Exosome-mediated crosstalk stimulated by liver fluke granulin promotes a 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 liver fluke Opisthorchis viverrini secretes a growth factor, termed liver fluke granulin (Ov-GRN-1), a homologue of the human progranulin (huPGRN). Secreted Ov-GRN-1 contributes significantly to biliary tract fibrosis and morbidity during infection. Here, exosome-mediated transfer of mRNAs from a human cholangiocyte cell line following exposure to Ov-GRN-1 to na ive recipient cells was investigated. In addition, aiming to minimize the effects of endogenous human GRN, the gene encoding human granulin was inactivated in H69 line cholangiocytes by genome editing, and several huPGRN-depleted cell lines, termed ΔhuPGRN-H69 cells, were established. These mutant H69 cell lines, termed ΔhuPGRN-H69, exhibited >80% reduction in huPGRN transcription and protein expression, both within cells and within secreted exosomes. Profiles of exosomal RNAs (exRNA) from ΔhuPGRN-H69 cells 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. Exposure to Ov-GRN-1 induced CCA-specific mRNAs including mRNAs encoding MAPK/AKT pathway members. 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, MAPK13 and SOX2 modified the microenvironment in na ive recipient cells co-cultured with exosomes from ΔhuPGRN-H69 exposed to Ov-GRN-1, and induced transcription of MAPK13 and SOX2 in naive H69 cells. Crosstalk in response to liver fluke granulin promoted a CCA-specific program through RTK signaling via MAPK and Wnt/β-catenin which, in turn, established a CCA-conducive milieu.