Bladder cancer (BC) mainly originates from the bladder urothelium. Animal models are used to study tumor initiation, invasion and metastasis, the tumor microenvironment, and therapeutic responses. Different models vary markedly in carcinogenic mechanism, immune background, tumor formation speed, and clinical translational value. Therefore, appropriate models should be selected according to the research objective.

Keywords: bladder cancer; urothelial carcinoma; animal model; BBN-induced model; orthotopic xenograft; xenograft model; genetically engineered model; tumor microenvironment

1 Research Positioning of Bladder Cancer Animal Models

1.1 Experimental Significance of Bladder Cancer Models

Bladder cancer animal models are used to simulate different stages of human bladder tumor development, from initiation and proliferation to invasion and metastasis. Compared with in vitro cell models, animal models can preserve bladder anatomy, urine stimulation, local immune responses, stromal cell participation, and angiogenesis. Therefore, they are more suitable for evaluating the tumor microenvironment, orthotopic growth, invasive progression, and therapeutic response.

Common research objectives include:

(1) Pathogenesis research

These models are used to analyze carcinogen exposure, gene mutations, inactivation of tumor suppressor pathways, activation of receptor tyrosine kinase signaling, and epigenetic abnormalities.

(2) Tumor microenvironment research

These models are used to observe immune cell infiltration, fibroblast activation, angiogenesis, inflammatory factor release, and immune escape.

(3) Pharmacodynamic evaluation

These models are used to evaluate chemotherapeutic drugs, intravesical therapy, immune checkpoint inhibitors, FGFR inhibitors, PI3K/AKT/mTOR pathway inhibitors, and combination treatment strategies.

(4) Molecular subtype and drug resistance research

These models are used to analyze luminal subtype, basal subtype, muscle-invasive bladder cancer, cisplatin resistance, immunotherapy response, and sensitivity to targeted therapy.

(5) Metastasis and recurrence research

These models are used to establish lung metastasis, liver metastasis, lymphatic metastasis, and orthotopic recurrence-related models.

1.2 Model Classification

Table 1 Key Points for Selecting Common Bladder Cancer Animal Models

2 Chemically Induced Bladder Cancer Models

2.1 BBN-Induced Model

The N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced model is a widely used chemical induction model in bladder cancer research. BBN can induce urothelial carcinoma through long-term drinking water exposure. The model usually preserves an immunocompetent background and is suitable for studying tumor initiation, inflammatory responses, immune escape, and therapeutic intervention.

(1) Modeling method

A common method is to dissolve BBN in drinking water at a concentration of approximately 0.05% and administer it continuously for 12–20 weeks. The modeling period, tumor incidence, and lesion severity are affected by animal strain, sex, age, BBN concentration, water intake, and observation endpoint. C57BL/6 mice are commonly used for immunotherapy and syngeneic tumor background studies.

(2) Pathological characteristics

The BBN model can induce urothelial hyperplasia, dysplasia, carcinoma in situ, papillary tumors, and invasive urothelial carcinoma. A longer induction period can increase the proportion of muscle-invasive lesions, but it also increases animal burden and individual variability.

(3) Mechanistic pathways

BBN metabolites can cause DNA damage and mutation accumulation, often involving HRAS/MAPK/ERK, p53, Nrf2/KEAP1, PI3K/AKT/mTOR, and immune checkpoint-related pathways. During tumor progression, PD-L1 upregulation, CD8+ T-cell functional suppression, myeloid cell infiltration, and increased inflammatory factors may occur.

(4) Applicable scenarios

The BBN model is suitable for studying the full process of bladder cancer development, carcinogen exposure-related mechanisms, the immune microenvironment, immune checkpoint inhibitors, chemotherapy combined with immunotherapy, and preventive interventions.

2.2 MNU-Induced Model

N-methyl-N-nitrosourea (MNU) is commonly administered by intravesical instillation to act directly on the urothelium and induce urothelial carcinoma. Compared with the BBN drinking water model, the MNU model provides more direct local exposure and is suitable for studying intravesical carcinogenesis and local urothelial lesions.

(1) Modeling method

MNU can be administered through transurethral intravesical instillation. A reference protocol is once weekly for 6–8 weeks. The specific dose and instillation volume should be optimized according to animal species, body weight, anesthesia method, and bladder capacity. Before instillation, the bladder is often emptied, and after instillation, the solution is retained for a certain period to increase contact with the urothelium.

