A method for constructing a BBN-induced orthotopic tumor model of upper urinary tract urothelial carcinoma in mice

Abstract

This invention relates to the field of animal model construction techniques, specifically to a method for constructing a BBN-induced upper urinary tract urothelial carcinoma animal model. This method involves administering BBN (N-butyl-N-(4-hydroxybutyl)nitrosamine) to mice at different stages and concentrations via drinking water to induce in situ tumorigenesis in the upper urinary tract urothelial carcinoma animal model. This technical solution overcomes the limitations of existing technologies where BBN can only induce urothelial carcinoma, primarily bladder cancer. The upper urinary tract urothelial carcinoma animal model constructed using this method allows observation of the entire developmental process of renal pelvis and ureteral cancer from early abnormalities to late-stage carcinogenesis, facilitating the study of mechanisms at different disease stages. This animal model holds promise for further elucidating the mechanisms of upper urinary tract urothelial carcinoma development and screening new therapeutic drugs, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention relates to the technical field of animal model construction methods, specifically to a method for constructing a BBN-induced in situ tumorigenesis model of upper urinary tract urothelial carcinoma in mice. Background Technology

[0002] Upper urinary tract urothelial carcinoma (UTUC) is a subtype of urothelial carcinoma that originates from the malignant transformation of the urothelial mucosa of the upper urinary tract. Here, "upper urinary tract" specifically refers to the entire anatomical region from the renal calyces and pelvis within the kidney to the distal ureter (near the bladder inlet), excluding the urothelial tissue of the bladder and urethra. Essentially, it is a tumor formed by the malignant proliferation of upper urinary tract mucosal cells. While it belongs to the same category of urothelial carcinoma as bladder urothelial carcinoma (the most common type of bladder cancer in the traditional sense), it is considered a separate disease entity due to differences in anatomical location and biological characteristics. The core differences between UTUC and bladder cancer can be explored from five dimensions: epidemiology and risk factors, genomic characteristics, diagnostic challenges, treatment strategies, and prognosis.

[0003] In bladder cancer, BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine) can induce in situ tumorigenesis in mice. This model, due to its high similarity to human bladder cancer in multiple dimensions, has become one of the experimental tools for bladder cancer research. However, due to the lack of animal models, basic research on the disease mechanisms of UTUC lags far behind that of bladder cancer. Although BBN has been shown to specifically induce urothelial tumors only and without causing systemic toxicity in mice, current experimental protocols can only induce urothelial carcinoma, primarily bladder cancer. The successful construction of a drug-induced in situ tumorigenesis model of UTUC will become a powerful experimental tool for conducting basic research on UTUC. Summary of the Invention

[0004] The present invention aims to provide a method for constructing a BBN-induced in situ tumorigenesis model of upper urinary tract urothelial carcinoma in mice, in order to solve the technical problem of low success rate in constructing animal models of in situ tumorigenesis of upper urinary tract urothelial carcinoma in the prior art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for constructing a BBN-induced upper urinary tract urothelial carcinoma animal model, the method comprising the following steps: administering different concentrations of BBN to animals at different time stages to induce the formation of an upper urinary tract urothelial carcinoma animal model.

[0006] Furthermore, the concentration of the BBN includes 0.025~0.05%.

[0007] Furthermore, the time phase includes a first time phase and a second time phase.

[0008] Furthermore, the first phase includes 0-1 month, 0-2 months, 0-3 months, 0-4 months, and 0-5 months.

[0009] Furthermore, the second phase includes 1-5 months, 2-5 months, 3-5 months, and 4-5 months.

[0010] Furthermore, the first phase of 0-1 months corresponds to the second phase of 1-5 months, the first phase of 0-2 months corresponds to the second phase of 2-5 months, the first phase of 0-4 months corresponds to the second phase of 4-5 months, and the first phase of 0-5 months does not have the second phase.

