|Year : 2022 | Volume
| Issue : 7 | Page : 1973-1980
Development of a Hepatic VX2 carcinoma model in rabbits using an improved minimally invasive method and evaluation with imaging examinations
Gang Yuan1, Yanneng Xu1, Yunqiao Wang2, Xun Zhang3, Weiming Wang4, Yanzheng He5, Xiaoming Zhu2
1 State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR; Department of Intervention Radiology, Traditional Chinese Medicine Hospital, Affiliated to Southwest Medical University, Luzhou, China
2 State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR, China
3 Department of Intervention Radiology, Traditional Chinese Medicine Hospital, Affiliated to Southwest Medical University, Luzhou, China
4 State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR; Department of General Surgery (Vascular Surgery), The Affiliated Hospital of Southwest Medical University, Luzhou, China
5 Department of General Surgery (Vascular Surgery), The Affiliated Hospital of Southwest Medical University, Luzhou, China
|Date of Submission||22-May-2022|
|Date of Decision||04-Jul-2022|
|Date of Acceptance||15-Jul-2022|
|Date of Web Publication||11-Jan-2023|
State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR
Department of General Surgery (Vascular Surgery), The Affiliated Hospital of Southwest Medical University, Luzhou - 646000
Source of Support: None, Conflict of Interest: None
Context: The hepatic VX2 carcinoma model in rabbits has been widely used in interventional diagnosis and treatment research for hepatocellular carcinoma (HCC). However, traditional methods for developing this model all have their shortcomings.
Aims: To develop an improved method to construct an animal model of hepatic VX2 carcinoma.
Settings and Design: The puncture technique was used to obtain the VX2 tumor tissue. A tumor puncture–inoculation kit was designed and modified to implant the tumor tissue into the recipient rabbit's liver.
Methods and Material: 18 New Zealand white rabbits were implanted with VX2 tumor tissue using the improved tumor puncture–inoculation kit under ultrasound guidance. Ultrasonography, contrast-enhanced computerized tomography, magnetic resonance imaging, and digital subtraction angiography were performed to evaluate tumor formation and imaging characteristics.
Statistical Analysis: Statistical analysis was performed using SPSS software. Two groups were compared using Student's t-test analysis.
Results: All rabbits tolerated VX2 tumor tissue implantation successfully. 17 out of the 18 experimental rabbits developed liver tumors, and one rabbit had abdominal tumor metastasis. The average volume of tumors was 39.47 mm3 and 460.1 mm3 (P < 0.001) on the 7th and 14th days after modeling, respectively. Imageological diagnosis showed that all tumors had abundant blood supply and typical imaging characteristics.
Conclusions: This improved modeling method is easy to operate and less traumatic, with a high tumor formation rate, low metastasis rate, prominent tumor imaging characteristics, and high detection rate, which is expected to become a promising method for constructing rabbit liver tumor model.
Keywords: Animal model, liver, methodology, VX2 tumor
|How to cite this article:|
Yuan G, Xu Y, Wang Y, Zhang X, Wang W, He Y, Zhu X. Development of a Hepatic VX2 carcinoma model in rabbits using an improved minimally invasive method and evaluation with imaging examinations. J Can Res Ther 2022;18:1973-80
|How to cite this URL:|
Yuan G, Xu Y, Wang Y, Zhang X, Wang W, He Y, Zhu X. Development of a Hepatic VX2 carcinoma model in rabbits using an improved minimally invasive method and evaluation with imaging examinations. J Can Res Ther [serial online] 2022 [cited 2023 Jan 27];18:1973-80. Available from: https://www.cancerjournal.net/text.asp?2022/18/7/1973/367451
Authors Gang Yuan, Yanneng Xu, Yunqiao Wang contributed equally to this work
| > Introduction|| |
Hepatocellular carcinoma (HCC) has become one of the leading causes of cancer-related death globally. Due to the poor diagnosis at the early stage, most patients are not suitable for surgical resection treatment. Interventional therapy such as transcatheter chemoembolization (TACE) has been recognized as one of the most common methods for patients with intermediate and advanced HCC., Hence, animal models used in interventional therapy experiments on HCC have played an irreplaceable role. Although the VX2 tumors in rabbits are not of hepatic origin, the VX2 liver tumor models of rabbits have similar pathological characteristics with human primary hepatic cancer; in particular, the tumor blood supply from the hepatic artery is very similar to HCC., In addition, rabbits are highly reproductive, cheap, and large enough to enable intra-arterial procedures. Therefore, rabbit VX2 liver tumor models are commonly used as animal models to study minimally invasive interventional therapy.,
