含EBV病毒產物之外泌小體經由活化纖維母細胞重塑腫瘤微環境;EBV Products-containing Exosomes Remodel the Tumor Microenvironment by Activating Stromal Fibroblasts

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题名:	含EBV病毒產物之外泌小體經由活化纖維母細胞重塑腫瘤微環境;EBV Products-containing Exosomes Remodel the Tumor Microenvironment by Activating Stromal Fibroblasts
作者:	李柏儒;Lee, Po-Ju
贡献者:	系統生物與生物資訊研究所
关键词:	EBV病毒;外泌小體;纖維母細胞;腫瘤微環境;EBV;exosomes;fibroblast;tumor microenvironment
日期:	2021-11-22
上传时间:	2021-12-07 11:54:45 (UTC+8)
出版者:	國立中央大學
摘要:	第三型鼻咽癌與EBV感染有密切關聯且此類型腫瘤組織常有大量免疫細胞浸潤。許多文獻已證實浸潤於鼻咽癌的免疫細胞與癌細胞的交互作用會影響病人預後。另一方面，腫瘤中的纖維化組織與較差的病人預後相關。然而，癌細胞與周邊纖維母細胞之間的相互作用機轉仍待深入探討。在本研究中，我們發現EBV+-鼻咽癌細胞所釋出的外泌小體含EBV潛伏膜蛋白-1(LMP1)及纖維化標誌分子FAP。此外泌小體可被周圍基質環境中的纖維母細胞吞噬，刺激纖維母細胞產生形態上的變化與收縮力的增強，並且活化纖維母細胞進而重塑微環境。轉錄體分析及經QPCR與ELISA驗證結果顯示鼻咽癌的外泌小體明顯影響與組織纖維化功能相關的基因群表現及促發炎細胞激素(IL-6, IL-8, MCP-1)的釋放。此外，細胞實驗結果顯示外泌小體的刺激使纖維母細胞的標誌FAP與活化型YAP1蛋白及其調控的下游分子(CYR61, CTGF, IGFBP3)表現量上升。小鼠腫瘤異體移植實驗結果發現被外泌小體刺激的纖維母細胞會促進腫瘤生長與微環境纖維化。同時，在小鼠腫瘤組織切片染色結果顯示外泌小體刺激使活化型YAP1在間質纖維母細胞中表現量上升，且與FAP表現有正相關性r=0.7671, p=0.0001, Spearman correlation test)。於鼻咽癌病人切片染色結果亦顯示活化型YAP1與FAP的正相關性(r=0.3749, p=0.0157, Spearman correlation test)。這意味著YAP1對調控外泌小體所引發的纖維母細胞活化扮演著重要的角色。使用YAP1的抑制劑Saracatinib 和Verteporfin於纖維母細胞可以有效降低因外泌小體的刺激而誘發的功能性變化，且可抑制纖維母細胞對癌細胞的促生長效應。綜合以上結果，本研究闡明腫瘤細胞藉由分泌外泌小體與纖維母細胞進行交互作用而導致腫瘤組織趨向一個有利於癌症進展的微環境，提供以YAP1抑制劑瓦解鼻咽腫瘤微環境而達到抑制癌細胞生長的理論基礎。 ;Type ? nasopharyngeal carcinoma is closely associated with EBV-infection and characterized by heavy lymphocyte infiltration. It is recognized that interactions between cancer cells and immune cells have profound effects on patients’ outcomes. Further, fibrotic response in the tumor tissue is correlated with poorer prognosis in NPC patients. Nevertheless, the intercommunications between stromal fibroblasts and NPC cells remain relatively unexplored. In the present study, we found that exosomes derived from EBV+NPC cells contained EBV-encoded latent membrane protein 1 (LMP1) and FAPprotein. Treating fibroblasts derived from NPC biopsies with these exosomes induced morphological changes and an enhanced contraction ability. Results of transcriptomic analyses revealed that exosome stimulation in fibroblasts predominantly affected expression of genes involved in function of fibrotic response. Fibrosis-associated genes such as IL-6, IL-8, and MCP-1 were validated by QPCR and ELISA assays. Exosome stimulation induced fibroblast activation as evidenced by increased expressions of FAP active YAP1, and YAP1 downstream molecules (CYR61, CTGF, IGFBP3). Results of mouse xenografted model demonstrated that exosome-stimulated fibroblasts promoted tumor growth. Immunohistochemical data showed a positive correlation between expressions of FAP and active YAP1 (r=0.7671 and p=0.0001, Spearman correlation test). An enhanced fibrotic response was noticed within exosome-treated tumors. The association among active YAP1, FAP, and fibrotic response were also found in human NPC biopsies (r=0.3749 and p=0.0157, Spearman correlation test). Targeting YAP1 by utilizing Saracatinib and Verteporfin markedly blunted EBV exosomes-mediated fibroblast activation and functional effects. Collectively, our data provide evidence to better understand the interactions among EBV, NPC cells, and stromal fibroblasts via exosomal transmission, which facilitates the establishment of a pro-tumor microenvironment. Furthermore, our data suggest that the use of YAP1 inhibitors might be a potentially effective strategy in treating NPC with desmoplastic response within tumors.
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