Liver fluke granulin promotes exosome-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with fish-borne liver flukes is a major risk factor for cholangiocarcinoma (CCA). The parasite secretes a growth factor termed liver fluke granulin ( Ov -GRN-1), a homologue of the human progranulin (huPGRN), which contributes significantly to biliary tract fibrosis and morbidity during infection. Here, exosome-mediated transfer of mRNAs from the human cholangiocyte cell line (H69) was investigated following exposure to Ov- GRN-1, to naïve recipient H69 cells. To minimize the influence of endogenous huPGRN, the gene encoding huPGRN was inactivated using CRISPR/Cas9-based gene knock-out. Several huPGRN-depleted cell lines, termed ΔhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of huPGRN transcripts and protein, both in gene-edited cells and within exosomes released by these cells. Profiles of exosomal RNAs (exRNA) 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 mRNAs including MAPK/AKT pathway members were induced by exposure to Ov -GRN-1. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 exposed to Ov -GRN-1. Of these, CCA-associated exRNAs modified the CCA microenvironment in naïve recipient cells co-cultured with exosomes from ΔhuPGRN-H69 exposed to Ov- GRN-1, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells comparing 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.