[0011] In this invention, the time stages refer to the different induction and development stages in the process of drug-induced animal disease model formation, based on pathological changes and disease progression within the animal, rather than being limited by specific administration days, weeks, or a single time point. The time stages are defined based on whether the animal exhibits histological, imaging, or biological characteristics consistent with the target disease, including but not limited to: abnormal tissue structure, cellular atypia, tumor formation, or other indicators characterizing disease occurrence. Therefore, the time stages in this invention can be appropriately extended or shortened according to different experimental conditions, individual animal differences, and the degree of induced response. As long as an animal model conforming to the expected disease characteristics is ultimately obtained, it should be considered to fall within the protection scope of this invention.

[0012] In a specific implementation of the present invention, the time period of month 0 represents starting from the beginning, month 1 represents a time period of one full natural month, and so on. The number of days in a natural month includes, but is not limited to, 28 days, 29 days, 30 days, and 31 days.

[0013] Furthermore, the concentration of the BBN is 0.025%-0.05%.

[0014] Furthermore, the BBN concentration in the first stage was 0.05%, and the BBN concentration in the second stage was 0.025%.

[0015] In this invention, the BBN concentration refers to the effective concentration of the drug used to induce a target disease model in animals within the drug administration system. Its value is not limited to a fixed or preset concentration range, but can be dynamically adjusted based on the animal's tolerance, induced response, and disease occurrence. The selection and adjustment of the BBN concentration are based on the principle of successfully inducing the animal to develop the target disease or form the target pathological state, including but not limited to achieving effective induction through increasing or decreasing the drug concentration, extending or shortening the drug administration period, and adjusting the drug administration method. As long as the drug concentration used can induce the animal to exhibit pathological characteristics consistent with the target disease, it is considered to conform to the technical solution of the drug concentration described in this invention, and is not excluded from this invention due to differences in specific values.

[0016] Furthermore, the upper urinary tract urothelial carcinoma includes renal pelvis urothelial carcinoma and ureteral urothelial carcinoma.

[0017] In this invention, the term "upper urinary tract urothelial carcinoma" refers to malignant tumors originating from the urothelial lining of the renal pelvis and ureter, with urothelial carcinoma being the most common histological type. This type of tumor shares similar molecular, morphological, and clinical characteristics with bladder urothelial carcinoma and is an important subtype of malignant tumors of the urinary system.

[0018] In this invention, the term "renal pelvis urothelial carcinoma" refers to a malignant tumor of the urothelial tract that occurs in the renal pelvis. The most common pathological type is urothelial carcinoma, which can manifest as papillary, solid, or invasive growth and is often accompanied by upper urinary tract obstruction or hydronephrosis.

[0019] In this invention, the term "ureteral urothelial carcinoma" refers to a malignant tumor that occurs in the ureteral epithelium (urothelium), often presenting as stenosis, wall thickening, or intraluminal mass, with urothelial carcinoma being the predominant pathological type.

[0020] Furthermore, the upper urinary tract urothelial carcinoma includes tumor-forming upper urinary tract urothelial carcinoma and non-tumor-forming upper urinary tract urothelial carcinoma.

[0021] In a specific embodiment of the present invention, the upper urinary tract urothelial carcinoma is a tumor-forming type of upper urinary tract urothelial carcinoma.

[0022] In this invention, the tumorigenic upper urinary tract urothelial carcinoma refers to UTUC cells or tissues that have the ability to form sustainably proliferating tumor tissue in immunocompetent animals. Typical characteristics include, but are not limited to: expressing a high proliferation index (e.g., high expression of Ki-67), carrying high-level tumor-associated molecules such as TP53, CDKN2A / B, and TERT-P, and exhibiting characteristics of cancer stem cells (e.g., high expression of CD44 and ALDH).

