Murine Liver Organoids as a Genetically Flexible System to Study Liver Cancer In Vivo and In Vitro

Abstract

The rising incidence of cholangiocarcinoma (CCA) coupled with a low 5‐year survival rate that remains below 10% delineates the urgent need for more effective treatment strategies. Although several recent studies provided detailed information on the genetic landscape of this fatal malignancy, versatile model systems to functionally dissect the immediate clinical relevance of the identified genetic alterations are still missing. To enhance our understanding of CCA pathophysiology and facilitate rapid functional annotation of putative CCA driver and tumor maintenance genes, we developed a tractable murine CCA model by combining the cyclization recombination (Cre)‐lox system, RNA interference, and clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology with liver organoids, followed by subsequent transplantation into immunocompetent, syngeneic mice. Histologically, resulting tumors displayed cytokeratin 19–positive ductal structures surrounded by a desmoplastic stroma—hallmark features of human CCAs. Despite their initial biliary phenotype in vitro, organoids retained the plasticity to induce a broader differentiation spectrum of primary liver cancers following transplantation into recipient mice, depending on their genetic context. Thus, the organoid system combines the advantage of using nontransformed, premalignant cells to recapitulate liver tumorigenesis as a multistep process, with the advantage of a reproducible and expandable cell culture system that abrogates the need for recurrent isolations of primary cells. Conclusion: Genetically modified liver organoids are able to transform into histologically accurate CCAs. Depending on the oncogenic context, they are also able to give rise to liver cancers that show features of hepatocellular carcinomas. The model can be used to functionally explore candidate cancer genes of primary liver cancers in immunocompetent animals and evaluate novel treatment regimens.

Cholangiocarcinoma 2020: the next horizon in mechanisms and management

“…Furthermore, CCA organoids have been shown to be a reliable system for drug testing and personalized medicine applications, and possess an almost negligible capacity to differentiate into hepatocytes 76,245 . As an alternative method, CCA organoids can be established by inducing genetic mutations in healthy organoids via viral transduction and CRISPR-Cas9 genome editing approaches, thus enabling the characterization and elucidation of the roles of oncogenes and/or tumour suppressor genes, either alone or in combination, in cholangiocarcinogenesis 76,246 .…”

Organoids to model liver disease

“…Using the CRISPR/Cas9 technology, normal intestinal organoids were transformed by sequential introduction of cancer driver gene mutations [90]. Two recent studies reported the generation of organoid models based on chol-orgs that acquire tumorigenic features following genetic modification to incorporate mutations commonly found in CCCs [91,92]. Remarkably, transplantation of such engineered organoids into immunodeficient mice gave rise to xenografts with carcinoma features with characteristics of either HCC or CCC depending on the inserted oncogenic driver.…”

Estrogen Acts Through Estrogen Receptor 2b to Regulate Hepatobiliary Fate During Vertebrate Development

“…To directly test the impact of E2/Esr2b signaling on hepatoblast differentiation and to determine the conservation of the roles of E2 signaling in hepatobiliary development across species, we utilized bipotent hepatoblasts derived from human induced pluripotent stem cells (iPSCs). These progenitor cells are capable of differentiating into hepatocytes and BEC/cholangiocytes as assayed by albumin and CK7 expression, ( 34 ) respectively (Fig. 5A).…”