【作者】 杨菲；

【导师】 王建莉；

【作者基本信息】 浙江大学 ， 免疫学， 2012， 博士

【摘要】 迄今为止,肿瘤的免疫治疗在临床中效果有限,究其原因,主要在于肿瘤免疫和免疫逃逸机制的复杂性,现在肿瘤复发与逃逸已经成为肿瘤免疫治疗的主要障碍,而肿瘤的远处转移是肿瘤复发、不易根治的最主要原因,因此深入研究肿瘤转移的相关机制,对于探索一种有效的肿瘤治疗方法具有非常重要的科学意义。肿瘤的侵袭能力指肿瘤细胞破坏基底膜及移行进入结缔组织的能力,肿瘤侵袭能力的强弱最终决定了肿瘤转移的程度。MMP9是基质金属蛋白酶(Matrix metalloproteinases, MMPs)中的一种,可以降解基底膜的主要成分—胶原,能够促进肿瘤细胞的侵袭。现在有研究表明,在炎症或者肿瘤发生时,高表达的FasL能够通过活化外周血髓系细胞或者肿瘤细胞的Fas信号,上调这些细胞的MMP9表达,加速它们向周围组织浸润、侵袭。这些结果提示细胞Fas信号的活化与MMP9的表达水平存在着某种重要的关联。Fas是经典的死亡受体,当Fas配体(Fas ligand, FasL)与Fas结合后,可以介导Fas活化,诱导细胞凋亡,从而维持机体的稳态。但是,在很多情况下,Fas可以引起非凋亡途径,包括促进炎症反应、神经突生长、肝组织再生、细胞增生及分化、血管再生、纤维化、细胞周期进展、细胞迁移等。虽然几乎所有的肿瘤细胞都表达Fas受体,但很大一部分肿瘤细胞中Fas信号的活化,不但不引起肿瘤细胞的凋亡,反而促进肿瘤的生长与转移。活化的肿瘤抗原特异性细胞毒性T细胞(Cytotoxic T lymphocytes, CTLs)是清除肿瘤细胞的最重要的效应细胞,活化的CTL能够高表达FasL。肿瘤发生时,虽然机体免疫被激发,肿瘤局部有活化CD8+T淋巴细胞(CTLs)聚集,但是其杀伤肿瘤的作用非常有限。因此,理解肿瘤逃逸和复发的机制可能为促进抗肿瘤T细胞治疗提供重要的思路。现在,肿瘤活化的CTL通过FasL/Fas信号对肿瘤产生了怎样的作用还不清楚。有文献报道活化的T细胞分泌大量表达FasL的具有生物活性的exosomes,这种exosomes能够诱导T细胞和树突状细胞(Dendritic cells, DCs)凋亡,从而发挥免疫抑制功能。Exosomes是由活细胞分泌的来源于多囊泡体的具有脂质双分子层膜结构的纳米级囊泡,其直径约为50-100nnm,密度范围从1.13g/ml到1.19g/ml,可经过多次密度梯度超速离心分离得到。来自不同细胞的exosomes具有生物功能的多样性。现在已经有大量的关于FasL表达的exosomes及其相关的免疫抑制功能的文献报道,但是,这些文献主要关注的是FasL表达的exosomes对T淋巴细胞及DC免疫功能的影响,而有关FasL表达的exosomes是否能够直接参与肿瘤Fas信号的活化,并促进肿瘤的转移,特别是与肿瘤转归密切相关的活化CTL分泌的FasL表达的Exosomes与肿瘤细胞转移是否具有相关性的文献报道很少。我们设计实验,制备了来自活化CTL的FasL阳性exosomes,研究其对肿瘤细胞侵袭的作用及其相关机制,并且证实了这样的exosomes在体内的存在及其生理意义。首先,我们通过OVA257-264多肽活化OT-I小鼠来源的CD8+T淋巴细胞,提取其分泌的活化CD8+T细胞来源FasL阳性的exosomes (EXO)。将EXO与表达Fas的B16小鼠黑色素瘤细胞或3LL小鼠肺癌细胞共培养,检测B16细胞或3LL细胞的增殖和凋亡情况,结果发现,B16细胞或3LL细胞表现出对EXO诱导凋亡抵抗,而EXO对B16细胞或3LL细胞的生长增殖没有影响。为了进一步观察EXO对于肿瘤细胞的影响,我们进行了体外侵袭实验。EXO可以促进B16小鼠黑色素瘤细胞以及3LL小鼠肺癌细胞的侵袭能力,当EXO用FasL中和抗体处理之后,它们对B16细胞和3LL细胞诱导侵袭能力的作用明显下降,表明EXO通过FasL/Fas信号发挥对肿瘤细胞的侵袭作用。由于MMP9与Fas信号的相关性以及MMP9对细胞侵袭的重要作用,我们检测了EXO作用于B16细胞之后,MMP9的表达变化。Real time及western blot结果显示,EXO能够通过Fas信号上调B16细胞MMP9的表达,当MMP9的作用被抑制之后,EXO诱导B16细胞侵袭的能力明显下降,证明EXO对B16细胞的侵袭作用是通过Fas信号上调MMP9表达来完成的。FasL能够活化肿瘤细胞的NF-κB和ERK信号通路,活化的NF-κB、ERK能够增加MMP9的表达。类似的,我们证明活化CD8+T细胞来源FasL阳性的EXO能够活化NF-κB和ERK信号通路,当NF-κB和ERK介导的信号通路被NF-κB抑制剂PDTC、ERK抑制剂U0126阻断后,EXO诱导B16肿瘤细胞上调MMP9以及侵袭的作用被有效的阻断,提示活化的NF-κB和ERK信号通路参与了EXO对B16细胞诱导MMP9表达及侵袭的作用。在对凋亡抵抗肿瘤细胞Fas信号通路的研究过程中人们发现,凋亡抵抗的肿瘤细胞能够通过上调Fas相关的死亡结构域IL-1p样转换酶抑制蛋白(Cellular FLICE inhibitory proteins, c-FLIP)来抑制经典凋亡通路的活化。活化CD8+T细胞来源FasL阳性的EXO作用于B16细胞5分钟之后,c-FLIPL水平增加,这种高水平可以持续到刺激之后24小时。以前报道c-FLIP能够促进NF-κB和ERK信号活化,而活化的NF-κB能够诱导c-FLIP的表达,并且与MMP9表达有关。所以我们检测了EXO作用B16细胞之后,c-FLIP与NF-κB、ERK信号通路的相互关系以及c-FLIP对MMP9的作用。结果我们证明,c-FLIPL介导了EXO促进B16细胞NF-κB和ERK磷酸化,反过来,NF-κB和ERK信号通路在EXO诱导B16细胞c-FLIPL水平升高中发挥了作用。同时,当B16细胞c-FLIPL的表达被siRNA干扰之后,EXO促进B16细胞上调MMP9及侵袭的能力也下降了,但是并没有逆转EXO对B16细胞的凋亡抵抗作用,表明c-FLIPL可能主要在EXO诱导肿瘤细胞上调MMP9表达及促进肿瘤细胞侵袭中有重要的作用。同时,我们还发现,EXO能够通过NF-κB和ERK信号促进B16细胞的凋亡保护蛋白Bcl-2的表达增加,Bcl-2与EXO凋亡抵抗关系密切。综合上述体外实验的结果,活化CTL能够分泌表达FasL的exosomes, EXO通过FasL/Fas作用于B16细胞后,c-FLIPL在较长时间内维持一个较高的水平,NF-κB和ERK信号通路被激活,MMP9表达上调,最终促进B16细胞的侵袭能力增加,有利于肿瘤细胞转移。在这个通过EXO上FasL引起的非凋亡途径中,c-FLIPL与NF-κB和ERK信号通路相互促进,Bcl-2表达升高,最终起到引起肿瘤细胞凋亡抵抗,侵袭能力增强的作用。为了验证体外制备的活化CD8+T细胞来源FasL阳性的EXO在体内对肿瘤侵袭的作用,我们设计了小鼠体内肺转移实验,通过尾静脉将B16肿瘤细胞和EXO共同注射入小鼠体内,14天后,解剖小鼠发现,EXO组B16肿瘤细胞肺转移增强,而EXOcont与对照相比没有明显差别,当FasL中和抗体作用EXO后,肺转移现象也得到了缓解。表明EXO通过FasL/Fas通路在体内促进B16肿瘤细胞侵袭。上述实验中FasL阳性的EXO都是体外培养OT-I小鼠来源的活化的CD8+T淋巴细胞纯化得到的,我们希望在体内可以找到生理存在的来自于CTL的表达FasL的具有促侵袭作用的exosomes.首先,我们通过皮下注射B16肿瘤细胞建立小鼠肿瘤模型,通过免疫荧光技术,证明在荷瘤小鼠的脾脏、淋巴结和肿瘤组织局部存在有CD8、FasL双阳性的细胞,而在对照小鼠中没有发现这样的细胞,确定了这种exosomes起源细胞在荷瘤小鼠中的存在。接着,我们从荷瘤小鼠的淋巴组织及肿瘤组织分离纯化得到exosomes,然后用CD8磁珠分选得到CD8+exosomes。CD8+exosomes体外与B16细胞共培养后,B16细胞的MMP9表达升高,侵袭能力增强。表明体内存在这样一群CD8+FasL+exosomes,能够促进肿瘤的侵袭。同时,我们用3LL肿瘤细胞建立肿瘤模型,验证了这一过程,表明体内这样一群具有肿瘤免疫逃逸作用的CD8+FasL+exosomes可能是一个较为广泛的现象。综上所述,本课题的研究完整的揭示经由活化CD8+T细胞来源的Exosomes所介导的肿瘤转移的全新机制,从而为肿瘤的治疗提供一种新的靶点与思路。更多还原

