【作者】 刘杨；

【导师】 余海； 王青青；

【作者基本信息】 浙江大学 ， 免疫学， 2011， 博士

【摘要】 肿瘤的免疫逃逸机制是肿瘤研究的热点问题,只有充分揭示肿瘤细胞在各个途径各个水平上逃避免疫攻击的机制,才有可能在设计和实施特异性的治疗方案上取得突破。执行负向免疫调控功能的抑制性细胞亚群在免疫应答的各个环节发挥着举足轻重的作用,比如肿瘤诱导的调节性T细胞(Treg)与肿瘤的发生发展密切相关,是肿瘤逃逸的重要环节,近年被广为研究。而越来越多的证据表明一群具有Gr-1和CD11b标志的髓系来源抑制性细胞(Myeloid derived suppressor cells, MDSCs)与肿瘤进展关系十分密切,近年来在肿瘤免疫学领域倍受关注。MDSCs是一个异质性的细胞群体,它主要由不成熟的粒细胞、单核细胞、树突状细胞和处于更早分化阶段的髓系前体细胞组成。在正常生理情况下,在小鼠骨髓中大约有20%比例的CD11b+ Gr-1+不成熟髓系细胞,在脾脏中也有少量分布,可迅速分化为成熟髓系细胞,也无免疫抑制功能。在荷瘤动物以及肿瘤患者体内可见到这群细胞大量产生,并在各种组织器官包括肿瘤局部聚集,并有很强的免疫抑制活性,促进肿瘤的免疫逃逸。小鼠来源的MDSCs共表达Gr-1和CD11b。已有的研究认为肿瘤产生的一些因子以及免疫细胞分泌的一些细胞因子对MDSCs的产生、活化和募集等过程发挥重要作用,主要包括VEGF、COX-2、GM-CSF、IL-4、IL-6等；而浸润于肿瘤组织的MDSCs能通过释放基质金属蛋白酶和分化为内皮细胞等方式促进肿瘤血管生成及肿瘤的浸润转移。MDSCs免疫抑制功能的研究主要集中在抗原特异性CD8+T细胞,主要机制为：增加精氨酸酶(Arginase 1, ARG1)的表达和活性从而耗竭L-精氨酸,下调TCR-CD3复合物ζ链的表达；或提高诱导性一氧化氮合酶(iNOS)活性,增加NO的合成和分泌,诱导T细胞凋亡；也可同时增加ARG1和iNOS的表达和活性,抑制T细胞；另有报道也可通过活性氧族(ROS)以及直接分泌抑制性因子如TGF-β。JAK/STAT是MDSCs扩增和活化的主要信号途径,包括STAT1, STAT3和STAT6和MDSC的聚集和抑制功能相关。目前对Gr-1+ CD11b+ MDSCs的负相免疫功能有所了解,但尚有许多问题需要进一步深入研究。在分子水平,对MDSCs异常聚集的机制及其发挥免疫抑制的主要效应分子如ARG1、iNOS等表达的调控机制(如表观遗传学调控机制)仍知之甚少。近年来,随着对基因的转录水平的调控机制认识的增加,基因的转录后调节机制被广泛关注,而microRNA (miRNA)作为主要的转录后调节因子更是成为了当前的研究热点。miRNA长度约为22个核苷酸,它通过结合在靶基因的mRNA的3’UTR抑制其翻译,也有报道可具有一定的降解靶基因mRNA的能力。miRNA参与了机体的各种的生理病理过程,如生长、发育、分化、炎症、肿瘤的发生。越来越多的研究显示miRNA也显著影响着免疫系统的各个方面,从造血、免疫细胞分化发育成熟以及免疫应答功能的发挥,都与miRNA的调控相关。对于这个重要的抑制性细胞亚群Gr-1+ CD11b+ MDSCs而言,miRNA调控很可能在其异常扩增和活化、发挥负向免疫调节中发挥着重要的作用,但目前相关的研究报道甚少。基于这一点,我们首先用miRNA表达谱芯片筛选了可能与调控Gr-1+ CD11b+ MDSCs功能相关的miRNAs。分选4T1乳腺癌荷瘤小鼠和对照小鼠骨髓中的Gr-1+CD11b+ MDSCs,通过应用miRNA表达谱芯片对比筛选得到8个miRNAs的表达水平存在显著性差异,我们选择了在肿瘤诱导的MDSCs中表达显著上调的miR-494作为本论文的研究对象。接着,我们进一步检测了来自于两个小鼠品系的共6个肿瘤模型小鼠体内的Gr-1+ CD11b+ MDSCs中miR-494的表达水平,结果显示相比于对照小鼠来源的Gr-1+ CDllb+细胞,所有这6种肿瘤模型诱导的MDSCs都高表达miR-494,提示miR-494表达增加是肿瘤相关的MDSCs的共同特征。先前的报道提示,肿瘤细胞通过分泌多种可溶性介质诱导MDSCs的聚集和活化,我们分离出正常小鼠骨髓来源的Gr-1+ CD11b+细胞,加入不同比例的肿瘤细胞培养上清(TCCM),结果显示TCCM可显著诱导miR-494在MDSCs中的表达上调,而且这种诱导作用呈现剂量和时间依赖性。同时,我们检测到伴随着miR-494的上调,MDSCs分泌的抑制性效应分子包括ARG1, iNOS2和促肿瘤转移相关的效应分子一金属蛋白酶(MMP2, MMP13和MMP14)表达剧烈增加。提示miR-494的上调可能和MDSC的聚集或活化相关。为了确认TCCM中何种介质参与诱导了miR-494的上调,我们使用了多种细胞因子和生长因子替代TCCM,结果发现TGF-β1可以部分重现TCCM对MDSCs中miR-494表达的诱导作用,而在TCCM培养组内加入TGF-β1阻断抗体可以部分阻断TCCM诱导的miR-494表达上调。我们进一步发现,相比于同窝对照野生型小鼠,Smad3-/-小鼠来源的Gr-1+ CD11b+细胞中的miR-494上调(TCCM介导)被显著地抑制。上述实验结果提示,肿瘤细胞分泌的可溶性因子尤其是TGF-β1可促进Gr-1+ CD11b+ MDSCs高表达miR-494,并且miR-494的增加可能与促进MDSCs效应分子的表达相关。为了探明miR-494在Gr-1+ CD11b+ MDSCs聚集和活化中发挥的可能作用,我们构建了编码pri-miR-494(Lv-494)和miR-494功能抑制序列(Lv-sponge)的慢病毒载体。用其进行体外转染MDSCs,我们发现过表达miR-494显著增加了MDSCs中效应分子的表达(ARG1,iNOS2, MMP2, MMP13和MMP14),而抑制miR-494则明显降低TCCM诱导的MDSCs效应分子的上调。为了确认miR-494介导的MDSCs免疫抑制效应的相关分子,以及MDSCs促肿瘤转移相关效应分子的上调是否直接与MDSCs的功能相关,我们检测了不同病毒处理后的MDSCs对CD8+ T细胞增殖和肿瘤细胞侵袭的影响。结果显示,过表达miR-494的Gr-1+CD11b+ MDSCs显著抑制CD8+ T细胞的增殖,还促进肿瘤细胞的侵袭,而这些效应都能被相关分子的抑制剂阻断。而且,过表达miR-494显著增加了MDSCs的存活,而抑制miR-494可诱导MDSCs发生凋亡；提示miR-494的上调可能是MDSCs大量聚集的一个重要机制。同时,进一步实验还发现过表达miR-494显著增加了MDSC对SDF-1(CXCL12)诱导的趋化的反应性。先前的报道已证实SDF-1介导的趋化是Gr-1+ CD11b+ MDSCs浸润到肿瘤局部的重要机制。上述结果显示miR-494的高表达在Gr-1+ CD11b+ MDSCs的聚集和活化、免疫抑制功能的维持中发挥着非常关键的作用,有可能是一个潜在的治疗相关的靶点。