舌鳞癌细胞远程塑造区域淋巴结“Bregs-Tregs”微环境的作用机制

One of the most important reasons for the diversity of lymphatic metastasis of TSCC (tongue squamous cell carcinoma) is the difference in lymph node microenvironment. Tumor microenvironment for metastasis can be regulated by tumor cells in primary lesion, but the mechanism is uncertain. Recently, it is reported that tumor-derived exosomes determine organotropic metastasis and regulate the pro-inflammatory gene expression to form pre-metastatic niche. Our previous study showed that TSCC cells-derived exosomes increased the killing efficiency of CTL, but at the same time, induced the proliferation of Tregs and then immunosuppression. We also demonstrated that the percentage of Bregs and Tregs increased significantly in metastatic lymph nodes of TSCC patients, and that Bregs could induced the proliferation of Tregs. Based on the studied above, we propose that TSCC cells can release exosomes to prepare“Bregs-Tregs” type of microenvironment in regional lymph nodes remotely for lymphatic metastasis. We will investigate the regulation mechanism in vitro and in vivo, then regulate the “exosomes-Bregs-Tregs pathway to demonstrate its critical role in TSCC lymphatic metastasis. This study will provide experimental evidence and new theory for TSCC target therapy.

舌鳞癌颈淋巴转移存在极大异质性，重要原因为淋巴结存在不同微环境。肿瘤转移微环境可以由原发灶肿瘤细胞远程塑造，机制未明。最新发现，肿瘤源性exosomes决定肿瘤的器官特异性转移；exosomes可能通过诱导慢性炎症塑造肿瘤转移微环境。我们发现，舌鳞癌细胞exosomes可诱导CTL靶向攻击，也能诱导Tregs扩增及免疫抑制；舌鳞癌转移淋巴结中Bregs、Tregs比例显著升高；Bregs可诱导Tregs扩增。据此率先提出：舌鳞癌细胞可能通过释放exosomes远程塑造区域淋巴结Bregs-Tregs型微环境，进而促进颈淋巴转移。拟在共培养体系、动物模型水平，荧光标记时空分布、细胞因子网络、免疫细胞极化、信号通路等方面探讨舌鳞癌细胞源性exosomes对颈淋巴微环境的调控机制；进而调控exosomes-Bregs-Tregs对话通路，证明对舌鳞癌颈淋巴转移的影响，为舌鳞癌生物治疗提供新的靶点

肿瘤源性外泌体（EXO）因其具有母体细胞“特征”而被认为在肿瘤微环境的调控和肿瘤转移微环境的塑造中扮演重要角色，并可作为液体活检的生物标志物用于早期诊断和治疗监测。本研究在揭示舌鳞状细胞癌源性外泌体（TEX）异质性的基础上，探讨TEX调控舌鳞癌恶性生物学行为和免疫抑制状态从而促进淋巴转移的机制，分析TEX源性miRNA与口腔鳞状细胞癌患者颈部淋巴状态的关系。结果显示：①细胞培养上清、OSCC患者血清和肿瘤组织块中可成功分离出具有特征性的EXO，超速离心法所得产物更纯净；②EXO可选择性包装母体细胞蛋白并被受体细胞摄入而引起变化；③TEX可增强正常上皮细胞增殖、迁移和侵袭能力；④TEX可增强舌鳞癌细胞的体外增殖、侵袭能力和体内转移能力，且高恶性程度来源TEX体外调控能力更强；⑤舌鳞癌患者循环外泌体miRNA表达谱与颈淋巴转移密切相关，其中血清外泌体源性hsa-miR-10b-5p在口腔癌和伴颈部淋巴转移的患者中含量高；⑥hsa-miR-10b-5p可增强舌鳞癌细胞的体外增殖和侵袭能力，外泌体是其可能调控载体；⑦TEX能抑制人PBMC的增殖，促进凋亡，上调Treg及Th1比例和免疫抑制相关蛋白表达，呈现免疫抑制状态；⑧TEX在小鼠体内靶向富集于肝、肺和脾，诱导肺部和脾脏成免疫抑制表型，增加肿瘤肺转移率。