INTRODUCTION

Hepatocellular carcinoma (HCC), the most common primary liver malignancy, accounts for 75-85% of liver cancer cases and is the third leading cause of cancer-related death.^1,2 Despite advances in diagnostic and therapeutic applications, HCC prognosis remains poor due to its typically late-stage diagnosis, high recurrence rates, and limited therapeutic options. The incidence of HCC is strongly linked to underlying chronic liver diseases, including hepatitis B virus and hepatitis C virus (HCV) infections, alcohol-associated liver disease, and metabolic dysfunction- associated steatotic liver disease (MASLD).^3,4 Additional risk factors such as aflatoxin exposure, obesity, and diabetes can also exacerbate HCC progression.

Animal models of HCC have been proposed in order to better understand its complicated pathogenesis and to explore novel therapeutic approaches. These models aimed to recapitulate the primary characteristics of human HCC, including genetic mutations, the tumor microenvironment, and disease progression. Among these models, diethylnitrosamine (DEN)-induced mouse model is one of the most extensively characterized and widely used systems to study HCC pathogenesis.⁵ Other commonly employed models include chronic carbon tetrachloride (CCl₄)-induced HCC models,⁶ combination of DEN+CCl₄-induced HCC models,⁷ MASLD-associated HCC (STAM^TM-HCC) models,⁸ alcohol-associated HCC models,⁹ hydrodynamics-based transfection (HBT) models,¹⁰ and orthotopic transplantation models.¹¹ Each of these models provide unique insights into specific facets of HCC pathogenesis, albeit with inherent strengths and limitations. However, no single animal model can fully represent the complexity of human HCC, which results from intricate genetic, environmental, and metabolic interactions.¹² Furthermore, translating animal study findings into human clinical applications remains a significant challenge due to interspecies variations in liver physiology and tumor biology. Therefore, to increase the translational relevance of preclinical studies, it is crucial to carefully select and combine appropriate animal models based on specific research questions.

Given the diversity and utility of existing models, this review aims to provide a comprehensive overview of the principal animal models employed in HCC research, with a particular focus on their advantages and limitations. By summarizing the current landscape of HCC modeling, this review aims to guide researchers in selecting the most appropriate models for their studies, and to facilitate the translation of experimental findings into meaningful clinical applications.

DEN-INDUCED HCC MOUSE MODEL

The carcinogenic effects of DEN are primarily driven by its metabolic activation, reactive oxygen species (ROS) production, and eventual DNA damage, which promotes genetic mutations. DEN is a potent alkylating agent that induces DNA damage by ethylating the N7 position of guanine, thereby forming N7-ethylguanine adducts. Such lesions compromise the integrity of DNA by causing depurination, generating apurinic sites, and promoting base mispairing. Modified guanine erroneously couples with thymine instead of cytosine, and the accumulation of such errors fosters hepatocellular carcinogenesis. CYP2E1, a key cytochrome P450 enzyme, metabolizes DEN to reactive intermediates and promotes ROS generation, including hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH·).^13,14 Exposure to DEN frequently induces mutations in the Ha-ras and B-raf oncogenes, and the Ctnnb1 gene, which plays a central role in the Wnt/β-catenin signaling pathway.^13,15,16 These mutations contribute to tumor initiation and progression by driving aberrant cell proliferation and survival, disrupting the normal regulatory mechanisms of hepatocytes. The extent of DEN-induced hepatocarcinogenesis is strongly influenced by the genetic background of the mouse strain, highlighting the importance of careful strain selection to ensure experimental relevance and reproducibility (Table 1).¹⁴ Furthermore, the age and sex of the mice significantly influence the progression of DEN-induced HCC. Due to the elevated hepatic DNA synthesis during this developmental stage, weanling mice exhibit heightened susceptibility to hepatocarcinogenesis.^13,14,17 Moreover, HCC develops at an early stage in all male mice (100%), whereas the incidence in female mice is limited to approximately 30%.¹⁸ These disparities are partly explained by the hepatoprotective effects of estrogens, which attenuate hepatocarcinogenesis via estrogen-dependent suppression of interleukin (IL)-6 production in Kupffer cells.¹⁹

