Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini: BrdU staining and fibrosis

Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini:

1. BrdU stained images for the detection of proliferation with manual counting of brown stained cell proportion. Each group in a seperate file BRDUxxx

2. Fibrosis local images stained with Sirius red with automated analysis with imageJ. Grouped into one zip file

3. Global analysis of liver fibrosis pathogenesis (Ishak scores) performed by 2 blinded pathologists. Grouped into one zip file

Methods in associated publication.  Briefly 3 groups of 15 hamsters were infected with 100 NEJ flukes that had been gene edited with CRISPR/Cas9 plasmids targeting either Ov-grn-1, Ov-tsp-2, or a scrambled sequence as the control group.  Images in these files are the micrographs from these three groups of hamsters.  Numbered 1-15 representing the hamster and left/middle/right representing the liver lobe the section was taken from.

Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini

ABSTRACT: 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.