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研究生： 葉姵暄
Ye, Pei-Hsuan
論文名稱： 針對精胺酸代謝的癌症療法
Targeting Arginine Metabolism for Cancer Therapy
指導教授： 賴明德
Lai, Ming-Derg
學位類別： 碩士
Master
系所名稱： 醫學院 - 生物化學暨分子生物學研究所
Department of Biochemistry and Molecular Biology
論文出版年： 2021
畢業學年度： 109
語文別： 英文
論文頁數： 97
中文關鍵詞： 癌症免疫療法 、精胺酸酶 、L-正纈胺酸 、ADI-PEG 20
外文關鍵詞： Cancer immunotherapy, Arginase, L-Norvaline, ADI-PEG 20
相關次數： 點閱：104  下載：0 
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    • 癌症免疫療法是一種藉由強化自身免疫系統的治療方法。然而，腫瘤細胞會透過許多不同的機制來躲避免疫系統的攻擊，例如發展出免疫抑制型的腫瘤微環境。腫瘤相關巨噬細胞和髓樣來源的抑制細胞皆屬於微環境中的免疫抑制型細胞，這些細胞會抑制CD8+ T細胞的毒殺作用。免疫抑制型細胞有一個共同點：大量表達精胺酸酶。精胺酸酶是尿素循環中的其中一個酵素，功能是將精胺酸水解成鳥胺酸和尿素。過多的精胺酸酶會使環境中的精胺酸不足，導致T細胞表面糖蛋白CD3 zeta鏈下調並進而抑制T細胞的活化。因此，精胺酸酶抑制劑被認為是一種治療癌症的策略。令人感到有趣的是，精胺酸剝奪法也可以是治療癌症的手段。精胺酸亞胺酶是其中一種可以降解精胺酸的酵素。ADI-PEG 20是精胺酸脫亞胺酶經過分子量20,000的聚乙二醇修飾後所產生的分子，目前正在針對精胺琥珀酸合成酶缺陷型的癌症進行臨床試驗。因此，我們假設L-正纈胺酸(一種非競爭性精胺酸抑制劑)和ADI-PEG 20的複合治療會有更好的抗癌效果。在本研究中，我們發現濃度低於0.1 mg/ml的L-正纈胺酸不會對體外培養的癌細胞株產生毒殺作用但卻能夠抑制體內的腫瘤生長。另外，透過生物資訊方法分析RNA定序的結果可以得知，細胞經過L-正纈胺酸處理後，那些差異表達的基因參與在許多和免疫相關的途徑，這些分析結果顯示L-正纈胺酸可能可以透過癌細胞來調控免疫反應。此外，使用L-正纈胺酸和ADI-PEG 20的複合療法治療黑色素瘤，可以產生更強的抗癌效果。透過流式細胞技術分析顯示，小鼠在接受複合治療後，會有較多的CD4+ T細胞和較少的Ly6G+ 髓樣來源的抑制細胞浸潤到腫瘤內。另外，透過單細胞RNA定序的技術更進一步分析複合療法對於腫瘤微環境中多種不同免疫細胞的影響，藉由分析定序資料得知，複合療法會增加CD4+和CD8+ T細胞浸潤到腫瘤，其他種類的免疫細胞在腫瘤中所佔的比例也會改變。本研究暗示L-正纈胺酸可能是抗癌免疫反應中的調節劑，並發現L-正纈胺酸和ADI-PEG 20複合療法的潛力。

    Immunotherapy is a type of cancer treatment that boosts patients' immune systems against malignant cells. Unfortunately, several studies have suggested that cancer cells are able to evade immune attack by a variety of mechanisms, including development of an immunosuppressive tumor environment. Numerous immunosuppressive cells present within the tumor microenvironment, such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) that inhibit cytotoxic activity of CD8+ T cells. These immunosuppressive cells express high levels of arginase in common. Arginase is an enzyme of the urea cycle that catalyzes the conversion of L-arginine to L-ornithine and urea. Arginase causes arginine insufficiency in the environment and impairs T cell activation by down-regulating CD3ξ chain on T cells. Hence, arginase inhibitor is considered to be a potential drug for cancer therapy. It is interesting to note that arginine deprivation is also a treatment strategy for cancer. PEGylated arginine deiminase (ADI-PEG 20) is an arginine-depleting enzyme used in clinical trial for ASS1-negative cancer. Consequently, we hypothesized L-Norvaline (a non-competitive arginase inhibitor) may trigger stronger anticancer response in combination with ADI-PEG 20. In this study, we found L-Norvaline showed no cytotoxic effect in cancer cell line at concentration lower than 0.1 mg/mL but inhibited tumor growth in vivo. Pathway analysis based on RNA-seq data showed that the differential expression genes (DEGs) were significantly enriched in some immune-related pathways. It was suggested that L-Norvaline possibly regulate immunity through cancer cell. In addition, treatment with L-Norvaline in combination with ADI-PEG 20 would generate stronger antitumor response against B16F10 melanoma. Combination therapy showed a trend of higher infiltration of CD4+ T cells and lower infiltration of Ly6G+ MDSCs by flow cytometric analysis. Furthermore, single-cell RNA-seq was performed to analyze in depth the changes of populations of tumor-infiltrating immune cells after combination treatment with L-Norvaline and ADI-PEG 20. The single-cell RNA-seq data demonstrated that tumor-infiltrating CD4+ and CD8+ T cells increased following the combination therapy. Besides, populations of other types of immune cells in the tumors are altered. This study implied the possibility of L-Norvaline as a modulator of immune response in cancer and provided a new potential therapy combined with ADI-PEG 20.

    中文摘要 I 英文摘要 II 誌謝 IV Table of Contents V Lists of Tables VII List of Figures VIII List of Supplementary Figures IX Introduction 1 I. Cancer immunotherapy 1 II. Tumor evasion and immunosuppression in the tumor microenvironment 1 III. Immunomodulatory role of arginases 2 IV. Arginase as a potential target in cancer treatment 3 V. Targeting arginine-dependent cancers with ADI-PEG 20 4 VI. Research aim 5 Materials and Methods 6 I. Cell culture 6 II. MTT assay 10 III. RNA sequencing 11 IV. Animal experiments 13 V. Flow cytometry 14 VI. Single-cell RNA-seq 17 VII. Statistical analysis 20 Results 21 I. L-Norvaline exerts no cytotoxic effect on tumor cells in vitro. 21 II. L-Norvaline treatment results in anti-tumor effects in vivo. 21 III. Anti-tumor effects of L-Norvaline need intact immune system. 22 IV. L-Norvaline treatment is involved in immune-related pathways. 22 V. The combination of L-Norvaline and ADI-PEG 20 significantly reduces the tumor growth. 23 VI. Elucidating distinct expression profiles of tumor-infiltrating immune cells after analysis with single-cell RNA-seq. 24 VII. Single-cell RNA-seq data reveals combined treatment of L-Norvaline and ADI-PEG 20 promotes tumor infiltration of CD4+ and CD8+ T cells. 25 Discussion 26 Conclusion 30 Reference 31 Tables 36 Figures 67 Supplementary figures 92

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