DEN can be administered to rodents via multiple routes, including drinking water, diet, oral gavage, or inhalation.^13,14,20,21 However, intraperitoneal administration is the most commonly employed protocol, as it enables precise dosage control and ensures consistency across experimental animals. Multiple intraperitoneal injections of DEN accelerate hepatic tumor proliferation (Table 1).^22,23 In a study exploring HCC induction via drinking water rather than the conventional intraperitoneal injection, mice received DEN-containing water (0.95 g/mL) for 6 consecutive days followed by a day of regular water, and this cycle was repeated for 24 weeks.²⁴

The DEN-induced HCC model provides the advantage of consistent liver tumor induction, particularly in male mice. While a single DEN administration in mice does not capture intermediate pathogenic processes, multiple DEN administrations have been demonstrated to induce fibrosis and chronic liver inflammation, more accurately reflecting human HCC progression.^14,25 Although the DEN-induced HCC model is widely used, tumor formation typically requires a prolonged period (32-48 weeks), and its translational relevance may be limited due to interspecies differences in immune responses between rodents and humans.²²

CCL₄-INDUCED HCC MOUSE MODEL

Chronic CCl₄-induced liver injury is recognized as a well-established experimental model for studying liver disease owing to its potent hepatotoxic effects. Administered CCl₄ is metabolized in the liver by CYP2E1, generating highly reactive trichloromethyl free radical (CCl₃·).^26,27 Produced reactive CCl₃· free radical damages the endoplasmic reticulum, leading to lipid accumulation, inhibition of protein synthesis, and disruption of mixed-function oxidase activity. The reactive CCl₃· free radical further undergoes further oxygenation to form trichloromethylperoxy radicals (CCl₃OO·), which exacerbate hepatocyte damage by promoting lipid peroxidation.^27,28 This leads to the degradation of polyunsaturated fatty acids in cellular membranes, resulting in increased membrane permeability. Additionally, the administration of CCl₄ activates Kupffer cell, leading to the secretion of cytokines such as tumor necrosis factor (TNF)-α, IL-1β, IL-6, and transforming growth factor (TGF)-β.²⁹ These cytokines, in turn, stimulate oxidant production in hepatocytes, resulting in increased ROS generation and thereby exacerbating oxidative stress and hepatic injury.^26,30 CCl₄-induced liver injury begins with an initial phase of predominant necrosis, characterized by elevated liver-specific enzymes and reduced pseudocholinesterase activity. This is followed by an intermediate phase of significant hepatic steatosis, marked by increased serum triglyceride and aspartate aminotransferase levels and progressive decline in liver function (intermediate phase). In the third phase, continued elevation in aspartate aminotransferase, hydroxyproline, and triglyceride levels indicates ongoing hepatic damage and fibrogenesis. In the final stage, further decline in pseudocholinesterase levels and the onset of liver atrophy reflect severe, progressive hepatic dysfunction linked to sustained fibrotic progression. Susceptibility to CCl₄-induced liver injury is significantly impacted on the genetic background of the mouse strain, which should be carefully considered when designing experimental models of hepatic fibrosis (Table 2).^26,31,32 Although a definitive link between age and susceptibility to CCl₄ exposure has not been firmly established, growing evidence suggests that the risk of hepatic injury increases with aging.³³ This upregulated inflammatory reaction accelerates the development of fibrosis in older animals, even while there is no alteration in the proliferative capacity of hepatocytes following CCl₄ injury.³³ A recent study revealed that ovariectomy exacerbated CCl₄-induced liver injury in mice, whereas estrogen supplementation mitigated the ovariectomy-associated hepatic damage.³⁴ The estrogen-mediated attenuation of CCl₄-induced liver injury suggests that the extent of hepatic damage may differ between sexes. Thus, to minimize the variability from hormonal fluctuations, liver injury studies commonly employ male rodents.

