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

靶向寄生虫-宿主通讯以对抗肝吸虫诱导的胆管癌 项目摘要 由猫尾后睾吸虫引起的肝吸虫感染在东亚仍然是个问题，并且在泰国是地方病 和老挝，约有1000万人感染。这种情况对公共卫生的影响是巨大的 因为在人类恶性肿瘤和真核病原体之间没有比 胆管癌（CCA）（胆管癌）和肝吸虫O.维韦里尼。泰国东北 报告了全球最高的CCA发病率，2014年CCA年龄标准化发病率（ASR）为 85/100，000，相当于每年26，000例CCA相关死亡。与没有肝脏的国家相比 考虑到其他地方CCA的发病率低于每10万例3例（美国，2014年ASR为1.67），吸虫病是显而易见的。 为了在宿主胆道的不利环境中生存，肝吸虫排泄/分泌（ES）蛋白， 细胞外囊泡（EV）用于宿主-寄生虫通信，以操纵宿主反应，并修饰 内环境稳定，导致恶性转化的变化。该提案针对肝吸虫的成分 驱动胆道癌症表型标志的ES：生长介质颗粒蛋白Ov-GRN-1 和细胞外囊泡（EV）及其囊泡表面四跨膜蛋白（TSP）。这些介质进入胆道 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。我们假设，阻断Ov-GRN-1和/或EV内化到胆管细胞中， 破坏宿主与寄生虫的交流，进而导致恶性转化。我们的目标是测试这一假设， 三个具体目标。目标1.评估使用CRISPR-Cas9敲除肝吸虫Ov-grn-1和 Ov-tsp基因对致病性和肿瘤形成的体外替代物的影响。目标2.描述发病机制， 基因编辑（敲除）寄生虫感染在信息啮齿动物模型中的胆管致癌性 肝吸虫感染和感染引起的胆管癌。目标3.确定亚基Ov-GRN-1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 功能性抗体最大限度地减少病理。我们将利用胆管癌发生的模型， 肝吸虫感染是确定的危险因素。该提案的创新包括编辑基因组 使用CRISPR-Cas9，用抗体靶向Ov-GRN-1和EV表面四跨膜蛋白， 方法来抗癌治疗，并结合这些创新的发现，以开发一种疫苗，以阻止 肝吸虫感染在宿主-寄生虫界面交流的Fluke蛋白可能代表了阿基里斯的弱点。 因此，以抗吸虫/抗癌疫苗的形式针对吸虫-宿主交流， 战胜疾病。 !

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. !