2014-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658546摘要：肺癌是台灣及其他工業化國家最常見之癌症死亡原因之一（第二位）。肺癌之預後極差，在目前之治療模式下，其五年存活率低於15%。因此找尋其他有效之輔助治療方法極為重要。越來越多支證據顯示，許多惡性腫瘤之形成是被慢性感染誘發，或是與慢性發炎有關。在腫瘤微環境中之發炎細胞被證實可促進癌細胞之細胞增生、細胞存活、細胞移行侵略以及血管新生，因此可促進腫瘤之發展。我們之前的研究也顯示發炎細胞素如COX-2及IL-8 在非小細胞肺癌中過度表現，以及巨噬細胞與肺癌細胞交互作用可調控肺癌細胞中之基因表現 (Yuan, A et al. Am J Respir Crit Care Med 2000; 162: 1957、 Int.J. Cancer 2005, 115, 545、Clin. Cancer Res. 2003: 9, 729, J. Clin. Oncol, 2005,23:953)。 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。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。 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。我們之前針對M1 或M2 (a, c) 型巨噬細胞對於肺癌細胞之腫瘤形成、侵略轉移等行為之影響，以及對肺癌細胞基因表現之調節功能，結果發現不同表現型之腫瘤巨噬細胞(M1 vs M2a/M2c)在實驗室內及活體內對調節肺腺癌細胞之增生、移行、侵略、血管新生、腫瘤生成及藥物抗藥性上具有不同及反相之功能。不同表現型之腫瘤巨噬細胞亦能活化或抑制肺腺癌細胞內不同之訊息傳遞路徑，而這些活化或抑制之基因產物可做為預測肺癌病患之臨床存活預後之有意義指標。這些結果顯示不同表現亞型之巨噬細胞對肺癌之生物行為及基因調控有不同之影響 (圖一～圖四)。最近之研究顯示巨噬細胞之表現型可被一些細胞素、化學素、酵素或介質改變。巨噬細胞之功能及表現型可反覆地及多方向地被微環境中之細胞素（如IFN-γ, IL-12, IL-4 or IL-10）變化來改變。Rauh 等人亦發現Src homology 2-containing inositol-5＇-phophatase (SHIP)可抑制巨噬細胞分化成M2亞型IL-10, TGF-β and，而能促進M1亞型巨噬細胞之形成。另外， phosphatidylinositol 3-kinase (PI3K)亦被發現可抑制LPS引發之M1亞型巨噬細胞之分化。其他之研究也顯示蛋白質、藥物或化學物亦可改變巨噬細胞之功能表現型。PPAR 蛋白(peroxisome proliferator-activated receptor-gamma) 可抑制巨噬細胞分化成M1 巨噬細胞，但可促成M2 巨噬細胞之分化。Benznidazole （一種殺錐蟲藥）可經由抑制巨噬細胞內NF-kB 之活化而抑制LPS （脂多醣）引起之巨噬細胞偏極化成M1巨噬細胞。Acetylbritannilatone 亦可抑制巨噬細胞內iNO 及COX-2 之表現及抑制巨噬細胞向M1 亞型巨噬細胞之分化。腎上腺皮質素則被顯示可造成巨噬細胞之不活化狀態。而最近之研究亦發現微小核醣核酸亦可影響巨噬細胞之偏極化。然而大規模之篩選可能引導巨噬細胞偏極分化為M1 或 M2 亞型巨噬細胞，或者可導致M1轉換成 M2 亞型巨噬細胞，或可導致M2轉換成M1亞型巨噬細胞之可能藥物，則從未被研究或執行。 因此我們在先前之計畫中，已使用cDNA 微陣列之方法分析出受不同亞型巨噬細胞影響之肺癌細胞株之基因表現剖面差異；並進一步利用生物資訊演算模式，與FDA 核准之藥物對肺癌細胞基因表現調控影響之資料庫(Connectivity Map Q2 website) 進行比對，將對肺癌細胞株之基因表現與不同亞型巨噬細胞對肺癌細胞株基因表現有相似影響之藥物，經由計分並選出分數最高的五十種藥物，成為可能對巨噬細胞產生偏極化或有相似效果之候選藥物 (圖五及表一)。 此計畫為一個二年計畫。在此計畫中我們將之前利用生物資訊篩選出之候選藥物，評估其對 巨噬細胞偏極化與基因表現之影響，並進一步研究候選藥物對共同培養之肺癌細胞株之生物行為特性與其基因表現之影響。在第一年計畫中，我們將上述之候選藥物，經由細胞表面抗原與細胞素表現分析來確認並找出可在實驗室內促進巨噬細胞分化成M1巨噬細胞、將M2巨噬細胞轉成M1巨噬細胞、或抑制M1巨噬細胞轉成M2 巨噬細胞之藥物。並進一步研究其對巨噬細胞基因表現之調控。在第二年計畫中，我們將評估這些藥物對與巨噬細胞共同培養之肺癌細胞株的生物行為特性如細胞增生、侵略、細胞凋亡、血管新生、上皮間質移行(EMT)及藥物抗藥性之影響。我們並進一步分析候選藥物對與巨噬細胞共同培養之肺癌細胞株基因表現之影響。此計畫已有助於找出對腫瘤巨噬細胞有偏極化作用並對肺癌細胞有直接或間接抑制作用之候選藥物，以提供之後選擇適當病患接受將來發展以巨噬細胞為標靶之新治療之依據。這些資訊將對以腫瘤微環境之腫瘤巨噬細胞為治療標的之肺癌治療新策略提供基礎，並對治療抗藥性肺癌提供一有潛力之治療模式。 <br> Abstract: Lung cancer is the most common cause of cancer-related death in Taiwan (top 2) and other industrialized nations, and it has poor prognosis with the five-year survival rate was less than 15% current treatment modalities. Therefore, the search for other effective adjuvant therapy is important for treatment of lung cancer. Recent data have established the concept that inflammation is a critical component of tumor initiation and progression. The inflammatory cells in tumor micro- environment had been shown to be able to enhance cancer cell proliferation, cell survival, cell migration, and angiogenesis, thereby promoting tumor development. Our previous studies also showed that the Inflammatory cytokines such as COX-2 and Interleukin-8 were over-expressed in NSCLC, and interaction between macrophage and lung cancer cell can regulate gene expression in lung cancer (Yuan, A et al. Am J Respir Crit Care Med 2000; 162: 1957、 Int. J. Cancer 2005, 115, 545、Clin. Cancer Res. 2003: 9, 729, J. Clin. Oncol, 2005, 23:953) Macrophages constitute a large proportion of the inflammatory cell infiltrate in tumor microenvironments. Tumor-associated macrophages (TAM) were previously regarded as potent immune cells that have anti-tumor activity. However, recent evidences