Targeting parasite-host communication to combat liver fluke-induced bile duct cancer

靶向寄生虫-宿主通讯对抗肝吸虫胆管癌变 项目总结 东亚地区肝吸虫感染仍有问题，泰国也有地方性感染 还有老挝，那里有大约1000万人感染。这种情况对公众健康的影响是巨大的。 因为人类的恶性肿瘤和真核病原体之间没有比两者之间的联系更强的联系 胆管癌(CCA)(胆道癌)和肝吸虫感染。泰国东北部 报告了全球CCA的最高发病率，2014年CCA年龄标准化发病率(ASR)为 每10万人中有85人死亡，这相当于每年有2.6万人死于与CCA相关的疾病。与没有肝脏的国家形成对比 考虑到其他地方CCA的发病率不到每10万人中有3人(美国，2014年为1.67 ASR)，吸虫是显而易见的。 为了在宿主胆道的恶劣环境中生存，肝吸虫排泄/分泌(ES)蛋白质和 胞外小泡(EVS)，用于宿主-寄生虫的交流，操纵宿主的反应，并修改 动态平衡，有利于恶变的变化。这项建议针对肝吸虫的成分 驱动胆道癌表型特征的ES：生长介质颗粒蛋白Ov-GRN-1 和胞外小泡(EVS)及其囊泡表面四肽(TSPs)。这些调解人进入胆道 上皮细胞，诱导增殖、迁移、血管生成、伤口愈合和促炎细胞因子IL- 6生产。我们推测，阻断Ov-GRN-1和/或EVS内化到胆管细胞将 破坏宿主与寄生虫的交流，进而恶性转化。我们的目标是用以下方法来检验这一假设 三个具体目标。目的1.评价CRISPR-Cas9基因敲除肝吸虫Ov-GRN-1和 OV-TSP基因对体外致病性和肿瘤发生的替代作用。目的2.描述发病机制和 在提供信息的啮齿动物模型中，基因编辑(敲除)寄生虫感染胆管致癌性 肝吸虫感染和感染诱发的胆道癌。目的3.确定Ov-Grn-1亚基和 EV TSP疫苗预防肝吸虫感染和感染诱导的癌症，并解决机制 功能性抗体通过这种方式将病理性降至最低。我们将利用一种胆管癌变的模型 哪种肝吸虫感染是确诊的危险因素。该提案的创新包括编辑基因组 应用CRISPR-Cas9，以抗体为靶点的Ov-Grn-1和EV表面四联蛋白 抗癌治疗的方法，并结合这些创新的发现开发出一种疫苗来阻断 肝吸虫感染。在宿主-寄生虫界面交流的吸虫蛋白可能代表阿喀琉斯病 因此，以抗吸虫/抗癌疫苗的形式针对吸虫-宿主通信可能最终 战胜疾病。 好了！

Targeting parasite-host communication to combat liver fluke-induced bile duct cancer PROJECT SUMMARY Liver fluke infection with Opisthorchis viverrini remains problematic in East Asia and is endemic in Thailand and Laos, where ~10 million people are infected. The public health implications of this situation are substantial since there is no stronger link between a human malignancy and a eukaryotic pathogen than that between cholangiocarcinoma (CCA) (bile duct cancer) and infection with the liver fluke O. viverrini. Northeast Thailand reports the highest incidence of CCA worldwide, with the 2014 CCA age standardized incidence rate (ASR) of 85 per 100,000, which equates to 26, 000 CCA-related deaths annually. The contrast to countries without liver flukes is stark given that incidence of CCA is less than 3 per 100,000 elsewhere (USA, 1.67 ASR in 2014). To survive in hostile environs of the host biliary tract, the liver fluke excretes/secretes (ES) proteins and extracellular vesicles (EVs) for host-parasite communication, to manipulate the host responses, and to modify homeostasis, changes conducive to malignant transformation. This proposal targets components of liver fluke ES that drive the phenotypic hallmarks of cancer in the biliary tract: the growth mediator granulin, Ov-GRN-1 and extracellular vesicles (EVs) and their vesicle surface tetraspanins (TSPs). These mediators enter biliary epithelial cells, inducing proliferation, migration, angiogenesis, wound healing and proinflammatory cytokine IL- 6 production. We hypothesize that blocking internalization of Ov-GRN-1 and/or EVs into cholangiocytes will disrupt host-parasite communication, and in turn malignant transformation. We aim to test this hypothesis with the three specific aims. Aim 1. Assess the impact of using CRISPR-Cas9 to knock out liver fluke Ov-grn-1 and Ov-tsp genes on in vitro surrogates of pathogenicity and neoplasia. Aim 2. Characterize pathogenesis and cholangio-carcinogenicity of infection with gene edited (knockout) parasites in an informative rodent model of liver fluke infection and infection-induced bile duct cancer. Aim 3. Determine whether subunit Ov-GRN-1 and EV TSP vaccines protect against liver fluke infection and infection-induced cancer, and address mechanisms by which functional antibodies minimize pathology. We will utilize is a model of cholangiocarcinogenesis in which liver fluke infection is the confirmed risk factor. Innovations of the proposal include editing of the genome of the liver fluke using CRISPR-Cas9, targeting Ov-GRN-1 and EV surface tetraspanins with antibodies as an approach to anti-cancer therapy, and combining the findings of these innovations to develop a vaccine to block liver fluke infection. Fluke proteins that communicate at the host-parasite interface likely represent an Achilles' heel, and so targeting fluke-host communication in the form of an anti-fluke/ anti-cancer vaccine may ultimately defeat the disease. !