Liver fluke granulin promote extracellular vesicle-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with the liver fluke is a major risk factor for cholangiocarcinoma (CCA). The liver fluke Opisthorchis viverrini secretes a growth factor termed liver fluke granulin, a homologue of the human progranulin, which contributes significantly to biliary tract fibrosis and morbidity. Here, extracellular vesicle (EV)-mediated transfer of mRNAs from human cholangiocytes to naïve recipient cells was investigated following exposure to liver fluke granulin. To minimize the influence of endogenous progranulin, its cognate gene was inactivated using CRISPR/Cas9-based gene knock-out. Several progranulin-depleted cell lines, termed ΔhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of specific transcript and progranulin, both in gene-edited cells and within EVs released by these cells. Profiles of extracellular vesicle RNAs (evRNA) from ΔhuPGRN-H69 for CCA-associated characteristics revealed a paucity of transcripts for estrogen- and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific evRNAs including MAPK/AKT pathway members were induced by exposure to liver fluke granulin. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 cells exposed to liver fluke granulin. Of these, CCA-associated evRNAs modified the CCA microenvironment in naïve cells co-cultured with EVs from ΔhuPGRN-H69 cells exposed to liver fluke granulin, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells in comparison with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCA-conducive disposition

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

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

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

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

Experiment 1: Transcript levels of gene edited adult flukes with bootstrapped population values.

Each group of the flukes was subjected to gene editing targeting Ov-grn-1 (ΔOv-grn-1 flukes), Ov-tsp-2 (ΔOv-tsp-2 flukes), or with an irrelevant guide RNA as a control (Control). Each panel shows ddCt (delta-delta cycle threshold) of individual flukes plotted relative to transcript levels of wild-type flukes for Ov-grn-1 (A) and Ov-tsp-2 (B). The dashed line purple box inset is an enlarged region of panel B, included for clarity. Resampling with replacement bootstrap analysis (B = 1000) of ddCT scores used to generate population average denoted by thick colored line and 95% confidence interval bars. (DOCX)</p

Reduced proliferation and minimal mutant p53 expression in cholangiocytes in hamsters infected with ΔOv-grn-1 genotype liver flukes.

Representative images of biliary cells that incorporated BrdU from regions proximal to flukes in control, ΔOv-grn-1 and ΔOv-tsp-2 groups (A). The boxed region in the upper image is expanded in the lower panel. The brown arrow highlights the positive BrdU-stained nuclei and the blue arrow highlights a bile duct cell that did not incorporate BrdU. BrdU index measured from cholangiocytes adjacent to where a fluke was located (n = 27 to 42 per group). (B). Representative micrograph of p53 immunohistochemical staining of biliary epithelium during infection with gene edited flukes (C). Anti-mutant p53 antibody stained the nuclei brown (brown arrows); blue arrows indicate negative cells. Black dashed box in upper wide-angle image magnified in the lower image to aid visualization. Positivity rate (percentages) of mutant p53-positive cholangiocytes (n = 29 to 39 images per group) (D). Where available, 500 to 800 cells were scored from sections of each of the left, middle, and right lobes of the liver marked by “X”. Fewer cholangiocytes (300–500) were available for assessment in several samples, denoted byⓧ Panels A and C: OV = Opisthorchis viverrini, H = hepatocytes, BD = bile duct, BE = biliary epithelium. Panels B, D: non-parametric Kruskal-Wallis test with Dunn’s multiple comparison correction compared against control: ns = not significant; ***, P ≤ 0.001, or against ΔOv-tsp-2: #, P ≤ 0.05; ###, P ≤ 0.001. Thick colored lines signify the median and the dashed black lines denote the inter-quartile range.</p

Programmed Knockout Mutation of Liver Fluke Granulin Attenuates Virulence of Infection-Induced Hepatobiliary Morbidity.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012) for cholangiocarcinoma (CCA) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam and Cambodia. We exploited this link to explore the role of the secreted growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions by undertaking programmed CRISPR/Cas9 knockout of the Ov-GRN-1 gene from the liver fluke genome. Deep sequencing of amplicon libraries from genomic DNA of gene-edited parasites revealed Cas9-catalyzed mutations within Ov-GRN-1. Gene editing resulted in rapid depletion of Ov-GRN-1 transcripts and the encoded Ov-GRN-1 protein. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, but the infection resulted in reduced pathology as evidenced by attenuated biliary hyperplasia and fibrosis. Not only does this report pioneer programmed gene-editing in parasitic flatworms, but also the striking, clinically-relevant pathophysiological phenotype confirms the role for Ov-GRN-1 in virulence morbidity during opisthorchiasis

Burden of disease in liver fluke infection-associated cholangiocarcinoma.