(2) Mechanistic pathways

MNU has alkylating activity and can induce DNA damage, mutation accumulation, and cell cycle abnormalities. Enhanced EGFR phosphorylation, activation of the PI3K/AKT/mTOR pathway, abnormal p53 pathway activity, and increased proliferation marker Ki-67 are commonly used to evaluate tumor biological changes.

(3) Applicable scenarios

The MNU model is suitable for studying local bladder carcinogenesis, early urothelial carcinogenesis, and preventive or therapeutic intravesical strategies. However, the model depends on intravesical instillation technique, and repeated instillation may also cause mechanical inflammation as an interfering factor.

2.3 MNNG-Induced Model

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) can be used for local intravesical induction of bladder tumors. This model is mainly used to study chemical carcinogenesis and local epithelial carcinogenic processes. Under some conditions, it can induce squamous differentiation or squamous cell carcinoma-like lesions.

(1) Modeling characteristics

MNNG is a highly carcinogenic alkylating agent, and experimental operation requires strict control of concentration, exposure time, and safety protection. Model reproducibility is affected by instillation conditions, degree of urothelial injury, and baseline animal status.

(2) Mechanistic pathways

MNNG mainly induces mutation and abnormal proliferation through DNA alkylation damage, often accompanied by abnormal p53 pathway activity, cell cycle dysregulation, EGFR-related signaling activation, and inflammatory responses.

(3) Applicable scenarios

The MNNG model is suitable for studying mechanisms of local chemical carcinogenesis. However, because of its operational risk and high model variability, stricter safety management and preliminary experimental validation are usually required.

Table 2 Comparison of Chemically Induced Bladder Cancer Models

3 Transplantable Bladder Cancer Models

3.1 Ectopic Cell Line-Derived Transplantation Model

Cell line-derived xenograft (CDX) models commonly inoculate bladder cancer cells subcutaneously into immunodeficient mice and are used to rapidly observe tumor growth and drug-mediated tumor inhibition. Common human bladder cancer cell lines include T24, 5637, J82, and UM-UC-3. The mouse syngeneic model commonly uses MB49 cells and can be applied under an immunocompetent mouse background.

(1) Modeling method

Subcutaneous inoculation usually involves mixing a defined number of tumor cells with extracellular matrix gel or buffer and injecting the mixture into the dorsal or axillary region of mice. Human-derived cells are mostly inoculated into nude mice, NOD/SCID mice, or NSG mice. Mouse MB49 cells can be inoculated into C57BL/6 mice. After tumor formation, animals can be randomized according to tumor volume and then treated with drug interventions.

(2) Model characteristics

Subcutaneous models show rapid tumor formation, simple operation, and easy tumor volume measurement, making them suitable for preliminary drug screening and mechanism validation. However, they lack the orthotopic urinary environment, urothelial barrier, local bladder immunity, and tissue-specific stromal responses, and therefore cannot fully represent intravesical tumor behavior.

(3) Key pathways

Different cell lines have different molecular backgrounds. T24 is commonly used for HRAS mutation and MAPK pathway research; 5637 is commonly used for FGFR3-related research; J82 is commonly used for p53 abnormalities and invasive phenotype research; UM-UC-3 is commonly used for migration, invasion, and EMT research.

3.2 Orthotopic Transplantation Tumor Model

The orthotopic transplantation tumor model inoculates bladder cancer cells into the bladder lumen or bladder wall, thereby better simulating tumor adhesion, growth, invasion, and therapeutic response in the local bladder mucosal environment.

(1) Modeling method

Common methods include transurethral intravesical injection of tumor cells, bladder wall injection of tumor cells, or inoculation after bladder mucosa pretreatment. Before intravesical inoculation, mild chemical or mechanical treatment of the bladder mucosa can be used to promote cell adhesion. The MB49 orthotopic model is commonly used for immunotherapy research under an immunocompetent mouse background.

(2) Model characteristics

The orthotopic model can reflect interactions among tumors, bladder mucosa, urine, local immune cells, and stromal cells. It is suitable for evaluating intravesical therapy, immunotherapy, local invasion, and the tumor microenvironment. Its limitations include higher technical difficulty and reliance on in vivo imaging, ultrasound, MRI, or pathological endpoints for tumor burden measurement.