Currently, there are three methods reported to construct the rabbit VX2 liver tumor model, including open surgery,, percutaneous injection, and intra-arterial or intraportal tumor cell injection.,, These methods have achieved varying degrees of success. However, they all have their defects: (1) Open surgical implantation modeling is reported with a high tumor formation rate and a low incidence of tumor metastasis, but it is highly traumatic, prone to bleeding and infection, and slow in postoperative recovery, as well as with strict requirement on researcher's surgical skills, which lead to the failure of its popularization. (2) Although percutaneous injection of the cell suspension or tissue fragments is less traumatic with lower infection risk, this method is very likely to cause abdominal tumor metastasis, often forming multiple tumors, which is not suitable for studying the pathological characteristics of a single tumor. (3) Cell suspension injection via the hepatic artery or portal vein is mainly used for the study of tumor metastasis and seldomly adopted for the development of the VX2 tumor model.
Therefore, this study aims to develop an improved method for constructing a rabbit VX2 liver tumor model, which overcomes the defects of previous modeling methods, such as trauma, bleeding, infection, and metastasis. This improved animal model will be valuable for the studies related to interventional therapy of HCC.
| > Materials and Methods|| |
This study was approved by the Institutional Animal Care and Use Ethical Committee of Southwest Medical University (IACUC No. SWMU 20210409). All animal experiments complied with the ARRIVE guidelines and were carried out according to the institutional guidelines. Eighteen New Zealand white male rabbits, aged from 2 to 3 months and weighing 2.5 − 3 kg, were purchased from the Experimental Animal Center, Southwest Medical University. Two VX2 tumor-bearing rabbits, weighing 2.5 kg, were purchased from Boyue Bioengineering Co., Ltd. (license number SCXK2019-0005, Jiyuan, China) as the source of VX2 tumor. All rabbits were brought to the research facilities in the animal house 2 weeks before the experiment, where they were handled daily to get accustomed to the new environment. Each rabbit was kept separately in a clean and ventilated steel cage, placed in a room with an open system at a temperature of 18−23°C and 60% humidity, and provided with artificial lighting for 12 hours a day to simulate the natural day and night alternation. Rabbits were fed with pellet feed (Tongwei, Chengdu, China) and allowed to drink freely.
To avoid asphyxiation during anesthesia, 12-hour fasting and 4-hour water deprivation on rabbits before anesthesia was guaranteed. All rabbits were anesthetized via intravenous injection of 3% pentobarbital sodium (30 mg/kg) through the auricular vein, and their vital signs, including respiration, heart rate, corneal reflexes, and pain stimuli, were under real-time monitoring by an anesthetist. After the experiment, the rabbits were placed on a warm and dry animal bed until they were fully awake and walked briskly before being transferred back to the cage.
Tumor puncture and inoculation kit
A semi-automatic biopsy needle (18 G, L-130 mm, TSK, Tessumi Co., Ltd, Tochigi, Japan) was used to obtain uniformly sized strips of VX2 tumor tissue. The tumor implantation kit included a push rod (from the TSK biopsy needle kit) and a syringe needle (16 G). The push rod was modified, with the inner sharp-headed needle being polished to be smooth and flat-headed, preventing the tumor tissue from remaining in the needle and protecting the liver tissue from accidental damage. In addition, a movable piston was assembled on the push rod to facilitate accurate positioning during operation [Figure 1].
|Figure 1: Puncture and tumor-inoculating kit. (a) TSK 18G semi-automatic biopsy gun kit with sharp-headed needles. (b) Tumor-inoculating push rod and 16G needle. (c) Partial close view of the biopsy gun, with approximately 2 cm concave at the top for sampling. (d) Partial close view of the push rod. The top has been polished into a flat head, and the green object is a movable piston used for positioning during tumor inoculation|
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Preparation of VX2 tumor fragments
The tumor-bearing rabbits were anesthetized, and then, under a sterile condition, the tumor was punctured with a semi-automatic biopsy needle (11 mm, specimen slot) under the guidance of ultrasound (M7, Mindray Medical Intl. Ltd., Shenzhen, China) to obtain sufficient VX2 tumor fragments, which were then placed in saline after the necrotic tissue was removed [Figure 2]a, [Figure 2]b, [Figure 2]c.
