Background : Cholangiocarcinoma (CC) is a fatal malignancy of the biliary epithelial cells in the liver, consisting of intrahepatic and extrahepatic biliary system. A vital discovery that significantly advances cancer research is the development of disease modelling technology like the cancer organoid technique. These organoids can be generated from CC patients’ tissue specimens but with limited efficiency. In addition, cancer organoids can be derived from normal adult stem cells subjected to CRISPR gene editing to simulate the gene mutation that occurred during early carcinogenesis.

Methods : To generate the iCC cancer organoid model, we co-transfected normal human chemically-derived hepatic progenitor cells (hCdHs) with CRISPR-Cas9 plasmid and gRNA plasmids for TP53 and BAP1. Following the transfection, we generated cancer organoids from these mutant cells and performed multiomics analysis (sanger sequencing, qPCR, and RNA-Seq) of this cancer disease organoid model.

Results : We successfully generated cancer organoid from hCdHs that can be massively expanded from a relatively small clinical sample by the knock-out of TP53 and BAP1, a well-established iCC cancer driver gene. These CRISPR-engineered cancer organoids showed comparable pathophysiological properties with the CC features. We observed cribriform feature on these organoid derived from intrahepatic biliary cell-derived adenocarcinoma (CK19+, ALB-), as well as mucin (MUC1+) overexpression in the organoid lumen by immunofluorescent staining. Transcriptomic profile by bulk RNA-Seq of this cancer organoid, as well as cell line, suggested malignant transformation (CEACAM5/S100P/CA9/TSPAN1+) that is significantly altered from the wildtype organoid.

Conclusions : These results demonstrated the capability of our CRISPR-engineered hCdHs-derived cancer organoid as a powerful cancer disease modelling platform.