惡性腦腫瘤至今仍是最難以治療的癌症之一，原因在於極其複雜的腦內腫瘤微環境，以及其對於輻射治療之抗性。 本研究室之前在攝護腺癌與腦癌的研究中發現在接受過輻射治療後的腦瘤微環境中，CD11b+髓狀細胞對於腫瘤的復發扮演相當重要之角色。本實驗利用CD11b-DTR 基因轉殖鼠來探討腦腫瘤接受HSV-Tk前驅藥物GCV的治療過程，這些CD11b+髓狀細胞在其中所扮演的角色。透過連續施打兩劑的Diphtheria toxin (白喉毒素)於基因轉殖鼠，我們幾乎可以完全移除腹腔內的巨噬細胞，並且讓血液中的單核球顯著的減少。 在小鼠原位腦腫瘤模組中發現，專一性的減少巨噬細胞以及單核球可以有效增強GCV的治療效果，讓老鼠的平均存活天數從30.6增加到37.6天，並伴隨著抑制腫瘤生長之效果。相反的，專一性的減少巨噬細胞以及單核球並沒有辦法延長未接受治療老鼠之生存天數(DT vs PBS : 22.4 vs 25 天)，反而有加速了老鼠死亡之頃向。應用免疫染色法分析腦腫瘤組織，結果指出，接受GCV治療之後，巨噬細胞大量的浸潤至腫瘤內部，並且伴隨著腫瘤微血管密度(MVD)的上升。然而，專一性的減少這群巨噬細胞，可以使MVD下降，並留下更多的iNOS+巨噬細胞以及大量iNOS+髓狀細胞的浸潤。另一方面，單單減少腫瘤內的巨噬細胞而未接受治療則會使腫瘤內部有更多的ARG-1+髓狀細胞的浸潤。綜合以上結果，我們可以發現在原位腫瘤中巨噬細胞是扮演對抗腫瘤生長的角色，而在治療後卻是一位協助腫瘤血管新生以及復發的幫兇。此外，我們也發現只有在腫瘤治療中同時減少巨噬細胞的數量才能發揮其最佳之結合療效。總結來說，本篇研究結果提出了一個可行且有效的傳統治療結合標靶巨噬細胞作為輔助治療，為之後更深入的臨床治療提供了一個有力的方向。

Malignant glioma is one of the toughest tumors to be treated at present due to the complexity of the tumor microenvironment and the intrinsic resistant to therapy. Previous studies have shown that CD11b+ myeloid cells play essential role in recurrent prostate and brain tumors following radiation therapy. In this study, the CD11b-diphtheria toxin receptor (CD11b-DTR) transgenic mouse model was used to evaluate the role of CD11b+ myeloid cells in TK/GCV suicide gene therapy for brain tumor using a murine astrocytomal tumor model, ALTS1C1-TK. The results show that the depletion of peritoneal macrophages (CD11b+F4/80+) and blood monocyte (CD11b+Ly6G-Ly6C-) could be achieved after two injections of DT, but the neutrophil (CD11b+Gr-1+) were increased transiently. Results also found that the depletion of CD11b+ myeloid cells enhanced the efficacy of TK/GCV therapy as shown by the increase of median surviving time of GCV-treated ALTS1C1-TK tumor-bearing mice from 30.6 days to 37.6 days with significant tumor growth reduction. Interestingly, the depletion of CD11b+ myeloid cells did not benefit the surviving time of tumor-bearing mice after receiving two doses of DT injections compared to the control PBS group (22.4 days vs 25.0 days, respectively). The immunohistological analysis of the tumor tissues revealed that F4/80+ macrophages were significantly increased after GCV administration associated with increasing micro-vascular density (MVD) of the tumor. Selective depletion of these macrophages resulted in reduced MVD and increasing iNOS+ macrophages and myeloid cells. On the other hand, selective depletion of the macrophages without GCV treatment resulted in the increase of the ARG-1+ myeloid cells in the tumor. These results indicate that macrophages could change their roles from the anti-tumor activity in the primary tumor to the pro-tumor function in the therapy-induced recurrent tumors. This study also found the best time for macrophages/monocytes depletion was performed during the administration of GCV, but not before or after GCV treatment. In summary, this study demonstrates that macrophages play different roles in the primary and the recurrent tumors. This study also provides a feasible strategy for combining conventional therapies with macrophage targeting for brain tumor therapy.