|Figure 2: Extraction and implantation of VX2 tumor fragments. (a) Ultrasound-guided puncture of the VX2 tumor in a tumor-bearing rabbit. (b) Biopsy needle entering tumor tissue under ultrasound monitoring (yellow arrow points to puncture needle). (c) The extracted VX2 tumor fragments are placed in sterile saline for later use. (d) Tumor fragments and hemostatic gelatin sponge are successively loaded into a 16G sterile needle, and the piston is fixed in place. (e) Puncture of the left liver lobe under ultrasound monitoring. (f) After the successful release of tumor tissues, the hyperechoic shadow seen on the ultrasound image in the liver parenchyma represents the mixture of hemostatic sponge and tumor tissues (as indicated by the yellow arrow)|
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Implantation of VX2 tumor fragments into rabbit liver
Firstly, a 1-centimeter gelatin sponge (Nanchang, China) was cut and kneaded into a cylindrical strip. Then, fresh tumor tissue strips were packed into a 16 G aseptic syringe needle. Next, the gelatin sponge strip was filled at the end of the needle [Figure 2]d. Finally, the VX2 tumor tissue and gelatin sponge were implanted into the rabbit liver. Before implantation, the assistant applied ultrasound to find a suitable planting location to ensure that the bile duct and blood vessels would not be damaged. The needle preloaded with tumor tissue and gelatin sponge was then carefully inserted into the target area of the liver, and the pushrod was fixed to complete the tumor tissue and gelatin sponge released into the liver parenchyma by withdrawing the needle. When the high echo mass was seen on the ultrasound display, indicating successful release of the tumor tissue and gelatin sponge, the needle and pushrod were pulled out, and the puncture point was pressed for 2–3 minutes [Figure 2]e, [Figure 2]f. Ultrasonography was performed again to examine whether there was active bleeding at the puncture site to assess liver damage.
Routine ultrasound examination was performed on the 7th and 14th days after VX2 tumor implantation. The tumors' shape and blood flow signal was observed with the superficial ultrasonic probe (thyroid mode). Then, the size, including the length and width at each tumor's greatest dimension imaging plane, was measured using conventional ultrasonography. To unify the standard, all ultrasound examinations were performed by the same ultrasound doctor. The tumor volume was calculated based on the formula (1), where “a” is the length of the tumor and “b” is the width.
Contrast-enhanced CT examination
14 days after VX2 tumor implantation, the rabbits were examined with contrast-enhanced computed tomography (CECT, Flash speed dual-source CT, Siemens, Germany). Before the examination, contrast agent iodixanol (iodine 320 mg/mL, Heng Rui, China) was injected into the abdominal wall vein of the anesthetized rabbits through a 24 G indwelling needle at the speed of 2 mL/s, with a total amount of 5 mL. CT scan was started 2 s after iodixanol injection, and images were entirely collected through continuous repeated multi-layer scanning mode. Scanning parameters: the tube current is 80 mA, the tube voltage is 120 kV, the layer thickness is 2.5 mm, the matrix is 512 × 512, and the image collection time is 30 s.
Magnetic resonance imaging examination
The anesthetized rabbits were fixed supinely to a special coil for animals. A clinical magnetic resonance imaging (MRI, MAGNETOM Verio 3.0T, Erlangen, Siemens, Germany) scanner was used to perform T1W, T2W, and SE sequence techniques. Images were acquired under axial T1-weighted turbo spin-echo (TR, 600 ms, TE, 11 ms) and T2-weighted turbo spin-echo (TR, 4820 ms, TE, 57 ms), with the slice thickness being 2.0 mm and field of vision being 130 × 130 mm.
