Table 3 Summary of the most representative in vivo CCA models based on genetically engineered mice

Alfp–Cre, Trp53^f/f

Advanced HCC–CCA (from LPCs)

Trp53 mutation found in human CCA

Long latency (mice 14–20 months of age), tumours of bilinear origin (combined HCC–CCA)

¹⁸³

Alb–Cre, Smad4^f/f, Pten^f/f

Multistep progression involving hyperplasia, dysplasia, carcinoma in situ, and well-established iCCA (from LPCs)

100% tumour penetrance

Cre activation during embryogenesis, long tumour latency (4–5 months) and lack of metastasis

¹¹⁴

Alb–Cre, Kras^LSL-G12D/+, Pten^f/f

Invasive iCCA with an abundant desmoplasia, primarily showing glandular morphology resembling well-differentiated human CCA (from LPCs)

100% penetrance, rapid development (7 weeks of age), abundant desmoplastic stroma, iCCA exclusive

Cre activation during embryogenesis, no apparent metastases or invasion to other organs

^117,118

Alb–Cre, Idh2^LSL-R172, Kras^LSL-G12D

Multifocal liver masses of iCCA (from LPCs)

100% penetrance, splenic invasion and peritoneal metastases

Cre activation during embryogenesis, long tumour latency (33–58 weeks)

¹¹⁹

Alb–Cre, NotchICD

Development of transplantable CCA, probably progenitor cell-derived (transplantation of cells from 8-month-old mice in immunodeficient animals gives rise to CCA) (from LPCs)

Notch expression is characteristic of human disease

Cre activation during embryogenesis, no obvious cancer development after 8 months in transgenic mice, requires additional transplantation model

¹⁸⁴

Alb–Cre, Trp53^f/f, NotchICD

Development of iCCA abortive glandular pattern (moderate to high pleomorphic nuclei with some atypical mitoses) and dense fibrous tissue with inflammatory cells (from LPCs)

100% penetrance, development of fibrous or inflammatory microenvironment

Long tumour latency (>8–9 months), no metastases

¹⁸⁵

Alb–Cre, Kras^LSL-G12D/+, Fbxw7^{LSL-R468C/LSL-R468C}

Dysplastic dust-like structures surrounded by fibrosis in all mice (only bile duct dilation and hyperplasia in some heterozygous Fbxw7^LSL-R468C mice at the age of 8 months) (from LPCs)

Low latency (2 months of age)

Cre activation during embryogenesis, homozygous Fbxw7 mutations not occurring in human disease

¹⁸⁶

Alb–Cre, Hspd1^f/f

Cholangiocellular lesions, characterized by irregular glands, loss of polarity, multilayering of cells and frequent mitosis resembling human BIN

Low latency, possibility of transplanting cholangiocellular lesions, activation of human CCA pathways

Not related to known oncogenic drivers of human disease, no metastases, not established iCCA

¹²³

Alb–Cre, Jnk1^f/f, Jnk2^−/−

JNK deletion causes changes in cholesterol and bile acid metabolism that foster cholestasis, bile duct proliferation and iCCA

iCCA exclusive

~95% penetrance, long tumour latency (14 months)

¹⁸⁷

Alb–Cre, NEMO^f/f, Jnk1^f/f, Jnk2^−/−

Hyperproliferative ductular lesions with atypia compatible with CCA

Elevated ROS associated with cholangiocellular proliferation

Not full penetrance, long latency (50 weeks)

¹⁸⁸

Alb–Cre, Kras^LSL-G12D/+, Trp53^f/f

Multistage progression including stroma-rich tumours and premalignant biliary lesions (IPBN) (from LPCs)

100% penetrance, average latency 16 weeks, metastatic lesions

Cre activation during embryogenesis, wide latency range, CCA in ~80% of mice

¹¹⁵

Kras^LSLG12D/+, Trp53^f/f infected with AAV8-TBG–Cre

Development of iCCA (40%), HCC (40%), combined HCC–CCA (20%) (from hepatocytes)

Recombination event in adult mice, higher CCA frequency in combination with DDC diet (all tumours ICC or combined HCC–CCA)

Cre-recombinase administration via AAV, large tumour latency range (12–66 weeks after AAV infection)

