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研究生：	葉裕民 Yeh, Yu-Min
論文名稱：	CSF-1R在癌症免疫系統的角色 The Role of CSF-1R in Cancer Immunity
指導教授：	沈孟儒 Shen, Meng-Ru 蘇五洲 Su, Wu-Chou
學位類別：	博士 Doctor
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	64
中文關鍵詞：	巨噬細胞、巨噬細胞群落刺激因子接受器、基因變異、生物指標、大腸直腸癌
外文關鍵詞：	macrophages, CSF-1R, germline genetic variant, biomarker, colorectal cancer
相關次數：	點閱：81 下載：2
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癌症免疫治療在最近這幾年有了突破性的進展，但是並非所有的腫瘤都對免疫治療有效，而且多數對治療有效的腫瘤後來也會產生抗藥性。詳細了解腫瘤的免疫微環境將有助於克服免疫治療的抗藥機轉並改善治療成效。
巨噬細胞是在腫瘤微環境中扮演重要角色的一種免疫細胞，受到巨噬細胞群落刺激因子接受器(Colony-Stimulating Factor 1 Receptor, CSF-1R)的調控，在CSF-1的刺激之下，CSF-1R會形成二聚體(dimer)、接著磷酸化、然後活化下游的重要的訊息傳遞路徑，藉由這些訊息傳遞路徑的活化，可以調控先天免疫系統(innate immunity)中單核球與巨噬細胞的分化、生長與存活，也會使巨噬細胞往M2的方向極化(polarization)。在本論文當中，旨在探討CSF-1R在腫瘤免疫系統中所扮演的角色。藉由分析140位癌症病患的CSF1R基因，我們發現在DNA序列第1085的位置常常可以觀察到由A變成G的單點變異(c.1085A>G)，這項生殖細胞系(germline)的單點變異會造成CSF-1R的第362個胺基酸從組氨酸(Histidine)變成精氨酸(Arginine; p.H362R)，變異發生的頻率(allele frequency)大約是43%。有趣的是這項基因變異發生的頻率有著人種上的差異，在台灣人體生物資料庫以及千人基因組計畫(1000 Genomes)中的資料顯示東亞的族群發生的頻率都在40%左右，而在其他的族群發生的比例則僅有7~11%。
分析大腸直腸癌病人的腫瘤檢體，我們發現：帶有此項基因變異的病人其腫瘤具有比較少的巨噬細胞，而當中M2巨噬細胞的比例比較少、與M2巨噬細胞相關的激素表現量也偏低。當進一步收集正常人的周邊血液、分離單核球、進一步用CSF-1刺激誘導分化為巨噬細胞時，我們也發現帶有此項基因變異的細胞，單核球分化成巨噬細胞、以及巨噬細胞往M2方向的極化的反應都比較差。這項變異所造成的胺基酸改變座落在CSF-1R要形成二聚體的結構域，推測有可能會因此影響CSF-1R的活化、訊息傳遞以及進一步影響到巨噬細胞的功能。因此，我們分析CSF-1所誘導的CSF-1R磷酸化以及活化後的內吞作用(endocytosis)，發現帶有此項基因變異的巨噬細胞不管在CSF-1R的磷酸化或內吞作用，和不帶有此項基因變異的細胞比較起來都比較不明顯，證實此項推測。此外，此項基因變異會影響藥物的敏感性：帶有基因變異的巨噬細胞對CSF-1R的抑制劑較為敏感，IC50大約在0.1~1 nmol/L左右；相反的沒有此項基因變異的巨噬細胞對於CSF-1R抑制劑的IC50則大約在10~100 nmol/L，此發現顯示CSF1R的基因變異或許有機會成為使用CSF-1R抑制劑的一項生物指標。
在我們分析的大腸直腸癌病人當中，除了看到帶有CSF1R c.1085A>G的基因變異的腫瘤具有比較少的M2巨噬細胞以及VEGF的表現之外，這些同為第三期大腸直腸癌的病人在臨床上不管是疾病無復發(recurrence-free survival)以及總存活時間(overall survival)都比不帶有這項基因變異的病人來的好。分析這兩群病人腫瘤當中與免疫相關的基因表現，我們發現CD40LG mRNA在帶有這項基因變異的腫瘤中表現量比較多，而腫瘤當中也的確觀察到比較多CD40L+的T細胞數量。而在帶有CSF1R c.1085A>G基因變異的大腸癌腫瘤組織中，除了有較多的CD40L+的T細胞，同時也伴隨著有較高的IL-2表現。根據過去文獻以及這些結果，推測：帶有CSF1R c.1085A>G基因變異的巨噬細胞應該是透過CD40與T細胞的CD40L結合，進一步刺激IL-2的表現而加強毒殺性T細胞(cytotoxic T cell)的功能，而使得帶有這項基因變異的腫瘤患者可以有比較好的臨床預後。
本論文成功的找到CSF1R c.1085A>G這一項重要的基因變異，此項基因變異會經由影響巨噬細胞的分化、極化、以及功能來改變腫瘤的免疫微環境；此外，這項變異也會透過CD40L與IL-2的表現，影響毒殺性T細胞的功能，進而影響病人的臨床預後；更重要的是此項基因變異有機會成為使用CSF-1R抑制劑的一項重要的生物指標。

There have been rapid advances in cancer immunotherapy recently. However, immunotherapy is not an effective treatment for all types of cancers and most of patients who initial respond to therapy develop progressive disease later. Better understanding of tumor immune microenvironment is needed to overcome the primary and secondary resistance and establish efficient cancer immunotherapies.
