Cell of origin in biliary tract cancers and clinical implications

Among BTCs, iCCA stands out due to a highly heterogeneous macro- and microscopic appearance. This is reflected in its histological classification which defines conventional tumours as well as rare variants.^, Based on the size of the affected duct, conventional iCCA tumours are sub-stratified into small and large bile duct iCCAs. Small bile duct iCCA mostly derives from interlobular and septal bile ducts and displays a mass-forming growth pattern. On the other hand, large bile duct iCCA arises in large intrahepatic ducts, presents increased mucin production and is more frequently preceded by precancerous lesions (i.e. biliary intraepithelial neoplasm or intraductal papillary neoplasm).^, It has been speculated that the different histopathology of these subtypes may be due to a different cell of origin and pathogenesis. For example, large bile duct iCCA shares phenotypic traits with pCCA and pancreatic cancer, perhaps suggesting a common origin.^, Similarly, given the association of small bile duct iCCA with chronic liver disease, it has been proposed that its onset may be linked to activation of hepatic stem cells and senescence of mature hepatocytes in chronic liver diseases. Nonetheless, conclusive data validating such hypotheses are lacking.

iCCA is the second most common primary liver cancer (PLC) after hepatocellular carcinoma (HCC). Even though the vast majority of iCCAs are sporadic, in recent years risk factors for chronic liver disease, which are conventionally associated with HCC (i.e. viral hepatitis, alcohol consumption, non-alcoholic steatohepatitis, etc.), have been shown to concomitantly increase the risk of iCCA (Table 1).^, The association between iCCA and chronic liver disease, as well as the the existence of mixed HCC-CCA tumours, has fuelled the hypotheses of a common cell of origin for these PLCs. Mixed HCC-CCA encompasses a group of rare (<1% of all PLCs) and histologically diverse tumours that, based on histopathological features, do not fit into either typical HCC or iCCA subtypes (Fig. 1). These tumours have attracted great attention due to their possible genesis from stem cells, although it should be noted that a stem/cell progenitor phenotype is not solid proof of their bona fide cell of origin. According to the latest World Health Organization classification, the term mixed HCC-CCA refers to tumours containing unequivocal intimately mixed components of both HCC and iCCA and/or bi-phenotypic stem-like cells. Cholangiolocarcinoma (CLC) is also grouped with mixed HCC-CCA, although it may represent a unique biliary subtype.^, CLC arises in small intrahepatic ductules and is characterised by low grade cytologic atypia, and anastomosing cords and glands resembling cholangioles or canals of Hering. From a histopathological point of view, CLCs are negative for hepatocyte markers such as HepPar1, but positive for biliary lineage markers including cytokeratin (CK)7, CK19 and the progenitor-like marker NCAM. Nonetheless, recent studies have reported that CLC and CK19+ HCC share clinico-pathological features, and thus may originate from a single hepatic progenitor cell (HPC).^,

The other CCA tumour types, pCCA and dCCA, are conventional mucin-producing adenocarcinomas, although several rare histological variants have been described. These tumours may display 4 main patterns of growth including: i) polypoid papillary tumours, ii) nodular tumours, iii) scirrhous constricting, the most common type, and (iv) diffusely infiltrating tumours. Predisposing factors for pCCA and dCCA revolve around chronic inflammation within the large bile ducts.^, This is frequently precipitated by primary sclerosing cholangitis (PSC) in Western countries and liver fluke infestation in Eastern countries. Histological analysis of samples containing extrahepatic or large intrahepatic bile duct tissue obtained from patients with PSC has revealed a key role of peribiliary glands (PBGs, Fig. 2) in the progression of bile duct lesions. PBGs are clusters of epithelial cells residing in the sub-mucosal compartment of large intrahepatic and extrahepatic bile ducts. Although the majority of cells contained in PBGs are mature epithelial cells, few populations expressing stem/progenitor cell markers and immature phenotypes have been identified.^, Interestingly, these cells proliferate in response to bile duct injury as observed in patients with PSC and liver fluke infection, thus functioning as a biliary stem cell niche. Accordingly, PBGs may represent the potential cell of origin of eCCA.

Unlike other BTCs, GBC is more common in women and often grows within the fundus of the gallbladder. At the histological level, adenocarcinomas constitute the most frequent phenotype of GBC; nonetheless, less frequent variants (for example, intestinal type, clear cell carcinoma and signet ring cell carcinoma) have recently been recognised by the WHO. Similar to eCCA, GBC is associated with underlying chronic inflammation of the large bile ducts.^, Overall, the gallbladder shares a common embryological origin with the liver and ventral pancreas. In a recent study, it has been demonstrated that while gallbladders do not have PBGs, pluripotent EpCAM+ endodermal stem/progenitors are present in the mucosal crypt of the human gallbladder. However, whether such cells could be a potential cell of origin of GBC remains to be explored.

