背景與目的：減緩甚至消除腫瘤生長和轉移一直是癌症治療的主要目標。腫瘤相關巨噬細胞（tumor-associated macrophages, TAM）的浸潤（infiltration）所誘發之發炎反應會促進腫瘤的生長和存活（proliferation and survival）並誘發腫瘤血管新生與轉移（angiogenesis and metastasis），其中巨噬細胞分泌的抗酒石酸性磷酸酶（tartrate-resistant acid phosphatase, TRAcP）可能扮演重要角色。因此本研究欲探討M2巨噬細胞是否經由調控抗酒石酸性磷酸酶的表現促進腫瘤生長速度，以及其相關機制。
方法：我們將M2巨噬細胞與JC乳腺癌細胞經由不同組合方式(單純注射JC、M2和JC一起注射，先注射M2 30分鐘後再注射JC)注射至小鼠的乳腺皮下，經過14天後測量其腫瘤生長的速度、大小和重量、腫瘤的抗酒石酸性磷酸酶表現和血清中抗酒石酸性磷酸酶5a的濃度。
結果：實驗結果發現預先注射M2巨噬細胞30分鐘後再注射JC乳腺癌細胞之組別與其他兩組（單純注射JC以及M2和JC一起注射）比較能顯著促進小鼠乳腺癌細胞之生長，而且這與腫瘤抗酒石酸性磷酸酶的表現增加有關。
結論：M2巨噬細胞可以藉由調節抗酒石酸性磷酸酶影響腫瘤相關環境，進而顯著促進小鼠乳腺癌細胞之生長。因此，抗酒石酸性磷酸酶可能可作為臨床監測乳癌癌症預後的生物指標或標靶治療的目標。

Background and purpose:
Alleviating or even eliminating tumor growth and metastasis has been the main goal of cancer treatment. Tumor-associated macrophages (TAM, also called M2 macrophage) infiltration-induced inflammatory responses not only promote tumor growth and survival, but trigger tumor angiogenesis and metastasis. The tartrate-resistant acid phosphatase (TRAcP) secreted by macrophages may play an important role. This study aimed to investigate whether M2 macrophages promoted the growth of tumor by regulating the expression of TRAcP and its mechanisms.

Method:
M2 macrophages and JC breast cancer cells were injected into the mammary glands of BALB/c mice via different combinations: 1) JC alone; 2) M2 and JC at the same time; 3) M2 was injected 30 min before the injection of JC. After 14 days, the rate and size of tumor growth, the body weight of the mice, the expression of TRAcP in tumors, and the level of TRAcP in serum were measured to compare the baseline data of above parameters.

Statistic Analysis:
All data showed by MEAN±SD, using Two-Way Repeated Measures ANOVA to compared different groups and different times for the body weight and subcutaneous tumor size of the mice. One the other hand, using One-Way ANOVA to compared different groups for tumor size of the mice. Statistical software is SPSS.

Result:
The results showed that the injection of M2 macrophages 30 min before the injection of JC significantly promoted tumor growth relative to other two groups (JC only or M2 and JC were injected at the same time), and this was associated with the expression of TRAcP in tumors and serum.

Conclusion:
M2 macrophages may regulate tumor-related environment by modulating the level of TRAcP in tumors and serum, and thereby have significant impacts on the growth of breast cancer cells in mice. Therefore, TRAcP may be a promising biomarker for clinical prognosis or a target for oncological therapy in mice breast cancer.