Table of contents
中文摘要 4
Abstract 5
致謝 7
Table of contents 8
Chapter I Introduction 11
1.1 Glioblastoma multiforme (GBM) 11
1.2 Tumor-associated macrophages (TAMs) 12
1.3 Genetic depleting model: CD11b-DTR transgenic mice 14
1.4 Suicide gene therapy model: HSV-tk/ GCV 15
Chapter II. Material and Methods 18
2.1 Mice 18
2.2 Cell line cultures 18
2.3 Cell transfection 19
2.4 RNA isolation and reverse-transcribe PCR 19
2.5 Cytotoxicity MTT assay of GCV prodrug 20
2.6 Peritoneal macrophage preparation 20
2.7 Facial peripheral blood cells collection 21
2.8 Spleen cells preparation 21
2.9 Brain mononuclear cells collection by percoll gradient 21
2.10 Intracranial injection of orthotopic tumor model 22
2.11 Drug preparation 23
2.12 Process of embedding brain tumor samples 23
2.13 Immunohistochemical analysis 24
2.14 H & E staining 24
2.15 Immunofluorescence staining 25
2.16 Flow cytometry 26
2.17 Statistics 27
Chapter III Results 28
3.1 Establishment of ALTS1C1-TK cell line 28
3.1.1 Characteristics of the HSV-sr39tk-transfected cell lines in vitro 28
3.1.2 In vivo anti-tumor effect of the TK/GCV suicide gene therapy system 29
3.2 The monocyte/macrophage depletion system: CD11b-DTR transgenic mice 30
3.2.1 Effect of DT treatment on CD11b positive peritoneal cells of CD11b-DTR mice 30
3.2.2 Effect of DT treatment on CD11b positive peripheral blood cells of CD11b-DTR mice 31
3.2.3 Effect of DT treatment on CD11b positive cells of the spleen and the brain of CD11b-DTR mice 32
3.2.4 Effect of DT administration on normal mice. 33
3.3 The roles of macrophages in HSV-tk/GCV therapy 34
3.3.1 Selective depletion of macrophages/monocytes benefits the HSV-tk/GCV therapy. 34
3.3.2 H & E staining revealed the progression of tumor growth 35
3.3.3 The distribution of peripheral blood myeloid cells during tumor progression 35
3.3.4 The correlation of peripheral blood myeloid cells with mice survival 36
3.3.5 Selective depletion of macrophages/monocytes in CD11b-DTR mice decreased the TAMs in the tumor microenvironment 37
3.3.6 Selective depletion of macrophages/monocytes did not change the Mean Vessel Density in the tumor microenvironment 38
3.3.7 Selective depletion of macrophages/monocytes affect iNOS, ARG-1 expressions in HSV-tk tumors 39
3.3.8 The time dependent effect of selective depletion of the macrophages/monocytes 40
3.3.9 Long-term cytotoxic effect of DT to the CD11b-DTR transgenic mice without tumor 41
Chapter IV Discussion 42
4.1 Selective depletion of monocytes/macrophage benefits HSV-tk/GCV therapy 42
4.2 Increased TAMs in tumor after GCV therapy lead to angiogenesis 43
4.3 Increasing Neutrophils is positive correlated to mice survival 44
4.4 The roles of Microglia to tumor are not clarified here 45
4.5 Cytotoxic effect of the diphtheria toxin to the CD11b-DTR mice 47
4.6 Role of T-cells in tk/GCV therapy and their association with TAMs 48
4.7 Macrophage targeting from theory to clinical 49
Figure and Diagrams 51
Reference 89

Reference
1. Ostrom, Q.T., et al., The epidemiology of glioma in adults: a "state of the science" review. Neuro Oncol, 2014. 16(7): p. 896-913.
2. Ostrom, Q.T., et al., CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007-2011. Neuro Oncol, 2014. 16 Suppl 4: p. iv1-63.
3. Louis, D.N., et al., The 2007 WHO Classification of Tumours of the Central Nervous System. Acta Neuropathologica, 2007. 114(2): p. 97-109.
4. Malmström, A., et al., Temozolomide versus standard 6-week radiotherapy versus hypofractionated radiotherapy in patients older than 60 years with glioblastoma: the Nordic randomised, phase 3 trial. The Lancet Oncology, 2012. 13(9): p. 916-926.