The liver is a unique organ with an extraordinary capacity to self-repair and regenerate upon various injuries. In homeostasis, adult hepatocytes are mostly quiescent with slow cell turnover; however, upon hepatic injury or partial hepatectomy, hepatocyte turnover accelerates to restore hepatic mass and function. Several studies using multiple lineage tracing of different hepatic cell lines have demonstrated that, after partial hepatectomy or in the context of acute liver injuries, regeneration predominantly relies on the self-renewal of hepatocytes rather than stem cell differentiation.^{,  ,  ,  ,}  Conversely, when hepatocyte proliferation is impaired, bipotential HPCs or oval cells, postulated to be localised in the canals of Hering (Fig. 2), are the cellular source of hepatocyte turnover.^,, To further complicate the scenario, 2 recent studies have demonstrated that cholangiocytes can also act as facultative liver stem cells and contribute to hepatocyte regeneration in the context of severe and prolonged liver damage.^, Indeed, Raven et al. demonstrated that the combination of liver injury (i.e. loss of β1-Integrin) with inhibition of hepatocyte proliferation (i.e. p21 overexpression) causes regeneration of functional hepatocytes from biliary cells. These findings have been further validated by Deng et al. who showed that biliary-derived hepatocytes replenish a large fraction of the liver parenchyma following severe chronic injuries induced by long-term thioacetamide or 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment. All together these data suggest that: i) the nature of the injury can determine the cellular source of epithelial regeneration; and ii) cholangiocytes represent a potential cellular source of hepatic regeneration. Although significant gaps exist between the animal models of liver regeneration and the complex clinical scenarios of chronic liver injuries, these studies provide insights into the potential mechanisms driving liver regeneration. Whether the injury-induced plasticity of hepatocytes and cholangiocytes could also occur in the setting of BTC tumorigenesis remains to be clarified.

Based on histological observations, the mature cholangiocyte within the intrahepatic small bile ducts has traditionally been considered the cell of origin of iCCA. Nonetheless, during the past decade, the use of sophisticated cell lineage tracing systems and GEMMs has provided compelling evidence supporting the hypothesis that iCCA may also originate from the malignant transformation of hepatocytes (Fig. 3). Most of these models are based on the use of hepatocyte-targeted Cre or overepression systems (Table 3). Among them, it should be noted that the activity of constitutive albumin (Alb)-Cre system in some instances is not restricted to fully differentiated hepatocytes (Fig. 4). In fact, depending on the developmental stage, the Alb-Cre allele may recombine floxed alleles in both hepatocytes, cholangiocytes and hepatoblasts.^, Alternatively, other investigators have adopted the hydrodynamic delivery of transposon-based system that is known to preferentially transduce 5−40% of hepatocytes in the adult liver.

To date, several studies have consistently shown that hepatocyte-specific activation of NOTCH, either alone^, or in cooperation with AKT activation,^, gain-of-function Kras or loss-of-function Tp53 mutations, results in the conversion of mature hepatocytes into malignant biliary cells. In order to provide the direct evidence that fully differentiated hepatocytes can give rise to iCCA, Fan et al. used a hepatocyte fate-tracing model and elegantly showed that NOTCH and AKT signalling cooperate to convert normal hepatocytes into biliary cells that act as precursors in iCCA development. Using both hepatic and biliary lineage tracing systems, Sekiya et al. have further demonstrated that, upon chronic liver injury, iCCA arises from biliary lineage cells derived from hepatocytes and not cholangiocytes, with NOTCH signalling being the key mediator of biliary differentiation. The tumour microenvironment may also play a role in determining iCCA growth from oncogenically transformed hepatocytes. Indeed, it has recently been demonstrated that in the presence of an apoptotic microenvironment, hepatocytes with aberrantly activated oncogenes (loss of p19Arf in combination with gain-of-function alterations including Myc and NrasG12V or Myc and AKT1) give rise to HCC whereas a necroptotic-associated cytokine microenvironment will lead to iCCA.

Hepatocytes are not always the cell undergoing the neoplastic transformation. Indeed, Guest et al. have demonstrated that in the context of chronic inflammation and biliary-specific (Ck19-Cre) activation of NOTCH and mutant TP53, cholangiocytes lead to iCCA formation. In addition, murine models based on the combination of Pten and Tgfbr2 deletions or Tp53 loss and Kras activation suggest that iCCA can derive from either biliary (Ck19-Cre or Sox9-Cre, respectively) or hepatic (Alb-Cre or hepatic-specific thyroid-binding globulin promoter [TBG]-Cre) compartments (Table 3).^{,  ,}  Interestingly, the use of an adeno-associated vector to express Cre recombinase only in adult hepatocytes under the TGB promoter (AAV8-TBG-Cre) suggested that Tp53 and Kras mutated adult hepatocytes are refractory to malignant transformation in the absence of liver injury.