[0023] The non-tumorigenic upper urinary tract urothelial carcinoma refers to UTUC cells or tissues that cannot form tumor tissue in immunocompetent mice and do not have the ability to initiate tumors in vivo. Its typical characteristics include, but are not limited to: mostly low-grade molecular subtypes (such as FGFR3 mutation-driven, TP53 non-mutated), low Ki-67 expression, limited proliferation capacity, high differentiation (strongly positive for Uroplakin I / II / III), and low EMT degree.

[0024] Furthermore, the animals include mice, rats, rabbits, and pigs.

[0025] Furthermore, the rat strains mentioned include, but are not limited to, commonly used laboratory rat strains in this field such as Sprague-Dawley (SD) rats, Wistar rats, Long-Evans rats, and Fischer 344 rats. These strains are characterized by a well-defined genetic background, stable physiological parameters, and a clear response to chemically induced or tumor-related stimuli, and are widely used in research on tumorigenesis mechanisms and the construction of disease models.

[0026] Furthermore, the rabbit breeds mentioned include, but are not limited to, experimental rabbit breeds such as the New Zealand White rabbit, the Japanese White rabbit, and the California rabbit. These breeds have moderate body size and clearly defined organ structures, and are commonly used for modeling urinary system diseases, imaging observation, and interventional procedures.

[0027] Furthermore, the pig breeds mentioned include, but are not limited to, miniature pigs, Göttingen miniature pigs, Yucatan miniature pigs, and Bamaminiature pigs, among other experimental pig breeds. These breeds are highly similar to humans in anatomical structure, physiological function, and metabolic characteristics, making them suitable for translational medicine and large animal tumor model research.

[0028] Furthermore, the animal in question is a mouse.

[0029] Furthermore, the mouse strains include C57BL / 6, BALB / c, FVB / N, and 129 / Sv.

[0030] In a specific embodiment of the present invention, the mouse is a 6-week-old female BALB / c strain mouse.

[0031] In this invention, the animal may also be selected from bovine animals, equine animals, feline animals, canine animals, lagomorph animals, suidae animals, cameloid animals, rodents, and primates, including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, and orangutans), and humans, preferably cattle, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys, and humans.

[0032] Furthermore, the drug administration treatment includes intraperitoneal injection, subcutaneous injection, oral administration, and gavage.

[0033] In a specific embodiment of the present invention, the drug administration treatment is administered via drinking water.

[0034] In this invention, the term "intraperitoneal injection" refers to a method of drug administration in which a drug or suspension is injected into the peritoneal cavity of an experimental animal. This method is commonly used for administering drugs to chemical carcinogens, screening drugs, and in tumor models. It enables rapid drug absorption and is widely applied in mouse tumor research.

[0035] The term "subcutaneous injection" refers to injecting drugs, cells, or reagents into the subcutaneous tissue space between the skin and muscles of an animal. It is often used in experiments such as drug sustained release and subcutaneous transplantation of tumor cells.

[0036] The term "water intake" refers to dissolving a drug in animal drinking water and allowing the experimental animal to ingest the drug through its own drinking water. This method is often used in long-term chronic exposure experiments.

[0037] The term "gavage" refers to an oral administration method that uses a gavage needle to deliver the drug solution directly into the animal's stomach. This method ensures accurate dosage intake and is often used for drugs that are difficult to dissolve in drinking water or for studies that require strict dosage control.

[0038] Furthermore, the BBN can be administered in any of the following forms: solution, suspension, or microparticles.

[0039] In a specific embodiment of the present invention, the BBN is administered in the form of a solution.

[0040] In this invention, the term "solution" refers to a homogeneous, transparent, and stable mixed system in which one or more solutes (such as drugs or chemical reagents) are completely dissolved in a solvent (such as water, physiological saline, or PBS) to form a single-phase liquid system. The solution contains no visible particles, precipitates, or dispersed phases and has the physical characteristics of being optically clear and having a uniform composition.

