鼻咽癌外泌小體藉由EB病毒潛伏膜蛋白1促進巨噬細胞免疫抑制型分化;Nasopharyngeal carcinoma-derived exosomes facilitate immunosuppressive macrophage polarization via Epstein-Barr virus-encoded latent membrane protein 1

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Title:	鼻咽癌外泌小體藉由EB病毒潛伏膜蛋白1促進巨噬細胞免疫抑制型分化;Nasopharyngeal carcinoma-derived exosomes facilitate immunosuppressive macrophage polarization via Epstein-Barr virus-encoded latent membrane protein 1
Authors:	劉子彤;Liu, Tzu-Tung
Contributors:	生物醫學工程研究所
Keywords:	Tumor-associated macrophages;Nasopharyngeal carcinoma;Epstein-Barr virus;tumor microenvironment;latent membrane protein 1;exosomes;腫瘤相關巨噬細胞;鼻咽癌;EB病毒;腫瘤微環境;EB病毒潛伏膜蛋白1;外泌小體
Date:	2023-08-16
Issue Date:	2024-09-19 15:19:59 (UTC+8)
Publisher:	國立中央大學
Abstract:	鼻咽癌以EB病毒的感染及嚴重的腫瘤免疫浸潤現象為特徵。在鼻咽癌中，腫瘤相關巨噬細胞在促進免疫抑制型態的微環境上扮演重要的角色，且被認為與不良預後密切相關。先前的許多研究指出，鼻咽癌細胞會透過外泌小體促進形成一個促進腫瘤生長的微環境，然而這些帶有EB病毒產物的外吐小體和免疫細胞之間的免疫調控關係仍待深入探索。此篇論文研究目的在於探討源於鼻咽癌細胞的外泌小體對巨噬細胞功能的調控。我們發現EB病毒產物可以透過外泌體傳遞給未感染之癌細胞及周遭巨噬細胞。質譜蛋白分析結果顯示鼻咽癌細胞的外泌小體帶有EB病毒產物，如EB病毒潛伏膜蛋白1 (LMP1)及EBV病毒胸苷激酶。我們藉由西方墨點法驗證外吐小體中含有EB病毒潛伏膜蛋白1 (LMP1)。實驗結果顯示，由人類周邊血單核球分化的巨噬細胞攝取這些含有LMP1的外泌小體後，會產生趨向免疫抑制型態的極化現象，並伴隨著相似M2型巨噬細胞的特徵，包括較高表現的CD206、ARG-1、VEGF及較少量的iNOS分泌。此外，受LMP1影響之巨噬細胞對初代癌細胞的毒殺作用表現較弱。細胞功能性實驗結果進一步顯示，巨噬細胞攝取這些外吐小體後，會幫助鼻咽癌細胞增生。RNA 定序的結果指出，含LMP1的外泌小體抑制了巨噬細胞之抗原呈現能力，並活化巨噬細胞MST1R (RON) 及IL-10訊號路徑。藉由後續深入的機制探討，我們發現LMP1的潛伏膜蛋白1的C端活化區2區段 (CTAR2) 與MST1R活化有關。除此之外，使用MST1R抑制劑可以部份抑制LMP1促發炎反應的抑制作用。透過鼻咽癌的公開RNA定序資料 (GSE102349) 分析，我們發現晚期鼻咽癌病人具有較高表現的MST1R (p < 0.001)。我們利用來自口腔癌病人的腫瘤組織處理過後得到的細胞進行單細胞定序，進一步探討了含LMP1的外泌小體在調控巨噬細胞免疫反應上的角色。分析結果顯示，將腫瘤微環境細胞群暴露於含LMP1的外泌小體會導致免疫抑制型態巨噬細胞的比例增加。分析細胞間的相互溝通訊號傳輸結果顯示，攝取外泌小體的巨噬細胞可能經由SPP1-ITGA4及Galectin-9-CD44/45交互作用調控T 細胞的毒殺能力。總結而言，我們發現來自鼻咽癌細胞含有LMP1的外泌小體能經由活化MST1R促進巨噬細胞免疫抑制型態的分化，進而建構有利於鼻咽癌生長的微環境。;Nasopharyngeal carcinoma (NPC) is characterized by Epstein-Barr virus (EBV) infection and severe immune infiltration within tumors. Tumor-associated macrophages (TAMs) are recognized as a crucial component contributing to an immune suppressive microenvironment and a poorer prognosis in NPC. Lines of evidence have shown that EBV+-NPC cells are capable of modulating tumor microenvironment (TME) via exosomes, resulting in a pro-tumoral niche. Nevertheless, the immunoregulatory link between the NPC-derived exosomes and macrophages remains a lot to be explored. The primary purpose of this study is to dissect how the NPC-derived exosomes regulate functions of macrophages. We demonstrated that EBV-encoded products could be transferred to uninfected cancer cells and surrounding macrophages via exosome cargos. Results of mass spectrometry analysis revealed that exosomes derived from NPC cells contain several EBV-encoded products, such as EBV-encoded latent membrane protein 1 (LMP1) and thymidine kinase. Western blotting further confirmed that NPC cells deposited LMP1 into exosomes. The uptake of these LMP1-containing exosomes (LMP1_exosomes) by human monocyte-derived macrophages led to an immunosuppressive polarization. Specifically, multiple properties of M2-like macrophages, including increased expressions of CD206, ARG-1, and VEGF, as well as a decreased expression of iNOS were found in LMP1_exosome-treated macrophages. Functionally, macrophages treated with LMP1_exosomes displayed a reduced cytotoxicity towards primary cancer cells compared to vehicle-treated macrophages. Treating macrophages with these exosomes also promoted NPC cells growth in an in vitro co-culture model. RNA sequencing data showed that LMP1_exosomes restrained the function of antigen presentation, while enhanced the macrophage stimulating 1 receptor (MST1R) and IL-10 signalings in macrophages. Mechanistic studies showed that LMP1-C-terminal activating region 2 (CTAR2) domain contributed to the activation of MST1R. Further, the transfer of LMP1 to macrophages enhanced pro-inflammatory signalings, which were partially suppressed by the use of MST1R inhibitor (SYN1143). Results of NPC tumor bulk RNA sequencing data demonstrated that higher level of MST1R was correlated with advanced stage of NPC (GSE102349) (p-value < 0.001). The role of EBV product-containing exosomes in modulating the immune responses of macrophages was further dissected using single-cell RNA sequencing (scRNA-seq) technique on exosome-treated cell suspension freshly isolated from oral tumors. Analytical results revealed that treating cells with EBV product-containing exosomes increased the proportion of immunosuppressive macrophages. Cell-cell communication analysis found that exosome-treated macrophages could modulate T cell cytotoxicity through ligand-receptor communications, crucial interactions are between SPP1 and ITGA4, as well as Galectin 9 and CD44/45. Collectively, our findings revealed that LMP1-containing exosomes facilitate immunosuppressive macrophage polarization via MST1R activation and contribute to NPC progression.
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