Macrophages, the pivotal cells of innate immunity, are the most dominant and important immune infiltrates in the tumor microenvironment. Colony-stimulating factor 1 receptor (CSF-1R) is the key regulator of macrophages. The binding of CSF-1 to CSF1-R induces a process of dimerization, autophosphorylation and subsequent activation of downstream signaling, which controls the differentiation, proliferation, polarization and function of macrophages. My thesis is designed to explore the role of CSF-1R in cancer immunity. By analyzing the whole genome sequencing data from 140 cancer patients, a CSF1R c.1085A>G germline genetic variant, causing the change of histidine to arginine in the domain of receptor dimerization of CSF-1R, was identified. The frequency of this germline variant had an ethnic difference. The alterative allele frequency of CSF1R c.1085A>G was around 40% in East Asians, but only 7% to 11% alternative allele frequency was observed in South Asia, Africa, America, and European population.
Colorectal cancer (CRC) patients harboring CSF1R c.1085A>G variant had less total tumor-associated macrophages and M2-like macrophages in tumor tissues which were accompanied by lower VEGF expression. When peripheral blood mononuclear cell (PBMC)-derived macrophages were used to study the impact of CSF1R c.1085A>G variant, the percentage of CSF-1-induced macrophage differentiation and M2 polarization was significantly lower in macrophages with CSF1R c.1085 genotype A_G compared to genotype A_A. Moreover, CSF-1 induced a less prominent phosphorylation and slow endocytosis of CSF-1R in macrophages with CSF1R c.1085 genotype A_G, suggesting the change of amino acid from histidine to arginine in the domain of dimerization caused by this genetic variant might be the mechanism mediating the differential biologic effects observed in vitro and in vivo. Moreover, macrophages with CSF1R c.1085A>G variant were sensitive to CSF-1R inhibitors with IC50 in the 0.1 to 1 nmol/L range. In contrast, the IC50 for macrophages with CSF1R c.1085 genotype A_A was around 10 to 100 nmol/L. This germline variant confers the sensitivity of macrophages to CSF-1R inhibitors, suggesting that this variant might a potential predictive biomarker while targeting CSF-1R signaling.
In this CRC cohort, patients harboring CSF1R c.1085A>G variant had a better overall survival and disease-free survival compared to patients with CSF1R c.1085 genotype A_A. Analysis of immune-related gene expression demonstrated CRCs with this variant had higher CD40LG and IL-2 expression than those with CSF1R c.1085 genotype A_A. More CD3+CD40L+ T cells and higher IL-2 expression observed in tumors with CSF1R c.1085 genotype A_G confirmed the finding of mRNA expression. These results together with prior reports suggest higher IL-2 expression generated by the interaction of CD40 ligand and CD40 between helper T cells and macrophages with CSF1R c.1085 genotype A_G might be the mechanism explaining why CRC patients with this germline variant had a better clinical outcome.
