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21-Day Rapid Modeling: Construction of an Orthotopic VX2 Rabbit Hepatocellular Carcinoma Model—Comprehensive Analysis of Pathological Findings, Tumor Growth Curves, and Imaging Features

Primary liver cancer (PLC) is one of the most common malignancies. The VX2 rabbit orthotopic liver cancer model exhibits tumor progression that closely resembles human hepatocellular carcinoma, making it an ideal experimental model for liver cancer research. Therapeutics and drugs developed in this model are of great significance for guiding clinical decision‑making in selecting appropriate treatment strategies.

The VX2 tumor was initially derived from rabbit squamous cell carcinoma induced by the Shope papillomavirus through repeated passages. It exhibits strong invasiveness and rapid proliferation, and can spontaneously metastasize via the portal vein or hepatic artery to the lungs, lymph nodes, and other sites, making it one of the few animal models capable of mimicking the metastatic behavior of hepatocellular carcinoma.

Construction of an Orthotopic VX2 Rabbit Hepatocellular Carcinoma Model

| Open-Access Implantation Method

① Laboratory animals

New Zealand White rabbits, weighing 2.5–3.5 kg and aged 5–6 months, have a moderate body size and excellent tolerance, making them well suited for surgical procedures.

② Experimental Methods

Tumor mass preparation: Thaw the VX2 tumor mass at 37°C, remove necrotic and muscular tissues, then mince the fish‑like tissue into 1–2 mm³ fragments and immerse them in sterile saline.

Surgical procedure

Experimental rabbits were fasted for 12 hours prior to inoculation, with free access to water.

Anesthesia was administered, and an abdominal incision was made to expose the left lobe of the liver. The tumor mass was injected into the left lobe via a puncture needle; after packing with gelatin sponge, hemostasis was achieved by applying pressure with sterile cotton swabs.

Reinsert the liver into the body and close the incision. Administer a prophylactic antibiotic (e.g., enrofloxacin) for 3 days postoperatively.

④ Precautions

01 Transplantation of tender, grayish-white, tumor‑adjacent tissue exhibiting vigorous growth can correspondingly enhance the survival rate of tumor cells.

02 Fasting the animals prior to modeling can reduce gastrointestinal reflux and prevent unnecessary adverse reactions.

03 Tumor tissue was implanted into the left lobe of the liver; the left lobe is highly vascularized and has a substantial volume, which facilitates tumor growth.

⑤ Model characteristics:

This method features a short tumor‑formation time, a high implantation success rate, localized tumor growth, and minimal ectopic implantation.

| Experimental Results

① Pathological tissue

A distinct tumor lesion is evident in the left lobe of the liver; upon incision, multiple foci of tumor‑active components are observed, with focal areas of minimal necrosis.

② Growth curve:

On day 14 post-transplantation, the tumor diameter reached 2.5 ± 0.3 cm, and by day 21, central necrosis had developed (with a CT value decrease of 30–40 HU).

‌ ② HE results:

Digital histological sections of VX2 tumors in the liver at different magnifications; image source [1].

As shown in the figure, the tumor area exhibits necrosis, cystic spaces, and a fibrous stroma, with large undifferentiated tumor cells characterized by a high nuclear-to-cytoplasmic ratio, eosinophilic or pale cytoplasm, and poorly defined borders; apoptotic cell debris is present within the necrotic regions (arrows).

③ Imaging features

CT :

Over time, the metastatic lesion increases in size and becomes more aggressive. As shown in the image, the lesion exhibits low attenuation; during the arterial phase, it demonstrates variable ring‑like enhancement, while in the portal venous phase, it remains hypodense with well‑defined borders relative to the surrounding hepatic parenchyma.

Image source [1]

Under light microscopy, the tumor cells are arranged in nests; they are large, irregularly shaped, and disorganized. Numerous blood sinusoids are present, with nuclei that are large and deeply stained, and scant cytoplasm. The tumor tissue is clearly demarcated from the surrounding hepatic parenchyma.

| Percutaneous Puncture and Injection Method Under Ultrasound/CT Guidance

Procedure:

Under image guidance, the puncture site in the left lobe of the liver was identified. After making a skin incision with a sharp blade, the tumor mass was pushed into the hepatic parenchyma using a puncture needle. Postoperatively, antibiotics (e.g., penicillin plus gentamicin) were administered by injection for three consecutive days.

Model features:

The procedure is minimally invasive and carries no risk of peritoneal implantation; the tumor models are typically solitary nodules, facilitating imaging‑based monitoring. The success rate exceeds 95%, comparable to that of the open‑abdominal approach. Larger rabbits should be selected to facilitate the procedure, and puncture must be precisely directed to avoid the interlobar spaces, thereby preventing inadvertent tumor implantation.

| Tumor cell suspension injection method

Operation Steps:

The tumor cell suspension was directly injected beneath the liver capsule or into the hepatic parenchyma.

Model features:

It has a low tumor‑formation rate (approximately 79%), readily leads to intrahepatic metastasis or needle‑track implantation, and is technically complex; therefore, it has been gradually replaced by the tumor‑mass method.

Summary

‌

Selecting an appropriate model is of great significance for investigating the pathogenesis of hepatocellular carcinoma and for drug screening. Aniphe BioLab offers a variety of approaches to establish animal models of liver cancer and provides pharmacological and efficacy evaluation services tailored to related disease models. We welcome all clients to contact us for more information!

References:

[1] Front Oncol. 2022 May 10;12:871829

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