截至2017年，頭頸鱗狀細胞癌(Head and neck squamous carcinoma, HNSCC)是台灣男性十大癌症之中高居第四名。在治療技術層面的蓬勃躍進下，相較於以往傳統放射治療，新穎的硼中子捕獲治療(Boron neutron capture therapy, BNCT)更能專一性的抑制癌細胞的生長。藉此，在探究BNCT的發展之下，透過動物實驗模型(Animal model)可作為驗證臨床試驗的最佳途徑，並觀察腫瘤微環境的變異來找尋適合的治療方針以提升治療效果。因此，本實驗主要利用致癌藥物4-NQO (4-Nitroquinolone-1-oxide)來誘發小鼠口腔腫瘤的生成，並找尋最佳的實驗方法以建立完整的小鼠頭頸癌實驗模型，以利於後續腫瘤微環境的探討研究及供後續研究人員之參考使用。同時，藉由非侵入式的血液採樣檢測方式來觀察經BNCT治療後的生物性指標(Biomarker)的變化，同時評估BNCT與免疫治療相結合之可行性。
從免疫組織染色(Immunohistochemical staining, IHC staining)結果顯示，當頭頸癌腫瘤隨著時間惡化增生，髓狀細胞(Myeloid cells)及巨噬細胞(Macrophages)有逐漸地增加趨勢並浸潤於腫瘤邊界(Tumor border)。另一方面，透過採集頭頸癌老鼠周邊血液(Peripheral blood mononuclear cells, PBMCs)於流式細胞儀(Flow cytometry)分析髓質性免疫抑制細胞(Myeloid-derived suppressor cells, MDSCs)中兩群細胞群系: 發炎性單核球(Monocytic MDSCs)及嗜中性球(Granulocytic MDSCs, G-MDSCs)的比例變化。結果顯示隨著腫瘤增生，兩種細胞族群皆呈現增加的趨勢，又以其中M-MDSCs和T細胞的一致動態變化為我們提供一種理想的腫瘤模型，並可揭示MDSCs所主導免疫逃逸和癌症的潛在機制在頭頸癌之腫瘤發展過程中。
利用已建立的頭頸癌老鼠模型應用於受BNCT與傳統光子治療後，觀察造成MDSCs與T細胞之影響變化。結果顯示在受到BNCT治療後的組別，在周邊血液內的G-MDSCs數量並沒有顯著的變化，相對於M-MDSCs的數量表現在治療後晚期變化有上升趨勢，其中又以照射後第七天最明顯。同時，在免疫組織染色也發現，在接受BNCT治療後一周的腫瘤組織，比起未治療的腫瘤內的血管密度與髓狀性細胞比例有顯著的增加差異，不僅如此，即便腫瘤大小有明顯減少，但在周邊血液內的髓狀細胞(CD11b+ cells)數量表現在治療後第七天皆有顯著的上升趨勢，這說明生物體仍處於免疫抑制的環境，因此仍有可能因受髓狀細胞的影響將導致腫瘤復發可能。總和以上結果發現，我們認為頭頸癌腫瘤在接受BNCT治療之後仍存在免疫抑制的腫瘤微環境，藉此免疫治療(抑制髓狀細胞增加)的結合是必然且需被執行的可能。

Head and neck squamous cell carcinoma (HNSCC) is the fourth among the top ten cancers for men in Taiwan. With the vigorous development of therapeutic techniques, many new specific therapeutic approaches for HNSCC have been developed. The boron neutron capture therapy (BNCT) is one of the novel treatments, which can be more specifically inhibiting the growth of tumor cells than traditional radiation therapy (RT). The animal model is useful on developing BNCT for HNSCC in verifying the findings from clinical trials. Here, we report an HNSCC model established by using carcinogen (4-Nitroquinolone-1-oxide, 4-NQO) to induce the development of oral squamous cell carcinoma (OSCC) in the mouse oral cavity. Application of 4-NQO in the mouse model has been shown to produce similar histological changes as that found in human oral epithelium neoplastic transformation, thus providing an ideal model for studying the response of oral cancer to therapy. However, the required exposure time of 4-NQO varied from different experimental setup. The prime aim of this study was to find the best experimental time and a consistent protocol for establishing an experimental murine model of head and neck cancer (HNC). Simultaneously, we also explored the tumor microenvironment (TME) of HNC and provided the reference for a future researcher. This study also investigated biomarkers and inflammatory factors in tumor and peripheral blood samples from BNCT-treated HNSCC mice by non-invasive liquid biopsy and studied their correlations with the disease prognosis.
