Cholangiocarcinomas can originate from hepatocytes in mice

Abstract

Intrahepatic cholangiocarcinomas (ICCs) are primary liver tumors with a poor prognosis. The development of effective therapies has been hampered by a limited understanding of the biology of ICCs. Although ICCs exhibit heterogeneity in location, histology, and marker expression, they are currently thought to derive invariably from the cells lining the bile ducts, biliary epithelial cells (BECs), or liver progenitor cells (LPCs). Despite lack of experimental evidence establishing BECs or LPCs as the origin of ICCs, other liver cell types have not been considered. Here we show that ICCs can originate from fully differentiated hepatocytes. Using a mouse model of hepatocyte fate tracing, we found that activated NOTCH and AKT signaling cooperate to convert normal hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Our findings suggest a previously overlooked mechanism of human ICC formation that may be targetable for anti-ICC therapy.

Figure 1. NICD/AKT-induced ICC formation.

(A) Photographic images of mouse livers taken at different time points after NICD/AKT plasmid injection. Small tumors (arrows) were visible as early as 3.5 weeks after injection. (B) H&E stainings of corresponding liver sections show rapidly expanding tumors replacing the normal liver parenchyma. Original magnification, ×40. At least 15 liver sections from 3 mice were analyzed for each time point.

Figure 2. Hepatocyte origin of NICD/AKT-induced ICCs.

(A) R26R-EYFP mice were intravenously injected with 4 × 10¹¹ viral genomes of AAV8-Ttr-Cre, followed by hydrodynamic tail vein injection of the NICD/AKT plasmids 1 week later. Tumors were analyzed 4.5 weeks after plasmid injection. (B) Coimmunostaining for EYFP (red) and Ck19 (green) 1 week after AAV8-Ttr-Cre injection shows that all hepatocytes, but no BECs, express EYFP. (C–E) Immunostainings of tumors for EYFP (red) show that they originated from hepatocytes. Additional immunostainings (all green) for Sox9 (C), Ck8 (D), and Mup (E) show that tumors express biliary markers but lack hepatocyte differentiation. Nuclei were stained with DAPI (blue). Original magnification, ×100; inset, ×200. At least 15 liver sections from 3 mice were analyzed for each immunostaining.

Figure 3. Conversion of hepatocytes into ICC precursors.

(A) R26R-EYFP mice were intravenously injected with 4 × 10¹¹ viral genomes of AAV8-Ttr-Cre, followed by hydrodynamic tail vein injection of the NICD/AKT plasmids 1 week later. Livers were analyzed 1.5 weeks after plasmid injection. (B) Coimmunostaining for EYFP (red), Ck8 (green), and Sox9 (white) shows a hepatocyte expressing the early BEC and LPC markers Ck8 and Sox9. (C) Coimmunostaining for EYFP (red), Ck8 (green), and HA (white) shows that hybrid cells express the NICD/AKT plasmids. (D) Coimmunostaining for EYFP (red), Mup (green), and Sox9 (white) shows decreased Mup expression in hybrid cells. Original magnification, ×400. At least 15 liver sections from 3 mice were analyzed for each immunostaining.