芳香族化合物受体的内源性配体ITE抗神经胶质瘤的药理研究

Aromatic Hydrocarbon Receptor (AhR) is known to not only binds to aromatic environmental toxins and activates their metabolism but also plays important roles in many physiological and pathological procedures such as development, immune response and carcinogenesis. Searching for endogenous ligands of AhR has been a hot research topic. In 2002，Song J et al has purified ITE, a small molecule from porcine lung and identified ITE as an endogenous ligand for AhR. In 2011, Christiane A. Opitz reported that Kynurenine, a metabolite of tryptophan in glioma, was an endogenous ligand of AhR that promote glioma carcinogenesis via binding to AhR. Kynurenine has been found to promote glioma development in two aspects: Inhibition of tumor immunology in a paracrine manner and increase tumor cell motility and clonogenicity in an autocrine manner. We propose that ITE, a tighter binder to AhR compared to kynurenine, can block the binding of kynurenine to AhR in glioma and thus block its tumor promoting effect. We plan to test this hypothesis in three aspects: First we are going to test the anti-tumor effect of ITE using mouse glioma models created by implanting murine glioma cell lines and compare the tumor growth between the ITE treatment and the control group；secondly we plan to investigate ITE's effects on cell proliferation, apoptosis, motility and clonogenicity using a panel of human glioma cell lines. Lastly we plan to investigate ITE's effects on tumor immunology utilizing mouse glioma model. We are going to compare between the ITE treatment and control group on the following aspects：tumor tissue immune cell infiltration，peripheral blood mononuclear cells proliferation and tumor bearing animals' T cell function.

芳香族化合物受体（AhR）介导机体对芳香族环境毒素做出反应，并在多种生理及病理过程中起重要作用,包括发育，免疫反应及肿瘤发生等。2002有报道从猪肺中获得小分子ITE，一个对AhR具有高亲和力的内源性配体。2011年有报道发现在神经胶质瘤中色氨酸的代谢产物犬尿氨酸作为AhR的配体而促进肿瘤发展。鉴于ITE对AhR的高亲和力及ITE在前期研究中对多种小鼠实体瘤模型表现出的抗肿瘤活性，我们认为在神经胶质瘤中ITE应能竞争性结合AhR从而抑制犬尿氨酸的肿瘤促进作用。 本项目旨在发现ITE是否对神经胶质瘤有效，并探讨其可能的作用机制，包含以下内容：1.以小鼠神经胶质瘤模型来检测ITE的抗肿瘤效果。2.以多种人神经胶质瘤细胞系来发现ITE对肿瘤细胞增殖，凋亡，运动性及集落形成能力的影响，并系统研究受ITE-AhR调控的基因。3多方面衡量ITE对肿瘤免疫的影响

中文摘要 (对项目的背景、主要研究内容、重要结果、关键数据及其科学意义等做简单概述，800字以内)：.神经胶质瘤中色氨酸--IDO/TDO--犬尿氨酸--AHR通路起到抑制肿瘤免疫的作用。针对这一通路的新药主要是IDO/TDO抑制剂，中期临床实验发现有些抑制剂无效。本项目的核心假说是利用AHR的高亲和力配体ITE竞争性结合AHR，解除这一抑制，激活肿瘤免疫。.在免疫健全的小鼠颅内移植鼠胶质瘤细胞系GL261形成胶质瘤模型，并利用这一模型检测了ITE与PD1及PDL1抗体联用的抗肿瘤效果。发现100ug PD1抗体能显著延长动物生存时间，ITE与PD1抗体联用有进一步延长生存时间的趋势。对脑肿瘤组织进行流式细胞分析表明，ITE与PD1联用可协同作用增加脑肿瘤组织中CD8+细胞的比例。除了CD3+CD4+及CD3+CD8+两种细胞，在小鼠胶质瘤组织存在CD3+CD4-CD8-细胞，ITE+PD1组这类细胞比例显著增加。利用IL-17、CD25及Foxp3抗体对肿瘤组织中CD4+T细胞进行了进一步分析，结果仍在分析统计。 .在小鼠脑瘤组织切片中发现ITE可令组织边缘变光滑，减少肿瘤向脑实质的侵袭。发现ITE可显著抑制体外培养的胶质瘤细胞的迁移，并可抑制3D培养的小鼠脑组织切片中肿瘤细胞的侵袭，与抗侵袭新药FAK抑制剂相比具有高效能。在胶质瘤细胞中以siRNA敲低AHR后ITE的迁移抑制作用消失。连续拍照发现在鼠尾胶原中ITE处理导致胶质瘤细胞丝状伪足对胞外基质的粘附减弱。分析ITE 处理细胞的转录组数据发现ITE可降低胶原蛋白、tenascin，HSPG2等多种胞外蛋白及蛋白聚糖的表达，并降低了胞膜定位的整合素、syndecan、glypican等的表达，提示ITE可通过降低上述基因的表达起到抑制肿瘤细胞迁移的作用。.本项目发现了ITE与PDL1及PD1抗体具有增强肿瘤免疫的协同作用，并可显著抑制肿瘤侵袭，为进一步将其开发为抗胶质瘤新药奠定了理论基础。其中ITE抑制胶质瘤细胞侵袭及迁移的研究已投稿，ITE与PDL1、PD1协同增强肿瘤免疫的论文正在撰写中。

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