Hepes

The mouse breast cancer cell line 4T1 and the colon carcinoma cell line CT26.WT were maintained in Dulbecco's modified Eagle's medium (DMEM, Biowest) supplemented with 10% fetal calf serum (FCS; Biochrom), 10 mM HEPES (Lonza), 2 mM L-glutamine (Lonza), and penicillin/streptomycin (Lonza). Cells were maintained at 37°C in a humidified 7.5% CO₂ atmosphere.
CD90.2 expressing stromal cells were isolated by MACS from BALB/c mouse mammary fat pad and skin or 4T1 or CT26.WT tumors generated by subcutaneous injection into BALB/c mice (Charles River). Isolated primary cells were plated on Matrigel (Corning) coated tissue culture ware and grown in DMEM supplemented with 8% FCS, 10 mM HEPES, 2 mM L-glutamine, P/S, and 1x non-essential amino acids (NEAA, Lonza) at 37°C in a humidified 7.5% CO₂ atmosphere.

C666-1 cells were cultured in RPMI-1640 with 25 mM Hepes (Lonza, Berlin, Germany) supplemented with 10% heat-inactivated FCS (Gibco, Carlsbad, CA), 2 mM L-Glutamine, 100 units/ml penicillin (Gibco), and 0.1 mg/ml streptomycin (Gibco). Cells were maintained at 37 °C, 5% CO₂ and passaged every 7 days at a 1:2 ratio using accutase (Sigma, St. Louis, MO, USA). Seventy-five percent of culture medium was replaced by fresh medium every 2–3 days. NPC43 and C17 cell lines were cultured in RPMI-1640 with 25 mM Hepes (Lonza, Berlin, Germany) supplemented with 10% heat-inactivated FCS (Gibco, Carlsbad, CA), 2 mM L-Glutamine, 100 units/ml penicillin (Gibco) and 0.1 mg/ml streptomycin (Gibco) and 4 µM Y27632 (Promocell, Heidelberg, Germany). Cells were maintained at 37 °C, 5% CO₂ and passaged 5 days at a 1:4 ratio using accutase (Sigma, St. Louis, MO, USA). Culture medium was replaced by fresh medium every 2 to 3 days.

Combined biopsies were washed in RPMI supplemented with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Lonza), 10% FBS (ThermoFisher) and 2% antibiotic-antimycotic (ThermoFisher). The epithelial layer was removed with RPMI supplemented with ethylenediaminetetraacetic acid (EDTA; Lonza), dithiothreitol (Chem-Lab, Belgium) and HEPES (Lonza) at 37°C on a magnetic stirrer at 400 revolutions/min (rpm) for 20 min. Biopsies were transferred to 5 mL of Hanks balanced salt solution with calcium and magnesium (HBSS^Ca2+/Mg2+; Lonza) supplemented with HEPES, FBS, 5.0 mg collagenase IV (Worthington Biochemical, cat. LS004188, USA) and 10 μL DNase I (Roche, Germany) at 37°C on a magnetic stirrer at 400 rpm for 40 min. The cell suspension was strained through a 70-μm filter (EASYstrainer™; Greiner), quenched with 5 mL of PBS with 2.5% BSA, centrifuged at 400g for 5 min. The remaining undigested tissue was again resuspended in 5 mL of HBSS with Ca and Mg supplemented with HEPES, FBS, 5.0 mg collagenase and 10 μL DNase I at 37°C on a magnetic stirrer at 400 rpm for 20 min. Combined cell suspensions were counted using an ABX counter.

Human melanoma cell lines A375, SK‐MEL5, and SK‐MEL28 were obtained from ATCC (Manassas, VA, USA) and maintained in DMEM (Lonza, Basel, Switzerland) supplemented with 10% (A375, SK‐MEL5) or 15% (SK‐MEL28) heat‐inactivated fetal bovine serum (Biovest, Riverside, MO, USA), 2 mm L‐glutamine, 10 mm HEPES, and 100 U·mL⁻¹ of penicillin/streptomycin (Lonza). The Jurkat cell line was obtained from ATCC (Manassas, VA, USA) and maintained in RPMI 1640 medium (Lonza) supplemented with 10% heat‐inactivated fetal bovine serum (Biovest), 2 mm L‐glutamine, 10 mm HEPES, and 100 U·mL⁻¹ of penicillin/streptomycin (Lonza). Cells were grown in a humidified atmosphere at 37 °C with 5% CO₂. The 293T cell line was purchased from ATCC (Manassas, VA, USA) and maintained in DMEM supplemented with 10% FBS. Vemurafenib, cobimetinib, trametinib, and SGI‐1776 were purchased from Selleckchem (Houston, TX, USA) and dissolved in sterile dimethyl sulfoxide (DMSO). Interferon‐γ (IFN‐γ) was obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Bone marrow was isolated from the femur and tibia of mice and the red blood cells were lysed with ACK lysis buffer (GIBCO). The remaining cells were then cultured in DMEM (GIBCO) (with 20% L929 cell media (in-house preparation), 10% FCS (Invitrogen), 1% L-Glutamine (GIBCO), 100 U/mL Penicillin and 100 μg/mL Streptomycin (GIBCO), 10 mM HEPES (Lonza) and 0.05mM 2-mercaptoethanol (GIBCO)) in 10mls at a density of 5x10⁵ cells/ml at 37°C. After 7 days of culture non-adhesive cells were removed before removing adherent BMDMs using 2.5mM edta (Invitrogen) in PBS (GIBCO) with 5% FCS (Invitrogen). Adherent BMDMS were washed and resuspended in DMEM (with 1% FCS (Invitrogen), 1% L-Glutamine (GIBCO), 100 U/mL Penicillin and 100 μg/mL Streptomycin (GIBCO), 10 μM HEPES (Lonza) and 0.05mM 2-mercaptoethanol (GIBCO)). Adherent BMDMs were then plated at a density of 2x10⁶ cells/ml for 24 hr. The adherent BMDMs were then stimulated for 24 hr with either 20ng/ml IL-4 (R&D) and 20ng/ml IL-13 (R&D) before RNA extraction.

Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (BioWhittaker, CC‐2519) and cultured on 0.5% gelatin‐coated plates (unless otherwise indicated) in medium 199 supplemented with 20% FBS, 100 U/ml penicillin (Lonza), 100 μg/ml streptomycin (Lonza), 2 mM l‐glutamine (Lonza), 10 mM HEPES (Lonza), and ECGS/H (Promocell, C‐30120). Cells were used up to passage 5. Mouse aortic endothelial cells (MAEC) were obtained from aortas of 4‐week‐old mice. Briefly, after fat removal under a microscope, aortas were incubated for 5 min at 37°C in collagenase solution (collagenase type I, 3.33 mg/ml, Worthington, LS004194), thus allowing removal of the adventitia with forceps. The aortas were then cut into small pieces (1–2 mm), and a cell suspension was obtained by incubation for 45 min at 37°C in 6 mg/ml type I collagenase and 2.5 mg/ml elastase (Worthington, LS002290) diluted in DMEM. Once EC colonies were visible, they were subjected to two rounds of positive selection with anti‐ICAM2 and magnetic beads. MAEC were cultured on 0.2% gelatin‐coated plates in DMEM/F12 medium supplemented with 20% FBS, 100 U/ml penicillin (Lonza), 100 μg/ml streptomycin (Lonza), 2 mM l‐glutamine (Lonza), 10 mM HEPES (Lonza), and ECGS/H (Promocell, C‐30120).