目錄
摘要 III
Abstract IV
關鍵字： VI
背景/簡介 1
實驗目的 4
實驗假說 4
實驗材料和方法 5
細胞培養 5
細胞轉型 5
流式細胞儀 6
蛋白質抽取 6
西方墨點轉漬法（Western blot） 7
動物實驗 9
腫瘤組織切片染色 10
酵素免疫分析法(ELISA) 11
統計分析（Statistic Analysis） 11
結果 12
討論 34
Reference 39
表目錄
表一、比較預先注射M2巨噬細胞是否會影響不同組別小鼠的體重(g)。 18
表二、比較預先注射M2巨噬細胞是否會影響小鼠乳腺癌腫瘤的惡性度。 19
表三、比較預先注射抗酒石酸性磷酸酶是否影響不同組別小鼠的體重(g)。 20
表四、比較預先注射抗酒石酸性磷酸酶是否會影響小鼠乳腺癌腫瘤的惡性度。 21
圖目錄
圖一、RAW264.7巨噬細胞（圖A）、M2巨噬細胞（圖B）與JC乳腺癌腫瘤細胞（圖C）之形態學差異。 22
圖二、利用流式細胞儀觀察RAW及M2巨噬細胞與JC腫瘤細胞之CD68、Arg-1、iNOS與TRAcP之表現差異 23
圖三、不同組別小鼠在第0、7、10、14天測得之皮下腫瘤體積(mm3)變化 24
圖四、不同組別小鼠（JC、RAW+JC、RAW 30 ´+JC、M2+JC和M2 30 ´+JC）在第14天犧牲後腫瘤體積（mm3）和腫瘤重量（g） 的變化 25
圖五、以西方墨點法觀察不同組別間（JC、RAW+JC 、RAW 30´+JC 、M2+JC、M2 30´+JC）腫瘤TRAcP蛋白質表現 26
圖六、以酵素免疫分析法觀察小鼠血清中TRAcP 5a蛋白質濃度表現 27
圖七、不同濃度抗酒石酸性磷酸酶蛋白質對小鼠腫瘤體積(mm3)（第14天）的影響 28
圖八、不同組別小鼠在第0、10、14天測得之皮下腫瘤體積(mm3)變化 29
圖九、不同組別小鼠（JC、750ng TRAcP +JC、750ng TRAcP 30 ´+JC、1.5μg TRAcP +JC和1.5μg TRAcP 30 ´+JC）在第14天犧牲後腫瘤體積（mm3）和腫瘤重量（g） 的變化 30
圖十、以西方墨點法觀察不同組別間（JC、750ng TRAcP +JC、750ng TRAcP 30 ´+JC、1.5μg TRAcP +JC和1.5μg TRAcP 30 ´+JC）腫瘤TRAcP蛋白質表現 31
圖十一、以酵素免疫分析法觀察小鼠血清中TRAcP 5a蛋白質濃度表現 32
圖十二、不同組別小鼠（JC、M2+JC、M2 30 ´+JC、750ng TRAcP +JC、750ng TRAcP 30 ´+JC、1.5μg TRAcP +JC和1.5μg TRAcP 30 ´+JC）在第14天犧牲後腫瘤體積（mm3）和腫瘤重量（g） 變化 33

