Roswell park memorial institute (rpmi)

K562, RAJI, 8866, C1R and 721.221 cells were maintained in RPMI (Sigma-Aldrich) supplemented with 10% fetal calf serum (Sigma-Aldrich), 2 mM glutamine (Biological Industries (BI)), 1 mM sodium pyruvate (BI), 1× nonessential amino acids (BI), 100 U/ml penicillin (BI), 0.1 mg/ml streptomycin (BI).
RKO, T-47D, and A549 cells were maintained in DMEM (Sigma-Aldrich) supplemented with 10% fetal calf serum (Sigma-Aldrich), 2 mM glutamine (Biological Industries (BI)), 1 mM sodium pyruvate (BI), 1× nonessential amino acids (BI), 100 U/ml penicillin (BI), 0.1 mg/ml streptomycin (BI).
NK-92 cells were maintained in two different media: RPMI (Sigma-Aldrich) supplemented with 10% fetal calf serum (Sigma-Aldrich), 2 mM glutamine (Biological Industries (BI)), 1 mM sodium pyruvate (BI), 1× nonessential amino acids (BI), 100 U/ml penicillin (BI), 0.1 mg/ml streptomycin (BI), and 200U/ml IL-2 (PeproTech); or MEM-alpha (BI) supplemented with 12.5% fetal calf serum (Sigma-Aldrich), 12.5% Horse serum (BI), 2 mM glutamine (BI), 1 mM sodium pyruvate (BI), 1× nonessential amino acids (BI), 100 U/ml penicillin (BI), 0.1 mg/ml streptomycin (BI), 200 U/ml IL-2 (PeproTech), 0.2 mM myoinositol, 0.02 mM folic acid, 0.1mM beta-mercaptoethanol, 0.2% Ribonucleosides and Deoxyribonucleosides for MEM-Alpha.

DCs were generated from mouse spleens and they were infected using lentiviral vectors as previously described (Chou et al., 2006 (link)). Briefly, spleens from 6- to 8-week-old SV129 mice were homogenised through a cell strainer to obtain a cell suspension. Cells were washed twice with RPMI (Sigma, UK) containing 1% heat-inactivated foetal bovine serum (FBS) and then resuspended in RPMI supplemented with 10% FBS, 1 mM pyruvate (Sigma, UK), 1× non-essential amino acids (Sigma, UK), 2 mM glutamine (Sigma, UK), 50 µM 2-ME (Gibco BRL), 20 ng/ml recombinant mouse GM-CSF (R&D Systems) and 1 ng/ml recombinant human TGF-ß (R&D Systems) and plated at a density of 2 × 10⁶ cells/ml in 75 cm² culture flasks at 37 °C in a 5% CO₂ atmosphere. After 5 days of culture, 5 ml fresh medium were added per flask and at day 8, the cells in suspension were collected, replated and kept in suspension in fresh medium. After a total of 17–18 days ex vivo, 80–90% of the cells in culture were DCs as determined by the expression of CD11c and DEC205 by FACS analysis. Cell viability before experimental assays was tested by Trypan Blue exclusion. The mouse microvascular endothelial immortalised cell line, SVEC 4-10 (O’Connell and Edidin, 1990 (link)) was obtained from the American Type Culture collection and cells were cultured using DMEM (Sigma, UK) supplemented with 10% FBS at 37 °C in a 5% CO₂ atmosphere.

The immortalized melanocyte cell lines Hermes 3C and 4C were purchased from the Wellcome Trust Functional Genomics Cell Bank (St George’s University of London, London, UK). To ensure melanocyte proliferation, cholera toxin was added to the media as recommended by Wellcome Trust. CT stimulates cyclic adenosine 3′,5′-monophosphate (cAMP) production within the cells. All Hermes lines and their derivatives were cultured as previously described [40 (link)], with the following modifications: RPMI was replaced by M254 medium (Cascade Biologics, Portland, OR, USA) and cells were incubated in 5% CO₂. SKMEL28 was purchased from ATCC and grown in RPMI (Sigma Aldrich, Saint Louis, MO, USA) with 8% FBS (PAA), 5% CO₂, at 37 °C. U2OS cells were obtained from ATCC and grown in RPMI (Sigma Aldrich) with 8% FBS (PAA), 5% CO2, at 37 °C. Further details about cloning, constructs, and lentiviral production, culture conditions, growth factor and anchorage independence assays, as well as siRNA experiments, isolation of RNA, and establishment of tumor xenografts are provided in the Supplementary Methods.

Lamina propria (LP) cells in the colon were isolated by a modified method described previously [18 (link)]. In brief, gut pieces were cut into 2 mm slices, and the epithelium was eliminated by stirring, first in PBS containing 3 mM EDTA for 10 min at 37°C (twice) and then in RPMI (Sigma Chemical Co., St. Louis, MO, USA) containing 1% FBS, 1 mM EGTA, and 1.5 mM MgCl2 for 15 min (also twice). Gut pieces were collected and stirred in RPMI containing 20% FBS, 100 U/mL collagenase (C2139; Sigma-Aldrich Corp., St. Louis, MO, USA), and 5 U/mL DNase 1 (Sigma-Aldrich Corp) for 90 min at 37°C. Halfway through the incubation and at the end of the incubation, the suspension was dissociated by multiple aspirations through a syringe for 2 min. The pellet was purified to LPL on a 45%/66.6% discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient at 600 ×g for 20 min. MLNs from individual mice were collected under sterile conditions in ice-cold PBS with 10% fetal calf serum (FBS). Then, the lymph nodes were gently disrupted with a sterile syringe plunger and filtered through a nylon cell strainer (40 μm mesh; BD Biosciences, San Jose, CA, USA). The cells were collected after centrifugation at 1500 rpm at room temperature for 5 min. The number of viable cells was counted by trypan blue staining.

The human lymphoblast derived K562 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and cultured at 37 °C in a humidified atmosphere (5% CO₂) in RPMI (Sigma–Aldrich, St. Louis, Missouri, USA) medium for 48 h. Oxytetracycline (OTC) from Sigma–Aldrich (St. Louis, Missouri, USA) was stocked at 100 mM in DMSO (Sigma–Aldrich, St. Louis, Missouri, USA). Bone powder, achieved from poultry raised with or without the administration of oxytetracycline, was extracted in cell culture medium as previously described [19 (link),20 (link)]. Briefly, the bone powder was dissolved in RPMI at the concentration of 124 mg/mL and kept under continuous stirring at 37 °C for 48 h. After that, the suspension was filtered, and the filtrate neutralized with KOH (Sigma–Aldrich, St. Louis, Missouri, USA) at pH 7.2–7.4. For compound treatment, 0.5–2.0 × 10⁶ cells/mL were supplemented with graded concentration of OTC or graded dilution of bone powder extracts as indicated. The viability of K562 cells was assessed before and after treatments by direct counting Trypan Blue-negative cells with a hemocytometer.