(3) Mechanistic pathways

Orthotopic models are commonly used to study the CXCL12/CXCR4 axis, MMP-2/MMP-9, VEGF, EMT markers, and immune checkpoint molecules. Interactions between tumor cells and stromal cells can promote upregulation of Vimentin, Snail, and Twist1 and downregulation of E-cadherin, indicating enhanced invasive and migratory capacity.

3.3 Patient-Derived Xenograft Model

Patient-derived xenograft (PDX) models are established by implanting patient bladder cancer tissues into immunodeficient mice. Compared with cell line-derived transplantation models, PDX models better preserve tumor tissue structure, molecular heterogeneity, and drug response characteristics.

(1) Modeling method

Fresh tumor tissue can be cut into small fragments and implanted subcutaneously, under the renal capsule, or at orthotopic sites in immunodeficient mice. NSG, NOG, or NOD/SCID mice are commonly used to improve tumor engraftment rate. After establishment, serial passaging, histopathological comparison, molecular subtyping, and drug sensitivity testing can be performed.

(2) Model characteristics

PDX models are suitable for individualized drug screening, validation of targeted therapy, analysis of drug resistance mechanisms, and molecular subtype research. Their main limitations include long modeling cycles, high cost, strong dependence of engraftment rate on sample quality, and the immunodeficient background, which limits direct evaluation of complete immunotherapeutic responses.

(3) Clinical translational value

PDX models can be used to predict sensitivity of patient-derived tumors to cisplatin, gemcitabine, FGFR inhibitors, immunotherapy combination strategies, and novel targeted drugs. PDX models have high application value for studies of FGFR3 alterations, luminal/basal subtypes, and cisplatin resistance.

4 Genetically Engineered Bladder Cancer Models

4.1 UPII-SV40T Transgenic Model

The UPII-SV40T model drives expression of SV40 large T antigen through a urothelium-specific promoter, inactivating the p53 and Rb pathways and thereby inducing high-grade urothelial lesions and bladder cancer. This model is suitable for studying tumorigenesis related to p53/Rb inactivation and progression of high-grade bladder cancer.

(1) Mechanistic characteristics

SV40 large T antigen can bind p53 and Rb, causing failure of cell cycle surveillance, DNA damage accumulation, and genomic instability. Increased Ki-67, abnormal p53/Rb axis activity, and high-grade urothelial carcinoma-like lesions are commonly observed in this model.

(2) Applicable research

This model is suitable for studying high-grade urothelial carcinoma, cell cycle dysregulation, genomic instability, and cisplatin resistance. When combined with drug intervention or immune background analysis, it can be used to evaluate DNA damage repair and chemotherapy response.

4.2 p53/Pten Conditional Knockout Model

Conditional knockout models such as Upk2-Cre;p53^fl/fl;Pten^fl/fl can specifically inactivate p53 and Pten in the urothelium, simulating loss of tumor suppressor pathways and enhancement of PI3K/AKT signaling.

(1) Mechanistic characteristics

p53 deletion weakens DNA damage responses and cell cycle checkpoints. Pten deletion activates the PI3K/AKT/mTOR pathway, promoting cell proliferation, survival, and invasion. Dual alterations more readily drive the formation of muscle-invasive bladder cancer.

(2) Applicable research

These models are suitable for studying muscle-invasive bladder cancer, the PI3K/AKT/mTOR pathway, cooperative inactivation of tumor suppressor genes, and targeted therapy.

4.3 Other Genetically Engineered Models

Common molecular alterations in bladder cancer also include FGFR3 abnormalities, STAG2 mutation, KDM6A mutation, ERBB2 activation, Wnt/β-catenin pathway abnormalities, and PPARγ-related pathway changes. Through CRISPR-Cas9, conditional knockout, or transgenic expression, bladder cancer models closer to specific molecular subtypes can be constructed.

Table 3 Common Pathways and Applications of Genetically Engineered Models

5 Metastasis Models and Tumor Microenvironment Models

5.1 Metastasis Models

Bladder cancer metastasis models are used to study tumor cell detachment from the orthotopic site, entry into circulation, distant organ colonization, and metastatic lesion formation. Common methods include tail vein injection, intracardiac injection, splenic injection, and screening of highly metastatic cells from orthotopic models.

