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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡幸真(Hsing-Chen Tsai) | |
| dc.contributor.author | Yu-Ting Lee | en |
| dc.contributor.author | 李侑庭 | zh_TW |
| dc.date.accessioned | 2023-03-19T21:30:29Z | - |
| dc.date.copyright | 2022-10-07 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-26 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84068 | - |
| dc.description.abstract | 肺癌為全球癌症死亡率之冠，即使現已開發免疫檢查點抑制劑，為肺癌治療帶來新曙光，仍有一定比例的患者因體內耗竭T細胞(Exhausted T cell, TEX)受到既有表觀遺傳基因體 (epigenetic landscapes)的限制，無法完全恢復成為功能T細胞，而導致其治療效果不彰。惡性胸水 (Malignant pleural effusion, MPE) 為晚期肺腺癌患者常見的併發症，胸水內含有轉移的癌細胞以及大量耗竭T細胞，為免疫失能之腫瘤微環境。本實驗室先前的研究中，透過145個表觀遺傳藥物進行藥物篩選，發現含溴結構域抑制劑 (bromodomain inhibitors, BETi) 中的JQ1在體外能夠顯著改善惡性胸水內耗竭性T細胞的功能。然而JQ1改善耗竭性T細胞功能的機制仍未清楚，因此本研究的目標為進一步探討JQ1調節耗竭性T細胞免疫功能的機制。首先，我們進行 RNA定序以鑑定在耗竭性T細胞中受JQ1影響的基因譜，從中發現JQ1治療後顯著上調耗竭性T細胞中代謝相關途徑的基因群，其中包括氨基酸代謝及五碳糖磷酸途徑等。我們進一步藉由不同的代謝抑制劑阻斷相關的代謝途徑，發現氨基酸代謝抑制劑Difluoromethylornithine (DFMO)可顯著降低JQ1提升T細胞的多功能性，表示JQ1可能是透過氨基酸代謝調控T細胞的功能。接著，我們透過即時聚合?連鎖反應驗證JQ1治療顯著上調編碼鳥胺酸脫羧? (ornithine decarboxylase, ODC)的基因ODC1，並利用小分子干擾RNA (small interfering RNA, siRNA)的方式抑制ODC1表現後，釐清JQ1確實透過提升ODC1基因表現而增加分泌細胞因子。最後，我們使用定序轉座?可接觸的染色質分析法(Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) 發現經JQ1處理後，惡性胸水中耗竭性T細胞的ODC1基因，其位於啟動子附近的染色質可及性增加，並由單細胞定序 (single-cell RNA sequencing, scRNA-seq) 發現JQ1可能透過組蛋白修飾影響衰竭性T細胞的基因表現。綜合上述結果，本研究探討JQ1透過表觀遺傳調控代謝重整的方式於晚期肺癌患者體內的耗竭性T細胞進行免疫調節作用，而可為發展肺癌惡性胸水的表觀遺傳治療提供新方向。 | zh_TW |
| dc.description.abstract | Lung cancer causes the leading mortality rate worldwide. Even if immune checkpoint inhibitors (ICIs) have been developed to bring light to lung cancer treatment, there are still patients experiencing poor therapeutic efficacy of ICIs, possibly due to static epigenetic landscapes in exhausted T cells (TEX). Malignant pleural effusion (MPE) is a common complication in patients with advanced lung adenocarcinoma. MPE is an immune dysfunctional tumor microenvironment containing metastatic cancer cells and TEX. In our previous study, we performed a drug screening of 145 epigenetic drugs and found that JQ1 and other bromodomain inhibitors (BETi) significantly improved the function of TEX in MPE ex vivo. However, the mechanism by which JQ1 improved the function of exhausted T cells remained unclear, and we aimed to further explore the mechanism in this study. First, we performed genome-wide RNA-seq analysis to investigate transcriptomic profiles affected by JQ1 in the TEX of MPE. Gene set enrichment analysis (GSEA) analysis revealed that several metabolic pathway-related gene sets were significantly upregulated by JQ1 treatment, including amino acid metabolism and pentose phosphate pathway, etc. Next, we blocked individual metabolic pathways with respective inhibitors and found that difluoromethylornithine (DFMO), an inhibitor for amino acid metabolism, significantly abolished the JQ1-mediated enhancement of T cell polyfunctionality. The data indicated that JQ1 may regulate the function of T cells through amino acid metabolism. We also verified that JQ1 significantly upregulated ornithine decarboxylase (ODC1), an enzyme involved in polyamine synthesis, in primary malignant pleural effusion T cells by real-time polymerase chain reaction (RT-PCR). Through siRNA-mediated knock-down experiments, we discovered that ODC1 enhanced effector functions in T cells. Finally, we dissected the epigenetic landscape of exhausted T cells in malignant pleural effusions after JQ1 treatment by performing an Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). We found that JQ1 increased chromatin accessibility at the promoter region in ODC1. Moreover, analysis of single-cell RNA sequencing (scRNA-seq) indicated that JQ1 may affect histone modifications in exhausted CD8 T cells. In summary, JQ1 exerted immunomodulatory effects on TEX in patients with advanced lung cancer through epigenetic regulation of metabolic reprogramming. This study may provide a new direction for the development of epigenetic immunotherapy for malignant pleural effusion in lung cancer. