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研究生： 陳宥蓁
Chen, Yu-Chen
論文名稱： 整合液體介電泳與光固化水膠之腫瘤免疫暨藥物微系統晶片研發
Tumor-lab-on-chip integrating liquid dielectrophoresis and hydrogel for cancer immunotherapy application
指導教授： 劉承賢
Liu, Cheng-Hsien
口試委員: 張晃猷
Chang, Hwan-You
徐琅
Hsu, Long
陳皇銘
Chen, Huang-Ming
盧向成
Lu, Shiang-Cheng
學位類別： 博士
Doctor
系所名稱： 工學院 - 動力機械工程學系
Department of Power Mechanical Engineering
論文出版年： 2023
畢業學年度： 111
語文別： 英文
論文頁數： 72
中文關鍵詞： 腫瘤晶片 、免疫治療 、化學治療 、3D 微環境
外文關鍵詞： Tumor-lab-on-chip, immunotherapy, chemotherapy, 3D microenvironment
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    • 化療/免疫療法的結合治療是臨床上具成效的治療方式。在KEYNOTE-189中所報導的III期臨床試驗結果顯示，將化療藥物與免疫治療藥物結合使用相比於單獨使用化療藥物後的死亡率顯著地降低，但目前對其機制尚未能清楚地被驗證，且治療的組合也需針對患者進行各別調整，即個人化醫療。腫瘤晶片（Tumor-lab-on-chip）的主要應用目標為透過體外的肺癌免疫模型來模擬腫瘤微環境（TME），進而為研究、治療提供具潛力的方案。腫瘤晶片中使用液體介電泳（LDEP）在氧化銦錫（ITO）電極軌道上驅動含有癌細胞的明膠甲基丙烯酰（GelMA），來形成腫瘤樣水凝膠。在其中，以Jurkat T 細胞包圍著凝固的GelMA-A549細胞來形成類似於癌症免疫的微環境。在腫瘤晶片上癌細胞（A549）會以IL-1β處理，進而誘導Jurkat T細胞進入GelMA的浸潤現象。此結果與單一使用化療藥物相比，在結合化療/免疫療法後的癌細胞死亡率顯著增加約35%。此外，結合化療/免疫療法所造成的癌細胞死亡分佈的趨勢也顯示其獨特的理論機制，這些結果顯示本研究之腫瘤晶片於癌症患者的個人化藥物治療中有著前景廣闊的應用性。

    The combo treatment of chemo/immunotherapy is a promising method in treating cancer patients. The results of the phase III KEYNOTE-189 clinical trial (previously untreated metastatic NSQ NSCLC) showed that chemotherapy drugs (carboplatin/cisplatin + pemetrexed) plus immunotherapy drug (pembrolizumab) reduced mortality compared with chemotherapy drug alone, in which the underlying mechanism has not been well demonstrated. The treatment combination also needs to be tuned for each patient, i.e., personalized medicine. Tumor-lab-on-chip aims to mimic the tumor microenvironment with an in-vitro lung cancer-immune model to provide a potential solution for this application. Gelatin methacryloyl-cancer cells were driven on the indium-tin-oxide electrode track using liquid dielectrophoresis to form the tumor-like hydrogel. Jurkat T cells surrounded the solidified GelMA-A549 cells resembling the cancer-immune microenvironment. The immune-cell infiltration phenomena of Jurkat T cells into the GelMA were induced via IL-1β-treated A549 cells on the tumor-lab-on-chip. The death of cancer cells significantly increased by ~35% after chemo/immunotherapy combination treatment compared with the single treatment. The distribution of dead cancer cells treated with chemo/immunotherapy revealed their distinct theoretical mechanisms, indicating the promising applications of this tumor-lab-on-chip for cancer patients in personalized medicine treatments.

    Abstract............................i 中文摘要............................ii Table of Contents..................iii List of Figures....................v List of Tables.....................vii 致謝...............................viii Abbreviations......................x Chapter 1 Introduction.............1 1.1 Current status of lung cancer treatment........3 1.1.1 Chemotherapy...................................4 1.1.2 Immunotherapy..................................5 1.2 Tumor microenvironment (TME)...................7 1.2.1 2-dimensional cell culture.....................9 1.2.2 3-dimensional cell culture.....................9 1.2.3 Cell culture on the chip.......................10 1.2.4 Cell co-culture................................11 1.2.5 Extracellular matrix – GelMA...................12 1.3 Bio-MEMS and lab on a chip.....................14 1.3.1 Electro-microfluidic platform application......15 1.4 Liquid dielectrophoresis.......................17 Chapter 2 Materials and methods.......................20 2.1 Cell line......................................20 2.2 Human lung adenocarcinoma cell line (A549).....20 2.3 Human immune cell, T cells (Jurkat T)..........21 2.4 Medium for A549 and Jurkat T cells.............22 2.5 Biocompatible material Gelatin methacryloyl (GelMA)......22 2.6 Cell tracker...................................24 2.7 LIVE/DEAD assay................................24 2.8 Fluorescence image analysis....................25 2.9 Immunotherapy drugs............................26 2.10 Chemotherapy drugs.............................26 2.11 Tumor-lab-on-chip fabrication..................27 2.12 Experimental equipment setup...................28 2.13 Tumor-lab-on-chip operation....................29 Chapter 3 Results.....................................31 3.1 The cancer cells mortality and T cells infiltration ability in GelMA..........................................31 3.1.1. Structure of GelMA.............................31 3.1.2. Infiltration of T cells in different concentrations GelMA....33 1.1.3. Mortality rate of cancer cells in GelMA........34 3.2 Cell culture in the tumor-lab-on-chip..........35 3.2.1. Tumor-lab-on-chip..............................35 3.2.2. Electrode pattern design on tumor-lab-on-chip..39 3.2.1. Electrode pattern..............................43 3.3 Interaction between infiltrating immune cells and cancer cells ...............................................46 3.3.1. Immune cells infiltration in the chip..........46 3.3.2. Mortality of cancer cells co-culture with immune cells.....53 3.3.3. Application of microfluidic device in clinical anti-cancer drug selection......................................57 Chapter 4 Conclusion and discussion...................62 Reference.............................................66

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