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

<p>Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini:</p> <p>1. BrdU stained images for the detection of proliferation with manual counting of brown stained cell proportion. Each group in a seperate file BRDUxxx</p> <p>2. Fibrosis local images stained with Sirius red with automated analysis with imageJ. Grouped into one zip file</p> <p>3. Global analysis of liver fibrosis pathogenesis (Ishak scores) performed by 2 blinded pathologists. Grouped into one zip file</p> <p>Methods in associated publication.&nbsp; 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.&nbsp; Images in these files are the micrographs from these three groups of hamsters.&nbsp; Numbered 1-15 representing the hamster and left/middle/right representing the liver lobe the section was taken from.</p> <p>&nbsp;</p> <p>Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini</p> <p>ABSTRACT: Infection with the food-borne liver fluke <em>Opisthorchis viverrini</em> 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 <em>Ov</em>-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (<em>&Delta;Ov-grn-1)</em> 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 <em>&Delta;Ov-grn-1</em> 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 <em>&Delta;Ov-grn-1</em> genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of <em>TP53</em> 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.</p>