The degree of hepatic fibrosis and the development of HCC are significantly influenced by the frequency and duration of CCl₄ administration. When administered intraperitoneally to C57BL/6 mice twice a week for 6 weeks or three times a week for 4 weeks, CCl₄ caused substantial intrahepatic collagen deposition that, according to the Desmet/Scheuer method, resembled stage 3 human fibrosis. Highly susceptible strains such as BALB/c mice can develop fibrosis after just 4 weeks of treatment. However, establishment of the CCl₄-induced HCC model is still challenging because of the complex pathogenic mechanisms of hepatotoxins, which typically require extended dosing (e.g., 0.2 mL/kg twice weekly for 28 weeks) to promote tumor formation (Table 2).³⁵ The subsequent administration of CCl₄ enables the study of intermediate stages such as inflammation, fibrosis, and necrosis without requiring transgenic mice, making the CCl₄-induced HCC mouse model highly cost-effective. Although CCl₄-mediated HCC models are simple and reproducible, the lack of established protocols (e.g., treatment period/ dose) and inconsistent outcomes pose limitations. Furthermore, CCl₄-induced HCC development requires multiple injections over extended periods (to 28 weeks), and the chemical’s high toxicity necessitates stringent safety precautions for researchers.

COMBINED DEN+CCL₄-INDUCED HCC MOUSE MODEL

Combined administration of DEN and CCl₄ accelerates HCC progression more effectively than administration of either agent alone. As previously mentioned, single dose DEN injection generates DNA adducts,^13,14,17 while subsequent administration of CCl₄ promotes ROS and endotoxin production, cumulatively aggravating hepatic pathology and more closely mirroring the clinical progression of human HCC.^13,14 In this setting, DEN is usually administered once at a dose ranging from 2.5 to 100.0 mg/kg, depending on the age and genetic makeup of the mice used in the experiment.³⁶ Research has revealed that even a small dose of DEN (1 mg/kg) combined with 0.2 mL/kg of CCl₄ could induce HCC by 5 months of age.⁷ Another study demonstrated that a single DEN injection (100 mg/kg) in 6- to 14-week-old mice, followed by 6-12 biweekly injections of CCl₄ (0.5 mL/kg) induced HCC development (Table 3).¹⁴ The combined administration of DEN and CCl₄ induces the full spectrum of hepatic pathophysiological stages including inflammation, fibrosis, and cirrhosis, which are characteristic features of human liver cancer. Given the inherently slow progression of hepatic tumor, however, the combined administration of DEN and CCl₄ provides the advantage of reducing the experimental time required to induce HCC. Moreover, this mouse model is also cost-effective than genetically engineered mouse models and achieves 100% liver tumor formation, ensuring high reproducibility and reliability. The relative simplicity of the experimental techniques boosts its practicality for researchers to understand HCC pathogenesis.