【Abstract】 To this day, the clinical effect of cancer immunotherapy is far from as we expected. The poor therapeutic effect may mainly result from complexity of tumor immunity and immune evasion. Tumor escape and recurrence set obstacle to tumor immunotherapy. The most important reason of tumor recurrence and difficult to cure is tumor distance metastasis. Therefore, it makes scientific sense to intensive study the related mechanism to explore an effective protocol to cure tumor.Tumor invasion indicates tumor cells can break basement membrane and transfer to connective tissue and the level of metastasis ultimately depend on the tumor invasive ability. MMP9, a member of the matrix metalloproteinases (MMPs), plays a critical role in breakdown of extracellular matrix and facilitating tumor invasion. It has been reported that Fas on peripheral blood myeloid cells or tumor cells stimulated by Fas ligand (FasL) can induce the expression of MMP9on these cells during inflammatory response and tumor development, subsequently lead to cell infiltration surrounding tissue and invasion increase. These results suggest there is a close relationship between Fas signal and the expression of MMP9.Fas is a classic death receptor, which can induce apoptosis triggered by FasL to maintain systematic homeostasis. However, under certain condition, Fas signal can exert non-apoptotic function, including inflammatory response, liver regeneration, increase branching of developing neurons, migration of cells, angiogenesis, fibrosis, proliferation and differentiation of cells and advancing the cell cycle. Therefore, although almost tumor cells express Fas receptor, Fas pathway may sever as a beneficial function for tumor cells survival rather than apoptosis.Activation of T cells is a pivotal step in the progress of host anti-tumor immunity. During this course, T cells increase the express of FasL. The tumor environment is infiltrated by tumor antigen-specific cytotoxic T lymphocytes (CTLs) that are, however, in an unresponsive status. It is not clear that FasL on CTL plays inhibitory or promoting role in tumor development via Fas signal. The exosomes secreted by activated T cells express bioactive FasL and can induce apoptosis of T cell and dendritic cells (DCs), suppressing immune response.Exosomes are nanoscale membrane vesicles (50-100nm) released by various live cells, which have a structure of lipid bi-layer. They are formed by membrane budding into the lumen of an endocytic compartment, leading to the formation of multivesicular bodies (MVB). Fusion of MVB to the plasma membrane leads to the extracellular release of exosomes. Exosomes, derived from various kinds of cells, have different bioactive function. FasL positive exosomes have reported to be an inhibitory effector in immune system, and these researches mostly focus on the roles in regulating T cells and DC function via apoptosis. However, the potential effects of expressed FasL in activated T cell exosomes on Fas