Digital subtraction angiography
The anesthetized rabbits were put supinely on the operating table. Under sterile conditions, the skin of the rabbits was cut 1–2 cm to fully expose the right or the left femoral artery, into which a 4-F vascular sheath (Terumo, Tokyo, Japan) was inserted. Under the monitoring of a biplane, flat panel angiographic system, and digital subtraction angiography (DSA, Axiom Artis Zee, Siemens, Germany), a 2.6-F microcatheter (Asahi, Japan) was accurately placed in the hepatic artery guided by a 0.018-inch guidewire (Asahi, Japan). Then, the location, size, and blood supply of the tumor were observed by angiography.
At the end of the experiment, all rabbits were euthanized by administering pentobarbital sodium through the marginal ear vein. The extracted tumor tissue was fixed with 10% neutral formaldehyde, embedded, and made into a wax block. The sections of the tumor were stained with hematoxylin–eosin (H&E), sealed with neutral gum, and then observed under an upright microscope (Olympus, Japan) equipped with a digital camera (Olympus DP70) and different objectives (4, 10, 20, and 40×). All tissues were observed in each section at 100×, and three images were collected at 100× and 400×, respectively. In addition, liver and lung samples were also stained with H&E to evaluate the occurrence of tumor metastasis.
The results are expressed as means ± standard errors of the means (SEM). Statistical analysis was performed using SPSS software (version 22.0), and Student's t-test analysis was conducted to compare the two groups if the tumor size data conformed to a normal distribution. Otherwise, using the Wilcoxon signed-rank test, a P value of <0.05 was considered statistically different.
| > Results|| |
The formation of VX2 tumor in the liver
All rabbits have implanted the VX2 tumor tissues under ultrasound guidance. There was no active bleeding or infection during and after implantation, and the survival rate within 14 days was 100%. By imaging and necropsy, this study defined the detection of tumors in the liver implant areas as successful model construction. During the 14-day follow-up, one rabbit was found with no tumor node in the liver; thus, the success rate of modeling is 94.4%. One rabbit was found with abdominal tumor metastasis during necropsy, with a metastasis incidence of 5.6% [Figure 3]a. The tumor size was measured on the 7th and 14th days after modeling, respectively, and the tumor volume was calculated. The results showed that the tumor grew slowly during the first week after implantation, with the volume being 39.47 ± 4.28 mm3, and the average tumor volume increased to 460.1 ± 13.46 mm3 14 days after the operation [Figure 3]b, P < 0.001.
|Figure 3: Formation of VX2 tumor in the liver. (a) Tumor formation and metastasis of the rabbit liver 2 weeks after modeling. (b) Liver tumor volume at 7 and 14 days after VX2 tumor tissue implantation, **** P < 0.001|
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Imaging features of liver VX2 tumors
Ultrasound scan showed that at 14 days after VX2 tumor tissue implantation, the tumors in the rabbit liver presented spherical or elliptical-like homogeneous hypoechoic nodes with clear contour and without capsule echo. Slightly dotted hyperechoic images shown in the center of some tumor tissue represented the unabsorbed hemostatic sponge [Figure 4]a. Color Doppler flow imaging showed obvious blood flow signals inside and around the VX2 tumors [Figure 4]b.
|Figure 4: Imaging examinations of the VX2 tumors 14 days after implantation. (a, b) Ultrasound examination shows the VX2 tumor (as indicated by the yellow arrow) as a homogeneous hypoechoic nodule (central punctate hyperechoic represents a hemostatic sponge that has not been fully absorbed). The tumor is single and located in the left lobe of the liver, with a clear contour and spherical shape. Color Doppler shows blood flow signals around and within the tumor. (c, d) CECT shows annular enhancement around the tumor in the arterial phase (as indicated by the yellow arrow) and the venous phase as a distinct low-density shadow (as indicated by the white arrow). (e, f) MRI demonstrates the low signal intensity of the tumor on T1WI (as indicated by the yellow arrow) and high signal intensity of the tumor on T2WI (as indicated by the white arrow). (g, h) DSA shows evident staining and abundant blood supply of the tumor, and the lesion is clearly visible in both subtraction (as indicated by the yellow arrow) and non-subtraction (as indicated by the white arrow) modes|
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CECT showed circular enhancement around the tumor in the arterial phase, whereas the tumor enhancement disappeared rapidly in the venous phase, showing a noticeable low-density shadow. The imaging feature of “fast-forward, fast-exit” was very similar to the CECT findings of HCC patients [Figure 4]c, [Figure 4]d.
