星狀膠質瘤是最常見的原發性顱內腫瘤，由於腫瘤微環境的複雜性，使得惡性膠質瘤至今仍是最難治療的腫瘤之一。先前的研究表明骨源性巨噬細胞在幾種腫瘤類型中起到了關鍵性作用。然而，腦源性的巨噬細胞（小膠質細胞）在星狀膠質瘤發展中的作用尚不清楚。在本研究中，我們使用星狀膠質瘤細胞株ALTS1C1與小膠質細胞株BV2在體外進行共培養，當ALTS1C1和BV2的比例為1:1時可以形成腫瘤集落。此外，我們建立了ALTS1C1單種細胞植入模型（A only）、ALTS1C1和BV2的比例為1：1的共植入模型（A+B）與ALTS1C1和BV2的比例為1：0.1的共植入模型（A+0.1B），並透過顱內注射(I.C.)以及肌內注射(I.M.)植入小鼠體內。與A only模型相比，A+B或A+0.1B模型中的小鼠存活時間較長，腫瘤的生長也受到抑制。此外，我們發現抗腫瘤狀態是由免疫系統引起的。 M-MDSC 是一種免疫抑制細胞，在 A+B 或 A+0.1B 模型中，無論是I.C.植入或 I.M.植入，M-MDSC在循環系統中的含量均被下調。 對T細胞的抑制是 M-MDSC 的主要功能，因此我們還觀察了 T 細胞的作用。有趣的是，在 A+B 或 A+0.1B 模型中，在循環系統和腫瘤微環境中觀察到更高豐度的胞毒性 T 細胞或更強的 T 細胞功能指標。因此，這項研究表明小膠質細胞可以引起抗神經膠質瘤的免疫反應。在臨床上，透過分析手術切除的惡性膠質瘤中的小膠質細胞以及小膠質細胞引起的免疫反應，可以製定更好的膠質瘤治療方案。

Malignant glioma is the most common primary intracranial tumor and it is one of the toughest tumors to be treated at the present due to the complexity of the tumor microenvironment. Previous studies have shown the critical role of the peripheral macrophage in several tumor types. However, the role of brain resident macrophage (microglia) has not been clear in the progression of astrocytoma. In this study, when the astrocytoma cell line, ALTS1C1, was co-cultured in a high-density manner with the microglia cell line, BV2, in a 1: 1 ratio in vitro, colonies could be formed. Furthermore, we compared the animal survival of mice receiving the ALTS1C1 single implantation (A only), 1:1 ratio of ALTS1C1 and BV2 co-implantation (A+B), and 1:0.1 ratio of ALTS1C1 and BV2 co-implantation (A+0.1B) via orthotopic intracranial (I.C.) implantation as well as intramuscular (I.M.) implantation. Results show that the surviving time of tumor-bearing mice was increased and the tumor growth was delayed in groups receiving A+B or A+0.1B model. Moreover, we found that the BV-2-associated anti-tumor effect was associated with the immune status. The abundance of M-MDSC (myeloid monocyte-derived suppressor cells) in the circulating system was down-regulated in A+B or A+0.1B model, no matter in I.C. implantation or I.M. implantation. On the other hand, the higher abundance of cytotoxicity T cells or stronger T cells functional factors were observed in the circulating system and tumor microenvironment in A+B or A+0.1B group. In summary, this study demonstrates that microglia could cause anti-glioma immunity, which is associated with the regulation of M-MDSC and T cell numbers. More studies are required to explore how BV2 regulates M-MDSC and T cell numbers during brain tumor progression.

able of content
中文摘要 1
Abstract 2
致謝 3
Table of content 4
Chapter I. Introduction 6
1.1 Glioma 6
1.2 Tumor-associated macrophages (TAMs) 7
1.3 Myeloid-derived suppressor cells (MDSCs) 8
1.4 T lymphocyte 9
1.5 Tumor microenvironment(TME) 10
1.6 Aim 11
Chapter II. Materials and Methods 12
2.1 Animals 12
2.2 Anesthetic 12
2.3 Blood Collection 12
2.4 Cell Culture 13
2.5 Splenocyte preparation 13
2.6 Orthotopic Intracranial and Intramuscular Tumor Implantation 14
2.6.1 Cell Preparation 14
2.6.2 Animal Procedures 15
2.7 Flow Cytometry 16
2.8 Process of embedding brain tumor samples 17
2.9 Immunohistochemical analysis 18
2.10 Immunofluorescence staining 18
2.11 Statistics 19
Chapter III. Results 20
3.1 Establishment of ALTS1C1 and BV2 co-culture model 20
3.1.1 Characteristics of ALTS1C1 and BV2 co-culture in vitro 20
3.1.2 Establishment of ALTS1C1 and BV2 orthotopic intracranial co-implantation model 21
3.2 The abundance and survival of MDSC in the ALTS1C1-BV2 co-implanting I.C. model 22
3.2.1 The abundance of circulating-MDSC in fully immunocompetent C57BL/6 I.C. model 22
3.2.2 The abundance and survival proportion of infiltrating-MDSC in fully immunocompetent C57BL/6 I.C. model 23
3.3 BV2-Mediated MDSCs depletion reverses tumor immune evasion 23
3.3.1 The abundance of circulating-T cells in fully immunocompetent C57BL/6 I.C. model 23
3.3.2 The abundance of infiltrating-T cells in fully immunocompetent C57BL/6 I.C. model 24
3.4 BV2-effect in exotopic-microenvironment 25
3.4.1 BV2 suppresses tumor growth in fully immunocompetent C57BL/6 I.M. model 25
3.4.2 BV2 suppresses the abundance of circulating-M-MDSC in fully immunocompetent C57BL/6 I.M. model 26
3.4.3 The abundance and survival proportion of infiltrating-MDSC in fully immunocompetent C57BL/6 I.M. model 27
3.4.4 The abundance of circulating-T cells in fully immunocompetent C57BL/6 I.M. model 27
3.4.5 The abundance of infiltrating-T cells in fully immunocompetent C57BL/6 I.M. model 28
3.5 BV2-effect in the lymphocyte lacking microenvironment 29
3.5.1 The abundance of circulating-MDSC in immunodeficient C.B17 I.C. model 29
3.5.2 The abundance of infiltrating-MDSC in immunodeficient C.B17 I.C. model 30
3.6 Microglia secreting Apolipoprotein E (ApoE) effect of ALTS1C1 progression 30
Chapter IV. Discussion 32
4.1 BV2 inhibits the growth of ALTS1C1 tumor 32
4.2 M-MDSCs are the main cells affected by BV2 on BV2-mediated ALTS1C1 tumor growth delay 33
4.3 Microglia inducing M-MDSC reduction leading to the higher abundance of CD8 cytotoxicity T cells 34
4.4 Different immune responses caused by the presence or absence of microglia in the tumor environment 35
4.5 Conclusion and future direction 36
Chapter V. Figures 38
Chapter VI. Reference 75

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