接下来,为了验证抑制MDSCs中的miR-494功能是否影响肿瘤的体内生长和转移,我们建立了4T1高转移乳腺癌小鼠模型,用Lv-sponge进行治疗并观察小鼠肿瘤生长、肺转移以及生存期。结果显示Lv-sponge治疗显著抑制原位肿瘤生长和肺转移,而Lv-sponge治疗的同时剔除CD8+T细胞虽然对原位肿瘤的生长无明显抑制作用,但Lv-sponge介导的抑制肺转移的作用仍很明显。荷瘤24天后,联合手术切除原位肿瘤显著提高了小鼠的存活率。而对肿瘤组织的流式和切片的免疫荧光染色分析显示，Lv-sponge显著减少了肿瘤组织内浸润的Gr-1+ CD11b+ MDSCs的数量。上述结果表明miR-494可能成为肿瘤免疫治疗的潜在靶点。microRNAs是通过调节靶基因的表达发挥其功能,我们应用了Targetscan和Pictar等对miR-494的靶基因进行分析。在数百个可能的靶基因中,PTEN引起了我们的注意,虽然尚没有报道显示PTEN在MDSCs中发挥调节功能,但是作为PI3K/Akt途径的重要调节分子,以往的报道显示PTEN负向调控SDF-1/CXCR4介导的中性粒细胞趋化和TGF-β1诱导的MMPs的表达。为了验证PTEN是否为miR-494调控MDSCs的功能靶点,我们首先比较了肿瘤小鼠和对照小鼠来源的Gr-1+ CDllb+ MDSCs中PTEN的表达。结果显示PTEN的mRNA水平没有显著性差异,但是肿瘤诱导的Gr-1+ CD11b+ MDSCs中PTEN的蛋白水平显著降低,这符合microRNA介导的转录后调节机制。而报告基因实验显示miR-494 mimic显著抑制PTEN 3’UTR荧光素酶报告基因的活性,转染Lv-494可有效降低PTEN在MDSCs中的表达,而Lv-sponge的作用正相反。以上结果表明miR-494靶向并抑制MDSCs中PTEN的表达。为了明确PTEN的降低是否介导miR-494对MDSCs功能的调节作用,我们构建了编码不含3’UTR区域的PTEN的慢病毒表达载体,通过转染MDSCs,我们发现在MDSCs中过表达PTEN不影响TCCM诱导的miR-494上调,但阻断了TCCM诱导的MDSCs中前述功能相关的所有效应分子的上调。上述结果表明PTEN是miR-494发挥调控MDSCs的功能靶点。PTEN是PI3K/Akt途径的主要的负性调节分子,我们进一步检测了Akt、mTOR和NF-κB的磷酸化水平,发现肿瘤相关的MDSCs显著高表达磷酸化的Akt、mTOR和NF-κB p65。TCCM也可诱导正常小鼠来源的Gr-1+ CD11b+细胞中该途径的显著活化。而使用P13K和mTOR及NF-κB的特异性抑制剂LY294002和雷帕霉素及BAY-117082则可阻断TCCM诱导的Gr-1+ CDllb+细胞的活化。综上,本研究表明肿瘤通过分泌可溶性介质,尤其是TGF-β1,诱导Gr-1+ CDllb+ MDSCs高水平地表达miR-494, miR-494通过靶向抑制PTEN,从而活化PI3K/Akt/mTOR信号途径,促进MDSCs的存活,诱导MDSCs功能相关的效应分子(ARG1,iNOS2, MMPs)的表达,从而促进MDSCs的大量聚集和活化,并趋化到肿瘤局部,通过抑制肿瘤特异性的CD8+ T细胞应答及降解细胞基质等机制促进肿瘤进展和转移。上述结果揭示了microRNA介导的调控机制和异常活化的PI3K/Akt信号途径在肿瘤相关MDSCs的聚集和活化中所发挥的重要调控作用,证实了miR-494作为一个关键分子调控Gr-1+ CD11b+ MDSCs的免疫相关和非免疫相关的促肿瘤效应,这可能为进一步深入阐明MDSCs在肿瘤免疫逃逸中的作用机制提出新的观点,并可能为研发针对这群细胞为靶点的治疗策略提供理论依据和实验基础,也可能为其它与MDSCs相关的疾病发病机制和治疗的研究提供启发。更多还原

【Abstract】 In 1863, Virchow hypothesized that the origin of cancer was at sites of chronic inflammation, in part based on the phenomena that inflammatory cells were present in biopsied samples from tumors. But the causal relationship between inflammation, innate immunity and cancer is generally accepted only in recent years. Mounting evidence indicates tumor-infiltrating inflammatory cells contribute significantly to tumor progression through suppressing the host immunity, facilitating tumor cells invasion and participating in the formation of the new blood vessels.The growth and metastasis of solid tumors are often associated with aberrant myelopoiesis, including the significant accumulation of myeloid-derived suppressor cells (MDSCs) in patients and various mouse tumor models that have the potential to promote tumor growth. MDSCs represent a heterogeneous population of myeloid cells comprising immature dendritic cells (DCs), macrophages, granulocytes, and other myeloid cells in early differential stages that can be identified in mice by expression of CD11b and Gr-1. Recent studies classified Gr1+CD11b+ cells as either Granulocytic MDSCs or Monocytic MDSCs based on nuclear morphology and the expression of Ly6G or Ly6C, respectively, and indicated both populations suppress antigen-specific