After resection of the livers at necropsy, a fragment of each lobe was either fixed in formalin for downstream thin sectioning or was manually disrupted to release flukes, which in turn were examined for gene editing events (Fig 1, Experiment 2). Gross anatomical appearance and histopathological results during induction of cholangiocarcinoma [31]. Multiple CCA nodules in the hamster liver were present on both diaphragmatic (A) and visceral surfaces (B). Micrographs of H&E-stained thin sections of liver highlighting foci of moderate dysplasia (C). This image shows bile ducts (blue #) encircled by dysplastic biliary epithelium (yellow arrow) surrounded by fibrosis (fb) with hepatocytes (h) to the left. H&E- stained images of CCA from each of the groups of hamsters group: control (D), ΔOv-grn-1 (E), and ΔOv-tsp-2 (F). Inflammation marked with green asterisk (*), cholangiocarcinoma labeled as CCA; other labels as in panel C. G. Assessment and scoring of lesions was undertaken independently by two co-authors (both veterinary pathologists) using anonymously labeled (blinded) micrographs. The severity of lesions increased from normal tissue (grey) to high grade CCA spanning multiple liver lobes (red). H. EPG from individual hamsters plotted against disease burden on a scale of zero (0, no lesion) to 6 (high CCA) scale. Data plots were slightly reformatted (nudged ± 0.1 on Y-axis) to enable display of overlapping points. Linear regression lines (which were not statistically significant) are shown in shaded color with 95% confidence intervals.</p

Liver fluke burden and levels of gene transcription.

Fecundity, worm numbers, and gene expression levels were determined at 10–14 weeks after infection of the hamsters with 100 gene edited juveniles, and from three hamsters per group (Experiment 1). Number of eggs per gram of feces (EPG) from each hamster at weeks 10 and 12 (A) and worm numbers at week 14 (B) showing mean (horizonal black line) and SEM bars. Each treatment group was compared to the control group in 2-way ANOVA with Holm-Sidak multiple comparison: ns = not significant; **, P ≤ 0.01; ***, P ≤ 0.001, and ΔOv-grn-1 against ΔOv-tsp-2: #, P ≤ 0.05. Gene transcript levels of Ov-grn-1 (C) and Ov-tsp-2 (D) were determined by qPCR for 10 to 13 flukes sampled from each animal (30–39 flukes total per group) and plotted with each datum point representing the transcript level of an individual fluke relative to wild-type flukes. Resampling with replacement bootstrap analysis (B = 1000) of ddCT scores (S3 Fig) was used to generate population average, as denoted by the thick, colored line and 95% confidence interval bars.</p

Cholangiocarcinoma model, fecundity, gene transcript and mutation rates.

Eggs per gram of feces (EPG) were assessed at week 23 prior to euthanasia at week 24 (Experiment 2). Panel A, EPG values of the three groups of hamsters. Violin plot denotes each hamster’s EPG with “x” symbols. Solid colored lines indicate the median values and dashed black lines indicate the quartiles. Kruskal Wallis with Dunn’s multiple comparison correction was used to compare EPG levels against control group: *, P ≤ 0.05; ns, not significant. At necropsy, a sample of 12 to 20 flukes were collected from each group, transcript levels determined for Ov-grn-1 (B) and Ov-tsp-2 (C), and plotted with each datum point representing the transcript level of the individual fluke relative to wild-type flukes. Resampling with replacement bootstrap analysis (B = 1000) of ddCT scores (derived from S4 Fig) was used to generate population average–denoted by the thick, colored line and 95% confidence interval bars.</p

Transcript levels of adult flukes from Experiment 2 with bootstrapped population values.

Each group of the 24-wk-old flukes was subjected to gene editing targeting Ov-grn-1 (ΔOv-grn-1 flukes), Ov-tsp-2 (ΔOv-tsp-2 flukes), or with an irrelevant guide RNA as a control (Control). Each panel shows ddCt (delta-delta cycle threshold) for individual flukes plotted relative to wild-type (WT) fluke transcript levels for both Ov-grn-1 (A) and Ov-tsp-2 genes (B). The dashed line purple box inset is an enlarged section of panel B for clarity. Resampling with replacement bootstrap analysis (B = 1000) of ddCT scores used to generate population average denoted by thick colored line and 95% confidence interval bars. (DOCX)</p