Fibronectin coated flasks

Human umbilical cords were obtained from the Leiden University Medical Center (Leiden, The Netherlands) after informed consent from the parents.
Human umbilical vein endothelial cells (HUVECs) were isolated according to Jaffe et al. 18, with minor modifications: trypsin/EDTA (Sigma‐Aldrich) was used to enzymatically detach the endothelial cells from the vein, and the endothelial cells were cultured on fibronectin‐coated flasks (isolated from bovine plasma; Sigma‐Aldrich) and refreshed twice a week with EC‐medium, consisting of M199 Earl's salt with L‐glutamine (Thermo Fisher), supplemented with 10% (v/v) fetal calf serum (PAA Cell Culture Company, Pasching, Austria, www.paa.com), penicillin/streptomycin (PAA Cell Culture Company), 1,000 IU of heparin (Leo Pharma), and 25 mg bovine pituitary extract (BPE) (Thermo Fisher). HUVECs were used at passages 2–3 18.
Stromal cells and HUVECS were cocultured in a 96‐well plate (Costar; Sigma‐Aldrich) for 1 week in a 4:1 ratio, as has been described previously 19. After 1 week, cells were fixated for 10 minutes with ice‐cold methanol (100%), and endothelial sprouting was visualized with CD31 immunofluorescence (Zeiss LSM500; BD Bioscience). The percentage capillary coverage was analyzed with ImageJ software. NG2 immunofluorescence (BD Bioscience) was determined (Zeiss LSM500) and quantified as mean fluorescent intensity in Image J.

SNU-719, a naturally derived EBV-infected gastric carcinoma cell line was maintained in RPMI1640 medium [37 (link)]. AGS cells were maintained in Ham’s F-12 nutrient mixture medium. EBV infected 293 cells, EBV-positive AGS B95.8 (gifts from dr. Henri-Jaques Delecluse, German Cancer Research Center, Heidelberg, Germany) and R-stop EBV have been described previously [38 (link),39 (link)] and were maintained under 100 µg/mL hygromycin B (Roche, Basel, Switzerland) selection. CNE-2 Akata cells (a gift from dr. Kwok-Wai Lo, Chinese University of Hong Kong, Hong Kong, China), a NPC cell line superinfected with the Akata strain of EBV, was maintained in RPMI1640 under 400 µg/mL G418 selection (Invitrogen, Carlsbad, CA, USA). C666.1, a NPC cell line consistently harboring EBV was cultured in DMEM in fibronectin-coated flasks (Sigma-Aldrich, Buchs, Switzerland). All media contained 10% FCS, 100 units/mL sodium penicillin, 100 µg/mL streptomycin sulphate and 2 mM L-glutamine.