T-cell responses, but through distinct effector molecules and signaling pathways. In addition to their depressive effect on host immune surveillance, MDSCs also facilitate tumor cell invasion and metastasis. We and others have previously reported that Tumor-derived factors mobilize MDSCs from bone marrow into the site of tumor where they produce multiple MMPs that contribute to tumor invasion.Previous studies have revealed that, in addition to the complex transcriptional programmes, the existence of potentially widespread post-transcriptional regulatory mechanisms that have crucial roles in regulating the development and function of immune cells. The mediators of these processes are known as microRNAs (miRNAs)—an abundant class of endogenous small non-coding RNAs of approximately 22 nucleotides in length that can form imperfect Watson-Crick base pairs at multiple sites within the 3’-untranslated region (UTR) of their cognate mRNA targets to repress their expression. miRNAs affect all aspects of immune system development from hematopoiesis to activation. At the same time, miRNA dysregulation in immune cells can lead to various immune-related pathological disorders, such as inflammation and cancer. Our previous studies shown microRNAs participate in the regulation of the function of macrophage. Mounting evidence indicates that MDSCs elicited by tumor-derived factors contribute significantly to tumor progression, however, it remains unclear about whether noncoding RNA is involved. In this study, we have reported the increased expression of miR-494 in MDSCs induced by tumor derived factors is critically involved in tumor growth and metastasis. First, we found the expression of miR-494 in MDSCs derived from all 6 tumor models on two different mouse strains was higher than MDSCs from naive mice, indicating the high level of miR-494 expression is the common characteristics of tumor-associated MDSCs. Second, our in vitro assays showed that tumor cells culture medium (TCCM) can induce the overtly level of miR-494 in MDSCs in a dose-dependent manner. Finally, we further found TGF-β1 in TDF was a main cytokine responsible for up-regulation of miR-494 in tumor-expanded MDSCs.We also observed that tumor-infiltrating MDSCs had a significant high level of miR-494 expression compared with splenic MDSCs, more intriguing, the expression level seemed be correlated between miR-494 and ARG1, iNOS2 and MMPs which known as closely related to the activity of MDSCs. Using overexpression or active inhibition of miR-494 approaches, we further determined miR-494 participated in regulating these genes expression and played a pivotal role in regulating both suppressive activity and the ability of facilitating tumor cell invasion and metastasis of MDSCs. The growth of the primary tumors was retarded and colonies of metastatic cells in the lungs were rarely detected when endogenous miR-494 activity in vivo was inhibited. Depletion of CD8+ T cells