5. Wong, E.T., et al., Outcomes and prognostic factors in recurrent glioma patients enrolled onto phase II clinical trials. J Clin Oncol, 1999. 17(8): p. 2572-8.
6. Ballman, K.V., et al., The relationship between six-month progression-free survival and 12-month overall survival end points for phase II trials in patients with glioblastoma multiforme. Neuro Oncol, 2007. 9(1): p. 29-38.
7. Brandenburg, S., et al., Resident microglia rather than peripheral macrophages promote vascularization in brain tumors and are source of alternative pro-angiogenic factors. Acta Neuropathol, 2016. 131(3): p. 365-78.
8. Chae, M., et al., Increasing glioma-associated monocytes leads to increased intratumoral and systemic myeloid-derived suppressor cells in a murine model. Neuro Oncol, 2015. 17(7): p. 978-91.
9. Wang, S.C., et al., Tumor-secreted SDF-1 promotes glioma invasiveness and TAM tropism toward hypoxia in a murine astrocytoma model. Lab Invest, 2012. 92(1): p. 151-62.
10. Gordon, S. and F.O. Martinez, Alternative Activation of Macrophages: Mechanism and Functions. Immunity, 2010. 32(5): p. 593-604.
11. Murray, P.J. and T.A. Wynn, Protective and pathogenic functions of macrophage subsets. Nature reviews. Immunology, 2011. 11(11): p. 723-737.
12. De Palma, M. and Claire E. Lewis, Macrophage Regulation of Tumor Responses to Anticancer Therapies. Cancer Cell. 23(3): p. 277-286.
13. Mantovani, A., et al., Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol, 2013. 229(2): p. 176-85.
14. Mantovani, A., et al., Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends in Immunology, 2002. 23(11): p. 549-555.
15. Zhou, M., et al., Serum macrophage-derived chemokine/CCL22 levels are associated with glioma risk, CD4 T cell lymphopenia and survival time. Int J Cancer, 2015. 137(4): p. 826-36.
16. Bingle, L., et al., Macrophages promote angiogenesis in human breast tumour spheroids in vivo. Br J Cancer, 2006. 94(1): p. 101-7.
17. De Palma, M., et al., Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell, 2005. 8(3): p. 211-226.
18. Squadrito, Mario L., et al., miR-511-3p Modulates Genetic Programs of Tumor-Associated Macrophages. Cell Reports, 2012. 1(2): p. 141-154.
19. Yuan, A., et al., Opposite Effects of M1 and M2 Macrophage Subtypes on Lung Cancer Progression. Scientific Reports, 2015. 5: p. 14273.
20. Lanciotti, M., et al., The role of M1 and M2 macrophages in prostate cancer in relation to extracapsular tumor extension and biochemical recurrence after radical prostatectomy. Biomed Res Int, 2014. 2014: p. 486798.
21. Laoui, D., et al., Tumor-associated macrophages in breast cancer: distinct subsets, distinct functions. Int J Dev Biol, 2011. 55(7-9): p. 861-7.
22. Liu, C.Y., et al., M2-polarized tumor-associated macrophages promoted epithelial-mesenchymal transition in pancreatic cancer cells, partially through TLR4/IL-10 signaling pathway. Lab Invest, 2013. 93(7): p. 844-54.
23. Rossi, M.L., et al., Immunocytochemical study of the cellular immune response in meningiomas. Journal of Clinical Pathology, 1988. 41(3): p. 314-319.
24. Morantz, R.A., et al., Macrophages in experimental and human brain tumors. Journal of Neurosurgery, 1979. 50(3): p. 305-311.
25. Simmons, G.W., et al., Neurofibromatosis-1 Heterozygosity Increases Microglia in a Spatially- and Temporally-Restricted Pattern Relevant to Mouse Optic Glioma Formation and Growth. Journal of neuropathology and experimental neurology, 2011. 70(1): p. 51-62.
26. Gutmann, D.H., et al., Somatic neurofibromatosis type 1 (NF1) inactivation characterizes NF1-associated pilocytic astrocytoma. Genome Research, 2013. 23(3): p. 431-439.
27. Hambardzumyan, D., D.H. Gutmann, and H. Kettenmann, The role of microglia and macrophages in glioma maintenance and progression. Nat Neurosci, 2016. 19(1): p. 20-7.
28. Saito, M., et al., Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice. Nat Biotechnol, 2001. 19(8): p. 746-50.
29. Van Ness, B.G., J.B. Howard, and J.W. Bodley, ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products. Journal of Biological Chemistry, 1980. 255(22): p. 10710-10716.
