2012-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658191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。最近研究提供了一個重要之觀念，那就是發炎是腫瘤形成與發展之重要步驟。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。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。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2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-containinginositol-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。另一方面，中草藥成分最近被發現具有免疫調節之功能。如Panax ginseng可活化巨噬細胞功能。Astragalus可刺激巨噬細胞及自然殺手細胞，與抗癌藥合用可增加鉑基化療藥對非小細胞肺癌之療效。然而中草藥成分對巨噬細胞偏極化之影響並未被研究。這些研究都顯示在體外及體內操控巨噬細胞之功能表現型之偏極化極為可能。然而大規模之篩選藥物、化合物或中草藥成分以找出對操控巨噬細胞功能表現型之偏極化有作用之藥物則未被進行。這些資訊對以腫瘤微環境細胞為治療標靶之肺癌治療新策略將有極大價值。此計畫為一三年計畫。在此計畫中我們將篩選並研發可操控巨噬細胞偏極化、可抑制與巨噬細胞共同培養之肺癌細胞之侵略性以及以腫瘤巨噬細胞為治療標靶之候選藥物。在第一年計畫中，我們將從FDA 核准之藥物組合中，如LOPAC、Chemodex以及Sigma 組共含1880 個藥物，篩選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 巨噬細胞之候選藥物。我們也篩選中草藥成分中可操控巨噬細胞偏極化之候選藥物。我們將進一步研究這些候選藥物與巨噬細胞上與巨噬細胞偏極化有關之接受體如toll-like、INF-γ、IL-4及IL-10 接受體之結合力。我們也將研究這些候選藥物對巨噬細胞內相關下游基因之調控作用，以確立這些候選藥物對操控巨噬細胞偏極化之作用機制。在第二年計畫中，我們將評估這些候選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。另外我們也將評估這些候選藥物在活體內(免疫不全鼠)對與巨噬細胞混和注入之肺癌細胞的腫瘤生成、轉移及血管新生之影響及作用。我們也將對有影響之肺癌細胞做基因表現分析，以確認候選藥物經由操控巨噬細胞功能對肺癌細胞生物特性調控之作用機制。在第三年計畫中，我們將分析候選藥物可有效抑制侵略力等生物特性之肺癌細胞株或肺癌腫瘤之基因表現，以建立對候選藥物有效反應之預測指標，並提供之後選擇適當3病患接受以巨噬細胞為標靶之新治療之依據。另外我們也將評估候選藥物是否可藉由操控巨噬細胞偏極化，在實驗室內及活體內可克服肺癌細胞對目前治療藥物如上皮細胞生長因子接受體抑制劑(EGFR-TKI)及化療藥物之抗藥性。最後我們將進行一臨床前期計畫已確認這些候選藥物對非小細胞肺癌在免疫不全鼠上之療效及副作用。此計畫將有助於篩選出可促進腫瘤巨噬細胞偏極化成M1 巨噬細胞或將M2 巨噬細胞反轉成M1 巨噬細胞之有效候選藥物，釐清候選藥物對操控巨噬細胞偏極化之作用機轉，並確認候選藥物對與巨噬細胞交互作用之肺癌細胞實驗室內及活體內之抑制作用。此計畫已有助於找出對候選藥物有效之預測指標，並提供之後選擇適當病患接受以巨噬細胞為標靶之新治療之依據。此計畫也將確立候選藥物是否可藉由操控巨噬細胞而促進對上皮細胞生長因子接受體抑制劑(EGFR-TKI)及化療藥物具抗藥性之肺癌細胞之抑制效果。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-yearsurvival rate was less than 15% current treatment modalities. Therefore, the searchfor other effective adjuvant therapy is important for treatment of lung cancer.Recent data have established the concept that inflammation is a criticalcomponent of tumor initiation and progression. The inflammatory cells in tumor microenvironmenthad been shown to be able to enhance cancer cell proliferation, cellsurvival, cell migration, and angiogenesis, thereby promoting tumor development. Ourprevious studies also showed that the Inflammatory cytokines such as COX-2 andInterleukin-8 were over-expressed in NSCLC, and interaction between macrophageand lung cancer cell can regulate gene expression in lung cancer (Yuan, A et al. AmJ 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 intumor microenvironments. Tumor-associated macrophages (TAM) were previouslyregarded as potent immune cells that have anti-tumor activity. However, recentevidences showed that macrophages can be modified by cancer cells andmicroenvironment, and are directed towards stimulating tumor growth and progression,and thus have pro-tumorigenesis activity. The possible function of TAM on progressionand invasion of human cancer, and the significance of TAM in patients’ prognosis arethe topics under intensive investigation recently. A high TAM density was reported tocorrelate with a high proliferation index, large tumor size, high tumor angiogenesis,high regional lymph node metastasis, and a poor patients’ prognosis in several humancancers including breast, cervix, melanoma, bladder, prostate cancer and lung cancer.However, several studies showed the opposite results as that high TAMs areassociated with less advanced clinical stage and decreased lymph node metastasis,well differentiated histological type, and favorable patient prognosis in prostate, gastricand lung cancers. In our previous studies, we also showed that TAM density andCOX-2 expression correlates with angiogenesis and adverse prognosis in patients withnon-small cell lung cancer, and macrophage and cancer cell interaction can stimulateIL-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 JRespir Cell Mol Biol 2005, J Clin Oncol 2005, Int J. Cancer 2005). Whether TAMsshow pro- tumorigenesis or anti-tumor activity depends on the interactions betweenTAMs and the cancer cells, other stromal cells, and the tumor microenvironment, andthe exact mechanism is still under investigation.2Recent evidence showed that depending on the activating stimuli, TAMscan differentiate into different subsets macrophage: classically (M1) or alternatively(M2) activated macrophages (including M2a, M2b and M2c), which has differentfunctions. 