【作者】 张俊；

【导师】 杨波； 何俏军；

【作者基本信息】 浙江大学 ， 药理学， 2014， 博士

【摘要】 第一部分低氧对巨噬细胞M2型极化的选择性促进作用及其机制研究目的：巨噬细胞具有很强的功能可塑性,根据诱导条件的不同,巨噬细胞可以极化成两种主要的功能表型：经典活化的巨噬细胞(M1型)和替代性活化的巨噬细胞(M2型)。肿瘤组织中的巨噬细胞被称为肿瘤相关巨噬细胞(TAM),其表型和功能与肿瘤微环境密切相关。低氧作为众多恶性肿瘤发生发展过程中所必然经历的微环境条件之一,已被证实在TAM的浸润中发挥着重要作用,但其在TAM极化表型转换中的作用则鲜有研究。本部分研究将以低氧和巨噬细胞为研究对象,系统探讨低氧对巨噬细胞表型极化的影响和相关作用机制。方法：本研究采用小鼠巨噬细胞系RAW264.7和原代巨噬细胞BMDM作为研究对象。(1)流式细胞术检测RAW264.7细胞和BMDM细胞膜表面抗原的表达；(2)免疫组化及免疫荧光检测瘤组织切片相关蛋白的表达；(3)RT-PCR检测M2型巨噬细胞相关mRNA的水平；(4)表达谱芯片分析相关差异基因的表达；(5)Western blot考察相关信号通路蛋白的表达。结果：首先,采用免疫荧光技术对Lewis肺癌细胞(LLC)移植瘤组织中M2型巨噬细胞的表达及分布进行检测,发现肿瘤中的巨噬细胞(F4/80+)大多表现为M2型(CD209+)且主要集中在低氧区域(PIMO+).这一结果提示,低氧可能对巨噬细胞的M2型极化有一定的调控作用。通过建立常压低氧动物模型,研究发现间歇性低氧处理C57BL/6小鼠能明显促进肿瘤组织中巨噬细胞的浸润,同时伴随着CD209+巨噬细胞比例的增加。进一步利用体外低氧共培养模型,研究发现低氧处理RAW264.7细胞能选择性地促进巨噬细胞极化因子(LLC-CM、IL6、IL4和IL13)诱导的M2型表面标记物CD209和CD206的表达,但对M1型表面标记物CD86的表达无明显影响。此外,RT-PCR结果显示低氧能显著增加IL6诱导的M2型特征性基因Arg1和Yml的mRNA水平。表达谱基因芯片结果显示低氧条件下IL6诱导的巨噬细胞表达高水平的M2型基因和低水平的M1型基因。在BMDM的原代巨噬细胞模型中,同样发现低氧能促进IL6诱导的CD209的表达。以上结果均表明,低氧能选择性地促进IL6诱导的巨噬细胞M2型极化。在机制研究方面,结合特异性抑制剂与分子生物学手段的应用,发现IL6/STAT3信号通路及低氧相关的HIF信号通路并不直接参与低氧对巨噬细胞M2型极化的选择性促进作用。进一步采用基因芯片技术分析不同诱导条件下获得的巨噬细胞的差异基因,结果发现多个差异基因在MAPK信号级联中富集。Western blot结果也显示低氧能显著增加巨噬细胞中p-ERK,p-p38,p-JNK的表达。通过采用特异性抑制剂,证实虽然低氧条件下MAPK的三条子信号通路都被激活,但只有抑制ERK信号后才能逆转低氧介导的巨噬细胞M2型极化。结论：低氧微环境可以选择性地促进巨噬细胞M2型极化,该选择性促进作用与ERK信号通路的激活密切相关。第二部分低氧介导的M2型巨噬细胞对肿瘤转移的影响目的：低氧是包括非小细胞肺癌(NSCLC)在内的多数实体瘤发展过程中所必然经历的微环境条件。长期以来,肿瘤低氧研究的重心是其对肿瘤细胞本身的影响,而忽视了低氧通过作用于非肿瘤细胞来推动肿瘤发生发展的可能性。TAM作为非肿瘤细胞的主要成分,被认为是肿瘤恶性行为的参与者。第一部分的研究已经证实低氧能选择性地促进巨噬细胞的M2型极化(将该条件下获得的M2型巨噬细胞定义为低氧介导的M2型巨噬细胞)。在本部分研究中,将以NSCLC为研究对象,考察低氧介导的M2型巨噬细胞对肿瘤转移的影响,进而探讨低氧对巨噬细胞M2型极化的作用与低氧介导的肿瘤恶性行为(转移)之间的相关性,有助于从新的角度完善肿瘤低氧研究领域,以期为肿瘤治疗提供新的理论基础和可能的治疗策略。方法：(1)免疫组化及免疫荧光检测临床病人样本和瘤组织切片相关蛋白的表达；(2)划痕修复和transwell小室法检测细胞的迁移运动能力；(3)管腔形成实验考察HUVEC细胞的管腔形成能力；(4)C57BL/6动物模型检测Lewis肺癌的肺转移率；(5)SRB染色法评价肿瘤细胞的增殖能力。结果：36例人肺组织样本基因芯片数据(GSE1987)分析和55例(其中20例为转移病人)临床NSCLC病人样本的免疫组化分析结果表明M2型TAM与NSCLC的转移密切相关。在此基础上,通过体内外实验评价第一部分中阐述的低氧介导的M2型巨噬细胞对NSCLC转移的影响。体外实验,采用条件培养基与LLC细胞或HUVEC细胞共孵育模型,通过细胞增殖实验、划痕修复实验、transwell实验和管腔形成实验等手段,发现低氧介导的M2型RAW264.7细胞上清可增加HUVEC细胞的管腔形成和LLC细胞的迁移运动,但对LLC细胞的增殖无影响。体内实验发现,将低氧介导的M2型RAW264.7细胞与LLC细胞的共接种可增加LLC的肺转移发生率(从22.6%增加到100%)和移植瘤肿瘤组织中的血管生成。以上结果表明,低氧介导的M2型巨噬细胞具有强有力的促进肿瘤血管生成和转移的能力。鉴于低氧介导的M2型巨噬细胞表现出与低氧类似的促进肿瘤演进的功能,进一步考察低氧对巨噬细胞M2型极化的促进作用与低氧介导的肿瘤恶性行为之间的相关性。结果显示,间歇性低氧处理C57BL/6小鼠可显著促进LLC的肺转移发生率,从20%增加到60%。免疫荧光实验结果表明低氧处理后,肿瘤组织中的M2型巨噬细胞显著增加。此外,本研究还发现ERK特异性抑制剂PD98059可通过靶向巨噬细胞,(而不是肿瘤细胞)抑制肿瘤细胞的迁移运动。结论：低氧介导的M2型巨噬细胞,在体内外均能显著促进肿瘤的转移(迁移)和血管生成。干扰ERK可通过靶向巨噬细胞,抑制肿瘤细胞的迁移运动。第三部分基于抑制TAMM2型极化的化合物M的发现及其抗转移作用研究目的：抑制TAM的M2型极化被认为是极具应用前景的抗肿瘤转移新策略,但相关小分子化合物的发现尚处于起步阶段。因此,寻找并发现特异性抑制TAMM2型极化的小分子化合物是开发以TAM为中心的治疗策略的一个关键方向。前期研究中,通过筛选发现了特异性抑制巨噬细胞M2型极化的化合物M。在本部分研究中,将进一步评价化合物M对肿瘤转移的作用并初步探讨其作用机制,确证通过小分子化合物干预M2型极化实现抗肿瘤转移的可行性,以期为设计全新抗肿瘤转移化合物或干预手段提供思路及潜在靶点。方法：(1)SRB染色法评价细胞增殖能力；(2)流式细胞术检测RAW264.7细胞和BMDM细胞膜表面抗原的表达；(3)免疫荧光检测瘤组织切片中相关蛋白的表达；(4)RT-PCR检测M1、M2型巨噬细胞特征性mRNA水平；(5) Transwell小室法检测肿瘤细胞的迁移运动；(6)C57BL/6动物模型考察LLC的肺转移情况；(7)HE染色检测肺部转移灶点；(8)Western blot考察相关信号通路蛋白的表达。结果：体外采用经典的IL13诱导的M2型巨噬细胞极化模型,从众多化合物中筛选得到可抑制巨噬细胞M2型极化的化合物M。在此基础之上,进一步应用IL4和IL6诱导的M2型极化模型,也得到了同样的结果。另一方面,通过对IFNy和LPS诱导的M1型巨噬细胞极化模型的考察,发现化合物M能选择性抑制巨噬细胞M2型极化,而对M1型极化无影响。LLC移植瘤组织切片的免疫荧光结果表明化合物M在体内也能抑制巨噬细胞的M2型极化。进一步考察化合物M的体外抗转移作用,研究发现化合物M可通过抑制巨噬细胞的M2型极化来影响肿瘤细胞的迁移运动。LLC自发肺转移动物实验结果显示化合物M具有体内抗肿瘤转移作用。通过引入巨噬细胞“清除剂”脂质体氯磷酸盐,研究发现化合物M对肿瘤肺转移的抑制作用可能是通过抑制肿瘤组织中巨噬细胞的M2型极化来发挥作用的。免疫荧光结果证实化合物M能减少移植瘤肿瘤组织中新生血管的形成。机制研究证实化合物M通过激活AMPK来发挥抑制巨噬细胞M2型极化的作用。结论：小分子化合物M能选择性地抑制巨噬细胞M2型极化,并基于此发挥体内外抗转移作用。化合物M对巨噬细胞M2型极化的选择性抑制作用依赖于AMPK信号通路的激活。更多还原

