腦瘤內部擁有複雜腫瘤微環境及腦內的血腦屏障會限制藥物治療以及其他治療的方法。先前實驗室的研究當中，在小鼠的腦癌顱內模型上建立了一個有效的腫瘤自殺基因系統:HSV-sr39tk/GCV system，經由HSV-sr39tk轉殖後的腦腫瘤細胞接受前驅藥物GCV，會將GCV磷酸化產生對細胞有毒殺性的產物而被毒殺，但是腫瘤自殺基因系統的效果僅限於限制腫瘤的生長。此次研究將探討並評估放射線結合自殺基因系統治療腦瘤的效果。本次研究中使用ALTS1C1-TK腦瘤細胞株，並在老鼠大腿上建立異位腫瘤模型。結果發現，經過單次25 Gy的照射之後，腫瘤內部的血管密度降低，並延緩腫瘤生長速度。經過GCV治療後，腫瘤內部血管密度降低幅度不明顯，但是具有延緩腫瘤生長的效果。不論單獨GCV或是放射線治療，仍然可以看見腫瘤復發的情形，並伴隨著大量巨噬細胞、髓狀細胞的浸潤。然而，單獨放射線治療的腫瘤和GCV組相比，有更多的CD8+ T細胞浸潤並能夠引起較強烈的免疫反應。結合兩者更能快速並有效地延緩腫瘤生長的速度，並有6/11的治癒率。同時在Re-challenge的實驗當中，結合治療組別的老鼠對於遠端的腫瘤具有抑制的效果，其中兩隻老鼠遠端的腫瘤生長則被完全抑制；而在近端(右腳)的腫瘤並無長期抑制的效果，在近端的復發腫瘤微環境當中發現有更高比例的髓狀細胞，而這群細胞中具有Ly6C表現的細胞有免疫抑制的傾向，並會影響到毒殺腫瘤的免疫效果。總結來說，這篇研究以多方面的角度評估了放射線結合GCV對於腦瘤治療的可行性。

The complicated tumor microenvironment (TME) and blood-brain barrier (BBB) of brain tumors limit the efficacy of drug delivery and treatment options for glioma in clinical. Previous study had established an effective suicide gene system, HSV-sr39tk/GCV system, in a murine astrocytoma model. HSV-sr39tk-transfected tumor cells were treated with GCV drug, following convert into cytotoxic product, causing transfected cells death in vitro. However, the efficacy of TK/GCV system was only limited to delay tumor growth in vivo. This study aimed to explore the potential of combining radiation therapy (RT) with TK/GCV therapy for brain tumor. To achieve this goal, ALTS1C1-TK IM model was utilized in this pilot study. Single treatment of 25 Gy radiation reduced microvascular density (MVD) and retarded tumor growth. On the other hand, GCV treatment alone protocol had less effect on reducing MVD, but induce rapid tumor growth delay. The analysis of tumor microenvironments shows the increase of infiltrating macrophages in recurrent tumor receiving either RT or GCV treatment. However, the increase of CD8+ T cells was only seen in radiation-treated tumor. Combining radiation with GCV treatment not only prolong tumor growth day for another 5-10 days, but also cured 6 out of 11 mice. The re-challenge experiment was performed on the right and left legs of 3 cured mice. A significant tumor growth delay was found for re-challenged tumor growing in both legs. However, the complete cure (two out of three) was only found in abscopal site (left leg). This result indicates that long-term immunity has developed in tumor cured mice following RT+ TK/GCV therapy, but the treatment has residual immune suppressing activity that hinders the anti-tumor immunity in treatment site (right leg). This study shows the potential of combining radiation therapy with TK/GCV for treating brain tumor.

Abstract i
中文摘要 ii
Acknowledgement iii
CONTENTS iv
LIST OF FIGURES vii
LIST OF TABLE ix
Chapter 1 Introduction 1
1.1 Astrocytoma 1
1.2 HSV-TK/GCV suicide gene therapy 2
1.3 Radiation therapy 3
1.4 Aim of this study 3
Chapter 2 Materials and Methods 5
2.1 Cell lines and in vitro experiments 5
2.1.1 Cell culture and transfection 5
2.1.2 Cytotoxicity assay of ALTS1C1-TK to GCV prodrug 5
2.2 Mice 6
2.3 Intramuscular (IM) tumor model establishment 6
2.4 Treatment 6
2.5 Tissue harvesting and processing 7
2.6 Flow cytometry 7
2.6.1 Peripheral Blood 7
2.6.2 Tumor 8
2.7 Immunohistochemistry staining 8
2.8 Statistics 9
Chapter 3 Results 10
3.1 In vivo response of SDRT and Hsv-sr39tk/GCV system on ALTS1C1-TK intramuscular model 10
3.1.1 The intramuscular tumor growth after GCV treatment and single-dose (SD) irradiation 10
3.1.2 The effect of GCV and SDRT treatment on CD11b positive peripheral blood mononuclear cells (PBMC). 10
3.1.3 CD11b positive MDC in peripheral blood versus tumor size 12
3.1.4 The effect of GCV and SDRT treatment on microvascular network in tumor. 13
3.1.5 The effect of GCV and SDRT on CD11b positive infiltrating myeloid-derived cells in tumor 13
3.1.6 The effect of GCV and SDRT on CD3 positive cells in tumor 14
3.2 Evaluate the potential to combine single-dose irradiation with Hsv-sr39tk/GCV system in vivo 15
3.2.1 Anti-tumor efficacy of combination treatment in ALTS1C1-TK IM model. 15
3.2.2 The effect of combination treatment on CD11b positive peripheral blood mononuclear cells. 16
3.2.3 The effect of combination treatment on tumor microvascular network. 17
3.2.4 The influence of combination treatment on infiltrating T lymphocytes and MDCs in tumor 18
3.2.5 Therapeutic efficacy differs from individuals 19
Chapter 4 Discussion and Conclusion 20
4.1 Microvascular density decrement following combination therapy 20
4.2 Radiation promotes CD8+ T-cells infiltration and benefits to combination therapy 21
4.3 T-cells variation related to high ratio of MDSC in tumor 22
4.4 The effect of tumor-associated macrophages (TAMs) are not clarified here. 23
4.5 The potential to combine with immunotherapy 24
4.6 Summary 24
Figure and diagram 26
Table 56
Reference 59

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