Further specificity can be achieved with tetracycline (Tet) expression systems that come in two forms, Tet-on or Tet-off, depending on whether promoter function is enabled or disabled by exogenous Tet administration. Both systems require the addition of either a tTA or rtTA allele, respectively. A significant advantage to using a Tet expression system is that induction via Tet is reversible (unlike CreERT), and combining this system with Cre-Lox technology through addition of an LSL between the promoter and start codon of the Tet-controlled transactivator can enable tissue specificity as well. One such example is the Tet-IDH2LSL-R140Q allele that when combined with Rosa26LSL-rtTA and Alb-Crerestricts Tet-inducible expression of mutant IDH1 to the liver.20 Another useful tool that can be combined with Cre-Lox is the flippase (FLP)- recombinase target (FRT) system, which functions analogously: an FLP recombinase excises DNA between two FRT sequences. It has been employed in the liver in combination with Cre-Lox to enable highly specific labeling of SOX9-positive hepatocytes, a unique compartment with potential to regenerate hepatocytes and BECs following injury.40 Thus, these systems, which allow for broad or specific manipulation of organs and cell types, are essential tools for modeling the various aspects of the human disease in mice.

There are several important considerations that investigators should bear in mind in applying these mutagenesis strategies. When using a Cre that is active in the liver progenitor population, such as the Alb-Cre allele, both liver cell types will be affected, limiting conclusions that can be drawn about the cell of origin. In addition, when using CreERT systems, the timing of tamoxifen administration may be critical. Alb-CreERT, for example, can label a small proportion of BECs, potentially confounding lineage tracing and cell of origin experiments.40,41 Also, all of the listed BEC-specific Cre drivers label epithelial cells across multiple organs including the lung, pancreas, and colon; in the absence of additional liver-specific oncogenic challenges, these experimental animals often develop tumors outside of the liver.41Nevertheless, significant advancements have been made in iCCA biology using the Cre-Lox system. In this review, we discuss more specifically how Cre-Lox-based GEMMs have advanced our understanding regarding three related topics: the cell of origin, the function of common mutations, and processes important for tumor development and maintenance.

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Cell of origin

Although the BEC has historically been the assumed cell of origin for iCCA, several observations suggest a more complex origin, including the existence of mixed HCC–iCCA tumors, an association with chronic hepatocellular injury, and the recently recognized capacity for hepatocytes to transdifferentiate into BECs.1,42 To test the possibility that hepatocytes can be the cell of origin for iCCA, one experiment lineage traced the hepatocyte compartment with an AAV8-Ttr-Cre and a Rosa26LSL-YFP reporter.43 Using sleeping beauty (SB) transposase (to be discussed) to express AKT and a constitutively active Notch1 intracellular domain (NICD) in the liver, iCCAs developed that expressed the YFP marker, indicating a hepatocyte origin. In another experiment, hepatocytes and BECs were lineage traced using the Alb-CreERT and Krt19-CreERT, respectively, with the Rosa26LSL-lacZ reporter.16 Following iCCA induction via the carcinogen TAA, iCCAs with β-gal activity were present only in the Alb-CreERT cohort, similarly illustrating hepatocytes as a possible cell of origin. On the other hand, when Krt19-CreERT is used to delete Tp53 and label BECs, thereby predisposing BECs to oncogenesis, it was demonstrated that TAA administration induces iCCA that is of biliary origin.17 Finally, in another key experiment demonstrating BECs as a cell of origin, induction of Kras and Pten mutations in BECs via Krt19-CreERT promoted iCCA development.41 Notably, mice in the latter experiment also exhibited significant extrahepatic tumor burden, illustrating a critical limitation of alleles such as Krt19-CreERT and Hnf1b-CreERT that are active in extrahepatic epithelial compartments as well. Altogether, the current evidence supports the notion that both hepatocytes and BECs can be a cell of origin in iCCA depending on the context (Figure 2). Understanding distinguishing biological features of BEC-derived and hepatocyte-derived iCCAs may prove to be relevant in the development of future therapeutic strategies.

Function of common mutations

In recent large-scale sequencing studies, the genetic landscape of iCCA has come into view. In non-liver fluke-related iCCA, the most common mutations are in IDH1/2 (22%), BAP1 (22%), TP53 (7%), KRAS (7%), and ARID1A (7%). Liver fluke-related iCCA, on the other hand, is distinguished by mutations in TP53 (45%), KRAS (19%), SMAD4 (16%), ARID1A (12%), and KMT2C (11%).44,45 Cre-Lox-based GEMMs have proven useful in discerning the functions of some of these mutations in iCCA. To explore the function of IDH1/2 mutations in iCCA, Alb-Cre was utilized to enable Tet-inducible expression of oncogenic IDH1/2 mutations in the liver, demonstrating that mutant IDH1/2 function to inhibit hepatocyte differentiation via epigenetic silencing of the major regulator of hepatocyte identity, Hnf4a, a key driver of hepatocyte differentiation.20 In a similar theme of mutations that impact cellular differentiation, Tp53 deletion in an Alfp-Cre-driven model leads to poorly differentiated liver tumors that have high expression of the stem cell marker Nestin and can be further directed toward a hepatocellular or biliary fate with additional lineage-specific mutations. Thus, Tp53 is an important suppressor of liver cell plasticity.46 Finally, Alb-Cre-driven KrasG12D expression and Tp53 deletion was shown to promote development of iCCAs that depend on autophagy, an important downstream process in many Kras-driven tumors.47 Despite significant progress in the past decade, much remains to be explored regarding the roles of these and many other oncogenic mutations found in iCCA.

Critical processes for tumor development and maintenance

Cre-Lox-based GEMMs are an essential tool to study and model the development of cancer in ways that accurately recapitulate the natural disease progression and tumor microenvironment. In addition to studying the importance of downstream pathways using genetic strategies, these models provide the opportunity to test potential therapeutic interventions in a relevant in vivo setting. In this regard, one group showed that iCCA in the Krt19-CreERT;Tp53f/f TAA model, a rat TAA model, and xenograft models are all sensitive to both macrophage-inhibiting therapies and Wnt-inhibiting therapies.48 The same research group again employed the Krt19-CreERT;Tp53f/f TAA and rat TAA models to demonstrate that the Notch pathway may be an additional therapeutic target.49 One strategy put the mouse model on a Notch3 null genetic background, finding that there is comparably minimal tumor development in this setting. Furthermore, pharmacologic inhibition of Notch signaling in the rat model similarly reduced tumor formation. These results suggest that further development of strategies to target these processes may be worthwhile. Finally, although not illustrated in the aforementioned examples, another clear advantage to GEMMs is the presence of an intact immune system. This important feature has been exploited in GEMMs of other malignancies such as pancreatic cancer to test T-cell-directed immunotherapies such as promising anti-PD-1/PD-L1 therapies.50