restored primary tumor growth, suggesting LV-sponge suppressed primary tumor growth mainly by blocking MDSCs-mediated tumor-specific T-cell tolerance. Whereas the phenomenon, that inhibition of miR-494 significantly reduced metastatic cancer cells in lung even CD8+T cells were depleted, in combination with the data from in vitro invasion assays suggested miR-494 regulated the ability of facilitating tumor cell invasion and metastasis of MDSCs via regulating MMPs expression. Interestingly, the number of tumor-infiltrating MDSCs significantly decreases in LV-sponge infected mice even combination of 2.43 mAb treatment that resulted in the primary tumor size comparable with LV-ctrl infected mice. We have further proven that the decrease number of tumor-infiltrating MDSCs due to the increased apoptosis and the decreased migration into tumor site of MDSCs induced by LV-sponge.Recent studies have determined that JAK/STAT signalling pathways play crucial roles in the suppressive activity of MDSCs. However, it remains unclear whether another signalling pathways are involved, especially the intracellular signal mechanisms responsible for Non-immunological functions of MDSCs. As a multifunctional tumor suppressor, PTEN is mutated or otherwise inactivated is frequently observed in a wide variety of human cancers. In addition to its tumor suppressive function, PTEN also play a critical role in the maintenance of the normal physiological functions of many organ systems. As far as immune system is concerned, published work has shown that depending on the cell type, PTEN is important for proper development, cell fate and cell function.Our study has now shown that PTEN is a functional target of miR-494 in MDSCs and PI3K/Akt pathway plays an "omnipotent" role of the regulation of MDSCs promoting tumor progression. As a functional miR-494 target, PTEN expression in MDSCs was suppressed by up-regulation of miR-494 induced by TDF that resulted in the enhanced ability of infiltrating into tumor site via SDF-1/CXCR4 axe. Moreover, down-regulation of PTEN activates PI3K/Akt pathway that alters the intrinsic apoptotic/survival signal to prolong the lifespan of MDSCs, which further contributed to the accumulation of MDSCs into tumor site. Furthermore, the activated PI3K/Akt pathway promotes the expression of ARG1, iNOS2 and MMPs via mTOR dependent pathway that results in the enhanced inhibitory activity and the ability of facilitating tumor cell invasion and metastasis that is attributed to tumor progress.In summary, our results indicate that upregulation of miR-494 promotes tumor growth and metastasis by enhancing the accumulation and activity of MDSCs in tumor tissue via suppressing PTEN expression, thereby resulting in increased Akt activity and subsequent activation of mTOR. By targeting miR-494, not only generated a strong antitumor immunity but also inhibited tumor metastasis, having the effect of "killing two birds with one stone".更多还原