Hmvec dly

Human adult dermal microvascular lymphatic endothelial cells (LEC) were purchased from Lonza (HMVEC-dLy; Braine-l′Alleud, Belgium) and used at passages 3 to 5. Cells were cultured at 37°C in a 5% CO₂ atmosphere in endothelial growth microvascular (EGM2-MV) medium (Lonza) composed of EBM2 medium with 5% FBS, hydrocortisone, h-FGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF and GA 1000 [9] (link). For drug treatments, cells were washed and cultured in EGM2-MV medium supplemented with 2% FBS, and half of the medium was renewed every 24 h. Adenosine (Sigma-Aldrich) was used at concentration ranging from 0.1 μM to 10 μM and the Adenosine deaminase inhibitor EHNA (Sigma) was used at a concentration of 10 μM to increase Adenosine half-life. CGS21680 and NECA (Sigma-Aldrich) were used as preferential A2a and A2b agonists, respectively.
Primary macrophages were isolated from peritoneal lavage of C57BL/6 mice intraperitoneally injected with 4% thioglycollate (Sigma-Aldrich, St. Louis, MO) 5 days earlier. After washing off non-adherent cells, macrophages were cultured in serum-free medium (RPMI-1640, Lonza) and conditioned medium was collected.

Luciferase-expressing Lewis Lung Carcinoma (LLC-Luc) cell line of C57BL/6 mouse origin was purchased from Caliper Lifesciences. LLC-Luc cells were cultured in DMEM (Gibco, Gent, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Gent, Belgium), 2 mM glutamine (Gibco, Gent, Belgium), 100 UI/ml penicilline/streptomycin (Gibco, Gent, Belgium), 1 mg/ml geneticin (Serva, Heidelberg, Germany) and maintained in a humidified incubator at 37°C and in a 5% CO₂ atmosphere. Two type of LEC were used in this study: HMVEC-dLy (Lonza, Braine-l'Alleud, Belgium) and Human telomerase-transfected dermal LECs (hTERT-HDLECs) [37] (link). LEC were cultured in EGM2-MV medium (Lonza, Braine-l'Alleud, Belgium) until confluence was reached.