The application of sophisticated approaches further supports the role of cholangiocytes in iCCA formation. For example, the use of a cell lineage visualisation system using tamoxifen-inducible Cre-loxP has recently demonstrated that Kras activation and Pten deletion in both adult hepatocytes or cholangiocytes leads to iCCA tumours from cholangiocytes rather than hepatocytes.^, Finally, using a technically challenging approach for specific intrabiliary instillation of a transposon system expressing activated forms of YAP and AKT coupled with IL-33 administration, Yamada et al. have further demonstrated the role of cholangiocytes in the onset of iCCA.

Overall, this evidence suggests that depending on the oncogenic insults, adult hepatocytes can undergo a phenotypic switch to induce iCCA development, while adult cholangiocytes can readily go through malignant transformation. Further research is needed to understand in which circumstances and under which oncogenic insults one cell type rather than the other will ultimately give rise to iCCA.

As previously mentioned, the existence of mixed HCC-CCA tumours suggests the possibility that a progenitor-like cell may represent the cell of origin of iCCA and other PLCs. Experimental models based on the activation of the HIPPO-YAP pathway via Alb-Cre derived knock-out of the neurofibromatosis type 2 (Nf2) gene, inactivation of Mst1/2 or Sav1, and ectopic expression of Yap, have demonstrated the expansion of atypical ductal cells or progenitor-like cells and the subsequent development of iCCA, HCC and mixed HCC-iCCA tumours.^{,  ,  ,}  Similarly, viral-related specific transduction of mouse HPCs/hepatoblasts with transgenes encoding oncogenic H-Ras and SV40LT or Bmi1 and mutated β-catenin results in the formation of a broad spectrum of liver tumours with iCCA and/or HCC features.^, However, due to the limitations of the Alb-Cre promoter, whether the HPCs are the actual cell giving rise to these tumours remains debatable. To date, the only study specifically pointing to the expansion of progenitor-like cells as a key mechanism contributing to iCCA development is based on the expression of gain-of-function Idh mutations. In this study, Saha et al. demonstrated that Idh mutations block hepatocyte lineage progression and enhance the expansion of HPCs which, upon acquisition of an oncogenic insult (i.e. Kras mutations), lead to the development of premalignant biliary lesions and progression to iCCA.

During embryogenesis, intrahepatic and extrahepatic bile ducts originate from the intrahepatic ductal plate and hepatic diverticulum, respectively, thus suggesting different tumorigenic processes for iCCA and eCCA subtypes. However, due to the lack of specific models of eCCA development, our understanding of the mechanisms underlying the onset of pCCA/dCCA remains scarce. Interestingly, well-differentiated eCCA tumours have been detected in the extrahepatic bile duct of a mouse model of iCCA generated by liver-specific Kras activation and Pten deletion and in transgenic mice over-expressing Erbb2 in the basal layer of the biliary tract epithelium (Table 3). Of note, in the latter study, mice also developed GBC suggesting that mature cholangiocytes may represent a common cell of origin for all BTC subtypes.

Recent efforts have been focused on generating a clinically relevant model of eCCA development by incorporating the most frequent genetic alterations identified in NGS-based studies.^,, In this regard, Nakagawa et al. developed a mouse model of injury-related eCCA through biliary-cell-specific Kras activation and deletion of TGFβ receptor type 2 (Tgfbr2) and E-cadherin (Cdh1). Remarkably, these mice fully recapitulate the characteristic features of the human disease, including thickening of the extrahepatic bile duct wall accompanied by a swollen gallbladder, and moderately-differentiated adenocarcinoma cells with periductal infiltrating growth patterns of pCCA/dCCA. Cell lineage tracing in these mice suggested biliary precursors of the PGB as the cell of origin of eCCA (Fig. 2, Fig. 3).

Similar to eCCA, only few murine models of GBC are currently available (Table 3). As discussed, overexpression of Erbb2 in the basal layer of the biliary tract epithelium under the bovine keratin 5 (BK5) promoter leads to tumour formation at various sites along the biliary tract. Interestingly, GBC develops in 90% of these transgenic mice suggesting a common cell of origin for GBC and CCA (Fig. 2). Data obtained in an independent investigation showed that targeted transduction of the gallbladder epithelium via Pdx1-Cre driven expression of oncogenic Kras leads to upregulated NOTCH signalling and gallbladder tumourigenesis, thus suggesting gallbladder epithelial cells, rather than cholangiocytes, are the direct target of carcinognesis. Similarly, malignant transformation of the gallbladder epithelium was observed in a knock-out model of the oxysterol receptor liver X receptor–β (LXRβ), which is involved in the control of lipid homeostasis and glucose metabolism. Carcinogenesis in this model was estrogen dependent, consistent with a higher incidence of GBC in women (Table 1).