【Abstract】 Section1The role of hypoxia in the selective promotion of M2macrophage polarization and its underlying mechanismsObjective:Macrophages display phenotypic and functional plasticity, as they can shift between different modes of activation and perform divergent functions according to the microenvironmental cues they receive. The macrophages have two well-established polarized phenotypes, the classically activated macrophages (M1) and alternatively activated macrophages (M2). The macrophages within the tumor are often called tumor-associated macrophages (TAMs). The activation and function of TAM is related to the microenvironment of tumor. As the hallmark feature of malignant tumors, hypoxia has been proved to play an important role in TAM infiltration into tumor tissue. However, the impact of hypoxia on the phenotype shift of TAM is still largely unknown. Thus, this study attempts to characterize the effect of hypoxia on macrophage phenotype shift and functional responses, as well as the underlying mechanisms involved.Methods:(1) Flow cytometric analysis was performed to analyze the expression of cell surface markers.(2) Immunostaining analysis was used to detect the expression of related proteins in tumor tissue.(3) The expression of M2phenotype genes was measured by RT-PCR.(4) A gene microarray approach was used to compare the expression of differential genes.(5) Western blot assay was employed to detect protein expression.Results:The previous study in LLC model has showed that nearly half of the macrophages (F4/80+) exhibited a M2phenotype (CD209+) and preferentially situated at hypoxic regions (PIMO+), indicating that tumor hypoxia is associated with M2macrophage polarization. To further clarify this issue, C57BL/6mice bearing LLC tumor were exposed to normoxia or normobaric hypoxia for4h every day. As expected, intermittent hypoxia exposure significantly increased the infiltration of macrophages (F4/80+) as well as the ratio of CD209+macrophages in primary tumor tissue. By using the in vitro co-culture model of hypoxia, we observed that hypoxia selectively promoted the expression of CD209and CD206(markers for M2macrophage) but not CD86(a marker for M1macrophage) in macrophages induced by stimulation (LLC-CM, IL6, IL4or IL13). Moreover, the mRNA expression of ARG1and YM1was significantly elevated in HC+IL6macrophages compared with that of NC+IL6macrophages. The microarray results showed that HC+IL6macrophages expressed low levels of M1phenotype related genes and high levels of M2phenotype related genes. We subsequently extended our study using a primary macrophages model based on the generation of BMDMs. Once again, IL6-treated BMDMs exposed to hypoxia expressed higher levels of CD209compared with the ones exposed to normoxia. The data above clearly demonstrate that hypoxia selectively promotes the M2polarization of macrophages triggered by IL6. Of note, HIF activity and STAT3signaling pathway did not participate in the M2macrophage polarization in response to IL6plus hypoxia. Furthermore, data from microarray assay and western blot analysis suggest that MAPK cascade is operative in our experimental model. While all three MAPKs, ERK, JNK and p38, are activated under hypoxia conditions, only the inhibition of ERK activation can block hypoxia-promoted M2macrophage polarization.Conclusion:Tumor