Immunohistochemical staining (IHC) results showed that this tumor has a cluster of a high density of CD68+ macrophages and Gr-1+ cells accumulated in tumor-normal tissue junction, where have high abundant cancer-associated fibroblast (CAF). We hypothesize that the CD11b+ myeloid cells are attracted to the tissue during the development HNSCC and finally accumulated in the CAFs region. Intriguingly, only a few tumor-associated macrophages (TAMs) could be identified in other tumor regions except the junction.
To predict the occurrence of HNSCC, the peripheral blood during tumor progression was analyzed. Preliminary result found that the population of monocytic and granulocytic myeloid-derived suppressor cells (M-MDSCs and G-MDSCs) in whole blood increases with tumor growth, which has a strong correlation with the decrease of CD8+ and CD4+ T cells. The unique spatial distribution of myeloid cells in tumor tissue and consistent dynamic change of MDSCs and T cells in the blood provides us an ideal tumor model for designing new strategies specific for HNC.
To our knowledge, there has thus far been relatively little research into the changes in immune response for BNCT-treated HNSCC mice. In our studies, the results demonstrated that
even if the HNSCC tumor was significantly decreased following the BNCT treatment, the absolute numbers of M-MDSCs and G-MDSCs increases on day seven after BNCT, but CD8+ and CD4+ T cell decreases after both BNCT treatment and photon therapy (RT). IHC staining results also showed that the HNSCC tumor had a cluster of a high density of CD31+ vascular cells and CD11b+ myeloid cells on day seven after BNCT. Taken together, we believed that HNC still maintained immunosuppressive TME after BNCT treatment. Noticeably, BNCT treatment did have a good effect on tumor treatment, but it could induce more significant side effects at the same time, thus the combination of immunotherapy in HNSCC tumor after BNCT treatment was inevitable and needed to be noticed in the future.

致 謝 I
摘 要 II
ABSTRACT IV
ABBREVIATIONS VI
LIST OF TABLES VII
TABLE OF CONTENTS VIII
CHAPTER I. INTRODUCTION 1
A. FORWARD 1
B. HEAD AND NECK SQUAMOUS CARCINOMA (HNSCC) 2
1) Incidence and epidemiology in Taiwan 3
C. TUMOR MICROENVIRONMENT (TME) 4
1) Circulating myeloid-derived suppressor cells (MDSCs) 5
2) Inflammatory factor 6
D. IN VIVO MODELS (4-NQO MODELS) 7
E. BORON NEUTRON CAPTURE THERAPY (BNCT) 8
1) Boron compounds 9
CHAPTER II. MATERIAL AND METHODS 11
A. MURINE MODEL 11
1) Animals 11
2) 4-NQO-induced spontaneous HNSCC murine model 11
B. BORON ANALYSIS AND BNCT STUDIES 12
1) BPA administration 12
2) Boron bio-distribution studies 12
3) Optimization of sample digestion and boron analysis 13
4) Geometry of holder and BNCT dosimetry 14
5) Photon irradiation/beam 15
6) Neutron irradiation/beam 15
C. IN VIVO STUDIES (OBSERVATION OF TME AND IMMUNE RESPONSE) 16
1) Murine orbital peripheral blood mononuclear cells isolation 16
2) Murine serum collection 16
3) Biochemistry analysis 17
4) Enzyme-linked immunosorbent assay (ELISA) 17
5) Flow cytometry analysis 18
