绿茶提取物EGCG对肿瘤“血管正常化”作用及其对肿瘤免疫微环境影响和机制探讨

The abnormal growth of tumor vessels leads to highly disorder of its structure and function, along with the disorder of metabolism and immune microenvironment. It has been demonstrated that appropriate anti-angiogenesis treatment tends to normalize previously torturous and malformed vessels, thus inducing a “vascular normalization” phenomenon, which then normalizing immune microenvironment. EGCG, a nature anti-angiogenesis agent refined from green tea, is defined to have multiple effects on angiogenesis factors. Our preliminary data suggested that EGCG could reduce lung cancer xenograft microvessel density (MVD), tissue hypoxia and interstitial fluid pressure (IFP) through regulating the balance between Angiopoietins-1 and 2. Therefore, in this proposal, we will characterize the function of EGCG in xenograft model, monitor tumor (MVD, microvessel pericyte coverage index(MPI)), function (perfusion function and vessel permeability), metabolic microenvironment (IFP and hypoxia) at multiple time points, and determine whether EGCG can induce tumor “vascular normalization ” along with its window phase if there is any. Next, we detect the index of immune microenvironment (tumor associated macrophage (TAM) and its M2 polarization, Dendritic cells (DC), etc.) in xenograft before, within and after the vessel normalization window phase induced by EGCG. Studies shown that hypoxia inducible factor (HIF-1 α) can be significantly up-regulated by hypoxia in tumor tissue, and decreased by EGCG treatment. While it has also been shown that HIF-1αhave effects on multiple factors toward the tumor immune microenvironment. Finally, we use hypoxia inducible factor-1α (HIF-1α) over-expressed A549 tumor cell line to build transplantation tumor xenograft model, observing the effect of HIF-1α on immune microenvironment under EGCG treatment, and its relationship with the time window of normalization. Which will provide novel evidence for the clinical application of EGCG as an adjuvant regimen of anti-tumor chemotherapy.

肿瘤血管异常生长会导致微血管结构功能、肿瘤代谢及免疫微环境紊乱。研究提示抗血管生成治疗可能引起肿瘤“血管正常化”并改善肿瘤免疫抑制微环境。我们前期研究发现绿茶提取物EGCG可降低肺癌移殖瘤微血管密度、组织间液压及乏氧。从而推测EGCG能诱导肿瘤“血管正常化”并影响免疫微环境。本研究拟建立移植瘤模型，取EGCG干预多时间点观察肿瘤血管结构、功能及代谢微环境，以明确其能否引起肿瘤“血管正常化”及其窗口期，然后在窗口期前、中、后观察肿瘤免疫微环境指标，明确EGCG对肿瘤免疫微环境的影响及其与正常化时间窗的关系。研究表明缺氧可以引起缺氧诱导因子（HIF-1α）上调，而EGCG可改善缺氧并降低HIF-1α，HIF-1α也被证明可影响肿瘤免疫微环境的多个因素。因此本研究建立HIF-1α过表达移植瘤模型，通过检测免疫微环境指标，探讨HIF-1α在EGCG对免疫微环境影响中的作用及其与正常化时间窗的关系。

背景：血管生成对于实体肿瘤的生长非常重要，而合理运用抗血管生成药物可使原来扭曲异常的肿瘤微血管出现一过性结构、功能正常化——肿瘤“血管正常化”现象。天然绿茶提取物表没食子儿茶素没食子酸[epigollatecatech in gallate, EGCG]已被证实具有很强的抗肿瘤血管生成作用。因此我们推测EGCG能引起肿瘤“血管正常化”现象。异常的肿瘤血管引起的高组织间液压（intestinal fluid pressure，IFP）、乏氧的微环境会影响免疫细胞的浸润和功能，进而促进免疫抑制微环境的形成，包括成熟树突状细胞（dendritic cells，DC）相对缺乏，肿瘤相关巨噬细胞（tumor associated macrophage，TAM）由M1亚型向M2转化。肿瘤组织缺氧还会引起缺氧诱导因子-1α（HIF-1α）表达上调，进而上调精氨酸酶1（Arg-1）和一氧化氮（NO）促进TAMs向M2极化，同时EGCG可降低多种实体瘤中HIF-1α的表达。因此我们推测，EGCG可以通过肿瘤“血管正常化”改善因乏氧而上调的HIF-1α来影响免疫抑制的肿瘤微环境。方法：建立人肺腺癌细胞株A549裸鼠移植瘤模型，在多时间点检测EGCG处理后的荷瘤裸鼠肿瘤微血管密度、周细胞覆盖率、基底膜结构、电镜结构、灌注及渗透功能指标、组织中氧分压、组织间液压、Pimonidazole标记乏氧水平、TAM累积量、评价TAM极化趋势、观察计算树突状细胞（DC）累积量及活化程度。建立慢病毒稳定转染HIF-1α过表达的A549细胞株，然后构建正常A549细胞株、慢病毒转染HIF-1α过表达细胞株及无意义序列转染细胞株裸鼠移植瘤模型，在EGCG处理不同时期检测免疫微环境指标及HIF-1α的表达情况。结果：EGCG处理荷瘤裸鼠可引起微血管密度（MVD）的下降，以及一过性的血管成熟度升高、灌注和渗透功能改善、肿瘤组织乏氧和IFP下降、TAM浸润增加、M2极化减弱、活化DCs浸润增加。过表达HIF-1α后，EGCG诱导的TAM在肿瘤组织中的浸润、M2极化的抑制、活化DCs浸润增加均不同程度受到抑制。结论：本研究首次证明EGCG可以引起人肺腺癌移植瘤肿瘤“血管正常化”现象， 并改善肿瘤免疫抑制微环境，该作用可能通过下调因乏氧而上调的HIF-1α介导，为EGCG临床应用于抗肿瘤辅助治疗提供更多依据。