hypoxia selectively promotes M2macrophage polarization through the activation of ERK. These observations highlight a novel concept of tumor hypoxia involved phenotype shift of macrophages.Section2Effects of hypoxia-promoted M2macrophages on tumor metastasisObjective:Hypoxia is a common phenomenon occurring in the majority of human tumors including NSCLC, and has been proved to play an important role in tumor progression. Most studies of tumor hypoxia focus on the effect of hypoxia on the inherent adhesive and invasive ability of tumor cells, but the possibility that hypoxia may aggravate tumor behaviors via effecting non-cancer cells is largely unknown. As the most prominent component of non-cancer cells, TAMs are associated with poor prognosis. Together with the fact that hypoxia selectively promotes the M2polarization of macrophages. In this section, we attempt to characterize the effect of hypoxia-promoted M2macrophages on tumor metastasis and angiogenesis, in the hope to determine whether the action of hypoxia on TAM is involved in hypoxia-driven tumor behaviors.Methods:(1) Immunostaining analysis was used to detect the expression of related proteins in tissue biopsies.(2) Wound healing assay and transwell assay were used to evaluate the effect of polarized macrophages on LLC cell migration.(3) The tube formation assay was employed to investigate the effect of polarized macrophages on tumor angiogenesis.(4) LLC spontaneous metastasis model was introduced to investigate the rate of metastasis.(5) SRB assay was used to examine the cell proliferation.Results:Through the analysis of microarray data (GSE1987) from36samples obtained from human lung tissue and the infiltration of M2macrophages in primary tumor tissues isolated from55NSCLC patients including20patients with metastasis, we observed that high density of M2TAM is associated with metastasis in NSCLC patients. Based on this finding, we prompted to evaluate the effect of hypoxia-promoted M2macrophages on the metastasis of LLC tumor both in vitro and in vivo. LLC cells or HUVEC cells were co-incubated with indicated macrophage-conditioned mediums, by employing cell proliferation assay, wound-healing assay, transwell assay and tube formation assay, the results suggested that the supernatant of hypoxia-promoted M2macrophages enhanced the formation of capillary-like structures in HUVEC cells and the migration of LLC cells, whereas showed no effect on LLC cell proliferation. Additionally, co-inoculation of hypoxia-promoted M2macrophages with LLC cells increased the metastasis of LLC transplanted tumors (from28.6%to100%) and the proportion of CD31-positive cells in tumor tissue. These results demonstrate that hypoxia-promoted M2macrophages could enhance the tumor metastasis and angiogenesis. Given that hypoxia-promoted M2macrophages exhibited similar function in cancer promotion as hypoxia, we thus hypothesized that hypoxia-promoted M2macrophage polarization might be involved in tumor hypoxia-driven tumorigenesis. By applying the in vivo hypoxia model, the results suggested that intermittent hypoxia significantly promoted the metastasis of LLC (from20%to60%), accompanied with increased CD209+macrophages infiltration in primary tumor tissue. Interestingly, by targeting to macrophages rather than tumor cells, the well-known ERK inhibitor PD98059could suppress the migration of LLC