MASLD-ASSOCIATED HCC MOUSE MODEL

MASLD-associated HCC mouse model was developed to more precisely recapitulate the clinicopathological spectrum from hepatic steatosis to metabolic dysfunction-associated hepatitis (MASH), fibrosis, and HCC in the presence of type 2 diabetes. This model was commercialized and registered by SMC Laboratories (Tokyo, Japan) and is frequently referred to as the STAM^TM model in scientific literature. This model addresses the limitations of conventional approaches, including genetically modified models, leptin-deficient models, methionine- and choline-deficient diet models, and long-term high-fat diet (HFD) models.³⁷ The STAM^TM mouse model was established by administering a single subcutaneous injection of 200 μg streptozotocin (STZ) to neonatal male C57BL/6J mice 2 days after birth, followed by an HFD starting at 4 weeks of age (Table 4).³⁸ Low-dose STZ induces mild islet inflammation and destruction, contributing to diabetic conditions within 4 weeks.³⁷ STZ is selectively transported into pancreatic β-cells via GLUT2, where it exerts cytotoxic effects through DNA alkylation, culminating in β-cell death.³⁹ STZ induced cytotoxicity causes metabolic disturbances, including hyperglycemia, impaired glucose tolerance, and reduced glucose responsiveness due to decreased insulin secretion.⁴⁰ Once these diabetes characteristics were established, the animals were subjected to an HFD to further accelerate disease progression. The composition of the HFD used in the STAM^TM model can differ based on the study’s purpose. In the STAM^TM model, HFDs typically account for 45% to 75% of the total calorie intake and may include additional components such as fructose or cholesterol.⁴¹ These HFDs usually consist of 60 kcal% fat, 20 kcal% protein, and 20 kcal% carbohydrates^42,43 or a diet that contains 45 kcal% fat, 20 kcal% protein and 35 kcal% carbohydrates.^41,44 Consequently, MASH develops by 8 weeks, followed by fibrosis and cirrhosis around 12 weeks, and HCC between 16 and 20 weeks of age.^45,46 In particular, hyperglycemia and impaired insulin secretion facilitate fatty acid esterification and subsequent triglyceride accumulation after 6 weeks of treatment.⁴⁷ Elevated triglyceride levels that overwhelm β-oxidation capability activate alternative metabolic pathways, resulting in the production of lipotoxic compounds such as ceramides and cholesterol, which subsequently promote ROS generation in hepatocytes.⁴⁰ Over the course of 8 weeks, persistent hepatic inflammation activates Kupffer cells via damage-associated molecular patterns and facilitates immune cell infiltration, which results in the release of pro-inflammatory cytokines such as IL-6, interferon-γ, TNF-α, and CCL2.⁴⁸ By weeks 10 to 12, this chronic inflammatory milieu triggers the expression of fibrogenic mediators, including TGF-β1, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, IL-4, and IL-10, which activate hepatic stellate cells (HSC) and advance hepatic fibrosis.^37,40,49 After 20 weeks of exposure, all mice developed HCC and aberrant activation of the PI3K/Akt/mTOR signaling pathway. This disruption activates downstream effectors such as nuclear factor-κB and the Wnt/β-catenin pathway, thereby promoting tumor cell proliferation, migration, and invasion.^40,50,51 Notably, there has been evidence of a sex-specific discrepancy in the STAM^TM model in mouse models. All male mice developed HCC, whereas female mice did not.³⁷ Female mice only exhibited fatty liver, mild liver inflammation, and decreased phagocytosis of macrophages. In addition, the female mice did not exhibit MASH-based persistent accumulation of macrophages and fibroblasts in the pericentral zones, indicating that the recruitment of fibroblasts around macrophages is the crucial characteristic for the advancement of diabetes-induced HCC.³⁷ Although the STAM^TM model effectively demonstrated HCC progression in patients with diabetes, it still has several limitations. The STAM^TM model is induced using STZ, which results in a near complete loss of pancreatic insulin production.⁴⁵ The model is frequently associated with weight loss and insulin deficiency due to the effects of STZ treatment features not typically observed in human MASH-HCC patients. In human MASH-HCC, obesity and insulin resistance boost de novo lipogenesis. However, reduced insulin secretion in the STAM^TM model may result in insufficient activation of de novo lipogenesis, restricting its ability to accurately replicate the metabolic factors implicated in disease progression.⁴⁷ Despite the limitations, the STAM^TM model demonstrates approximately 100% reproducibility with a well-defined disease progression in a comparatively brief experimental period.³⁷ Given its high reproducibility and ability to replicate the clinicopathological process in male MASH-HCC patients, this model offers valuable insights into the link between metabolic disorders and carcinoma, serving as a platform for developing novel therapeutic strategies.

ALCOHOL-ASSOCIATED HCC MOUSE MODEL

Alcohol is a well-established etiological factor implicated in a broad spectrum of liver disorders, ranging from simple steatosis to liver cancer. Alcohol-associated HCC is caused by a variety of mechanisms, including the dysregulation of alcohol metabolism, induction of CYP2E1, impaired hepatocyte regeneration, and associated nutritional inadequacies.^52,53 Imbalanced hepatic homeostasis promotes lipid peroxidation and structural/functional damage to cellular membranes, nucleic acids, and proteins, which ultimately results in hepatocyte death. Chronic alcohol exposure further amplifies the CYP2E1 expression and enzymatic activity, increasing the accumulation of toxic metabolites and promoting progressive hepatic injury.^54,55 Additionally, ethanol-induced hypoxia and activation of the hypoxia-inducible transcription factors further induce hepatocyte death, escalating profibrotic responses such as HSC activation.⁵⁶ The ongoing hepatocyte death and production of reactive metabolites accelerate genomic instability, accelerating the progression from dysplastic lesions to malignant tumors.⁵⁷ Beyond the direct effects of alcohol, alcoholic liver disease (ALD) development is further impacted by gut barrier dysfunction, which promotes the translocation of endotoxins into the circulation.⁵⁸ The elevated endotoxin level further promotes the activation of HSCs, causing excessive extracellular matrix deposition, fibrosis, cirrhosis, and eventually HCC.⁵⁷