Reference
1.Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat Immunol. 2010 Oct;11(10):889-96.
2.Bull H, Murray PG, Thomas D, Fraser AM, Nelson PN. Acid phosphatases. Mol Pathol. 2002 Apr;55(2):65-72.
3.Candido J, Hagemann T. Cancer-related inflammation. J Clin Immunol. 2013 Jan;33 Suppl 1:S79-84.
4.Chao TY, Wu YY, Janckila AJ. Tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) as a serum maker for cancer with bone metastasis. Clin Chim Acta. 2010 Nov 11;411(21-22):1553-64.
5.Chao TY, Yu JC, Ku CH, Chen MM, Lee SH, Janckila AJ, Yam LT. Tartrate-resistant acid phosphatase 5b is a useful serum marker for extensive bone metastasis in breast cancer patients. Clin Cancer Res. 2005 Jan 15;11(2 Pt 1):544-50.
6.Chen YG, Janckila A, Chao TY, Yeh RH, Gao HW, Lee SH, Yu JC, Liao GS, Dai MS. Association of Tartrate-Resistant Acid Phosphatase-Expressed Macrophages and Metastatic Breast Cancer Progression. Medicine (Baltimore). 2015 Dec;94(48):e2165.
7.Chi KH, Wang HE, Chen FD, Chao Y, Liu RS, Chou SL, Wang YS, Yen SH. Preclinical evaluation of locoregional delivery of radiolabeled iododeoxyuridine and thymidylate synthase inhibitor in a hepatoma model. J Nucl Med. 2001 Feb;42(2):345-51.
8.Condeelis J, Pollard JW. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell. 2006 Jan 27;124(2):263-6. Review.
9.Crusz SM, Balkwill FR. Inflammation and cancer: advances and new agents. Nat Rev Clin Oncol. 2015 Oct;12(10):584-96.
10.Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K. Development of monocytes, macrophages, and dendritic cells. Science. 2010 Feb 5;327(5966):656-61.
11.Goede V, Brogelli L, Ziche M, Augustin HG. Induction of inflammatory angiogenesis by monocyte chemoattractant protein-1. Int J Cancer. 1999 Aug 27;82(5):765-70.
12.Gordon S. Alternative activation of macrophages. Nat Rev Immunol. 2003 Jan;3(1):23-35.
13.Hagemann T, Wilson J, Kulbe H, Li NF, Leinster DA, Charles K, Klemm F, Pukrop T, Binder C, Balkwill FR. Macrophages induce invasiveness of epithelial cancer cells via NF-kappa B and JNK. J Immunol. 2005 Jul 15;175(2):1197-205.
14.Janckila AJ, Nakasato YR, Neustadt DH, Yam LT. Disease-specific expression of tartrate-resistant acid phosphatase isoforms. J Bone Miner Res. 2003 Oct;18(10):1916-9. Review.
15.Janckila AJ, Neustadt DH, Yam LT. Significance of serum TRACP in rheumatoid arthritis. J Bone Miner Res. 2008 Aug;23(8):1287-95.
16.Janckila AJ, Parthasarathy RN, Parthasarathy LK, Seelan RS, Hsueh YC, Rissanen J, Alatalo SL, Halleen JM, Yam LT. Properties and expression of human tartrate-resistant acid phosphatase isoform 5a by monocyte-derived cells. J Leukoc Biol. 2005 Feb;77(2):209-18.
17.Janckila AJ, Slone SP, Lear SC, Martin A, Yam LT. Tartrate-resistant acid phosphatase as an immunohistochemical marker for inflammatory macrophages. Am J Clin Pathol. 2007 Apr;127(4):556-66.
18.Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J, Harris AL. Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res. 1996 Oct 15;56(20):4625-9.
19.Lin EY, Gouon-Evans V, Nguyen AV, Pollard JW. The macrophage growth factor CSF-1 in mammary gland development and tumor progression. J Mammary Gland Biol Neoplasia. 2002 Apr;7(2):147-62.
20.Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature. 2008 Jul 24;454(7203):436-44.
21.Mantovani A, Bottazzi B, Colotta F, Sozzani S, Ruco L. The origin and function of tumor-associated macrophages. Immunol Today. 1992 Jul;13(7):265-70.
22.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. 2002 Nov;23(11):549-55.
23.Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol. 2011 Oct 14;11(11):723-37.
24.N Volodko, A Reiner, M Rudas, R Jakesz. Tumour-associated macrophages in breast cancer and their prognostic correlations. The Breast. 1998 Apr;7(2):99-105.
25.Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell. 2010 Apr 2;141(1):39-51.
26.Raposo TP, Beirão BC, Pang LY, Queiroga FL, Argyle DJ. Inflammation and cancer: till death tears them apart. Vet J. 2015 Aug;205(2):161-74.
27.Riabov V, Gudima A, Wang N, Mickley A, Orekhov A, Kzhyshkowska J. Role of tumor associated macrophages in tumor angiogenesis and lymphangiogenesis. ront Physiol. 2014 Mar 5;5:75.
28.Sica A. Role of tumour-associated macrophages in cancer-related inflammation. Exp Oncol. 2010 Sep;32(3):153-8. Review.
29.Solinas G, Germano G, Mantovani A, Allavena P. Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J Leukoc Biol. 2009 Nov;86(5):1065-73.
30.Ueno T, Toi M, Saji H, Muta M, Bando H, Kuroi K, Koike M, Inadera H, Matsushima K. Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res. 2000 Aug;6(8):3282-9.
31.Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003 Dec;112(12):1796-808.
32.Wu YY, Chao TY, Liu HY, Huang TC, Chen JH, Dai MS, Janckila A, Lai SW, Chang PY. The correlation between a chronic inflammatory marker Tartrate-resistant acid phosphatase 5a with cancer cachexia. J BUON. 2015 Jan-Feb;20(1):325-31.
33.Wu YY, Janckila AJ, Slone SP, Perng WC, Chao TY. Tartrate-resistant acid phosphatase 5a in sarcoidosis: further evidence for a novel macrophage biomarker in chronic inflammation. J Formos Med Assoc. 2014 Jun;113(6):364-70.
34.Zenger S, He W, Ek-Rylander B, Vassiliou D, Wedin R, Bauer H, Andersson G. Differential expression of tartrate-resistant acid phosphatase isoforms 5a and 5b by tumor and stromal cells in human metastatic bone disease. Clin Exp Metastasis. 2011 Jan;28(1):65-73.
35.衛生福利部國民健康署. 2013年癌症登記報告. 台灣癌症登記資料庫.