cells.Conclusion:Hypoxia-promoted M2macrophages enhance LLC metastasis and angiogenesis both in vitro and in vivo. By targeting macrophage polarization, the blockage of ERK could serve as a promising lung cancer therapeutic strategy. This study unveils a novel concept of tumor hypoxia and provides evidence for lung cancer intervention through modulating the phenotype of macrophages.Section3Compound M selectively inhibits TAM M2polarization and its effects on tumor metastasisObjective:Since TAM depolarization from M2phenotype is considered to be a promising anti-metastasis strategy, the development of specific small-molecule inhibitors for TAM M2polarization have been taken into concern. We have previously screened series of compounds and found that compound M can selectively reduce M2macrophage polarization. Based on this finding, we will further investigate the role of compound M in anti-metastasis and evaluate its underlying mechanisms.Methods:(1) SRB assay was used to examine the cell proliferation.(2) Flow cytometric analysis was performed to analyze the expression of cell surface markers.(3) Immunostaining analysis was used to detect the expression of related proteins in tumor tissues.(4) The expression of M1/M2phenotype genes was measured by RT-PCR.(5) Transwell assay was used to evaluate the migration of LLC cell.(6) LLC spontaneous metastasis model was introduced to investigate the tumor metastasis.(7) HE staining was used to evaluate lung metastasis.(8) Western blot assay was employed to detect protein expression.Results:By using IL13induced-M2macrophages, we successfully found compound M from series of small-molecule compounds, which could significantly suppress the M2polarization of macrophages. In addition, compound M decreased the M2polarization of macrophages induced by IL4and IL6. Subsequently, we extended our study into IFN-y and LPS-mediated Ml macrophage polarization model. Intriguingly, compound M has no effect on IFN-γ and LPS-triggered M1polarization. The data above clearly demonstrate that compound M selectively inhibits the M2polarization of macrophage. Immunostaining derived from LLC tumor tissues further validate that compound M can also inhibit M2macrophage polarization in vivo. Next, in order to clarify the role of compound M in inhibition of metastasis, transwell assay and LLC spontaneous metastasis assay were performed. As expected, compound M blocked the migration-promotion effect of M2macrophages on LLC cells and effectively prevented the metastasis of LLC in vivo. Furthermore, C57BL/6mice were depleted of macrophages by clodronate liposomes2days before inoculation with LLC cells, and then treated with or without compound M. The results from lung metastasis suggested that the anti-metastasis effect of compound M is through the inhibition of M2polarization. In addition, compound M could reduce the formation of new blood vessels in LLC-transplantated tumor tissue. We further demonstrated that reeducation of M2macrophage polarization by compound M relies substantially on activation of AMPK signaling.Conclusion:Compound M selectively suppresses the M2polarization of TAM through the activation of AMPK to prevent tumor metastasis. The study confirms the feasibility of small molecule compounds to inhibit tumor metastasis by targeting macrophage ploarization and provides potential therapeutic targets for anti-metastasis therapy.更多还原