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研究生：	王明恩 Wong, Ming-Ern
論文名稱：	探討巨噬細胞“勿吃我”訊號對瀰漫大B細胞淋巴瘤發展所扮演的角色 Studying the role of macrophage “don't eat me” signals in diffuse large B cell lymphoma
指導教授：	洪良宜 Hung, Liang-Yi
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技與產業科學系 Department of Biotechnology and Bioindustry Sciences
論文出版年：	2023
畢業學年度：	111
語文別：	英文
論文頁數：	63
中文關鍵詞：	骨髓、CD24 、Siglec-10 、腫瘤微環境、腫瘤相關巨噬細胞
外文關鍵詞：	CD24, Siglec-10, BM, TME, tumor-associated macrophage
相關次數：	點閱：168 下載：0
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DLBCL（弥漫性大B细胞淋巴瘤）屬於侵袭性B细胞非霍奇金淋巴瘤（NHL），可分為ABC亞型和GCB亞型。一般來說，GCB-DLBCL患者的預後比ABC亞型要好，DLBCL的骨髓（BM）受累往往導致預後更差。儘管R-CHOP化療免疫療法是治療DLBCL的標準療法，但30~40%的患者最终会復發或對治療没有反應。癌细胞在腫瘤微乎環境（TME）中通過調控因子和與周圍细胞的相互作用而產生的免疫耐受是癌症復發或治療失效的可能原因。根據先前的研究顯示，癌细胞可能表達CD24和CD47，分别通过Siglec-10和SIRP-α相互作用誘導巨噬细胞發出 "不要吃我 "的信號。這裡，我們研究了巨噬细胞和DLBCL在BM中的相互作用。我們的研究結果表明，CD24和CD47在DLBCL细胞中上調，而當用從DLBCL细胞系收集的條件培養基培養M0巨噬细胞時，DLBCL會使肿瘤相關巨噬细胞（TAMs）更趨向於M2， Siglec-10的表達會增加。那在骨髓受累的患者中的骨髓和淋巴結具有TAMs浸潤的情形。M2-TAMs與DLBCL共培養後, TAMs的吞噬能力下降。基於這些觀察，我們認為有BM受累的DLBCL可以影響巨噬细胞的行為，並誘發 "不吃我 "的途徑來逃避免疫攻擊, 並且是透過腫瘤細胞表達CD24以及巨噬細胞過表達的Siglec-10此途徑幫助腫瘤細胞逃離免疫監控。

DLBCL (diffuse large B-cell lymphoma) belongs to the aggressive B-cell non-Hodgkin lymphoma (NHL). It can be divided into two types which are GCB and ABC type. There are some standard chemoimmunotherapies called R-CHOP for DLBCL, it is efficient but still have 30% of patients have dismal prognosis. The tumor microenvironment (TME) recruits abundant tumor-associated macrophages, which play a crucial role in tumor progression and malignancy. These studies showed cancer cells that express CD24 induce the “don’t eat me” signaling in macrophages via interacting with Siglec-10. Our findings suggest that within the tumor microenvironment the presence of M2 phenotype tumor associated macrophages (TAMs) assumes a supportive role in facilitating pro-tumor functions, thereby promote malignancy of B cells. Furthermore, our research found that TAM markers and inhibitor ligand from SUDHL-8 are increased, to gain insights further compare with lower expression of TAMs and inhibitor ligand from cell line HT by using phagocytosis assay to identify macrophages phagocytosis activity. Indeed, M2 macrophages exhibit a decreasing phagocytic activity when co-cultured with DLBCL with higher expression levels of CD24 and Siglec-10 in the cell line. Subsequently, CD 24 monoclonal antibodies and CD24 knockdown assay treat in lymphoma successfully rescue the phagocytosis ability of M2 macrophage. Moreover, our results demonstrate that macrophages play several functional roles in TME such as migration, phagocytic activity, infiltration and polarization ability but not in proliferation. Also, from the clinical sample analysis, higher expression of TAMs and Siglec-10 is found in patient who’s within aggressive bone marrow involvement. From IHC analyze, we found that macrophage infiltration in organ tissue as well which is bone marrow and lymphatic system. In vivo, the bone involvement in xenograft model is successfully established and reveal the expression of Siglec-10 was upregulated. Unexpectedly, these studies indicate there are no differences observed between two subtypes, both exhibit similar functionality in various aspects. Our results indicate that DLBCL with BM involvement can affect the behaviors of macrophages and induce the don’t eat me pathway to escape the immune attack through CD 24-Siglec-10 pathway.

Chinese abstract (中文摘要) I
Abstract II
Acknowledgements VI
Table of Contents VIII
Content of Tables XI
Content of Figures XII
Content of Appendices XIII
Abbreviation list XIV
1 Research Background 1
1-1 DLBCL lymphoma 1
1-2 Mutations of DLBCL 2
1-3 The treatment of DLBCL 3
1-4 CD 24 and CD 47 in DLBCL 3
1-5 Don’t eat me signalings 4
1-6 Anti-blockade effect in monoclonal antibody 4
1-7 Bone involvement of DLBCL. 5
1-8 The correlation between lymphoma and TAM 6
1-9 Macrophage phagocytosis7
1-10 TAMs proliferation 7
1-11 Research objective 7
2. Materials and Methods  10
2-1 Cell culture 10
2-2 CCK 8 assay 11
2-3 Ki-67 staining 11
2-4 TAMs migration assay 11
2-5 Clinical specimen 12
2-6 RNA extraction 12
2-7 Reverse transcriptase 13
2-8 Real time polymerase chain reaction (RT-qPCR) 13
2-9 Preparation of conditioned medium 14
2-10 Isolation of PBMC 14
2-11 M1 and M2 differentiated 14
2-12 Phagocytosis assay 14
2-13 Knockdown assay 15
2-14 CD24 antibodies blockage 16
2-15 IHC staining 16
2-16 In vivo experiment 17
2-17 Statistical analysis 19
3. Results 20
3-1 Macrophages can be slightly recruited to the tumor microenvironment in DLBCL 20
3-2 DLBCL drives macrophage to M2 phenotype 20
3-3 DLBCL does not affect the proliferative capacity of TAMs 21  
3-4 The CD 24 and CD 47 mRNA expression are elevated in DLBCL cancer cell lines 22
3-5 The Siglec-10 mRNA expression is elevated in TAMs 23
3-6 SUDHL 8 cell decreases M1 and M2 macrophage phagocytosis ability 23
3-7 CD24 blockade successfully enhances the engulfment of macrophages through CD24-Siglec-10 signaling pathway 24
3-8 CD24 knockdown promote macrophages phagocytotic ability through CD24-Siglec-10 signaling pathway 25
3-9 The establishment of bone involvement xenograft mice model 25
3-10 CD20 level is upregulated in BM from lymphoma model 26
3-11 Higher expression of Siglec-10 in U2932 lymphoma transplant mice within bone involvement 26
3-12 conclusion 27
4. Discussion 28
4-1 The molecular classification and genetic alternation of DLBCL 28
4-2 “Don’t eat me signaling” pathway involved in DLBCL 28
4-3 The content regulated in BM involvement  29
4-4 The factors are involved in TME regulating the crosstalk between macrophage and lymphoma 29
4-5 The phagocytotic activity of macrophages involves interaction with other immunosuppressive checkpoints 30
4-6 CD24 downstream signaling regulate in lymphoma 31
4-7 DLBCL is involved in high immune tolerance expression 32
4-8 Future research 33
References 34
Tables 38
Figures  41
Appendix 61
                                    

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