Previous studies have shown that aging is a critical determinant in the experimental models of alcohol exposure. Adolescent mice demonstrate elevated ethanol intake and tolerance, whereas older mice are more vulnerable to ALD, which is attributed to the diminished CYP2E1 enzymatic activity and the downregulation of SIRT1 in both hepatocytes and HSCs.⁵⁹ Therefore, to optimize metabolic responsiveness while minimizing age-related susceptibility, the use of 8- to 11-week-old mice is recommended in rodent models to reduce alcohol-induced mortality.⁵⁷ Additionally, due to differences in alcohol metabolism, hormone regulation, and antioxidant defense, mouse strain and sex differences also exert a substantial impact on the development of alcohol-associated HCC (Table 5).^60-62 Females are typically more susceptible to alcohol-induced hepatotoxicity due to elevated endotoxin levels, potentially driven by estradiol-mediated reductions in lipoprotein levels. However, estrogen also raises hepatic glutathione levels, providing defense against oxidative stress. In contrast, males may experience more rapid alcohol-associated HCC progression because of lower antioxidant capacity, highlighting a complex interplay of hormonal effects. Given the complexity of the many contributing factors, females may experience a more pronounced liver injury due to heightened susceptibility to advanced HCC progression. This complexity makes it especially challenging to make direct comparisons of sex-based vulnerability in ALD-associated HCC.

Based on previous studies, the combined exposure of alcohol and carcinogens synergistically drives liver injury, progressing from inflammation to liver fibrosis and HCC. To investigate alcohol-associated liver carcinogenesis, several mouse models have been developed by integrating ethanol administration with agents such as DEN, CCl₄, or HCV, thereby closely simulating the pathological characteristics of alcohol-associated HCC.^52,63-69 In the DEN+alcohol model, C57BL/6 male mice receive intraperitoneal injections of DEN (75 mg/kg for 3 weeks, followed by 100 mg/kg for another 3 weeks), with a 4% Lieber-DeCarli ethanol diet introduced at week 8. This regimen induces early HCC development as early as week 12, significantly earlier than in DEN-only models.⁶⁸ An alternative protocol employs a single low-dose DEN injection (10 mg/kg) at 2 weeks of age, followed by 4.8% ethanol feeding for 3-7 weeks, leading to visible tumor formation by weeks 19-20.^66,69 In the DEN+CCl₄+alcohol model, mice are sequentially administered DEN (100 mg/kg via intraperitoneal injection, followed by 50 mg/kg via oral administration), CCl₄ (up to 8 mL/kg by oral gavage), and 9% ethanol in drinking water, resulting in progressive induction of hepatitis (by day 60), fibrosis (by day 90), cirrhosis (by day 120), and HCC (by day 150).⁶⁶ In the HCV+alcohol model, transgenic mice expressing the HCV NS5A gene under the apoE promoter are chronically administered a 3.5% ethanol Lieber-DeCarli diet (Table 5).⁶³ This mild but sustained alcohol exposure amplifies NS5A-mediated Toll-like receptor 4 signaling and promotes liver tumorigenesis, with 23% of mice developing HCC within a year. These mouse models effectively provide a valuable platform for elucidating alcohol-associated molecular and cellular mechanisms, especially the interplay of environmental carcinogens, genetic alterations, and alcohol-induced oxidative injury.^63-70

