Table_1_Extracellular Vesicles Released by Tumor Endothelial Cells Spread Immunosuppressive and Transforming Signals Through Various Recipient Cells.docx

Head and neck squamous cell carcinoma (HNSCC) has a high recurrence and metastatic rate with an unknown mechanism of cancer spread. Tumor inflammation is the most critical processes of cancer onset, growth, and metastasis. We hypothesize that the release of extracellular vesicles (EVs) by tumor endothelial cells (TECs) induce reprogramming of immune cells as well as stromal cells to create an immunosuppressive microenvironment that favor tumor spread. We call this mechanism as non-metastatic contagious carcinogenesis. Extracellular vesicles were collected from primary HNSCC-derived endothelial cells (TEC-EV) and were used for stimulation of peripheral blood mononuclear cells (PBMCs) and primary adipose mesenchymal stem cells (ASCs). Regulation of ASC gene expression was investigated by RNA sequencing and protein array. PBMC, stimulated with TEC-EV, were analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting. We validated in vitro the effects of TEC-EV on ASCs or PBMC by measuring invasion, adhesion, and proliferation. We found and confirmed that TEC-EV were able to change ASC inflammatory gene expression signature within 24–48 h. TEC-EV were also able to enhance the secretion of TGF-β1 and IL-10 by PBMC and to increase T regulatory cell (Treg) expansion. TEC-EV carry specific proteins and RNAs that are responsible for Treg differentiation and immune suppression. ASCs and PBMC, treated with TEC-EV, enhanced proliferation, adhesion of tumor cells, and their invasion. These data indicate that TEC-EV exhibit a mechanism of non-metastatic contagious carcinogenesis that regulates tumor microenvironment and reprograms immune cells to sustain tumor growth and progression.

头颈部鳞状细胞癌（Head and neck squamous cell carcinoma, HNSCC）具有较高的复发与转移率，但其肿瘤扩散的具体机制尚未阐明。肿瘤炎症是肿瘤发生、增殖及转移的核心生物学过程。本研究假设：肿瘤内皮细胞（tumor endothelial cells, TECs）释放的细胞外囊泡（extracellular vesicles, EVs）可诱导免疫细胞与基质细胞发生重编程，进而构建有利于肿瘤扩散的免疫抑制微环境，我们将该机制命名为非转移性传染性致癌作用。本研究从原代头颈部鳞状细胞癌来源的内皮细胞中收集细胞外囊泡（记为TEC-EV），并将其用于刺激外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）与原代脂肪间充质干细胞（primary adipose mesenchymal stem cells, ASCs）。通过RNA测序与蛋白质芯片技术探究ASCs的基因表达调控模式；采用酶联免疫吸附试验与荧光激活细胞分选术分析经TEC-EV刺激后的PBMC。我们通过检测细胞侵袭、黏附与增殖能力，在体外验证了TEC-EV对ASCs或PBMC的调控效应。研究发现并证实，TEC-EV可在24~48小时内改变ASCs的炎症基因表达特征。TEC-EV还可增强PBMC分泌转化生长因子-β1（transforming growth factor-β1, TGF-β1）与白细胞介素-10（interleukin-10, IL-10），并促进调节性T细胞（T regulatory cell, Treg）的扩增。TEC-EV携带有介导Treg分化与免疫抑制的特异性蛋白质与RNA分子。经TEC-EV处理后的ASCs与PBMC可增强肿瘤细胞的增殖、黏附与侵袭能力。上述实验数据表明，TEC-EV可通过调控肿瘤微环境、重编程免疫细胞以维持肿瘤的生长与进展，展现出非转移性传染性致癌的作用机制。