resistant tumor cells in tumor metastasis are still less understood. To illustration this issue, we isolated activated CD8+T cell exosomes (termed as EXO). The effect and related mechanism of promoting tumor invasion were investigated and the counterpart of EXO in vivo was identified, which may provide a novel view to understand the tumor escape in immune system.Firstly, we purified FasL positive exosmoes (EXO) derived from OVA-specific active CD8+T cells of OT-I mice. Then we detected whether EXO could induce the apoptosis of Fas expressing B16or3LL tumor cells. The results showed that FasL positive EXO has little effects on B16or3LL tumor cells apoptosis. In addition, prolonged treatment time or increased concentration, EXO did not affect the proliferation of B16or3LL compared with control. We further investigated whether EXO can cause other functional changes in tumor cells, and we performed the invasiveness assays in vitro. These results suggest that EXO may increase invasive ability of tumor cells via Fas signaling in vitro. It has been reported that CD95L mediates migration of myeloid cells via MMP9activation. We assume that the effect of EXO on B16tumor cells invasion is also related to MMP9activation. The results of real time PCR and western blot provide solid evidence that up-regulation of MMP9induced by EXO is Fas/FasL dependent. To further confirm the relation between EXO mediated promotion of tumor cell invasion and MMP9up-regulation, MMP9inhibitor was added to B16tumor cell invasiveness assays in vitro. These results indicate that the tumor cell invasion induced by FasL positive EXO was associated with MMP9expression via Fas signaling.The stimulation of CD95ligand (CD95L) on tumor cells can activate NF-κB and ERK pathways and the activation of NF-κB and ERK increases MMP9expression. Similarly, EXO stimulation activated the NF-κB and ERK pathways in B16cancer cells. To further reveal the roles of activated NF-κB and ERK in MMP9expression and tumor cell invasion, specific inhibitors for NF-κB and ERK signaling pathways were used to pre-treat B16cancer cells before EXO stimulation and then MMP9expression was examined. We found both NF-κB and ERK inhibitors (PDTC and U0126) markedly suppressed the EXO-induced MMP9expression and tumor cell invasion. Collectively, these results showed that FasL positive EXO activated NF-κB and ERK signaling pathway was responsible for the elevated MMP9expression and tumor cell invasion. Cellular FLICE inhibitory proteins (c-FLIP) is an endogenous inhibitor of death receptor-induced apoptosis and Fas signal can up-regulate the lever of c-FLIP in apoptosis-resistant tumor cells. In B16tumor cells, we found that Fas engagement with EXO led to the increased accumulation of c-FLIPL-The increase of c-FLIPL could be detected at5minutes after EXO stimulation and the high level of c-FLIPL could maintain until to24h. It has been reported that c-FLIP can promote activation of ERK and NF-κB signaling pathways and NF-κB signals can induce the expression of c-FLIP, which is related to the expression of MMP9. We therefore investigated the relation of c-FLIP and ERK or NF-κB signaling and the effect of c-FLIP on MMP9in B16tumor cells upon EXO stimulation. Results showed that the phosphorylation of ERK and NF-κB was significantly decreased