Despite these advantages, several limitations hinder the complete replication of human ALD-associated HCC pathology in these mouse models. Significantly, mice metabolize alcohol seven times faster than humans and tend to lower their intake once acetaldehyde accumulates, making it difficult to establish a mode of chronic alcohol consumption.⁷¹ Additionally, the forced administration of alcohol -often through liquid diets- can induce metabolic alterations unrelated to alcohol exposure.⁵⁷ Disease progression modeling is further constrained by the inability to maintain long-term alcohol consumption.⁷⁰ Importantly, in humans, alcohol consumption is frequently sporadic and modulated by individual dietary patterns and lifestyle, which contrasts with the sustained and standardized alcohol exposure protocols commonly employed in animal models. Furthermore, although these models replicate alcohol-induced liver damage leading to HCC, the underlying genetic mutations and molecular pathways often diverge from those observed in humans, raising concerns about their translational relevance. Variability in alcohol dosing protocols, administration methods, and interstrain genetic differences further complicates reproducibility and undermines experimental reliability (Table 5).

HBT-INDUCED HCC MOUSE MODEL

HBT is a gene delivery technique involving the rapid intravenous administration of a large volume of naked DNA plasmids, typically via the tail vein. The resulting hydrodynamic pressure transiently disrupts the capillary endothelium and induces pore formation in the plasma membranes of adjacent parenchymal cells, thereby facilitating the entry of plasmid DNA into the intracellular compartment. Subsequently, the membrane pores reseal, effectively trapping the plasmid DNA within the cells. The internalized plasmids are subsequently transcribed, resulting in the expression of oncogenes or other functional genes that promote tumorigenesis.¹⁰ Notably, the liver preferentially uptakes the majority of the injected plasmid DNA, leading to effective transfection in approximately 10-40% of hepatocytes. This strategy has been successfully utilized for efficient hepatic gene delivery in mice and has enabled the in depth investigation of liver cancer through the HBT-induced HCC mouse model. Although plasmid DNA is degraded within a few days following hydrodynamic transfection, sustained transgene expression is crucial for effective tumor induction. Using the Sleeping Beauty (SB) transposon method, which promotes stable transgene incorporation into the host genome, long-term gene expression was achieved (Table 6).^10,72 This integration enables persistent oncogene expression, thereby promoting tumor development. The resulting model replicates the key genetic alterations observed in human HCC, offering a robust and biologically relevant platform for investigating HCC pathogenesis and evaluating potential therapeutic interventions. Specifically, DNA plasmids are prepared by recombinant DNA technology by constructing and amplifying plasmids containing the target gene.⁷³ The DNA plasmid solution, corresponding to 8-10% of the mouse’s body weight, is rapidly injected via the tail vein.^10,73 When combined with the plasmid, the SB transposon technology allows for stable integration and sustained expression of the target gene in hepatocytes for extended transgene expression.¹⁰ The onset and progression of liver tumor development vary based on the specific gene introduced. For instance, Fah-deficient mice administered with the corresponding plasmid begin to develop liver nodules approximately 20 weeks following injection, while those receiving a plasmid expressing Myc generally develop liver tumors within 5 to 8 weeks.⁷²

Compared to transgenic mice, the HBT-induced HCC model is highly efficient and relatively simple to establish. The DNA plasmid can be easily reconstructed through the recombinant DNA technique, which takes a shorter period of time. Because of its great reproducibility, this model enables rapid assessment of the experimental findings. One of the primary limitations of the HBT-induced HCC model is the transient expression of non-integrating DNA plasmids. However, this limitation can be addressed by employing the SB transposon system, which facilitates stable genomic integration and prolonged transgene expression. Furthermore, the successful establishment of HCC models using this system requires careful selection of oncogenes or tumor suppressor genes that accurately recapitulate the molecular pathogenesis of HCC.

ORTHOTOPIC HCC IMPLANTATION MOUSE MODEL

The orthotopic HCC implantation mouse model is a widely employed preclinical platform that closely recapitulates the tumor microenvironment characteristic of human liver cancer.⁷⁴ The orthotopic HCC mouse model is established by implanting HCC cells or tumor tissue directly into the liver of mice, enabling tumor growth within its native microenvironment.^74,75 In this model, either HCC cell suspensions or tumor tissue fragments are directly implanted into the liver of mice, enabling tumor growth within the native hepatic architecture. There are two main implantation techniques: cell suspension injection, where cultured HCC cells (HepG2, Huh7, Hepa1-6, or HCA-1) are directly transplanted into the liver parenchyma, and tumor fragment transplantation, which involves surgically embedding pieces of pre-existing tumors into the hepatic tissue. While the former provides a simpler and more rapid approach to induce tumor formation (typically within 1-2 weeks), the latter better preserves tumor heterogeneity and stromal architecture.⁷⁶

