人中性粒细胞多肽-1（HNP1）作用肿瘤微血管促进免疫细胞侵润的机制研究

Immunotherapy has made great progress in cancer treatment. Howerer, the abnormality of tumor microvessel frequently hinder the effects of immunotherapy via blocking entrance of immune effective cells into tumors. We previouly reported that human neutrophil peptide-1 (HNP1) can mediate anti-tumor immunity, and enhance the infiltration of immune cells into tumors via affecting tumor microvessels. As known, vessel normalization can enhance the therapeutic effects through increasing immune cell infiltration. The vessel normalization can be drived by anti-angiogenic treatment and M1 polarization tumor associated macrophages（TAM）. A serial of investigations including our studies have suggested that HNP1 could inhibit angiogenic signals, such as VEGF, fibronectin and integrin, induce M1 polarization of TAM, and affect cell junction. Therefore, it is speculated that HNP1 could promote vessel normalization. The current study aims to investigate the effects of HNP1 on microvessl and the underlying mechanisms. It is expected to confrim our hypothesis that HNP1 can promote tumor vessel nomalization through impairing the angiogenic signalings of VEGF, fibronectin and integrin, and promote the M1 polarization of TAM, and elucidate the molecular machanisms. Besides, we plan to verify that HNP1 may help immune cells infitrate into tumors via affecting cell junction of endiethelial cells. Execution of the program will be helpful to understand the molecular mechanisms by which HNP1 enhances the effects of immunotherapy, and provide basic research data for further clinical application of HNP1.

免疫治疗在肿瘤治疗中取得重大进展，但肿瘤血管异常，可阻止免疫效应细胞进入肿瘤实质这一关键步骤，削弱免疫治疗疗效。我们前期发现中性粒细胞多肽HNP1能诱导肿瘤免疫，并可能作用于肿瘤血管，促进免疫细胞向肿瘤实质浸润，具体机制未明。研究证实，血管正常化可促进免疫细胞侵润，抗血管生成治疗和肿瘤相关巨噬细胞M1极化都能促进肿瘤血管正常化。我们和国外研究提示：HNP1可抑制VEGF、纤连蛋白和整合素等血管生成信号，促进巨噬细胞M1极化，并可能影响细胞连接，进而促进肿瘤血管正常化。本项目将从HNP1作用微血管的效应和机制入手，证实HNP1可通过影响VEGF、纤连蛋白和整合素信号及诱导巨噬细胞M1极化促进肿瘤血管正常化，阐明其机制。并尝试证明HNP1还可通过影响血管内皮细胞连接而有助免疫细胞进入肿瘤实质。本项目的实施，将有助深入理解HNP1增强免疫治疗疗效的机制，并为其用于临床免疫治疗提供研究基础。

本项目前期证实防御素alpha-1（HNP1）通过局部基因导入肿瘤组织，可以诱导特异抗肿瘤免疫反应，并可能影响肿瘤微血管，促进免疫细胞浸润。本项目立足证实防御素alpha-1（HNP1）在肿瘤微环境中能够通过诱导肿瘤血管正常化，促进免疫细胞浸润，并在研究中进一步阐明相关机制。研究按照计划实施，证实了防御素HNP1能够作用于肿瘤微血管，影响微血管内皮细胞连接，促进血管正常化（Oncology Reports，2014），我们进一步观察到HNP1表达能够促进瘤内巨噬细胞向M1型转化，但未能有效证实HNP1对VEGF及以及纤连蛋白和整合素α5β1的信号的调节作用。我们还证实了防御素beta在瘤内的表达能够诱导特异的抗肿瘤免疫，并可以通过局部的趋化效应，促进免疫细胞的聚集，增强局部的抗肿瘤效应（Hum Gene Ther, 2014）。为进一步阐明HNP1局部的免疫和作用微血管的机制并推动临床转化，我们在食蟹猴体内尝试开展了局部治疗研究，通过HNP1脂质体复合物局部注射，尽管观察到潜在的全身免疫调节作用，但存在严重毒性，这一毒性成为HNP1临床转化主要障碍。. 此外，在项目支持下，我们还结合本项目相关问题和临床实践，探讨了局部给药或穿刺导致瘤内出血，红细胞对局部微环境的影响和对肿瘤的促进作用，证实出血可导致局部微环境巨噬细胞M2转化，直接促进肿瘤生长和治疗抵抗（J Immunol，2015）；证实了在恶性浆膜腔积液环境下，VEGF可促进肿瘤细胞的EMT转化和获得肿瘤干细胞特性，导致局部化疗和免疫治疗抵抗（JBC，2016）；证实了炎症抑制剂苏林酸能够通过调节免疫，诱导特异免疫反应发挥抗肿瘤作用（Sci Rep, 2016）；通过多靶点抑制剂苏尼替尼联合抑制VEGF上游调控因子HIF联合治疗肿瘤，证实可以抑制血管生成，增加治疗疗效（Oncology Research，2014）。这些研究可能为临床干预提供新的思考。.在本项目支持下，课题组发表SCI论文10篇，其中2篇IF＞5，申请专利1项，支持了2名博士后研究工作，培养博士研究生2名，硕士研究生3名，实现了预期目标。