MRI imaging revealed that the tumor showed a low signal on T1WI and a high signal on T2WI and that the tumor was regular in shape and clearly demarcated from the surrounding liver tissue [Figure 4]e and [Figure 4]f.
DSA showed that the tumor was stained obviously and the blood supply was abundant and that the blood supply arteries around the tumor, mainly including the left branch of the hepatic artery and its branches, showed spherical distribution [Figure 4]g, [Figure 4]h.
The gross pathology features of VX2 tumors were as follows. The tumors were spherical or ellipsoidal-spherical and hard; the tissues had a grayish-white and solid nodule appearance after incision, with a small amount of necrotic tissue in the center, and the boundary between all tumors and the surrounding liver tissue is evident [Figure 5]a. The H&E staining of the tumors' pathological sections showed an irregular, dense, and nest-like morphology of the cells (×100), with large nuclei and deep staining [Figure 5]b. At high magnification (×400), the tumor cells showed glandular arrangement, irregular shape, large nuclei, uneven staining, and abundant cytoplasm, and vacuolar changes in the cytoplasm were observed in some cells [Figure 5]c. Necropsy revealed one rabbit with abdominal metastasis. The metastatic tumor was single, located on the omentum surrounding the liver, closely adjacent to the liver tumor, and was 5 × 5 mm in size. In addition, no macroscopic tumor metastases were found in the remaining rabbits, and no tumor cells were found in the pathological sections of liver and lung tissue specimens outside the tumor area [Figure 5]d, [Figure 5]e.
|Figure 5: Pathological examination of the VX2 tumor, liver, and lung tissues. (a) 14 days after the implantation of VX2 tumor tissues, the cut surface of rabbit liver VX2 tumor has a grayish-white and solid nodule appearance, with a small amount of necrotic tissue in the center (white arrowhead) and a clear boundary between the tumor and the surrounding liver tissue. (b and c) Histopathological images (100× and 400×) of tumor tissues stained with H and E. The tumor cells showed glandular arrangement, irregular shape, large nuclei, uneven staining, abundant cytoplasm, and vacuolar changes in the cytoplasm (black arrowheads). H and E staining of the liver (d) and lung (e) tissues (no tumor cells). Scale bar is 50 μm in (b), 10 μm in (c), 100 μm in (d) and (e)|
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| > Discussion|| |
HCC is a malignant tumor with a poor prognosis, seriously threatening human health. Establishing an animal model with a stable and reliable orthotopic liver tumor is the basis for studying HCC diagnosis and treatment. The VX2 tumor, first proposed by Shope and Hurst, is an anaplastic squamous cell carcinoma originating from hare virus-induced dermal papilloma, which can be implanted into a rabbit's head and neck, liver, lungs, kidneys, uterus, and other organs to build tumor models similar to human orthotopic tumors., Due to its characteristics of hepatic artery blood supply and fast growth, the rabbit liver VX2 tumor model is widely applied in the experimental study of imaging diagnosis and interventional therapy of HCC.,,
Previous studies used percutaneous cell suspension injection and open surgery to construct liver VX2 tumor models.,, However, percutaneous cell suspension injection has been gradually eliminated due to its tedious process of cell suspension preparation process, low success rate, and susceptibility to tumor metastasis. Although open surgery is reported with a high tumorigenesis rate and low metastasis rate. It takes a long operating time and causes great surgical trauma, likely to induce postoperative complications such as bleeding, bile fistula, and infection, thus bringing great trouble to researchers. Therefore, researchers have tried several minimally invasive approaches to avoid these deficiencies. In the past 2 decades, researchers began to use an automatic biopsy gun to extract tumor samples from the peritoneal tumor, directly insert the biopsy gun into the liver under ultrasound guidance, and release the tumor tissue through a 180° rotating biopsy gun. Compared with traditional methods, this method is simple, time-saving, and less invasive. However, the rotating biopsy gun will inevitably cause damage to the liver, leaving potential risks such as bleeding or bile fistula post-operation. Some studies on rabbit VX2 tumor model construction under the guidance of ultrasound,, CT, and other imaging equipment have been reported in recent years.
