基于类器官培养骨肉瘤来源的SNORA12外泌体上调Treg细胞TIGIT表达诱导免疫抑制的机制研究

本研究应用单细胞测序技术分析骨肉瘤免疫微环境发现部分患者TIGIT+Treg细胞比例高，抗体封闭TIGIT后原代CD3+ T细胞对骨肉瘤细胞株杀伤率显著提高。患者血清及类器官培养上清中外泌体和可溶性蛋白分别加入健康人原代CD3+细胞培养体系，发现外泌体组上调表达TIGIT。构建外泌体中测序丰度最高的5种ncRNA质粒转染T细胞发现仅SNORA12上调TIGIT表达，转录组分析发现MEK/ERK/ATF3信号通路活化。提出“骨肉瘤细胞来源SNORA12外泌体通过活化MEK/ERK通路促Treg细胞TIGIT表达诱导免疫耐受”的假说。拟将1) 探索SNORA12是直接结合靶基因mRNA还是通过结合核糖体蛋白或者是作为miR的ceRNA分子活化MEK/ERK/ATF3的具体机制；2）完善骨肉瘤类器官培养作为免疫治疗筛选平台；3）探索干涉等离子体显微成像技术捕获分析骨肉瘤外泌体预测预后的可行性。

We explored the characters of osteosarcoma immune microenvironment by single cell sequencing and found the proportion of TIGIT+Treg cells are more than 10% in some patients. After blocking the TIGIT by antibody, the cytotoxicity of primary CD3+T cells from healthy donor on OS cell line significantly increased. The exosome and macromolecular protein conponent from serum and supernatant of organoid culture were added into the culture system of healthy primary T cells respectively. It was the exosome that upregulated TIGIT expression. We constructed 5 ncRNA plasmids which were the highest abundance in exosomes and transfected them into the T cells from healthy donors respectively. Only SNORA12 up-regulated TIGIT expression, and activated MEK/ERK pathway by mRNA array. We hypothesized that "exosome containing SNORA12 discharged from osteosarcoma cells induced immune tolerance through promoting TIGIT expression on Treg cells by activating the MEK/ERK pathway" was proposed. Further 1) explored how did SNORA12 ur-regulate the TIGIT, by directly binding to target gene mRNA, or working as a factors of ribosomal protein compound or combined with miR as ceRNA; 2) established the osteosarcoma organoid database for screening the precision immunotherapy; 3) predicted the significance of osteosarcoma exosomes detecting by interferometric plasma microscopy as a prognostic factor. Our study will provide a theoretical basis of the new immunotherapeutic target on osteosarcoma.