Experimental VX2 Rabbit Liver Tumor Model in Carbon Tetrachloride-Induced Cirrhosis of the Liver

Abstract

Liver cirrhosis is a major underlying factor in the development of hepatocellular carcinoma. Currently, there is an unmet need for midsize experimental vertebrate models that would offer reproducible implantable liver tumors in a cirrhotic liver background. This study establishes a protocol for a syngeneic rabbit model of VX2 liver cancer with underlying liver cirrhosis induced using carbon tetrachloride (CCl₄). Male New Zealand white rabbits (n = 3) received CCl₄ by intragastric administration once weekly. Concentrations started at 5% v/v CCl₄ dissolved in olive oil. CCl₄ dosing was progressively increased every week by 2.5% v/v increments for the duration of treatment (16 weeks total). VX2 tumors were then orthotopically implanted into the left hepatic lobe and allowed to grow for 3 weeks. Cross-sectional imaging confirmed the presence of hepatic tumors. Gross and histopathological evaluations showed reproducible tumor growth in the presence of liver cirrhosis in all animals.

Figure 1.

Timeline for carbon tetrachloride (CCl₄)–induced cirrhosis in rabbits. The schematic represents the animal experiment schedule in this study. The initial dose consisted of 5% CCl₄ (v/v) dissolved in 1 mL of olive oil (Day 0) and progressively increased by 2.5% for the duration of treatment (16 weeks). At Week 16, VX2 tumors were implanted into animals’ livers and grown for 3 weeks (Week 19).

Figure 2.

Axial abdominal computed tomography in a cirrhotic rabbit 3 weeks after tumor implant. (a) Unenhanced, (b) 15-second arterial phase, (c) 90-second portal venous, and (d) 5-minute delayed phase images illustrate the 2.5-cm VX2 tumor mass with arterial phase hyperenhancement (oval) showing washout on portal venous phase and delayed imaging.

Figure 3.

Digitized hematoxylin-eosin histology samples of VX2 tumors from carbon tetrachloride (CCl₄)–treated animals at different magnifications. (a) Cirrhotic liver parenchyma (CL) and tumor (T) (left) and areas of viable tumor (VT), fibrotic stroma (FS), and pseudocapsule with ill-defined borders (right). (b) Histopathological evaluation of cirrhosis was assessed with Masson’s trichrome (left) and sirius red staining (right). (c) Gross pathology of CCl₄-treated rabbit showed the anterior (left) and posterior (right) images of VX2 tumor in a cirrhotic liver.

Figure 4.

Liver biopsies stained using sirius red and trichrome for both the control and carbon tetrachloride–treated rabbits. A Meta-analysis of Histological Data in Viral Hepatitis score of F0 indicated no fibrosis activity for the control, and a score of F4 indicated cirrhosis. Fibrosis index (FI) revealed higher percentage of collagen per area in the treated group using both staining methods.