膠質瘤是致命的癌症，由於其異質性腫瘤微環境而具有很高的復發能力。細胞外囊泡（Extracellur Vescles）在腫瘤微環境扮演的角色起著越來越重要,尤其是外泌體(Exosome)做為細胞間通信的媒介是最為常見的細胞外囊泡。我們實驗室之前的研究發現老鼠星形細胞瘤細胞ALTS1C1與BV2細胞或骨髓來源的M2巨噬細胞能相互作用，例如化學抗性和集落形成。因此，我們專注於研究兩種腫瘤相關巨噬細胞的外泌體對於ALTS1C1細胞的作用。我們使用沉澱法的方式純化外泌體，並通過奈米粒子跟蹤分析儀，西方墨點法和穿透式電子顯微鏡來確認外泌體的特性。我們的結果發現Raw264.7和BV2細胞所分泌的外泌體都可以被ALTS1C1細胞攝取，但只有BV2外泌體對ALTS1C1細胞具有劑量反應，包括細胞形態變化，誘導細胞增殖和遷移能力以及改變ALTS1C1細胞中DEDD和VEGFa的表現水平。此外，BV2細胞所分泌的外來體可增加腫瘤血管的功能並減少缺氧區域，但在肌肉內模型腫瘤中卻意外地延遲了腫瘤的生長，並增加了胞毒性T細胞(CD8+)的比例。

Glioma is deadly cancer and has a high recurrence ability due to its heterogeneous tumor microenvironment (TME). Extracellular vesicles (EVs) play a more and more important role in TME. The exosome, which is the most common EVs, can act as a cargo between cell to cell communication. Previous studies of our laboratory have found that ALTS1C1, a murine astrocytoma cell line, had some interactions with BV2 or bone marrow-derived M2 macrophage, such as chemoresistant and colony formation. Thus, we focused on comparing the response of ALTS1C1 in interacting with two types of tumor-associated macrophage-derived exosomes. We used precipitation based protocols to purify exosomes and checked the characterization of exosomes by Nanoparticle Tracking Analyzer, Western blot, and Transmission Electron Microscopy. Our results showed that both Raw264.7 and BV2 cell-derived exosomes could be uptaken by ALTS1C1, but only BV2 exosomes had a dose-dependent effect on ALTS1C1 cell, including cell morphology change, cell proliferation ability, and migration ability as well as the change of the expression level of DEDD and VEGFa in vitro. Moreover, BV2 derived exosomes could increase the tumor vessels function and reduce the hypoxia area, but un-expectely delayed tumor growth in the intramuscular model tumor associated the increase of CD8+ T cell percentage.

致謝 ........................................................................................................................................................1
中文摘要 ................................................................................................................................................2
Abstract..................................................................................................................................................3
Chapter1: Introduction ........................................................................................................................7
1.1 Glioma..........................................................................................................................................7
1.2 Tumor microenvironment..........................................................................................................7
1.3 Extracellular vesicles ..................................................................................................................8
1.4 Tumor-associated macrophages ................................................................................................9
1.5 Aim.............................................................................................................................................10
Chapter2: Materials and Methods ....................................................................................................11
2.1 Cell line cultures ........................................................................................................................11
2.2 Exosome isolation ......................................................................................................................11
2.3 Exosome characterization..........................................................................................................12
2.3-1 Nanoparticle Tracking Analyzer (NTA) ............................................................................12
2.3-2 Exosomal protein extraction and BCA assay ....................................................................13
2.3-3 Western blot ........................................................................................................................13
2.3-4 Transmission Electron Microscopy sample prepare .........................................................15
2.4 In vitro study...............................................................................................................................16
2.4-1 Fast Dio labeled exosome...................................................................................................16
2.4-2 Immunofluorescence staining............................................................................................16
2.4-3 MTT assay...........................................................................................................................17
2.4-4 Cell growth curve and cell cycle analyze ...........................................................................18
2.4-5 Wound healing assay..........................................................................................................18
2.4-6 Total RNA extraction and real-time polymerase chain reaction (RT-PCR) ....................19
2.5 Animal experiments ...................................................................................................................20
2.5-1 Mice.....................................................................................................................................20
2.5-2 Intramuscular tumor implantation ....................................................................................21

2.5-3 Tumor collection.................................................................................................................21
2.5-4 Flow cytometry analysis......................................................................................................22
2.5-5 Immunohistochemistry analysis.........................................................................................23
2.6 Statistics......................................................................................................................................24
Chapter3: Result .................................................................................................................................25
3.1 Exosome characterization ........................................................................................................25
3.2 In vitro test .................................................................................................................................26
3.2-1 Exosomes purified from ALTS1C1 or macrophages can be uptaken by ALTS1C1, BV2, and RAW264.7...................................................................................................................26
3.2-2 BV2 exosomes have dose-response to ALTS1C1 ............................................................27
3.2-3 ALTS1C1 cell morphology can be changed by BV2 exosome .......................................29
3.2-4 BV2 Exosome can induce ALTS1C1 Cell proliferation and migration ability ............29
3.2-5 BV2 Exosome can induce ALTS1C1 VEGFa and reduce DEDD expression level. .....30
3.3 In vivo test ..................................................................................................................................31
3.3-1 BV2 exosome can delay tumor growth in I.M. model ....................................................31
3.3-2 BV2 exosome reduce hypoxia area and increase the ratio of the functional vessel .....33
Chapter4: Discussion..........................................................................................................................34
4.1 Brief introduction of what important results found in this study. .......................................34
4.2 The size and amount of exosome isolation depended on the isolation and quantitative methods. ..........................................................................................................................................34
4.3 ALTS1C1 used different pathways to uptake BV2 versus Raw264.7 exosomes. ................36
4.4 BV2 exosome-mediated ALTS1C1 proliferation and migration may be associated with miR24-3p..........................................................................................................................................38
4.5 BV2 exosome could cause tumor vessel normalization and delay tumor growth in an intra-muscular tumor model. ..................................................................................................................39
4.6 Summary...................................................................................................................................40
4.7 Future plan ................................................................................................................................41
Chapter5: Figures ...............................................................................................................................42
Figure1 .........................................................................................................................................42

Figure2 .........................................................................................................................................47
Figure3 .........................................................................................................................................49
Figure4 .........................................................................................................................................51
Figure5 .........................................................................................................................................53
Figure6 .........................................................................................................................................55
Figure7 .........................................................................................................................................57
Figure8 .........................................................................................................................................58
Figure9 .........................................................................................................................................60
Figure10 .......................................................................................................................................63
Figure11 .......................................................................................................................................65
Figure12 .......................................................................................................................................67
Supporting data 1........................................................................................................................70
Supporting data 2........................................................................................................................71
Chapter6: References .........................................................................................................................73

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