30. Robinson, E.A., O. Henriksen, and E.S. Maxwell, Elongation Factor 2: AMINO ACID SEQUENCE AT THE SITE OF ADENOSINE DIPHOSPHATE RIBOSYLATION. Journal of Biological Chemistry, 1974. 249(16): p. 5088-5093.
31. Honjo, T., et al., Diphtheria Toxin-dependent Adenosine Diphosphate Ribosylation of Aminoacyl Transferase II and Inhibition of Protein Synthesis. Journal of Biological Chemistry, 1968. 243(12): p. 3553-3555.
32. Stoneman, V., et al., Monocyte/Macrophage Suppression in CD11b Diphtheria Toxin Receptor Transgenic Mice Differentially Affects Atherogenesis and Established Plaques. Circulation research, 2007. 100(6): p. 884-893.
33. Fillat, C., et al., Suicide Gene Therapy Mediated by the Herpes Simplex Virus Thymidine Kinase Gene / Ganciclovir System: Fifteen Years of Application. Current Gene Therapy, 2003. 3(1): p. 13-26.
34. Moolten, F.L., Tumor Chemosensitivity Conferred by Inserted Herpes Thymidine Kinase Genes: Paradigm for a Prospective Cancer Control Strategy. Cancer Research, 1986. 46(10): p. 5276-5281.
35. Duarte, S., et al., Suicide gene therapy in cancer: Where do we stand now? Cancer Letters, 2012. 324(2): p. 160-170.
36. Takamiya, Y., et al., Gene therapy of maliganant brain tumors: A rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells. Journal of Neuroscience Research, 1992. 33(3): p. 493-503.
37. Freeman, S.M., et al., The “Bystander Effect”: Tumor Regression When a Fraction of the Tumor Mass Is Genetically Modified. Cancer Research, 1993. 53(21): p. 5274-5283.
38. Moolten, F.L. and J.M. Wells, Curability of Tumors Bearing Herpes Thymidine Kinase Genes Transfered by Retroviral Vectors. Journal of the National Cancer Institute, 1990. 82(4): p. 297-300.
39. Caruso, M., et al., Regression of established macroscopic liver metastases after in situ transduction of a suicide gene. Proceedings of the National Academy of Sciences of the United States of America, 1993. 90(15): p. 7024-7028.
40. Mesnil, M. and H. Yamasaki, Bystander Effect in Herpes Simplex Virus-Thymidine Kinase/Ganciclovir Cancer Gene Therapy: Role of Gap-junctional Intercellular Communication1. Cancer Research, 2000. 60(15): p. 3989-3999.
41. van Dillen, I.J., et al., Influence of the Bystander Effect on HSV-tk / GCV Gene Therapy. A Review. Current Gene Therapy, 2002. 2(3): p. 307-322.
42. Chiang, C.S., et al., Irradiation promotes an m2 macrophage phenotype in tumor hypoxia. Front Oncol, 2012. 2: p. 89.
43. Marilena Campanella, C.S., Glauco Tarozzo, and Massimiliano Beltramo,
Flow cytometric analysis of inflammatory cells in ischemic rat brain. Stroke. 2002;33:586-592, doi:10.1161/hs0202.103399, 2002.
44. Romero, I.L., et al., Molecular pathways: trafficking of metabolic resources in the tumor microenvironment. Clin Cancer Res, 2015. 21(4): p. 680-6.
45. Frieler, R.A., et al., Depletion of macrophages in CD11b diphtheria toxin receptor mice induces brain inflammation and enhances inflammatory signaling during traumatic brain injury. Brain Res, 2015. 1624: p. 103-12.
46. Duffield, J.S., et al., Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. The Journal of Clinical Investigation. 115(1): p. 56-65.
47. .
48. Leek, R.D., et al., Association of Macrophage Infiltration with Angiogenesis and Prognosis in Invasive Breast Carcinoma. Cancer Research, 1996. 56(20): p. 4625-4629.
49. Lamagna, C., M. Aurrand-Lions, and B.A. Imhof, Dual role of macrophages in tumor growth and angiogenesis. J Leukoc Biol, 2006. 80(4): p. 705-13.
50. OHNO, S., et al., Correlation of Histological Localization of Tumor-associated Macrophages with Clinicopathological Features in Endometrial Cancer. Anticancer Research, 2004. 24(5C): p. 3335-3342.