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T21:30:29Z (GMT). No. of bitstreams: 1 U0001-2908202210354000.pdf: 8776957 bytes, checksum: 2c7071ff2d9a32e7fa535b26a85de87f (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 序言及謝辭 1 中文摘要 2 Abstract 3 List of Figures 10 List of Tables 12 List of abbreviations 13 1. Introduction 14 1.1. Lung cancer 14 1.1.1. The epidemiology of lung cancer 14 1.1.2. Types of lung cancer 14 1.2. Tumor immune microenvironment 15 1.2.1. Tumor microenvironment in MPE 15 1.2.2. Characterization of exhausted T cells 16 1.2.3. Immune checkpoints inhibitors therapy in NSCLC 16 1.2.4. Current challenges of immune checkpoint inhibitors therapy in NSCLC 17 1.3. Epigenetic therapy in immune-oncology 18 1.3.1. Epigenetic regulation 18 1.3.2. The epigenetic landscape of T cell exhaustion 19 1.3.3. Epigenetic regulation in T cell exhaustion 19 1.3.4. Bromodomain and extra-terminal inhibitors (BETi) 20 1.4. Immunometabolism 21 1.4.1. Definition of immunometabolism 21 1.4.3. Metabolic features of T cell activation 23 1.4.4. Metabolic reprogramming in T cell exhaustion 24 1.4.5. Pentose phosphate pathway 25 1.4.6. Polyamine metabolism 26 1.5. Previous studies from our laboratory 27 2. Aim of the study 29 3. Materials and Methods 30 3.1. Malignant pleural effusion (MPE) 30 3.2. Lymphocytes isolated from MPE 30 3.3. Cell culture 30 3.3.1. Cryopreservation of lymphocytes 30 3.3.2. Ex vivo expansion of primary T cells 31 3.3.3. Jurkat cell 31 3.3.4. T cell Exhaustion model in Jurkat cells 31 3.4. Drug treatment on T cells 32 3.5. siRNA transfection 33 3.6. Flow cytometry analysis 33 3.6.1. PMA and ionomycin T cell stimulation 33 3.6.2. Intracellular cytokine staining 33 3.6.3. Inhibitory receptors staining 34 3.6.4. Staining of Ki-67 34 3.7. IL-2 Enzyme-linked immunosorbent assay (ELISA) 35 3.8. Quantitative RT-PCR analysis 36 3.8.1. RNA extraction 36 3.8.2. DNase I treatment 37 3.8.3. Reverse transcription (RT) reaction 37 3.8.4. Real-time PCR 37 3.8.5. RNA sequencing data analysis 38 3.8.6. Gene set enrichment analysis (GSEA) 39 3.8.7. Functional enrichment analysis 39 3.9. Single-cell RNA sequencing (scRNA-seq) 39 3.10. Bioinformatic analysis of the scRNA-seq data 40 3.10.1. Pre-processing of Single-cell RNA sequencing raw data 40 3.10.2. Gene marker analysis 41 3.11. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) 41 3.11.1. Bioinformatic analysis of the ATAC-seq 42 3.12. Quantification and statistical analysis 43 4. Results 44 4.1. Gene characterization of tumor-reactive T cells in malignant pleural effusions 44 4.1.1. T cells in MPE exhibit a widely altered gene profile after JQ1 treatment 44 4.1.2. JQ1 upregulates gene expression related to metabolic pathways 44 4.1.3. JQ1 reinvigorated T cell exhausted-related gene expression 45 4.2. Metabolic inhibition assay for targeting the potential metabolic pathways on which JQ1 elevates T cell polyfunctionality 46 4.2.1. Metabolic pathways and their inhibitors involved in this study 46 4.2.2. The efficacy of metabolic inhibitors maintained for 48 hours in general 47 4.2.3. Block of polyamine biosynthesis suppressed JQ1 from elevating T cell polyfunctionality 48 4.3. Genome-wide mRNA-sequencing validation on MPE T cells 48 4.3.1. Leading-edge genes from GSEA represent the most contribution of enrichment scores specific to the dataset 48 4.3.2. JQ1 significantly upregulated ODC1 in amino acid metabolism in MPE T cells 49 4.3.3. Genes in the pentose phosphate pathway and fatty acid metabolism were not upregulated after JQ1 treatment 49 4.4. ODC1 gene knockdown assay to specify the immunomodulatory effect between JQ1 and polyamine metabolism 49 4.4.1. JQ1 exerts its immunomodulatory effects through upregulating ODC1 49 4.5. The epigenetic landscape of MPE in bromodomain inhibitor JQ1 intervention 50 4.5.1. Bromodomain inhibition induces epigenetic patterns in tumor-reactive T cells in MPE 50 4.6. The single-cell transcriptomics of CD8+ T cells in MPE after JQ1 intervention 51 4.6.1. Different subsets were expressed in CD8+ T cells of MPE 51 4.6.2. JQ1 upregulated ODC1 and effector cytokine genes in the subset of CD8+ effector T cells and exhausted T cells 53 4.6.3. Histone modification drove the transcriptional regulation of JQ1 53 5. Discussion 55 6. Conclusion 60 7. Reference 84 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討含溴結構域抑制劑對肺癌反應T細胞的代謝重整作用 | zh_TW |
| dc.title | Metabolic reprogramming effect of bromodomain inhibitor JQ1 on tumor-reactive T cells in lung cancer patients | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林妙霞(Miao-Hsia Lin),邱彥霖(Yen-Ling Chiu) | |
| dc.subject.keyword | 惡性胸水,表觀遺傳學,耗竭性T細胞,代謝重整, | zh_TW |
| dc.subject.keyword | Malignant pleural effusion,Epigenetics,T cell exhaustion,Metabolic reprogramming, | en |
| dc.relation.page | 92 | |
| dc.identifier.doi | 10.6342/NTU202202911 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2022-09-26 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 毒理學研究所 | zh_TW |
| 顯示於系所單位： | 毒理學研究所 | |
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