when the expression of c-FLIPL was inhibited by siRNA, in turn, the inhibition of NF-κB and ERK signaling pathways decreased the expression of c-FLIPL on B16cells induced by EXO. Meanwhile, the expression of MMP9and the ability of cell invasion were also inhibited when c-FLIPL expression was knocked down. We found that the apoptosis of c-FLIPL knock down B16cells showed no difference to control cells. These results suggest that c-FLIPL play an important role in the increase of MMP9and tumor invasion induced by EXO. In B16cells stimulated by EXO, we found that the expression of Bcl-2obviously increased and specific inhibitor of ERK and NF-κB could inhibit the up-regulation of Bcl-2.After inhibited by Bcl-2inhibitor, we found that EXO could induce apoptosis of B16cells by FasL. These results suggest that Bcl-2is closely related to the Fas resistance of B16tumor cells induced by FasL expressed EXO.These results in vitro indicated that activated CTLs could release FasL positive exosomes (EXO). Furthermore, the high level of c-FLIPL could maintain until24h, the NF-κB and ERK signaling pathways were actived, the expression of MMP9was up-regulated and the ability of invasion was improved on B16tumor cells induced by EXO via FasL/Fas system, facilitating tumor cells invasion. In the non-apoptotic Fas signaling pathway, c-FLIPL actived NF-κB and ERK signaling, in turn, NF-κB and ERK signaling was involved in up-regulated the expression of c-FLIPL and the expression of Bcl-2was increased leading to tumor cells resistant apoptosis and improving cells invasion.To further clarify the effect of EXO on tumor invasion in vivo, we performed tumor invasion analysis with the B16tumor cell and EXO inducing murine lung cancer model through tail vein injection. We found that EXO greatly increased the tumor cell lung metastasis after14days. After pre-incubated with FasL mAbs, the ability of EXO to promote tumor lung metastasis was markedly decreased. These results strongly support that EXO increase the tumor cell lung invasion via Fas signaling in vivo.To further confirm the existence of CD8and FasL positive exosomes and their effects on tumor invasion in tumor mice, we established B16tumor model. Firstly, the result of immunofluorescence showed that there were CD8and FasL positive cells in spleens, draining lymph nodes and tumor tissue of tumor mice, indicating it exists the such cells which releasing CD8+and FasL+exosomes in vivo. Then we isolated CD8positive exosomes from spleens and draining lymph nodes in control and tumor mice by CD8positive magnetic beads. The CD8positive exosomes from tumor tissues were also isolated. After stimulated with exosomes isolated from tumor but not control mice, B16tumor cells showed increased expression level of MMP9and enhanced ability of invasion. These results indicate that CD8and FasL positive exosomes are naturally generated with tumor progression and exerted an exacerbated effect in tumor invasion. Meanwhile, we established3LL tumor model to test such the phenomenon. And the results suggested CD8and FasL positive exosomes may not be limited in particular tumor.Our results reveal a novel mechanism of tumor immune escape and suggest that strategies to inhibit rather than to promote Fas activity should be considered during cancer therapy.更多还原