My PhD study identified an important germline genetic variant, CSF1R c.1085A>G. This variant regulates tumor immunity by altering the differentiation, polarization and function of macrophages and is associated with clinical outcome of CRC patients. Moreover, the different response to CSF-1R inhibitors mediated by this genetic variant suggests this germline variant might be an important predictive biomarker when targeting CSF-1R signaling.

ABSTRACT IN CHINESE	1
ABSTRACT	3
ACKNOWLEDGEMENT	5
CONTENTS	6
1.	INTRODUCTION	9
1.1 Cancer immunotherapy	9
1.2 Colorectal cancer	9
1.3 Tumor-associated macrophages	10
1.4 Colony stimulating factor 1 receptor and macrophages	11
1.5 Targeting CSF-1R signaling	12
1.6 Unmet clinical needs	13
1.7 Specific aims and study designs	14
2.	MATERIALS AND METHODS	15
2.1 Databases for analyzing CSF1R genetic variants	15
2.2 Polymerase chain reaction (PCR) and Sanger sequencing	16
2.3 RNA sequencing	16
2.4 Chemicals and antibodies	17
2.5 Immunofluorescence staining and confocal images	18
2.6 Immunohistochemical staining of IL-2	18
2.7 Isolation and culture of human peripheral blood mononuclear cell (PBMC)-derived macrophages	19
2.8 Analysis and sorting by flow cytometry	20
2.9 Determination of CSF-1R phosphorylation	21
2.10 Monitoring of CSF-1R endocytosis	21
2.11 Measurement of cell viability	22
2.12 Statistical analysis	22
3.	RESULTS	23
3.1 The frequency and distribution of CSF1R germline genetic variants	23
3.2 Global distribution of CSF1R c.1085A>G genetic variant	24
3.3 CSF1R c.1085 genotype is associated with polarized macrophages and cytokines	24
3.4 CSF1R c.1085 genotype determines macrophage differentiation and polarization	25
3.5 CSF1R c.1085 genotype impacts CSF-1R phosphorylation and endocytosis	26
3.6 CSF1R c.1085A>G genetic variant has an impact on the sensitivity of macrophages to CSF-1R inhibitors	28
3.7 CSF1R c.1085 genotype is associated with clinical outcome	28
3.8 Immune response genes differentially expressed in CRCs with different CSF1R c.1085 genotypes	30
3.9 CRCs with CSF1R c.1085A>G variant contain higher number of CD40L+ T cells	30
3.10 IL-2 expression is higher in CRC with CSF1R c.1085A>G variant	31
4.	DISCUSSION	33
5.	TABLES	42
Table 1. Comparison of the variant allele frequency of CSF1R gene between cancer patients and normal population	42
Table 2. Clinical characteristics of 60 CRC patients	43
Table 3. The number of genes across 36 functional groups in Oncomine Immune Response Research Assay	44
Table 4. Summary of the impact of germline CSF1R c.1085A>G variant	45
6.	FIGURES	46
Figure 1. The frequency and distribution of germline CSF1R genetic variants.	46
Figure 2. The alterative allele frequency of CSF1R c.1085 in different ethnic groups	47
Figure 3. The distribution of total, M1 and M2 macrophages in CRC with different CSF1R c.1085 genotypes	48
Figure 4. VEGF expression levels in CRC with different CSF1R c.1085 genotypes	49
Figure 5. The impact of CSF1R c.1085 genotypes on CSF-1-induced macrophage differentiation and M2 polarization	50
Figure 6. The impact of CSF1R c.1085 genotype on CSF-1-induced CSF-1R phosphorylation	51
Figure 7. The impact of CSF1R c.1085 genotype on CSF-1-induced CSF-1R endocytosis	52
Figure 8. The impact of CSF1R c.1085 genotype on the sensitivity to CSF-1R inhibitors	53
Figure 9. Patients used to analyze the clinical outcome and gene expression	54
Figure 10. The clinical outcome of CRC patients with CSF1R c.1085 genotype A_A and A_G	55
Figure 11. Immune response genes differentially expressed in CRCs with CSF1R c.1085 genotype A_A and A_G	56
Figure 12. The distribution of CD40L+ T cells in CRCs with CSF1R c.1085 genotype A_A and A_G	57
Figure 13. IL-2 expression in CRCs with CSF1R c.1085 genotype A_A and A_G	58
Figure 14. CSF1R c.1085 genotype regulates tumor immunity and affects clinical outcome by altering the proliferation, polarization and function of macrophages	59
7.	REFERENCES	60
8.	APPENDIX	64
8.1 Publication List for Dissertation	64
                                    

1. Rotte, A. Combination of CTLA-4 and PD-1 blockers for treatment of cancer. J Exp Clin Cancer Res 38, 255 (2019).
2. Sharma, P., Hu-Lieskovan, S., Wargo, J.A. & Ribas, A. Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell 168, 707-723 (2017).
