Cell lineage tracing reveals a biliary origin of intrahepatic cholangiocarcinoma

Abstract

Intrahepatic cholangiocarcinoma is a treatment refractory malignancy with a high mortality and an increasing incidence worldwide. Recent studies have observed that activation of Notch and AKT signaling within mature hepatocytes is able to induce the formation of tumors displaying biliary lineage markers, thereby raising the suggestion that it is hepatocytes, rather than cholangiocytes or hepatic progenitor cells that represent the cell of origin of this tumor. Here, we use a cholangiocyte-lineage tracing system to target p53 loss to biliary epithelia and observe the appearance of labeled biliary lineage tumors in response to chronic injury. Consequent to this, upregulation of native functional Notch signaling is observed to occur spontaneously within cholangiocytes and hepatocytes in this model as well as in human intrahepatic cholangiocarcinoma. These data prove that in the context of chronic inflammation and p53 loss, frequent occurrences in human disease, biliary epithelia are a target of transformation and an origin of intrahepatic cholangiocarcinoma.

Figure 1. Transgenic system of tamoxifen inducible, Cre mediated cell tracking with Trp53 deletion in CK19CreER^TeYFP^R26p53^f/f mice

(A) Transgenic construct of fluorescent labelling and tumour suppressor deletion in CK19 positive cells in response to tamoxifen in 6 week old mice. (B) In the presence of Cre and tamoxifen, eYFP activity is seen within small ductules as well as large bile ducts. The eYFP⁺ population expands following 14 days of dietary 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (Scalebars: 50μm). Quantitative analysis of Cre efficiency 72 hours post injection in Cre negative mice exposed to tamoxifen (n=5), Cre positive mice without tamoxifen (n=3) and Cre positive mice exposed to tamoxifen (n=8). (C) Following tamoxifen injection eYFP positivity is seen only in CK19 expressing cells. These are cholangiocytes that also express the biliary markers Sox9. No co-localisation is seen with the mature hepatocyte marker Cyp2D6 (Scalebars: 50μm).

Figure 2. ICC is derived from CK19 positive cholangiocytes

(A) Experimental strategy of tamoxifen induction in CK19CreER^TeYFP^R26p53^f/f mice followed by oral administration of 600mg/ml thioacetamide for 26 weeks. (B) Multifocal tumours developed only in CK19CreER^TeYFPp53^−/− (homozygous for p53 deletion) (n=5) and not CK19CreER^TeYFP^R26p53^+/− (n=14) or CK19CreER^TeYFP^R26p53^+/+ (n=5) animals, and only following TAA administration. (C) Co-immunofluorescent staining of eYFP with the biliary lineage markers CK19 and Sox9. All eYFP⁺ cells were seen to be CK19 positive. eYFP positivity did not overlap with the mature hepatocyte marker Cyp2D6. Nuclei are stained with DAPI (Scalebars: 50μm).

Figure 3. Native Notch signalling is activated in ICC

(A) Immunofluorescent staining of activated Notch1 in the membranes of malignant ductules of TAA-induced ICC in CK19CreER^TeYFPp53^−/− mice frequently co-localises with eYFP positivity (filled arrowheads) (Scalebars: 50μm). (B) Immunostaining of activated Notch1 within nuclei of peri-tumoral hepatocytes (open arrowheads) (Scalebars: 50μm and 125μm (Second photomicrograph taken under oil). (C) Activated Notch1 immunostaining in uninjured mouse liver; CCl₄ induced fibrosis (16weeks) and DDC diet (Scalebars: 50μm) (D) Activated Notch1 immunostaining in human ICC specimens. Staining in malignant biliary epithelia (filled arrowheads) and peri-tumoral hepatocytes (open arrowheads) (Scalebars: 50μm).