EBV+ Nasopharyngeal Carcinoma Spheroids

An EBV+ nasopharyngeal cell line (NPC43)^{20 (link)} was used in this study. NPC43 cells, stably expressing Lifeact-mCherry,^{4 (link)} were cultured in RPMI medium 1640 (1X, Gibco) supplemented with 10% fetal bovine serum, 1% antibiotic antimycotic (Gibco; 100 units per ml penicillin G sodium, 100 mg ml⁻¹ of streptomycin, and 0.25 mg ml⁻¹ of amphotericin B), and 0.2% 2 mM rock inhibitor Y-27,632 (ENZO) at 37 °C in a 5% CO₂. NPC43 cells were maintained in 2D culture, passaged every 3 days in 1 : 3 ratio until 80% confluence. Cells cultured for more than 20 passages were discarded. To generate NPC tumour spheroids, NPC43 cells were harvested by trypsinization and resuspended in ice-cold tumour spheroid medium (cell culture medium containing 2% (v/v) Matrigel (CORNING, cat no. 354270)) at a concentration of 10⁶ cells per ml. 100 μl of the cell suspension was mixed in 2.9 ml of cold tumour spheroid medium and seeded on low attachment plate (CORNING, cat no. 3471) at 37 °C in a 5% CO₂. Spheroids were harvested after 4 days for dissemination assays but were observed for up to 7 days in some experiments.