3. Dekker, E., Tanis, P.J., Vleugels, J.L.A., Kasi, P.M. & Wallace, M.B. Colorectal cancer. Lancet 394, 1467-1480 (2019).
4. Andre, T., et al. Improved overall survival with oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial. J Clin Oncol 27, 3109-3116 (2009).
5. Yoshino, T., et al. Pan-Asian adapted ESMO consensus guidelines for the management of patients with metastatic colorectal cancer: a JSMO-ESMO initiative endorsed by CSCO, KACO, MOS, SSO and TOS. Ann Oncol 29, 44-70 (2018).
6. Ganesh, K., et al. Immunotherapy in colorectal cancer: rationale, challenges and potential. Nat Rev Gastroenterol Hepatol 16, 361-375 (2019).
7. Chen, F., et al. New horizons in tumor microenvironment biology: challenges and opportunities. BMC Med 13, 45 (2015).
8. Roussos, E.T., Condeelis, J.S. & Patsialou, A. Chemotaxis in cancer. Nat Rev Cancer 11, 573-587 (2011).
9. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. Int Immunol 30, 511-528 (2018).
10. Noy, R. & Pollard, J.W. Tumor-associated macrophages: from mechanisms to therapy. Immunity 41, 49-61 (2014).
11. Nucera, S., Biziato, D. & De Palma, M. The interplay between macrophages and angiogenesis in development, tissue injury and regeneration. Int J Dev Biol 55, 495-503 (2011).
12. Edin, S., et al. The distribution of macrophages with a M1 or M2 phenotype in relation to prognosis and the molecular characteristics of colorectal cancer. PLoS One 7, e47045 (2012).
13. Sherr, C.J., et al. The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell 41, 665-676 (1985).
14. Verstraete, K. & Savvides, S.N. Extracellular assembly and activation principles of oncogenic class III receptor tyrosine kinases. Nat Rev Cancer 12, 753-766 (2012).
15. Lin, H., et al. Discovery of a cytokine and its receptor by functional screening of the extracellular proteome. Science 320, 807-811 (2008).
16. Li, W. & Stanley, E.R. Role of dimerization and modification of the CSF-1 receptor in its activation and internalization during the CSF-1 response. EMBO J 10, 277-288 (1991).
17. Yeung, Y.G. & Stanley, E.R. Proteomic approaches to the analysis of early events in colony-stimulating factor-1 signal transduction. Mol Cell Proteomics 2, 1143-1155 (2003).
18. Pixley, F.J. & Stanley, E.R. CSF-1 regulation of the wandering macrophage: complexity in action. Trends Cell Biol 14, 628-638 (2004).
19. Van Overmeire, E., et al. M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment. Cancer Res 76, 35-42 (2016).
20. Biswas, S.K., et al. A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation). Blood 107, 2112-2122 (2006).
21. Mantovani, A., Sozzani, S., Locati, M., Allavena, P. & Sica, A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23, 549-555 (2002).
22. Ries, C.H., Hoves, S., Cannarile, M.A. & Ruttinger, D. CSF-1/CSF-1R targeting agents in clinical development for cancer therapy. Curr Opin Pharmacol 23, 45-51 (2015).
23. Pyonteck, S.M., et al. CSF-1R inhibition alters macrophage polarization and blocks glioma progression. Nat Med 19, 1264-1272 (2013).
24. Strachan, D.C., et al. CSF1R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8(+) T cells. Oncoimmunology 2, e26968 (2013).
25. Ries, C.H., et al. Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy. Cancer Cell 25, 846-859 (2014).
26. DeNardo, D.G., et al. Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer Discov 1, 54-67 (2011).
27. Zhu, Y., et al. CSF1/CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models. Cancer Res 74, 5057-5069 (2014).
28. Mok, S., et al. Inhibition of CSF-1 receptor improves the antitumor efficacy of adoptive cell transfer immunotherapy. Cancer Res 74, 153-161 (2014).
