacine (Sigma-Aldrich, #I7378), 10 μg/ml insulin (Sigma-Aldrich #I2643), 1 μM dexamethasone (Sigma-Aldrich #D2915), and 0.5 mM Isobutylmethylxanthine (Sigma-Aldrich, #I7018), to induce the differentiation into adipocytes. Medium was replenished every three days. For cell growth measurements, cells were detached with TrypLE Express (Gibco, #12304-021, Thermofisher) and were counted with a Cell Countess II FL (Thermo-Fisher Scientific).

Four canine lymphoid tumour cell lines (CLBL-1, GL-1, UL-1, and Ema) were used in this study: CLBL-1, a canine B-cell lymphoma cell line [42 (link)]; GL-1, a canine B-cell leukaemia cell line [43 (link)]; UL-1, a canine T-cell lymphoma cell line [44 (link)]; and Ema; a canine T-cell lymphoma cell line [45 (link)]. UL-1 and Ema were established from dogs with lymphoma showing drug resistance after chemotherapy, whereas CLBL-1 and GL-1 were established from dogs with leukaemia or lymphoma who were not subjected to chemotherapy. CLBL-1, GL-1, and Ema were kindly provided by Dr. Rütgen, University of Veterinary Medicine Vienna, Austria, Dr. Nakaichi, Yamaguchi University, Japan, and Dr. Mizuno, Yamaguchi University, Japan, respectively. Our group established UL-1 previously [44 (link)]. Our previous study reported that CLBL-1 and GL-1 were sensitive to vincristine, and UL-1 and Ema were resistant to vincristine [46 (link)]. These cell lines were cultured in RPMI-1640 medium at 37°C, with 10% foetal bovine serum (Biowest, Nuaille, France) in a humidified atmosphere containing 5% CO₂.

Tumours were minced prior to enzymatic dissociation using 4 mg mL⁻¹ collagenase type IV (Thermo Fisher, cat. no. 17104019) in DMEM/F12, at 37 °C for 2 h. Cells were washed using cyclical treatment of pelleting and resuspension in phosphate-buffered saline (Thermo Fisher, cat. no 14190235) for three cycles. The final cell suspensions were strained through 70 µm cell strainers (Falcon, cat. no. 352350), prior to pelleting and resuspension in RPMI (Thermo Fisher, cat. no 61870036), supplemented with 10% foetal bovine serum (Biowest, cat. no. S181B) and 1% penicillin-streptomycin (Thermo Fisher, cat. no. 15140122). Cells were kept in a humidified atmosphere of 5% CO₂ at 37 °C. Cell line identity was authenticated by comparing the STR profile (Indexx BioResearch) of each cell line to its original tumour. Cells were routinely screened for mycoplasma contamination using Venor^®GEM OneStep mycoplasma detection kit (Minerva Biolabs, cat. no. 11-8100).

The MG-63 cell line was used in this study because it can retain the differentiated phenotype over consecutive subcultures and shows a faster growth than the primary bone-forming lines, which makes it a good in vitro model. MG-63 cells (Sigma-Aldrich, Darmstadt, Germany) were cultivated in T75 culture flasks with Dulbecco’s Modified Eagle Medium (Biowest, Nuaillé, France) enriched with a 1% antibiotic (glutamine-penicillin-streptomycin [Biowest, Nuaillé, France]) and 10% foetal bovine serum (Biowest). After performing two cell subcultures and achieving an 80% confluence, the cells were seeded on the disc surfaces in a 24-microwells plate at a previously calculated concentration of 2 × 10⁴ cells. Three discs were employed for each type of surface and the experiments were triplicated. Microwells without discs were used as negative controls. After 4 h of incubation, the cell culture was controlled by an Olympus CKX41SF2 phase contrast microscope (Olympus, Shinjuku-ku, Tokyo, Japan). Then, the microwells were completely filled with Dulbecco’s Modified Eagle Medium and incubated for a period of 24 h.