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 thegene expression regulation in lung cancer cell. The results showed that the differentphenotype macrophages (M1vs M2a/M2c) have different and opposite in vitro and invivo effects on regulation of lung cancer cell behaviors, such as proliferation, migration,invasion, angiogenesis, tumorigenesis and drug resistance. In addition, M1 andM2a/M2c macrophages can activate or inhibit different gene expression signaltransduction pathways in lung cancer cells, and these activated or inhibited geneexpression signals can be used as significant indicators for patients’ survival in lungcancers. These results implied that different phenotype TAMs have different impacts onregulation of cancer cell progression and gene expression in lung cancer (Fig 1~Fig 4).Recent studies showed that macrophage phenotype can be changed bycytokines, chemikine, and matrix metalloprotease and mediator secretion. Macrophagecan reversibly and progressively change the pattern of functional phenotype through amultitude 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-containinginositol-5’-phophatase (SHIP) can repress the macrophage differentiating into M2phenotype macrophage, and can promote differentiation in M1 phenotype macrophage.In contrast, IL-10, TGF-β and phosphatidylinositol 3-kinase (PI3K) can restrainsmacrophage differentiating into M1 phenotype macrophage after lipopolysaccaridestimulation .Recent investigation also showed that several proteins or drugs have beenreported to be able to regulate the phenotype change of macrophage. Thepharmacological activation of PPARs attenuated expression of macrophageinflammatory programs, and the peroxisome proliferator-activated receptor-gamma is anegative regulator of classically activated macrophage (M1) activation. Benznidazole, atrypanocidal drug, was shown to inhibit Lipopolysaccharide induction of nitric oxidesynthase gene tanscription through Inhibition of NF-kB Activation in macrophage, andthen suppress the macrophage polarized to M1 phenotype. Acetylbritannilatone wasalso shown to suppresses NO and PGE2 synthesis in macrophages through theinhibition of iNOS and COX-2 gene expression, and inhibit macrophage activation toM1 phenotype. Glucocorticoids are potent stimulators of macrophage deactivation, asevidenced by down-regulation of MHC class II molecules, inhibition of antigenpresentation and suppression of inflammation. However, large-scale screening ofdrugs for their activity for modulating the phenotype polarization of macrophage to M13or M2 (a/c), or switch macrophage between M1 and M2 (a/c) phenotype has not beperformed before.On the other hand, medicinal plants like Chinese herb drugs used asimmunomodulation can provide an alternative to conventional chemotherapy for humancancer. Some Chinese herb drugs such as Panax ginseng (39), Echinaceapurpurea ,and Astragalus may activate host defense mechanism by enhancemacrophage activity. Astragalus-based Chinese herbs combined with platinum basedchemotherapy improved the survival in NSCLC patients than chemotherapy along andstimulation of macrophages and NK cells by Astragalus memranaceus is a possiblemechanism (42). How about the effects of Chinese herbs in TAMs (M1, M2a, M2b orM2c)? This warranted