2011-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658217摘要：巨噬細胞為腫瘤微環境(tumor microenvironment)中發炎細胞之多數成員。癌細胞可藉由分泌化學吸引物(chemoattractants)來吸引血液中巨噬細胞聚集至腫瘤組織中，而形成腫瘤巨噬細胞。雖然巨噬細胞之前被認為具有抑制腫瘤作用之免疫細胞，最近之證據顯示巨噬細胞可被癌細胞或腫瘤微環境更改，轉而變為具有刺激腫瘤生長及擴散等促腫瘤作用之細胞。關於腫瘤巨噬細胞在人類癌症之進展轉移上扮演之功能，以及對癌症預測病人預後之重要性最近被熱烈的研究。高腫瘤巨噬細胞密度被報告在乳癌、子宮頸癌、黑色素瘤、膀胱癌、攝護腺癌及肺癌中，與癌細胞增生指數、腫瘤大小、高度血管新生、高度淋巴結轉移以及病患不良預後成正相關。然而在攝護腺癌、胃癌及肺癌中，也有報告指出高腫瘤巨噬細胞密度與早期臨床分期、較少淋巴結轉移、分化較好組織型以及較佳病患預後有關。在我們先前之研究中發現，腫瘤巨噬細胞密度及COX-2 表現與肺癌血管新生及病患不良預後成正相關，而巨噬細胞與肺癌細胞株交互作用後，會刺激肺癌細胞IL-8 及其他五十個基因之表現，其中包括與血管新生、發炎及代謝等相關基因(Yuan A et al. Clin Cancer Res 2003, Am J Respir Cell Mol Biol 2005, J ClinOncol 2005, Int J. Cancer 2005)。然而決定腫瘤巨噬細胞扮演抑制腫瘤作用或是促腫瘤作用之確切機轉仍未清楚，但可能受巨噬細胞與癌細胞、間質細胞以及腫瘤微環境間交互作用之影響。最近證據顯示巨噬細胞在不同刺激下，可分化成不同表現型之 M1(第一型，典型活化巨噬細胞)及M2 (第二型，另類活化巨噬細胞，又包括M2a, M2b 及M2c) 巨噬細胞，並可能擁有不同之功能。IFN-γ、lipopolysacharide (LPS) 及TNF 可促成第一型巨噬細胞 (M1)之分化，它可能具有引發Th1 反應及第一型發炎作用、殺菌及毒殺細胞等作用；然而IL-4、IL-13、immunocomplex、toll-like 接受體刺激物及IL-10 可促成第二型巨噬細胞 (M2a, M2b, M2c)之分化，並可能具有引發Th2 反應、過敏、抗寄生蟲、免疫調節、基質重組及免疫抑制與促腫瘤等作用。這些不同表現型之巨噬細胞具有不同之表面接受體、不同cytokine 及chemokine 表現、以及不同代謝方式，如此讓鑑定這些不同表現型之巨噬細胞，在現今變為可能。然而 M1 或M2 (a, b, or c) 型巨噬細胞對於肺癌細胞之腫瘤形成、侵略轉移等行為之確切影響及功能，以及對肺癌細胞基因表現之影響仍不清楚。M1 或M2 (a, b, or c)型巨噬細胞與血管內皮細胞交互作用對血管內皮細胞血管新生能力及血管新生相關基因之表現仍然未知。在肺癌或惡性胸水中不同表現型（M1 或M2）之巨噬細胞數目，與病患預後之關連性尚未被研究。在肺癌組織不同位置如間質或癌細胞間，浸潤之巨噬細胞其表現型之分類也未曾被研究。對於在體外或體內調控巨噬細胞分化為M1 或M2 型巨噬細胞，對腫瘤形成及轉移之影響，以及其具潛力之診斷及治療上之應用仍不清楚。這個子計畫是一個3 年之計畫。在此計畫中我們將首先研究M1 或M2 型巨噬細胞在體外對肺癌細胞之表現型(包括增生、移行及侵略能力)以及基因表現之影響，以及對血管內皮細胞之血管新生力及血管新生相關基因表現之影響 (第一年)。我們接著將在免疫不全鼠之腫瘤異體腫瘤移植模式及角膜血管新生模式中，研究M1 或M2 型巨噬細胞對肺癌腫瘤形成、轉移及血管新生之影響，以及對血管內皮細胞血管新生力之影響。我們也將研究肺癌組織中或惡性胸水中，M1 或M2 型巨噬細胞數目與病患臨床預後之相關性。另外我們也將鑑定肺癌組織中不同位置巨噬細胞之表現型及其功能 (第二年)。我們將在體外或體內促進巨噬細胞分化為M1 或M2 型巨噬細胞，並研究其在免疫不全鼠模式中抑制腫瘤生長及轉移之效果。另外，我們將發展一個敏感性的磁核共振影像模式，在活體內評估不同表現型巨噬細胞在腫瘤內之分佈。最後我們也將篩選數種中藥，評估其在體外及體內對巨噬細胞分化M1 或M2 型巨噬細胞之影響 (第三年)。表<br> Abstract: Macrophages constitute a large proportion of the inflammatory cell infiltrate inmany tumor microenvironments. Cancer cells can secrete a variety ofchemoattractants which attract macrophages and cause them to accumulate in thetumor tissue, where the macrophage becomes a tumor-associated macrophage(TAM). Although previously regarded as potent immune cells that have anti-tumoractivity, recent evidences showed that macrophages can be modified by cancer cellsand microenvironment, and are directed towards stimulating tumor growth andprogression, and thus have pro-tumorigenesis activity.The possible function of TAM on progression and invasion of human cancer,and the significance of TAM in patients’ prognosis are the topics under intensiveinvestigation recently. A high TAM density was reported to correlate with a highproliferation index, large tumor size, high tumor angiogenesis, high regional lymphnode metastasis, and a poor patients’ prognosis in several human cancers includingbreast, cervix, melanoma, bladder, prostate cancer and lung cancer. However,several studies showed that high TAMs are associated with less advanced clinicalstage and decreased lymph node metastasis, well differentiated histological type,and favorable patient prognosis in prostate, gastric and lung cancers. In our previousstudies, we also showed that TAM density and COX-2 expression correlates withangiogenesis and adverse prognosis in patients with non-small cell lung cancer, andmacrophage and cancer cell interaction can stimulate IL-8 and about 50 genesexpression (involved in angiogenesis, inflammation, metabolism etc) in lung cancercell (Yuan A et al. Clin Cancer Res 2003, Am J Respir Cell Mol Biol 2005, J ClinOncol 2005, Int J. Cancer 2005). Whether TAMs show pro- tumorigenesis