29. Tap, W.D., et al. Pexidartinib versus placebo for advanced tenosynovial giant cell tumour (ENLIVEN): a randomised phase 3 trial. Lancet 394, 478-487 (2019).
30. https://clinicaltrials.gov/ct2/show/NCT02481336?term=meng-ru+shen&rank=1.
31. Fan, C.T., Lin, J.C. & Lee, C.H. Taiwan Biobank: a project aiming to aid Taiwan's transition into a biomedical island. Pharmacogenomics 9, 235-246 (2008).
32. Genomes Project, C., et al. A global reference for human genetic variation. Nature 526, 68-74 (2015).
33. Lou, J., Low-Nam, S.T., Kerkvliet, J.G. & Hoppe, A.D. Delivery of CSF-1R to the lumen of macropinosomes promotes its destruction in macrophages. J Cell Sci 127, 5228-5239 (2014).
34. Zhang, Q.W., et al. Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature. PLoS One 7, e50946 (2012).
35. Hollingsworth, J.W., et al. CD44 regulates macrophage recruitment to the lung in lipopolysaccharide-induced airway disease. Am J Respir Cell Mol Biol 37, 248-253 (2007).
36. Daoussis, D., Andonopoulos, A.P. & Liossis, S.N. Targeting CD40L: a promising therapeutic approach. Clin Diagn Lab Immunol 11, 635-641 (2004).
37. Loskog, A.S. & Eliopoulos, A.G. The Janus faces of CD40 in cancer. Semin Immunol 21, 301-307 (2009).
38. Maston, G.A., Evans, S.K. & Green, M.R. Transcriptional regulatory elements in the human genome. Annu Rev Genomics Hum Genet 7, 29-59 (2006).
39. Huang, T., Shu, Y. & Cai, Y.D. Genetic differences among ethnic groups. BMC Genomics 16, 1093 (2015).
40. Yoo, S.S., et al. Effects of polymorphisms identified in genome-wide association studies of never-smoking females on the prognosis of non-small cell lung cancer. Cancer Genet 212-213, 8-12 (2017).
41. Egan, K.M., et al. Cancer susceptibility variants and the risk of adult glioma in a US case-control study. J Neurooncol 104, 535-542 (2011).
42. Tu, Z., Young, A., Murphy, C. & Liang, J.F. The pH sensitivity of histidine-containing lytic peptides. J Pept Sci 15, 790-795 (2009).
43. Zhao, J., Zhu, J. & Thornhill, W.B. Spinocerebellar ataxia-13 Kv3.3 potassium channels: arginine-to-histidine mutations affect both functional and protein expression on the cell surface. Biochem J 454, 259-265 (2013).
44. Tang, X. & Bruce, J.E. Chemical cross-linking for protein-protein interaction studies. Methods Mol Biol 492, 283-293 (2009).
45. Crow, M.J., Seekell, K., Ostrander, J.H. & Wax, A. Monitoring of receptor dimerization using plasmonic coupling of gold nanoparticles. ACS Nano 5, 8532-8540 (2011).
46. Neher, E. & Marty, A. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc Natl Acad Sci U S A 79, 6712-6716 (1982).
47. Pradel, L.P., et al. Macrophage Susceptibility to Emactuzumab (RG7155) Treatment. Mol Cancer Ther 15, 3077-3086 (2016).
48. Poh, A.R. & Ernst, M. Targeting Macrophages in Cancer: From Bench to Bedside. Front Oncol 8, 49 (2018).
49. Khorana, A.A., Ryan, C.K., Cox, C., Eberly, S. & Sahasrabudhe, D.M. Vascular endothelial growth factor, CD68, and epidermal growth factor receptor expression and survival in patients with Stage II and Stage III colon carcinoma: a role for the host response in prognosis. Cancer 97, 960-968 (2003).
50. Forssell, J., et al. High macrophage infiltration along the tumor front correlates with improved survival in colon cancer. Clin Cancer Res 13, 1472-1479 (2007).
51. Ruffell, B. & Coussens, L.M. Macrophages and therapeutic resistance in cancer. Cancer Cell 27, 462-472 (2015).

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