The orthotopic mouse model for HCC induction offers critical advantages, including rapid and reproducible tumor formation due to direct hepatic injection of cancer cells, comparatively low technical difficulty, and accurate recapitulation of the human tumor microenvironment (Table 7). Furthermore, a key advantage of the orthotopic model is its ability to reflect the vascular, stromal, and immune components of the hepatic microenvironment, rendering it more physiologically relevant than subcutaneous xenograft models. This model is particularly useful for investigating tumor progression, angiogenesis, immune cell interactions, and metastasis.^75,77,78 However, the model necessitates substantial surgical expertise to ensure precise injection and to prevent tumor cell leakage into the peritoneal cavity, which might result in ectopic tumor growth. Furthermore, tracking tumor progression in vivo can be challenging unless the tumor cells are genetically labeled with bioluminescent or fluorescent reporters. Despite these limitations, the orthotopic HCC model remains a translationally relevant tool for investigating liver cancer biology and evaluating therapeutic strategies in a setting that closely mirrors human disease.

DISCUSSION AND CONCLUSION

Given the ethical and practical limitations on human experimentation, the development of optimal animal models is critical for elucidating the pathophysiology of liver cancer. Mice are widely employed due to the substantial similarity between mouse and human pathogenic pathways, enabling the study of disease progression and the evaluation of therapeutic strategies with high translational relevance. Mouse models produce highly reproducible results, ensuring consistent and reliable data for preclinical research. Moreover, their relatively short lifespan allows for rapid observation of disease progression, thereby reducing the overall duration of experiments. In this review, we compared various mouse models, including those induced by DEN, CCl₄, DEN+CCl₄, STZ+HFD, chronic alcohol feeding, hydrodynamic transfection, as well as orthotopic implantation (Table 8; Fig. 1). Each model presents distinct advantages and offers insights into diverse aspects of HCC pathophysiology. Given the heterogeneity of disease progression and underlying mechanisms, the selection of an appropriate model customized to the specific research objective is essential for yielding relevant and interpretable results.

As no single animal model perfectly recapitulates all human HCC characteristics, meticulous assessment of each model’s strengths and limitations is necessary in both basic and translational research. Ultimately, the strategic selection of HCC mouse models enables the comprehensive exploration of disease mechanisms and therapeutic interventions, thereby advancing the overall understanding of HCC.

Article information

Acknowledgements

The figures were generated using BioRender.com (http://www.biorender.com).

Conflicts of Interest

The authors have no conflicts of interest to disclose.

Ethics Statement

This review article is fully based on articles that have already been published and does not involve additional participants. Therefore, IRB approval is not necessary.

Funding Statement

This work was supported by grants from the National Research Foundation of Korea (NRF) (No. 2021R1A6C101A442 and 2022R1C1C1008912), the Supporting Program of the Korean Association for the Study of the Liver, and the Korean Liver Foundation.

Data Availability

Not available.

Author Contributions

Conceptualization: SHP

Supervision: WS

Writing - first draft: DK, JJ

Writing - review & editing: GYK

Figure 1.

A comprehensive overview of HCC mouse models. Schematic illustration of representative experimental systems used to study HCC pathogenesis and therapeutic strategies. Models include DEN-induced HCC mouse model, CCl4-induced HCC mouse model, combined DEN+CCl4-induced HCC mouse model, MASLD-associated HCC mouse model, alcohol-associated HCC mouse model, HBTinduced HCC mouse model, and orthotopic HCC implantation mouse model (using either HCC cell line suspension or tumor tissue). These systems differ in their etiological relevance, pathological progression, and applicability for mechanistic and preclinical studies. DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; HBT, hydrodynamics-based transfection; CCl4, carbon tetrachloride; MASLD, metabolic dysfunction-associated steatotic liver disease.

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