51. Coffelt, S.B., R. Hughes, and C.E. Lewis, Tumor-associated macrophages: effectors of angiogenesis and tumor progression. Biochim Biophys Acta, 2009. 1796(1): p. 11-8.
52. Redente, E.F., et al., Tumor progression stage and anatomical site regulate tumor-associated macrophage and bone marrow-derived monocyte polarization. Am J Pathol, 2010. 176(6): p. 2972-85.
53. Lin, E.Y., et al., Macrophages Regulate the Angiogenic Switch in a Mouse Model of Breast Cancer. Cancer Research, 2006. 66(23): p. 11238-11246.
54. Hochweller, K., et al., A novel CD11c.DTR transgenic mouse for depletion of dendritic cells reveals their requirement for homeostatic proliferation of natural killer cells. Eur J Immunol, 2008. 38(10): p. 2776-83.
55. Jung, S., et al., In Vivo Depletion of CD11c+ Dendritic Cells Abrogates Priming of CD8+ T Cells by Exogenous Cell-Associated Antigens. Immunity, 2002. 17(2): p. 211-220.
56. Ma, Y., et al., Autophagy and Cellular Immune Responses. Immunity. 39(2): p. 211-227.
57. Dijkgraaf, E.M., et al., Chemotherapy Alters Monocyte Differentiation to Favor Generation of Cancer-Supporting M2 Macrophages in the Tumor Microenvironment. Cancer Research, 2013. 73(8): p. 2480-2492.
58. DeNardo, D.G., et al., Leukocyte Complexity Predicts Breast Cancer Survival and Functionally Regulates Response to Chemotherapy. Cancer Discovery, 2011. 1(1): p. 54-67.
59. Shree, T., et al., Macrophages and cathepsin proteases blunt chemotherapeutic response in breast cancer. Genes & Development, 2011. 25(23): p. 2465-2479.
60. Paulus, P., et al., Colony-Stimulating Factor-1 Antibody Reverses Chemoresistance in Human MCF-7 Breast Cancer Xenografts. Cancer Research, 2006. 66(8): p. 4349-4356.
61. Mantovani, A. and P. Allavena, The interaction of anticancer therapies with tumor-associated macrophages. The Journal of Experimental Medicine, 2015. 212(4): p. 435-445.
62. Hanahan, D. and Robert A. Weinberg, Hallmarks of Cancer: The Next Generation. Cell, 2011. 144(5): p. 646-674.
63. Coffelt, S.B., et al., Elusive Identities and Overlapping Phenotypes of Proangiogenic Myeloid Cells in Tumors. The American Journal of Pathology, 2010. 176(4): p. 1564-1576.
64. Piao, Y., et al., Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype. Neuro-Oncology, 2012. 14(11): p. 1379-1392.
65. Lu-Emerson, C., et al., Increase in tumor-associated macrophages after antiangiogenic therapy is associated with poor survival among patients with recurrent glioblastoma. Neuro-Oncology, 2013. 15(8): p. 1079-1087.
66. Kolaczkowska, E. and P. Kubes, Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol, 2013. 13(3): p. 159-175.
67. Nauseef, W.M. and N. Borregaard, Neutrophils at work. Nat Immunol, 2014. 15(7): p. 602-611.
68. Kruger, P., et al., Neutrophils: Between Host Defence, Immune Modulation, and Tissue Injury. PLoS Pathogens, 2015. 11(3): p. e1004651.
69. Dvorak , H.F., Tumors: Wounds That Do Not Heal. New England Journal of Medicine, 1986. 315(26): p. 1650-1659.
70. Lakshman, R. and A. Finn, Neutrophil disorders and their management. Journal of Clinical Pathology, 2001. 54(1): p. 7-19.
71. Bekes, E.M., et al., Tumor-Recruited Neutrophils and Neutrophil TIMP-Free MMP-9 Regulate Coordinately the Levels of Tumor Angiogenesis and Efficiency of Malignant Cell Intravasation. The American Journal of Pathology, 2011. 179(3): p. 1455-1470.
72. Powell, D.R. and A. Huttenlocher, Neutrophils in the Tumor Microenvironment. Trends in Immunology, 2016. 37(1): p. 41-52.
