腫瘤外質體對調節型T 細胞的代謝與分化上之影響

Abstract

在有氧氣環境中，癌細胞仍偏好使用糖解作用來產生能量。當葡萄糖因癌細胞使用糖解作用而消耗時，微環境中的的免疫淋巴球的葡萄糖來源即受到影響，進而影響到免疫細胞抗癌的功能。目前許多研究指出，細胞代謝路徑的改變，例如糖解作用、有氧磷酸化、脂肪酸代謝等皆會影響輔助型T細胞 (T helper cells) 的分化並影響他們的免疫功能。在癌症的研究上，發現癌細胞所分泌的外質體會先至其預轉移的部位進行微環境的調節，使癌細胞在後續轉移的過程中更順利。在之前的研究中，我們已經證實癌細胞所分泌的外質體可以促進調節型T細胞 (regulatory T cells) 的產生，而調節型T細胞正是使癌細胞逃離免疫系統監控的關鍵。 本研究中，我們假設，胃癌分泌的外質體可能會透過調節naïve T細胞的代謝途徑使其分化成FOXP3+調節型T細胞。我們首先將胃癌病人血液中的外質體分離出來，並透過檢測細胞的糖解能力狀況，探討胃癌所分泌的外質體對於T 細胞在分化過程中的代謝影響。細胞代謝的狀況由螢光偵測，同時也透過西方墨點法觀察T細胞中跟代謝相關的酵素與蛋白的表現，包括己糖激酶(Hexokinase II)， 肉鹼棕櫚醯基轉移酶 (CPT1A) 以及葡萄糖載體蛋白 (GLUT1)。 實驗結果發現，無論是由乙型轉化生長因子(rhTGF-β)或胃癌病患血液分離出來之外質體所誘導的調節性T細胞，均有較高的糖解速率。在西方墨點法的實驗中，由乙型轉化生長因子所誘導的調節性T細胞，其己糖激酶，肉鹼棕櫚醯基轉移酶以及葡萄糖載體蛋白都明顯高於未分化的naïve T細胞。而由胃癌病患血液分離出來之外質體體誘導的調節性T細胞組別中，其己糖激酶與肉鹼棕櫚醯基轉移酶的表現均高於乙型轉化生長因子所誘導的調節性T細胞。 吾人的結果顯示，癌細胞外質體不僅與乙型轉化生長因子有相同的作用能將T細胞誘導成為調節性T細胞，同時也提升其糖解代謝能力，甚至在糖激酶與肉鹼棕櫚醯基轉移酶的表現均高於乙型轉化生長因子所誘導的調節性T細胞。因此，癌細胞所分泌的外質體中，或許含有能夠影響調節性T細胞代謝能力的因子。

Cancer cells regularly use glycolysis despite the presence of oxygen, which promotes the depletion of extracellular glucose in tumor microenvironment and causes tumor infiltrating T cells (TILs) dysfunction. Recently, many studies indicated the modulations of major intracellular metabolic pathways, such as glycolysis, oxidative phosphorylation and fatty acid oxidation in T cells could influence the differentiation of the T helper subtype and shaped their function. In our previously study, we have showed tumor-derived exosomes could promote regulatory T cells expansion, facilitate tumor cells to escape from immune surveillance. In this study, we hypothesis that tumor-derived exosomes promote naïve T cells to differentiate into FOXP3+ regulatory T cells by reprogramming the metabolism pathway. The tumor-derived exosomes were isolated from the peripheral blood of gastric cancer (GC) patients. The metabolic phenotypes of glycolysis of T cells with or without GC-derived exosomes treatment were detected by the time-resolved fluorescence probes. The expression of metabolic enzymes, including Hexokinase II, CPT1A and GLUT1 in Treg cells and GC-derived exosomes-induced Treg cells were analyzed by Western blot. In our results, the expression of both rhTGF-β and GC-derived exosomes treated T cells have higher glycolysis rate and Hexokinase II, CPT1A expression than naïve T cells. Compared to rhTGF-β induced regulatory T cells, GC-derived exosomes induced more expression of HK2 and CPT1A in regulatory T cells. Therefore, GC-derived exosomes may regulate cellular metabolism to affect the differentiation of FOXP3+regulatory T cells.