(1) Tail vein injection model

Tail vein injection is commonly used to establish lung metastasis models and is suitable for evaluating hematogenous dissemination, lung colonization, and anti-metastatic drug efficacy. This model bypasses orthotopic invasion and intravasation and is more suitable for studying metastatic colonization after tumor cells enter circulation.

(2) Orthotopic metastasis model

When local invasion and distant metastasis occur after orthotopic inoculation, the model more closely resembles clinical metastasis. It can be used to study EMT, matrix remodeling, angiogenesis, and immune escape. However, the modeling cycle is long, and the incidence and sites of metastasis are affected by cell line and animal condition.

(3) Key indicators

Commonly detected indicators include MMP-2, MMP-9, VEGF, CXCR4, CXCL12, Snail, Twist1, Vimentin, E-cadherin, and Ki-67. MMPs and VEGF can reflect matrix degradation and angiogenesis, while EMT markers can indicate enhanced invasive and migratory capacity.

5.2 Tumor Microenvironment Models

The bladder cancer tumor microenvironment includes tumor cells, immune cells, fibroblasts, endothelial cells, extracellular matrix, and inflammatory factors. Immunocompetent models, orthotopic models, BBN models, and humanized mouse models are more suitable for studying the tumor microenvironment.

(1) Immune cells

CD8+ T cells, Treg cells, macrophages, myeloid-derived suppressor cells, and dendritic cells can all affect antitumor immune responses. PD-1/PD-L1, CTLA-4, IFN-γ, Granzyme B, and CD206 are commonly used to evaluate immune status.

(2) Fibroblasts and stroma

Cancer-associated fibroblasts can promote tumor invasion through TGF-β, CXCL12, IL-6, and extracellular matrix remodeling. α-SMA, Fibronectin, Collagen I, and Vimentin can be used to evaluate stromal activation.

(3) Angiogenesis

VEGF, CD31, HIF-1α, and MMP-9 are commonly used to evaluate hypoxia, angiogenesis, and invasion-related processes.

6 Model Selection According to Different Research Objectives

6.1 Research on Bladder Cancer Initiation Mechanisms

BBN, MNU, or genetically engineered models should be prioritized. If carcinogen exposure and multi-stage carcinogenesis are the focus, the BBN model is more suitable. If local urothelial carcinogenesis is the focus, MNU or MNNG intravesical instillation models are more direct. If specific driver genes are the focus, GEM models should be selected.

6.2 Drug Screening and Tumor Proliferation Research

Subcutaneous CDX models should be prioritized. These models show rapid tumor formation, simple operation, and convenient tumor volume measurement, making them suitable for preliminary screening of antitumor drugs. However, further validation using orthotopic models, PDX models, or immunocompetent models is required.

6.3 Intravesical Therapy Research

Orthotopic transplantation tumor models or chemically induced orthotopic models should be prioritized. These models preserve the intravesical drug delivery environment and can be used to evaluate drug retention in the bladder, local antitumor effects, and urothelial toxicity.

6.4 Immunotherapy Research

Immunocompetent background models, such as BBN-induced models or MB49 syngeneic orthotopic models, should be prioritized. If human immune therapy antibodies, CAR-T therapy, or strategies involving human immune cells are being evaluated, humanized mouse models may be considered.

6.5 Targeted Therapy and Individualized Therapy Research

For studies related to FGFR3 abnormalities, PI3K/AKT activation, p53/Rb inactivation, or specific molecular subtypes, corresponding cell lines, PDX models, or genetically engineered models can be selected. PDX models are more suitable for drug sensitivity prediction and drug resistance mechanism analysis.

Table 4 Selection of Bladder Cancer Animal Models According to Different Research Objectives

Table 5 Products Related to Bladder Cancer Animal Model Evaluation and Mechanistic Research

Bladder cancer animal models should be selected according to research objective, immune background, tumor location, molecular characteristics, and evaluation endpoint. Chemically induced, orthotopic transplantation, ectopic transplantation, PDX, and genetically engineered models each have applicable boundaries. Rational combination of multiple models helps improve the reliability of mechanistic research and drug translation.

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[4] Methods for Establishing Animal Models of Cardiovascular Diseases

[5] Methods for Establishing Models of Nervous System Diseases

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