73. Nozawa, H., C. Chiu, and D. Hanahan, Infiltrating neutrophils mediate the initial angiogenic switch in a mouse model of multistage carcinogenesis. Proceedings of the National Academy of Sciences of the United States of America, 2006. 103(33): p. 12493-12498.
74. Rao, H.-L., et al., Increased Intratumoral Neutrophil in Colorectal Carcinomas Correlates Closely with Malignant Phenotype and Predicts Patients' Adverse Prognosis. PLoS ONE, 2012. 7(1): p. e30806.
75. Li, Y.-W., et al., Intratumoral neutrophils: A poor prognostic factor for hepatocellular carcinoma following resection. Journal of Hepatology, 2011. 54(3): p. 497-505.
76. Jensen, T.O., et al., Intratumoral neutrophils and plasmacytoid dendritic cells indicate poor prognosis and are associated with pSTAT3 expression in AJCC stage I/II melanoma. Cancer, 2012. 118(9): p. 2476-2485.
77. Jensen, H.K., et al., Presence of Intratumoral Neutrophils Is an Independent Prognostic Factor in Localized Renal Cell Carcinoma. Journal of Clinical Oncology, 2009. 27(28): p. 4709-4717.
78. Trellakis, S., et al., Polymorphonuclear granulocytes in human head and neck cancer: Enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease. International Journal of Cancer, 2011. 129(9): p. 2183-2193.
79. Prinz, M. and J. Priller, Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease. Nat Rev Neurosci, 2014. 15(5): p. 300-312.
80. Frieler, R.A., et al., Depletion of macrophages in CD11b diphtheria toxin receptor mice induces brain inflammation and enhances inflammatory signaling during traumatic brain injury. Brain Research, 2015. 1624: p. 103-112.
81. Ueno, M., et al., Layer V cortical neurons require microglial support for survival during postnatal development. Nat Neurosci, 2013. 16(5): p. 543-551.
82. Ding, Z., et al., Antiviral drug ganciclovir is a potent inhibitor of microglial proliferation and neuroinflammation. The Journal of Experimental Medicine, 2014. 211(2): p. 189-198.
83. Zhao, L., et al., Recruitment of a myeloid cell subset (CD11b/Gr1 mid) via CCL2/CCR2 promotes the development of colorectal cancer liver metastasis. Hepatology, 2013. 57(2): p. 829-39.
84. Probst, H.C., et al., Histological analysis of CD11c-DTR/GFP mice after in vivo depletion of dendritic cells. Clinical and Experimental Immunology, 2005. 141(3): p. 398-404.
85. Hochweller, K., et al., A novel CD11c.DTR transgenic mouse for depletion of dendritic cells reveals their requirement for homeostatic proliferation of natural killer cells. European Journal of Immunology, 2008. 38(10): p. 2776-2783.
86. Männ, L., et al., CD11c.DTR mice develop a fatal fulminant myocarditis after local or systemic treatment with diphtheria toxin. European Journal of Immunology, 2016.
87. Munn, D.H., et al., Inhibition of  T Cell Proliferation by Macrophage Tryptophan Catabolism. The Journal of Experimental Medicine, 1999. 189(9): p. 1363-1372.
88. Rodriguez, P.C., et al., l-Arginine Consumption by Macrophages Modulates the Expression of CD3ζ Chain in T Lymphocytes. The Journal of Immunology, 2003. 171(3): p. 1232-1239.
89. Sharda, D.R., et al., Regulation of Macrophage Arginase Expression and Tumor Growth by the Ron Receptor Tyrosine Kinase. Journal of immunology (Baltimore, Md. : 1950), 2011. 187(5): p. 2181-2192.
90. Kuang, D.-M., et al., Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1. The Journal of Experimental Medicine, 2009. 206(6): p. 1327-1337.
91. Zhu, Y., et al., CSF1/CSF1R Blockade Reprograms Tumor-Infiltrating Macrophages and Improves Response to T Cell Checkpoint Immunotherapy in Pancreatic Cancer Models. Cancer research, 2014. 74(18): p. 5057-5069.
92. Pyonteck, S.M., et al., CSF-1R inhibition alters macrophage polarization and blocks glioma progression. Nature medicine, 2013. 19(10): p. 1264-1272.
93. Ries, Carola H., et al., Targeting Tumor-Associated Macrophages with Anti-CSF-1R Antibody Reveals a Strategy for Cancer Therapy. Cancer Cell, 2014. 25(6): p. 846-859.