Objective: To investigate the effect of exosomes derived from Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) cells on lymphangiogenesis and lymph node metastasis of NPC.
Methods: Exosomes from NP69 cells and EBV-positive HK1 (HK1-EBV) cells were obtained by ultracentrifugation and identified by Western blotting and nanoparticle tracking analysis. Dio dye phagocytosis test was performed to observe exosome uptake by lymphatic endothelial cells. Lymphatic endothelial cells were treated with exosomes from nasopharyngeal epithelium (NP69), HK1-EBV, and C666-1 cells or exosome-free supernatant of HK1-EBV and C666-1 cells, and tube formation and migration of the cells were observed. In a nude mouse model of popliteal lymph node metastasis of NPC, the effects of normal saline, NP69 cell-derived exosomes, HK1-EBV cell-derived exosomes, exosome-free supernatant of HK1-EBV cells, and HK1-EBV exosome-free supernatant protein on lymphangiogenesis and lymph node metastasis of the tumor were observed.
Results: The exosomes obtained by ultracentrifugation contained abundant exosome-specific proteins and showed a normal size range. The exosomes from NPC cells and NP69 cells could be taken up by lymphatic endothelial cells. Compared with the blank control and exosomes form NP69 cells, exosomes derived from HK1-EBV and C666-1 cells significantly promoted tube formation and migration of lymphatic endothelial cells (P < 0.05), and the exosomes and exosome-free supernatant of HK1-EBV and C666-1 cell produced similar effects (P > 0.05). In the tumor-bearing nude mice, exosomes derived from HK1-EBV cells significantly promoted metastasis of NPC cells and local lymphangiogenesis compared with the blank control, NP69 cell-derived exosomes and exosome-free supernatant of HK1-EBV cells (P < 0.05).
Conclusions: Exosomes from EBV-positive NPC cells can significantly promote lymphangiogenesis and lymph node metastasis of NPC.
目的: 探讨EB病毒(EBV)阳性鼻咽癌细胞外泌体对淋巴管新生与鼻咽癌淋巴结转移的影响。
方法: 利用超速离心获得细胞外泌体，对外泌体进行Western blot与纳米颗粒追踪分析(NTA)鉴定。利用Dio染料吞噬实验，验证外泌体可以被淋巴管内皮细胞摄取。离体条件下，分别利用等体积培养基以及20 μg/mL鼻咽上皮细胞(NP69)、HK1-EBV、C666-1细胞外泌体和除去外泌体的HK1-EBV与C666-1上清蛋白预处理淋巴管内皮细胞，检测淋巴管内皮细胞成管与迁移情况；在体实验，首先建立裸鼠腘窝淋巴结转移模型，负瘤裸鼠被随机分成4组，3只/组。对照组：腘窝处注射生理盐水(30 μL)；NP69外泌体组：10 μg/30 μL NP69外泌体；HK1-EBV除去外泌体上清组：10 μg/30 μL HK1-EBV除去外泌体上清蛋白；HK1-EBV外泌体组：10 μg/30 μL HK1-EBV外泌体。每2 d注射1次，共4次。
结果: 超速离心获得外泌体富含外泌体特异性蛋白，同时NTA鉴定表明其粒径范围正常；鼻咽癌细胞与NP69外泌体可以被淋巴管内皮细胞摄取；HK1-EBV与C666-1外泌体较空白对照组与NP69外泌体可显著促进淋巴管内皮细胞成管与迁移(P < 0.05)，HK1-EBV与C666-1外泌体组与对应除去外泌体上清蛋白组间无显著性差异(P > 0.05)；腘窝淋巴结转移实验发现：HK1-EBV外泌体较空白对照组、NP69外泌体以及除去外泌体的HK1-EBV上清蛋白可显著促进鼻咽癌细胞淋巴结转移以及局部淋巴管新生(P < 0.05)。
结论: EBV阳性鼻咽癌细胞外泌体能促进鼻咽癌淋巴管新生与淋巴结转移。
Keywords: Epstein-Barr virus; exosome; lymph node metastasis; lymphangiogenesis; nasopharyngeal carcinoma; tumor microenvironment.