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系統識別號 U0026-2305202016021500
論文名稱(中文) 探討腫瘤相關纖維細胞如何藉由細胞外囊泡影響肺癌細胞對表皮生長因子受器標靶藥物之抗藥性
論文名稱(英文) The role of cancer associated fibroblasts (CAF) extracellular vesicles (EVs) in mediating resistance of EGFR-TKI in NSCLC
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 108
學期 1
出版年 108
研究生(中文) 施乃育
研究生(英文) Nai-Yu Shih
學號 T16061100
學位類別 碩士
語文別 英文
論文頁數 46頁
口試委員 指導教授-蘇五洲
口試委員-林建中
口試委員-洪澤民
中文關鍵字 表皮生長因子受體-酪氨酸激酶抑製劑  腫瘤相關纖維細胞  細胞外囊泡  抗藥性 
英文關鍵字 EGFR-TKI  cancer associated fibroblasts  extracellular vesicles  drug resistance 
學科別分類
中文摘要 腫瘤微環境由細胞外基質(ECM)、腫瘤相關的正常上皮和基質細胞、免疫細胞和血管細胞等所組成。腫瘤相關纖維細胞(CAF)是腫瘤基質的主要成分,且被發現會通過腫瘤與基質之間的相互作用,進而影響表皮生長因子受體-酪氨酸激酶抑製劑(EGFR-TKI)的藥物敏感性。 在腫瘤微環境中,數種生長因子會分泌,如TGF-β和PDGF等,同時也可以通過外吐小體(exosomes)遞送。儘管大多數研究集中於癌細胞分泌的外吐小體,但最近的研究強調了CAF分泌的外吐小體。然而,目前仍很少使用系統性方法研究腫瘤細胞與CAF之間所有可能途徑的相互作用。
  細胞外囊泡(EVs)是由多種細胞類型所分泌,可以調節免疫功能、影響血管生成、細胞增殖以及癌細胞的侵襲和轉移。近年來也發現CAF所分泌的細胞外囊泡會使結直腸癌,乳腺癌和胰腺癌細胞對化療藥物出現抗藥性。
我們假設CAF會透過細胞外囊泡內物質來影響肺癌細胞對EGFR-TKI藥物敏感性。首先我們證明了,CAF所分泌的細胞外囊泡可以被肺癌細胞(PC9)吸收。接著,進一步證明CAF分泌的細胞外囊泡會使肺癌細胞對EGFR-TKI有抗性。我們先將CAF處理EGFR-TKI,接著從培養液中分離出細胞外囊泡,並發現藥物處理過的細胞外囊泡會更顯著降低肺癌細胞對EGFR-TKI的敏感性。這之後我們從CAF,DMSO處理過的CAF和TKI處理過的CAF中分離出細胞外囊泡,並對它們的miRNA含量進行了比較分析。結果顯示,在TKI處理過的CAF細胞外囊泡中,鑑定出含量最豐富的miRNA為miR-423-5p,且大量表現miR-423-5p增加了肺癌細胞對EGFR-TKI的抗藥性。透過以上實驗,我們初步發現了CAF細胞可透過透過外囊泡miR-423-5p是來影響肺癌細胞EGFR-TKI抗藥性。
英文摘要 Tumor microenvironment consists of the extracellular matrix (ECM), tumor-associated normal epithelial and stromal cells, immune cells, and vascular cells. Cancer associated fibroblasts (CAF), which are the major components of cancer stroma, were found mediating epidermal growth factor receptor - tyrosine kinase inhibitor (EGFR-TKI) resistance via tumor-stroma interactions. Growth factors like TGF-β and PDGF can be either freely secreted in the tumor microenvironment or delivered via exosomes. Although most studies focus on cancer cell-secreted exosomes, recent study highlighted the role of CAF-secreted exosomes. However, the study using systemic approach for all possible pathways interacting between tumor cell and CAF remained rare.
Extracellular vesicles (EVs) including exosome secreted by a variety of cell types modulate immune function, angiogenesis and cell proliferation, as well as tumor cell invasion and metastasis. EVs from cancer associated fibroblasts were also found promoting chemoresistance in colorectal, breast and pancreatic cancer cells.
