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Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis. Author summary: Infection with the human liver flukes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis remains a public health concern in regions where these parasites are endemic. O. viverrini is endemic in the Mekong River drainage countries including Thailand and the Lao People's Democratic Republic. Infection follows the consumption of undercooked freshwater fish harboring the parasite. Liver fluke infection, opisthorchiasis, is associated with diseases of the liver and bile ducts including cancer of the biliary tract, cholangiocarcinoma, a cancer with a poor prognosis. This report characterizes, for the first time, experimental infection with gene-edited O. viverrini liver flukes during concurrent exposure to a dietary nitrosamine in a rodent model of liver fluke infection-associated cancer. Cancer development was slowed in hamsters infected with the parasite following CRISPR-based knock-out mutation and loss of a parasite gene known to stimulate growth of cells lining the bile ducts. These findings definitely link a parasite factor to cancer etiology, and present a new laboratory model to investigate risk factors for infection-associated cholangiocarcinoma and to assess efficacy of anti-infection/anti-cancer vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world. [ABSTRACT FROM AUTHOR]