showed that macrophages can be modified by cancer cells and microenvironment, and are directed towards stimulating tumor growth and progression, and thus have pro-tumorigenesis activity. A high TAM density was reported to correlate with a high proliferation index, large tumor size, high tumor angiogenesis, high regional lymph node metastasis, and a poor patients’ prognosis in several human cancers including breast, cervix, melanoma, bladder, prostate cancer and lung cancer. However, several studies showed the opposite results as that high TAMs are associated with less advanced clinical stage and decreased lymph node metastasis, well differentiated histological type, and favorable patient prognosis in prostate, gastric and lung cancers. In our previous studies, we also showed that TAM density and COX-2 expression correlates with angiogenesis and adverse prognosis in patients with non-small cell lung cancer, and macrophage and cancer cell interaction can stimulate IL-8 and about 50 genes expression (involved in angiogenesis, inflammation, metabolism etc) in lung cancer cell (Yuan A et al. Clin Cancer Res 2003, Am J Respir Cell Mol Biol 2005, J Clin Oncol 2005, Int J. Cancer 2005). Whether TAMs show pro- tumorigenesis or anti-tumor activity depends on the interactions between TAMs and the cancer cells, other stromal cells, and the tumor microenvironment, and the exact mechanism is still under investigation. Recent evidence showed that depending on the activating stimuli, TAMs can differentiate into different subsets macrophage: classically (M1) or alternatively (M2) activated macrophages (including M2a, M2b and M2c), which has different functions. We have previously studied the effects and possible functions of M1 or M2 (a, b or c) macrophage subsets on the lung cancer cells’ biologic behaviors and on the gene expression regulation in lung cancer cell. The results showed that the different phenotype macrophages (M1vs M2a/M2c) have different and opposite in vitro and in vivo effects on regulation of lung cancer cell behaviors, such as proliferation, migration, invasion, angiogenesis, tumorigenesis and drug resistance. In addition, M1 and M2a/M2c macrophages can activate or inhibit different gene expression signal transduction pathways in lung cancer cells, and these activated or inhibited gene expression signals can be used as significant indicators for patients’ survival in lung cancers. These results implied that different phenotype TAMs have different impacts on regulation of cancer cell progression and gene expression in lung cancer (Fig 1~Fig 4). Recent studies showed that macrophage phenotype can be changed by cytokines, chemikine, and matrix metalloprotease and mediator secretion. Macrophage can reversibly and progressively change the pattern of functional phenotype through a multitude of patterns in response to changes in cytokine environment such as IFN-γ, IL-12, IL-4 or IL-10. Rauh et al. also showed that Src homology 2-containing inositol-5’-phophatase (SHIP) can repress the macrophage differentiating into M2 phenotype macrophage, and can promote differentiation in M1 phenotype macrophage. In contrast, IL-10, TGF-β and phosphatidylinositol 3-kinase (PI3K) can restrains macrophage differentiating into M1 phenotype macrophage after lipopolysaccaride stimulation. Several proteins or drugs have also been reported to be able to regulate the phenotype change of macrophage recently. The pharmacological activation of PPARs attenuated expression of macrophage inflammatory