6) Process of embedding tongue tumor samples 19
7) Immunofluorescence staining 19
8) Harris’s hematoxylin and Eosin staining (H&E staining) 20
D. STATISTICAL ANALYSES 21
CHAPTER III. RESULTS 22
A. ESTABLISHMENT OF 4-NQO-INDUCED MURINE SPONTANEOUS ORAL CANCER MODEL 22
1) The Carcinogen of 4-NQO in Drinking Water Causes Tumor Formation on Mice Tongues 22
2) The Time Dependent Effect of Inducing Spontaneous HNSCC Tumor Formation 22
B. CHARACTERISTICS OF THE 4-NQO-INDUCED SPONTANEOUS HNSCC IN VIVO 23
1) AST/ALT Ratio Could Serve as a Prognostic Factor in Spontaneous HNSCC mice 23
2) Increase of Macrophage and Myeloid Cells in Mouse HNSCC Tumor and the Preference to Accumulate in the Tumor Invasive Front Region 24
3) Increased Frequency of Circulating M-MDSCs and G-MDSCs in 4-NQO-induced HNSCC Mice 25
4) Circulating M-MDSCs Correlate with Tumor Progression in the 4-NQO-induced Spontaneous HNSCC Murine Model 26
5) Decreased Frequency of Circulating CD3+CD4+, and CD3+CD8+ T cells in the HNC mice, and Correlate with Tumor Progression and G-MDSC, and M-MDSC Subsets 27
6) The Level of IL-1β, IL-6, CCL2, and TNF-a in Serum with HNSCC Tumor Progression 28
C. THE METABOLISM OF BORON-10 COMPOUNDS IN 4-NQO-INDUCED SPONTANEOUS HNSCC MURINE MODEL 29
1) Pharmacokinetics of 10B Concentration After Intraperitoneal Injection of BPA 29
2) High Boron Concentration Accumulation in Mucosal and Salivary Gland Tissues 30
D. COMPARISON OF BNCT AND PHOTON THERAPY (RT) IN HNSCC MICE 31
1) Effect of BPA-mediated BNCT Treatment on Tumor Growth of 4-NQO-induced HNSCC Mice 31
2) Evaluation of Liver Injury by Plasma Analysis 32
3) Circulating M-MDSC Increased in 4-NQO-induced Spontaneous HNSCC Mice Subjected to BNCT 32
4) Dynamic Circulating G-MDSC in 4-NQO-induced Spontaneous HNSCC Mice Subjected to BNCT or RT 33
5) The Change of Circulating Immune T-cells in Spontaneous HNSCC Murine Model Following BNCT or RT 34
6) BNCT-Induced the Increase in Microvascular Density and CD11b Myeloid Cells in 4-NQO-induced Spontaneous HNSCC Murine Model 35
8) Changes in Serum Levels of Immune Mediators Following BNCT or Photon Therapy 36
CHAPTER IV. DISCUSSIONS 38
A. AGE WOULD NOT AFFECT THE STUDIES OF 4-NQO-INDUCED SPONTANEOUS HNSCC TUMOR MODEL 38
B. THE CARCINOGENESIS OF 4-NQO INDUCED-MOUSE HNSCC TUMOR 39
C. DYNAMICS OF CD11B+ MYELOID CELLS WERE ASSOCIATED WITH 4-NQO-INDUCED HNSCC TUMOR PROGRESSION 41
D. MDCS DURING HNSCC TUMOR PROGRESSION AND THE EFFECT OF IMMUNE T CELLS WITH AGING 42
E. MUCOSA AND SMG HAD HIGH CONCENTRATIONS OF BORON-10 AND MIGHT CAUSE SIDE EFFECTS TO HAPPEN FOR BNCT 44
F. BNCT CAUSES CHANGES IN VASCULAR AND IMMUNE CELLS OF SPONTANEOUS HNSCC TUMORS 45
G. IL-6 COULD BE A POTENTIAL BIOMARKER FOR ORAL SQUAMOUS CELL CARCINOMA AND THE COMBINATION WITH BNCT 46
CHAPTER V. CONCLUSIONS 48
FIGURES AND DIAGRAMS 50
A. ESTABLISHMENT OF 4-NQO-INDUCED SPONTANEOUS HNSCC MURINE MODEL AND OBSERVATION OF THE CHANGES OF TUMOR PROGRESSION WITH IMMUNE RESPONSE 50
B. THE CHANGES OF TUMOR MICROENVIRONMENT FOLLOWING BNCT AND PHOTON THERAPY (RT) 75
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