粒線體Lon蛋白誘導的腫瘤細胞外泌囊泡對於樹突細胞免疫抑制的角色

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研究生：	李語婕 Lee, Yu-Chieh
論文名稱：	粒線體Lon蛋白誘導的腫瘤細胞外泌囊泡對於樹突細胞免疫抑制的角色 The Role of Mitochondrial Lon-induced Extracellular Vesicles from Tumor in the Immunosuppression of Dendritic Cells
指導教授：	李岳倫 Lee, Alan Yueh-Luen 汪宏達 Wang, Horng-Dar
口試委員:	賴品光 Lai, Charles Pin-Kuang 楊立威 Yang, Lee-Wei
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2024
畢業學年度：	112
語文別：	英文
論文頁數：	62
中文關鍵詞：	Lon蛋白酶、外泌囊泡、樹突細胞、腫瘤微環境、代謝體
外文關鍵詞：	Lon, extracellular vesicles, dendritic cells, tumor microenvironment, metabolites
相關次數：	點閱：58 下載：0
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粒線體Lon是一種壓力蛋白，在癌症中對於在不同壓力下維持粒線體功能及細胞存活扮演重要的角色。Lon的上調透過影響細胞凋亡、線粒體自噬、代謝和腫瘤微環境來促進腫瘤的生長和轉移。先前的研究已證實，Lon上調的癌細胞可釋放細胞外泌囊泡(Extracellular Vesicles, EVs)到腫瘤微環境，會塑造出抗發炎環境。EVs中的物質，包含蛋白質、miRNA以及DNA等，可能對免疫細胞產生免疫抑制的反應。在本研究中，我們想要找到來自Lon過度表達的癌細胞所分泌的EVs的新穎分子，能夠調節樹突細胞（DCs）免疫反應。首先，我們證實了來自過表現Lon蛋白的癌細胞所分泌的EVs會導致樹突細胞的抗發炎表現。接著，我們透過對EVs進行代謝體分析，發現苯丙胺酸，酪胺酸以及色胺酸生物合成和其他胺基酸生物合成相關的代謝物，在過量表現Lon蛋白的癌細胞所分泌的EVs中會增加。我們進一步發現，在增加的代謝物中，L-酪胺酸對BMDCs產生了抗炎效果。總結，本研究發現對樹突細胞造成抗炎表型的潛在關鍵代謝物，這個由過量表現Lon的癌細胞所分泌的代謝體，可能進一步幫助腫瘤形成支持其生長的腫瘤免疫環境。

Mitochondrial Lon is a stress protein which plays a crucial role in maintaining mitochondria function under different stress in cancer. Upregulation of Lon is involved in tumor growth, metastasis and proliferation by affecting the apoptosis, mitophagy, metabolism and tumor microenvironment (TME). Extracellular vesicles (EVs) released by cancer cells shape an anti-inflammatory environment in the TME. Previous studies have demonstrated that the substance within EVs, including protein, miRNA, DNA, cause immunosuppressive effects on immune cells. In this study, we aimed to find a new biomolecule within EVs from Lon-overexpressed cancer cells that plays a crucial role in modulating the immune response of dendritic cells (DCs), which are key antigen-presenting cells in the TME. First, we showed that EVs from Lon-overexpressed cancer cells led to anti-inflammatory phenotypes of dendritic cells. Next, by conducting metabolomics analysis from EVs, we found that the metabolites involved in phenylalanine, tyrosine, and tryptophan biosynthesis and other amino acids biosynthesis were increased in EVs from Lon-overexpressed cancer cells. We further discovered that among the increased metabolites in EVs, L-tyrosine cause an anti-inflammatory effect on BMDCs. In sum, this study shed a light on the potential metabolite secreted by cancer cells which may contribute to the anti-inflammatory phenotype of DCs, which might further assist tumor to form a pro-tumor immunity environment to support its growth.

摘要  i
Abstract  ii
1. Introduction  1
1.1 Tumor microenvironment  1
1.2 Mitochondrial Lon protease  2
1.3 Extracellular vesicles in tumor progression  3
1.4 Immune response in the TME  4
1.5 Dendritic cells in the TME  6
2. Rationale and specific aims  8
2.1 Rationale  8
2.2 Specific Aims  8
3. Materials and methods  9
3.1 Materials  9
3.2 Methods  12
4. Results  25
4.1 Overexpression of mitochondria Lon in B16F10 and MC38  25
4.2 Identification of extracellular vesicles (EVs)  25
4.3 The effect of Lon-induced EVs from B16F10 on the function of DC2.4  27
4.4 DC2.4 showed anti-inflammatory phenotype after treating Lon-induced EVs from MC38.  28
4.5 Treatment of Lon-induced EVs from cancer cells caused regulatory phenotype of BMDCs  29
4.6 KEGG enrichment analysis of Lon overexpression MC38-secreted EVs showed upregulation in metabolites involving in multiple metabolism pathways.  31
4.7 Treatment of metabolites upregulated in Lon-induced EVs to BMDCs revealed preliminary information about the cause of phenotype changes in BMDCs.  33
4.8 Conclusion  34
5. Discussion  35
5.1 Limitation of DC2.4 in the experiments  35
5.2 Quantification methods for EVs treatment on dendritic cells  35
5.3 The unknown mechanism behind the increase of EVs secretion from cancer cells after Lon overexpression  37
5.4 The evidence of anti-inflammatory effects of L-tyrosine on DCs remains to be reinforced.  37
6. Figures and Table  39
Figure 1. Expression of Lon plasmid transfected in B16F10 and MC38 cell lines.  39
Figure 2. Identification of purified EVs from B16F10.  40
Figure 3. Identification of purified EVs from MC38.  41
Figure 4. The effect of Lon-induced EVs from B16F10 treatment on the function of DC2.4.  43
Figure 5. The effect of Lon-induced EVs from MC38 on the function of DC2.4.  45
Figure 6. The effects of Lon-induced EVs from B16F10 on the phenotype of BMDCs.  47
Figure 7. The effects of Lon-induced EVs from MC38 on the phenotype of BMDCs.  49
Figure 8. KEGG enrichment analysis of differentially expressed metabolites in EVs from MC38 transfected with control vector or Lon plasmids.  50
Figure 9. Detailed analysis of metabolites contributed to upregulated metabolism pathway within EVs secreted from MC38.  52
Figure 10. Preliminary findings on the functional changes of BMDCs caused by metabolites treatment  56
Table 1. The list of primers.  57
7. References  58


                                

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