oranti-tumor activity depends on the interactions between TAMs and the cancer cells,other stromal cells, and the tumor microenvironment, and the exact mechanism isstill under investigation.Recent evidence showed that depending on the activating stimuli, these cellscan develop into different subsets: classically (M1) or alternatively (M2) activatedmacrophage (including M2a, M2b and M2c), which has different function. Evidencesshowed that IFN-γ with LPS or TNF can polarize macrophage into M1 phenotypemacrophage, which may induce Th1 response, type 1 inflammation, and involved inkilling of pathogen or tumor cells. In contrast, IL-4, and IL-13, immunocomplex andtoll-like receptor ligand, and IL-10 can polarize macrophage into M2a, M2b and M2cphenotype macrophages respectively, which may involves in the Th2 response,allergy, killing of parasite, immunoregulation, matrix deposition, remodeling,down-regulation of immune responses and tumor promotion. The differentexpression of surface receptors, cytokine, chemokine and metabolism makeidentification of M1, M2a, M2b or M2c phenotype macrophages possible currently.The exact effects and possible functions of M1 or M2 (a, b or c) macrophagesubsets on the lung cancer cells’ biologic behaviors such as tumorigenesis, invasionand metastasis, and on the gene expression regulation of lung cancer cell areunclear. The effect of interaction between M1 or M2 (a, b, or c) macrophage andvascular endothelial cell on the angiogenesis capacity and angiogenesis-relatedgene expression is still unknown. The prognosis role of M1 or M2 macrophageaccumulation in tumor or malignant pleural effusion on the clinical outcome of lungcancer patients was not investigated before. The molecular characterization of TAMsin stroma or in tumor nest as M1 or M2 (a, b, or c) macrophage was not studiedbefore. The effect of in vitro or in vivo modulation of TAMs polarization into M1 or M2macrophage on the tumor progression and metastasis in lung cancer, and theirpotential diagnostic and therapeutic application are still unclear.This subproject is a 3-year project. In this study, we will investigate the effectof M1 or M2 macrophage on the phenotype (proliferation, migration and invasion)changes and gene expression profile changes of lung cancer cell, on theangiogenesis activity and angiogenesis-related genes expression of vascularendothelial cell using in vitro assess models (in the first year). We will also assessthe effect of M1 or M2 macrophage on the tumorigenesis, metastasis andangiogenesis capacity of lung cancer cell or HUVEC in in vivo tumor xenograft SCIDmice models and corneal angiogenesis models. We also investigate the prognosticeffect of M1 or M2 TAMs presentation on patient’s clinical outcome in lung cancerand malignant pleural effusion patients. We will identify the phenotype (M1 or M2) ofstromal or infiltrating TAMs in lung cancer tumor specimens (in the second year).We will try to modulate the M1 or M2 polarization of TAM in tumor in vitro and in vivoto investigate their effect on inhibition of lung cancer progression and metastasis inanimal models. We will also develop a sensitive MRI imaging method for evaluationof TAMs number, in M1 or M2 phenotype in vivo in lung tumor transplant in animalmodel. Finally, we will also screen herb drugs and evaluate the possibility to convertthe tumor associated macrophage from M2 phenotype to M1 phenotype or form M1to M2 and then proved by mice tumor xenograft model (in the third year).腫瘤相關巨噬細胞M1M2aM2c巨噬細胞非小細胞肺癌Tumor associated macrophageM1M2a and M2c macrophagenon-small cell lung cancer