This study further proposed an improved method for rabbit liver VX2 tumor model construction, which has several advantages compared with previous methods: (1) The inoculation kit was modified and redesigned. The inner sharp-headed needle of the push rod was polished to be smooth and flat-headed, which can prevent the tumor tissue from remaining in the needle and protect the liver tissue from accidental damage. Besides, a movable piston was assembled on the push rod to facilitate accurate positioning during operation. (2) The method of obtaining VX2 tumor tissue was improved. In some previous studies,,, ultrasound was applied just in the process of tumor tissue implantation. The traditional incision method was still used to obtain tumor tissue, which not only resulted in waste and unnecessary sacrifice of experimental animals but also obtained uneven sizes of tumor tissue, resulting in great differences among the established tumor models. In our study, ultrasound was used both in VX2 tumor tissue acquisition and implantation. In the process of obtaining VX2 tumor tissue, the application of ultrasound ensures that all operations are minimally invasive and can reduce the suffering of experimental rabbits. The donor rabbit can be protected by puncture sampling under ultrasound guidance, and sampling can be done multiple times, avoiding unnecessary sacrifice. The equal size of tumor tissue obtained by a biopsy needle can ensure the uniformity of the tumor after implantation. Moreover, under high-resolution ultrasound guidance, the tumor necrosis area can be avoided during sampling to ensure the viability of the tumor tissue obtained and improve the tumorigenesis rate after implantation. In the process of VX2 tumor tissue implantation, the application of ultrasound not only provides helpful guidance for puncture operation and improves the success rate of implantation but also can monitor the occurrence of postoperative liver bleeding for the early diagnosis of bleeding complications. (3) Application of hemostatic gelatin sponge: In previous studies, the gelatin sponge was merely used in open surgery to prevent bleeding of liver incision and leakage of VX2 tumor suspension.,, This study reported for the first time the simultaneous implantation of gelatin sponge and VX2 tumor tissue strips into the liver by a minimally invasive puncture method. Applying a gelatin sponge reduced the risk of bleeding and implantation metastasis. Also, it was conducive to intraoperative and postoperative image follow-up.
Encouragingly, using this improved method to construct a rabbit liver VX2 tumor model, the tumor-forming rate was as high as 94.4%, and only one of the 18 rabbits failed to develop a liver tumor. Necropsy revealed no liver or lung metastasis of the tumor except for one case of abdominal metastasis (presumably because the implantation location was near the liver edge and the tumor protruded out of the liver, leading to abdominal implantation metastasis). Hence, based on the improvements, this method caused a lower distant metastasis rate than the previously reported ultrasound-guided rabbit liver VX2 tumor modeling. Moreover, the liver tumor constructed was single and could be clearly imaged through ultrasound, CECT, and MRI, facilitating imaging follow-up of the development of tumor lesions or tumor outcomes after different interventions. DSA angiography is the basis of TACE therapy for HCC, and whether it can clearly show the tumor supplying artery is the key to the success of treatment. As expected, DSA showed apparent staining and abundant tumor blood supply in this study. The advantages of high tumor formation rate, low metastasis rate, rich blood supply, and clear DSA image indicate that the model can be well applied to the study of TACE treatment.
| > Conclusions|| |
In conclusion, this study developed a hepatic VX2 carcinoma model in rabbits using an improved minimally invasive method, which is safe, reliable, and easy to operate. The established liver tumor models are suitable for conventional imaging examinations such as ultrasound, CT, and MRI. DSA imaging can also clearly display the blood supply and angiogenesis of VX2 tumors. This improved method for constructing rabbit hepatic VX2 tumor models can provide reliable support for the further study of interventional treatment for HCC.
CT and MRI examinations were performed by the Department of Radiography at the Affiliated Hospital of Traditional Chinese Medicine of Southwest Medical University. The authors would like to acknowledge the Department of Radiography for providing these resources for this study. The authors thank Ms. Liu Li (School of Foreign Languages, Southwest Medical University) for her English language editing assistance.
Financial support and sponsorship
Macau Science and Technology Development Fund (Project No.: 0168/2019/A3, SKL-QRCM (MUST)-2020-2022).
Conflicts of interest
There are no conflicts of interest.
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