Carcinogenic Liver Fluke Secretes Extracellular Vesicles That Promote Cholangiocytes to Adopt a Tumorigenic Phenotype.

BACKGROUND: Throughout Asia, there is an unprecedented link between cholangiocarcinoma and infection with the liver fluke Opisthorchis viverrini. Multiple processes, including chronic inflammation and secretion of parasite proteins into the biliary epithelium, drive infection toward cancer. Until now, the mechanism and effects of parasite protein entry into cholangiocytes was unknown. METHODS: Various microscopy techniques were used to identify O. viverrini extracellular vesicles (EVs) and their internalization by human cholangiocytes. Using mass spectrometry we characterized the EV proteome and associated changes in cholangiocytes after EV uptake, and we detected EV proteins in bile of infected hamsters and humans. Cholangiocyte proliferation and interleukin 6 (IL-6) secretion was measured to assess the impact of EV internalization. RESULTS: EVs were identified in fluke culture medium and bile specimens from infected hosts. EVs internalized by cholangiocytes drove cell proliferation and IL-6 secretion and induced changes in protein expression associated with endocytosis, wound repair, and cancer. Antibodies to an O. viverrini tetraspanin blocked EV uptake and IL-6 secretion by cholangiocytes. CONCLUSIONS: This is the first time that EVs from a multicellular pathogen have been identified in host tissues. Our findings imply a role for O. viverrini EVs in pathogenesis and highlight an approach to vaccine development for this infectious cancer.This work was supported by a Project Grant (APP1085309) from the National Health and Medical Research Council of Australia (NHMRC). AL is supported by a NHMRC principal research fellowship. SC was supported by the Thailand Research Fund (TRF)-the Royal Golden Jubilee PhD scholarship (RGJ) through Dr. Banchob Sripa.This is the final version. It was first published by OUP at http://dx.doi.org/10.1093/infdis/jiv29

Carcinogenic parasite secretes growth factor that accelerates wound healing and potentially promotes neoplasia

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

<i>Ov-</i>GRN-1 stimulated wound repair <i>in vivo</i>.

<p><b>(A)</b> Sequential images over four days of healing wounds revealed the response of mice to treatment with recombinant r<i>Ov-</i>GRN-1 or rTRX control; skin-deep wounds made with a 5 mm diameter biopsy punch between the ears of Balb/c mice. Minor modifications (brightness, contrast, cropping) were made to aid viewing. <b>(B)</b> The rate of wound healing over four days was measured; wound closure was determined electronically from photographs by measuring wound areas with ImageJ software. To aid viewing, curves have been shifted left or right marginally to minimize error bar overlap. <b>(C)</b> Assessment of the angiogenic properties of recombinant <i>Ov</i>-GRN-1 in the chorioallantoic membrane (CAM) assay. The numbers of blood vessels in quail eggs that grew on 0.5 cm² filter paper soaked in r<i>Ov</i>-GRN-1 or vehicle (control) were ascertained after incubation for 15 hours. Data points are the averages of two experiments with 3–5 biological replicates displayed with SEM bars. * = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001, **** = <i>P</i><0.0001, ns = not significant.</p

<i>Ov-</i>GRN-1 stimulated wound repair <i>in vitro</i>.

<p><b>(A)</b> Cholangiocytes exposed to ES products from flukes where <i>Ov-grn-1</i> had been silenced by RNA interference displayed significantly reduced proliferation over 36 h of co-culture. ES products (10 μg/ml) were derived from flukes that were exposed to dsRNAs for 5 days. Cell proliferation was monitored in real time using xCELLigence; every tenth data point is shown to aid visualization. Statistical comparisons were between <i>Ov-grn-1-</i> and <i>luc-</i>dsRNA-treated parasites. <b>(B)</b> Images of the scratch assay involving H69 cholangiocyte monolayers co-cultured in Transwell plates with <i>Ov-grn-1</i> or <i>luc</i>-dsRNA-treated. Dotted lines denote wound edges over time. <b>(C)</b> Selected time points were measured from the photographs in (B); statistical comparisons were between cells cultured with <i>Ov-grn-1-</i>and <i>luc</i>-dsRNAs. <b>(D)</b> Wound healing scratch assay as shown in panel c but using the CCA cell line M214 (D). <b>(E)</b> Wound healing scratch assay as shown in panels (C) and (D) but recombinant protein applied to cells instead of co-culturing cells with live flukes. Statistical comparisons were between 20 nM rTRX and r<i>Ov</i>-GRN-1 treatments or rTRX and PBS treatments. For all panels, data points represent the averages of two or three biological replicates with 3–5 biological replicates displayed with SEM error bars (some bars masked by data points). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001, ns = not significant. Additional data shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005209#ppat.1005209.s002" target="_blank">S2 Fig</a>.</p

Liver fluke granulin internalized by H69 cholangiocytes.

<p><b>(A)</b> Widefield (deconvolved) micrographs showing the lateral (xy) overview of live H69 cholangiocytes imaged after 18 h incubation with Alexa Fluor 488-conjugated r<i>Ov</i>-GRN-1 (green) and Hoescht nuclear stain (blue). <b>(B)</b> With further magnification of fixed cells the labeled r<i>Ov</i>-GRN-1 was evident among the cytoskeletal actin network (red) of numerous cells with DAPI (blue) stained nuclei. <b>(C)</b> 3D-SIM lateral (xy) overview image of a well-separated individual cholangiocyte stained as in panel B. <b>(D)</b> Rendered axial (yz) view of boxed inset in (C) showing r<i>Ov</i>-GRN-1 (green) present between the apical and basal actin filaments (red) of the cholangiocyte (DAPI channel omitted). Additional material shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005209#ppat.1005209.s009" target="_blank">S1 Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005209#ppat.1005209.s009" target="_blank">S1 Movie</a>.</p