¹¹⁶

Ah–Cre^ERT, Kras^G12V/+, Pten^f/f

Multifocal non-invasive papillary neoplasms in the intrahepatic biliary tract (from major interlobular bile ducts to small bile duct radicles in portal tracts)

100% penetrance, low latency (43 days), tumour development starts in adult mice

Not specific to liver tissue, lack of invasive tumour or metastasis

¹⁸⁹

Sox9–Cre^ERT2, Kras^LSL-G12D/+, Trp53^f/f

iCCA tumours accompanied by adjacent extensive ductular reactions and desmoplasia, with areas resembling BIN (from cholangiocytes)

100% penetrance, iCCA exclusive, recombination in mature cholangiocytes

30 weeks average latency

¹¹⁶

Ck19–Cre^ER, Kras^LSL-G12D, Tgfbr2^flox/flox, Cdh1^flox/flox

Markedly thickened extrahepatic bile duct wall with a swollen gallbladder involving invasive periductal infiltrating-type eCCA and lymphatic metastasis (from biliary cells)

Low latency (4 weeks), eCCA exclusive

Concurrent development of lung adenocarcinomas leads to asphyxiation of mice

¹²⁴

Pdx1–Cre, Pik3ca^LSL-H1047R/+

Adult mice develop enlarged extrahepatic bile duct and BIN with complete penetrance leading to eCCA (from well-differentiated, stroma-rich ductal adenocarcinomas to more undifferentiated)

eCCA exclusive, only one genetic hit driving CCA

~40 weeks average latency, 90% penetrance, wide tumour latency range

¹⁹⁰

LPCs from Alb–Cre, Kras^LSL-G12D, Trp53^{LSL-R172H/lox +/−}, FIG–ROS fusion

Allografted tumours resemble advanced CCA

Quick model, orthotopic implantation in the liver, iCCA exclusive, stroma presence

Requires technical training to isolate LPCs

¹⁰⁰

LPCs or cholangiocytic progenitor cells or hepatocytes from Trp53^−/− mice

Tumours exhibit a high stromal content and a mixed hepatocellular and cholangiocellular differentiation

Quick model

Not CCA exclusive

¹⁸³

Adult liver organoids from Kras^LSL-G12D, Trp53^f/f mice

Kras-driven organoids lead to CCA, while Myc expression in wild-type organoids induces HCC formation

Tumour latency of 6–8 weeks for Kras-mutated and Trp53-knockout organoids

Requires training in organoid isolation, growth and manipulation

¹²

Cholangiocytes from Kras^LSL-G12D, Trp53^f/f mice

Tumours with a high stromal component expressing CCA markers

Quick and reproducible model, orthotopic implantation in the liver, iCCA exclusive, stroma presence

Requires technical training to isolate mouse cholangiocytes

⁷³

Alb–Cre^ERT2, R26^RlacZ/+ or Ck19–Cre^ERT2, R26^RlacZ/+ mice treated with TAA

Macronodular liver cirrhosis containing cells the typical histology of CCA

100% penetrance, iCCA exclusive

Long latency (30 weeks)

¹⁹¹

Ck19–Cre^ERT/eYFP, Trp53^f/f mice treated with TAA

Treatment with TAA generates oncogenic stress yielding multifocal invasive iCCA

iCCA exclusive

80% penetrance, long latency (>6 months)

¹²⁵

Trp53^−/− mice treated with CCl₄

Bile duct injury or necrosis, proliferation and fibrosis development triggered by CCl₄

Exclusive iCCA

50% of mice develop tumours, metastatic lesions rarely observed

¹²⁶

Gsta3^−/− mice treated with aflatoxin B1

Macroscopic and microscopic liver cysts, hepatocellular nodules, cholangiomas, iCCA, and oval cell proliferation

Participation of oval cells in tumorigenesis

Long latency (12 and 24 weekly aflatoxin B1 injections followed by a rest period of 12 and 6 months)

¹⁹²

Alb–Cre, Jnk1^f/f, Jnk2^−/− mice treated with DEN

Cystogenesis and cholangioma-like structures in liver parenchyma with strong infiltration of immune cells

Participation of inflammatory insult

No established CCA, long latency

¹⁸⁸