Recent Advances in Implantation-Based Genetic Modeling of Biliary Carcinogenesis in Mice

Abstract

Epithelial cells in the biliary system can develop refractory types of cancers, which are often associated with inflammation caused by viruses, parasites, stones, and chemicals. Genomic studies have revealed recurrent genetic changes and deregulated signaling pathways in biliary tract cancer (BTC). The causal roles have been at least partly clarified using various genetically engineered mice. Technical advances in Cre-LoxP technology, together with hydrodynamic tail injection, CRISPR/Cas9 technology, in vivo electroporation, and organoid culture have enabled more precise modeling of BTC. Organoid-based genetic modeling, combined with implantation in mice, has recently drawn attention as a means to accelerate the development of BTC models. Although each model may not perfectly mimic the disease, they can complement one another, or two different approaches can be integrated to establish a novel model. In addition, a comparison of the outcomes among these models with the same genotype provides mechanistic insights into the interplay between genetic alterations and the microenvironment in the pathogenesis of BTCs. Here, we review the current status of genetic models of BTCs in mice to provide information that facilitates the wise selection of models and to inform the future development of ideal disease models.

Keywords: biliary tract cancer; genetically engineered mouse; hydrodynamic injection; implantation; nude mouse; organoid; orthotopic model; syngeneic.

Figure 1

Malignancy of the hepatobiliary system. Anatomical classification of the biliary system (left). The corresponding tumors are listed (right). Biliary tract cancers (BTC) (blue) and liver cancer (black) are not necessarily mutually exclusive.

Figure 2

Options in implantation-based modeling of BTC. (See Table 1 for the details of each study.) As examples of options for modeling BTC, target cell selection (A) and host selection (B) are illustrated. Abbreviations are: IHBD, intrahepatic bile duct and EHBD, extrahepatic bile duct. Nude mice and C57BL/6J strain mice were used as representatives for immunodeficient mice and immunocompetent mice, respectively.

Figure 3

In vivo genetic engineering. (A). Genetically engineered mice (GEM). By crossing conditional GEM for the “gene X” and “gene Y”-Cre mice, which allow biliary system-specific recombination, resultant mice are supposed to be genetically engineered only in the biliary system (left). Note that, in reality, Cre expression could be broader (right). (B). Hydrodynamic gene delivery (HGD). Tail vein injection is the major means of gene delivery, while injection via GB is also reported.

Figure 4

Implantation of organoids and derived tumor bud into mice. (A). Subcutaneous implantation. For C57BL/6J, shaving hair is not mandatory on injection, but strongly recommended upon sacrifice. (B). Orthotopic implantation. Only one-step injection is illustrated. Note that direct injection to EHBD might be technically feasible, but has never been reported. Splenic injection can transfer cells to the liver via splenic vein and the portal vein.

Figure 5

The two-step implantation model of GBC. Genetically engineered organoids were inoculated into the subcutis of syngeneic mice. In the second step, the IoTB approach facilitated accurate preclinical studies in a large cohort.

Figure 6

Flow chart for selection of the BTC model. Depending on the purpose of study and affordable efforts, researchers can select the most suitable experimental system for BTC modeling.