Fig. 3

Exosomes containing EBV products promote fibroblast activation and NPC cell growth. A Phenotypic changes in fibroblasts. Primary fibroblasts were treated with PBS or exosomes (10 μg/ml) derived from HK1 cells or HK1EBV cells. Images were captured 24 hours post-exosome stimulation. Scale bar: 50 μm. B Real-time monitoring of changes in the fibroblasts stimulated with exosomes derived from HK1 cells, HK1EBV cells, or PBS (control). The black arrow indicates the time of exosome addition. Data are presented as the mean and SD of triplicate experiments. C Assessment of exosome-mediated effects on fibroblast contractility. Fibroblasts were treated with exosomes derived from HK1 or HK1EBV cells or medium only (control) for 3 days (left). Quantification of contractility was determined by measuring the gel area using ImageJ. Data are presented as the mean and SD (*p < 0.05, **p < 0.01, ***p < 0.001; paired t test). Scale bar, 2 mm. D Real-time proliferation assay of the NPC cells treated with cancer cell culture medium, culture supernatants harvested from the fibroblasts treated with HK1EBV cell-derived exosomes (Exo-Tx Fibro^CM), or untreated fibroblasts (Fibro^CM). Data are presented as the mean and SD of triplicate experiments. E Analysis of EdU incorporation in the NPC-TW06 cells treated with supernatants harvested from the fibroblasts treated with HK1EBV cell-derived exosomes or control medium for 10 hours, followed by EdU labeling (green) for 4 hours. Scale bar, 100 μm. EdU⁺ cells were quantified using CellSens software. (**p < 0.01; ***p < 0.001; Bonferroni’s multiple comparison test). F Analysis of pro-proliferative signaling pathways in NPC-TW06 cells. Protein lysates were harvested 30 minutes after exosomal treatment. GAPDH was used as a loading control. G Stimulation with IL6 (20 ng/ml), IL8 (50 ng/ml), or CCL2 (100 ng/ml) enhances NPC cell proliferation (right) and DNA synthesis (left). EdU⁺ cells were quantified using CellSens software (Olympus) (**p < 0.01; ***p < 0.001; Bonferroni’s multiple comparison test). H. Depletion of IL6, IL8, or CCL2 by the addition of cytokine-specific neutralizing antibodies suppresses Exo-Tx Fibro^CM-mediated NPC cell proliferation. NPC cells were pretreated with antibodies for one hour and then stimulated with conditioned medium. Data are presented as the mean and SD of triplicate experiments