programs, and the peroxisome proliferator-activated receptor-gamma is a negative regulator of classically activated macrophage (M1) activation. Benznidazole, a trypanocidal drug, was shown to inhibit Lipopolysaccharide induction of nitric oxide synthase gene tanscription through Inhibition of NF-kB Activation in macrophage, and then suppress the macrophage polarized to M1 phenotype. Glucocorticoids are potent stimulators of macrophage deactivation, as evidenced by down-regulation of MHC class II molecules, inhibition of antigen presentation and suppression of inflammation. In addition, microRNAs are also reported to play an role in polyrizatoin of tumor associated macrophage recently. However, large-scale screening of drugs for their activity for modulating the phenotype polarization of macrophage to M1 or M2 (a/c), or switch macrophage between M1 and M2 (a/c) phenotype has not be performed before. In our previous studies, we had used cDNA microarrya to identify the different gene expression profile between lung cancer cells cocultured with different phenotype macrophages (M1 vs M2). We further use bioinformative algorithm to compared the gene expression profile between lung cancer cocultured with different phenotype macrophage with the gene expression profile of lung cancer affected by large scale FDA approved drugs in data bank of Connectivity Map Q2 website. We select the potential drugs which have the similar effects of different phenotype macrophage on regulation of lung cancer gene expression. We rank the score of each potential drugs, and select the top 50 rank drug as the candidate drugs which may have the potential effects on polarization of macrophage or similar effects on regulation lung cancer cell gene expression as different macrophage (Figure 5 and Table 1). This project is a two-year project. In this project, we will evaluated the effect of these candidate drugs on regulation of polarization of macrophage and regulation of the gene expression of macrophages. We will further evaluate the effects of these candidate drugs on modulation of the biologic behavior and gene regulation of lung cancer. In the first year, we will use the surface marker assay and cytokine expression assay to evaluate and select the drugs which can induce the macrophage to differentiate to M1 or M2 phenotype macrophage, or to convert M2 macrophage to M1 macrophage, or to inhibit the conversion of M1 macrophage to M2 macrophage in vitro. We will also evaluate the effect of these drugs on gene expression of macrophages. In the second year, we will evaluate the effects of candidate drugs on modulation of biologic behaviors such as cell proliferation, invasion, apoptosis, angiogenesis, epithelial-mesenchymal transition (EMT) and drug sensitivity to currently used chemotherapy drugs of lung cancer cocultured with macrophages. We will also evaluates the effects of these candidate drugs on regulation of gene expression of lung cancer. This project is helpful to select the potential candidate drugs which can modulate the polarization of macrophage, or can directly or indirectly inhibit lung cancer cell progression. The results of this project also can provide important information for developing a new immunotherapy strategy to target the tumor associate macrophages in tumor microenvironment as a new treatment modality for lung cancer in the future.候選藥物不同表現型巨噬細胞以巨噬細胞為治療標靶腫瘤擴展腫瘤微環境非 小細胞肺癌candidate drugdifferent phenotypemacrophagetargeting TAMstumor progressiontumor microenvironmentnon-small cell lung cancers