further studies.This project is a three-year project. In this project, we will screen and developcandidate drugs that have effect to modulate TAMs polarization, to regulateinvasiveness of lung cancer cell cocultured with macrophage, and to treat lung cancerand TKI resistance lung cancer by targeting TAMs. This project is a three-year project.In the first year, we plan to screen the LOPAC panel including 1280 FDA approveddrugs, and Chemodex and Sigma panel including 600 FDA approved drugs for theireffects to polarize TAMs from the M2 to M1 macrophage or to inhibit TAMs changingfrom M1 to M2 in vitro. We will also screen herb drugs compounds panel and evaluatetheir effect to convert the tumor associated macrophage from M2 phenotype to M1phenotype or to inhibit TAMs switching from M1 to M2 phenotype in vitro. We willevaluate the affinity of these drugs and biochemical effects (such as docking) of newdeveloped drugs to activate toll-like receptor or IFN-gamma receptors or to inhibit IL-4and IL-10 receptors, or their effects on the downstream signals in macrophages. In thesecond year, we will assess the in vitro effects of the new developed drugs inregulation of behaviors of lung cancer cells coculture with different phenotype TAMs,including prolifereation, invasion, apoptosis, angiogenesis, epithelial-mesenchymaltransition (EMT) and drug resistance when lung cancer cells are cocultured withmacrophages. We will also verify the biologic effects of these candidate drugs inregulation of cancer progression in vivo including tumorigenesis, metastasis andangiogenesis using SCID mice tumor xenograft models using lung cancer cells mixedwith different phenotype macrophage. We will also identify the activated or inhibitedgene expression in lung cancer or lung cancer cells interacted with different phenotypemacrophage and treated by these candidate drugs to elucidate the mechanism of theeffects of candidate drugs. In the third year, we will characterize the TAMs inducedsignals in lung cancer cells or lung cancer tumor xenograft specimen to establish thepredictors for treatment response to these candidate drugs in vitro and in vivo and thiswill help to select candidate for treatment targeting TAMs. We will evaluate the in vitroand in vivo effects of candidate drugs in overcoming EGFR TKI resistance or4chemotherapy resistance in resistant lung cancer cells by modulating TAMs. Finally, wewill conduct a pre-clinical trial to evaluate the therapeutic effects of FDA approveddrugs or herb drug compounds that has effects to switch M2 to M1 macrophage ontreatment of lung cancer in a murine model.The efforts of this project will help to identify the potential candidate drugs orherb drug compounds that will modulate TAMs switching to M1 phenotypedifferentiation or convert M2 to M1 phenotype macrophage, to elucidate the possiblemechanism of these drugs effects on modulating TAMs, and to evaluate and confirmthe effects of these candidate drugs in inhibition of aggressiveness of lung cancer cellinteracted with different TAMs in vitro and in vivo models. This project will also identifythe predictors for treatment response by these candidate drugs in lung cancer cellsinteracted with TAMs, and this will help to select appropriate lung cancer for usingtherapy targeting in TAMs in tumor microenvironment. This project also help touncover the possibility to use drugs targeting TAMs in overcoming drug resistance toEGFR TKI or currently used chemotherapeutic agents in treatment of lung cancers.This information will provide the bases for developing drugs targeting TAMs as a newtherapeutic strategy in improving treatment of lung cancer or overcoming drugresistance of lung cancer in the future.不同表現型巨噬細胞治療標靶腫瘤生成血管新生轉移藥物非小 細胞肺癌targeting TAMsphenotypescreendrugstumorigenesisangiogenesismetastasisnon-small cell lung cancers