We proposed that CAF may mediate EGFR-TKI resistance by transferring exosomal cargo, and EVs from TKI-treated CAF may enhance the drug resistance. We planned to verify our hypothesis in vitro and identify the cargo from CAF derived EVs and associated mechanism contributing EGFR-TKI resistance. Our data first showed that CAF derived EVs can be uptaken by PC9 cells. We further demonstrated PC9 cells were resistant to EGFR-TKI after CAF EVs treatment. We pre-treated CAF with gefitinib and isolated EVs from culture medium, the sensitivity to EGFR-TKI of PC9 cells were decreased. Next, we isolated EVs from CAF, DMSO-treated CAF and TKI-treated CAF and performed a comparative analysis of their miRNA content. Among the miRNA expression panels, the miR-423-5p was the most abundant miRNA identified in TKI-treated CAF EVs. The overexpression of miR-423-5p increased PC9 cells resistance to gefitinib. We demonstrated that CAF EVs regulated drug resistance via transferring miR-423-5p to PC9 cells.
論文目次 Acknowledgements ................................................................................... iii
中文摘要...........................................................................................................................iv
Abstract ...........................................................................................................................vi
Abbreviation list .........................................................................................................viii
Contents............................................................................................................................ix
Chapter 1: Introduction...............................................................................................1
1-1 Tumor mircroenvironment..................................................................................2
1-2 General biology of cancer associated fibroblasts (CAF) ................................3
1-3 Biology of secreted membrane vesicle................................................................4
1-4 The impact of extracellular vesicle on cancer...................................................5
1-5 Exosomes in Cancer Resistance ..........................................................................5
1-6 Lung cancer and target therapy...........................................................................6
1-7 The importance of EGFR pathways, EGFR-TKI in lung cancer and associated resistance mechanism....................................................................................7
1-8 The correlation between CAF and resistance to chemotherapy or targeted therapy ……………………………………………………………………………………9
1-9 Rationales...............................................................................................................11
1-10 Specific aims........................................................................................................12
Chapter 2: Materials and Methods ........................................................................13
2-1 Materials................................................................................................................14
2-1-1 Drugs and reagents..................14
2-1-2 Antibodies...................................14
2-1-3 Cell lines.....................................14
2-2 Methods..........................................15
2-2-1 Cell culture.............................15
2-2-2 Cell count ......................................15
2-2-3 The ultrafiltration method for isolating EVs................. 16
2-2-4 Transmission Electron Microscopy (TEM).............16
2-2-5 Nanoparticle tracking analysis using Nanosight system ...........17
2-2-6 EV staining............................... 18
2-2-7 EVs uptake................................. 19
2-2-8 Cytotoxic analysis-MTT assay..................... 19
2-2-9 Collection do whole cell/EV lysates............... 20
2-2-10 Western blotting .............................. 21
2-2-11 MicroRNA transfection and MTT assay............ 21
2-2-12 Statistical analysis....................... 22
Chapter 3: Results ....................................23
3-1 Characterization of extracellular vesicles (EVs) from lung cancer cells and cancer associated fibroblasts (CAF) using the ultra-filtration (UF) method……………….......24
3-2 The uptake of cancer associated fibroblasts (CAF) derived EVs in recipient cells…………………………………………….25
3-3 The cytotoxicity to EGFR-TKI in PC9 cells and CAF .....25
3-4 The uptake of CAF derived EVs in PC9 cells increase resistance to gefitinib ......26
3-5 MicroRNA expression profiles are significantly different between EVs from CAF and gefitinib -treated CAF................27
3-6 The transfection of MiR-423-5p enhancing gefitinib resistance of PC9 cells...28
Chapter 4: Discussions and Conclusion ...................29
4-1 Discussion.................................30
4-2 Conclusion .................................34
References..........................................35
Figures ................................................39

Figure 1. The ultra-filtration (UF) method could be a feasible method for the isolation of exosomes from lung cancer and the other normal cell.................................................39
Figure 2. The labeling of EVs of lung cancer cells and the uptake of theses EVs were imaged under immunoflurocent microscope....................................................................40
Figure 3. The MTT assay of PC9 cells and cancer associated fibroblasts (CAF) in response to gefitinib…………………………………......................................................42
Figure 4. The effect of CAF-EV and CAF-EV after gefitinib treatment on cell viability and on EGFR downstream signaling pathway of PC9.....................................................43
Figure 5. Differential microRNA expression in EVs from CAF compared to those from CAF after gefitinib treatment ..................................................................................44
Figure 6. The effect of transfection of mir-423 in combination of gefitinib on cell viability of PC9 cell............................................................................................................45
Figure 8 Conclusion....................................................................................................46
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