[0041] The term "suspension" refers to an insoluble solid microparticle (drug, chemical carcinogen, or other powder) dispersed in a liquid medium in the form of microparticles, forming a heterogeneous two-phase system. The particles in the suspension are suspended in the liquid and may gradually settle over time, but can be redispersed after gentle shaking. It is often used for the administration of insoluble drugs, intraperitoneal injection of chemical carcinogens (such as certain poorly soluble small molecules), or experiments that require the preservation of the original particle structure.

[0042] The term "microparticles" refers to discrete small particles, typically ranging in size from about 1 nm to 100 μm, in solid or semi-solid form. These particles can be composed of drugs, polymers, crystals, nanomaterials, or other chemical entities. Microparticles can be dispersed in liquids to form suspensions or used as solid powders. Typical forms used in animal experiments include, but are not limited to: solid powder particles of carcinogens or drugs, polymer-encapsulated drug microparticles (such as PLGA microparticles), crystalline particles of poorly soluble compounds, particulate compounds commonly used in drug induction models, and microparticles that are typically not completely soluble in water, requiring suspension, homogenization, or other dispersion techniques for administration.

[0043] The second aspect of the present invention provides the application of BBN in constructing an animal model of upper urinary tract urothelial carcinoma. The application involves administering BBN at a concentration of 0.025% to animals via drinking water for the first 0-2 months and at a concentration of 0.05% for the next 2-5 months to induce an animal model of in situ tumor formation of upper urinary tract urothelial carcinoma.

[0044] Furthermore, the animal in question is a mammal.

[0045] Furthermore, the mammals include mice, rats, rabbits, and pigs.

[0046] Furthermore, the mammal in question is a mouse.

[0047] Furthermore, the mouse strains include C57BL / 6, BALB / c, FVB / N, and 129 / Sv.

[0048] In a specific embodiment of the present invention, the mouse is a 6-week-old female BALB / c strain mouse.

[0049] Furthermore, the BBN is used for drinking water treatment in the form of a solution.

[0050] In this invention, the term "in situ tumorigenesis" refers to the process by which tumors form in experimental animals at their original anatomical location (in situ) in a target organ or tissue without exogenous tumor cell transplantation or tissue inoculation, induced by drugs, chemicals, or other non-cell injection methods. "In situ" means the tumor originates from the abnormal proliferation and malignant transformation of the animal's own tissue cells, rather than being artificially implanted, and occurs at an anatomical site corresponding to a human disease; "tumorigenesis" refers to a neoplastic lesion that can be clearly identified at the histological, imaging, or molecular level. This type of tumorigenesis process can realistically simulate the natural occurrence, progression, and microenvironmental evolution of human tumors. Compared with cell transplantation models or artificial inoculation models, the in situ tumorigenesis model can more realistically reflect the temporality of tumor development, the tissue microenvironment, and the immune status, and has higher clinical relevance and research value.

[0051] The advantages and beneficial effects of this invention are as follows: 1. This invention constructs a drug-induced in situ tumorigenesis mouse model for upper urinary tract urothelial carcinoma (including renal pelvis carcinoma and ureteral carcinoma), overcoming the technical bottleneck of the lack of existing UTUC animal models. In current clinical and basic research, the incidence of UTUC is significantly lower than that of bladder cancer, resulting in a long-standing lack of reproducible and quantifiable animal models. Most current studies rely on cell line xenograft models or genetically engineered mice, but the former cannot simulate the tumorigenesis process and must be established using immunodeficient animals, failing to reflect the influence of the body's immunity on tumor progression; the latter has a long construction cycle, high cost, and can only reflect tumor progression caused by mutations in some major oncogenes, resulting in a narrow patient coverage. This model uses chemical carcinogens to induce in situ tumorigenesis in the renal pelvis or ureteral epithelium, achieving in situ chemical induction of renal pelvis and ureteral carcinoma for the first time. This provides an animal model with a location consistent with human UTUC, realistically representing the natural evolution of the tumor. Furthermore, using immunocompetent animals without a transgenic background for modeling better reflects the true carcinogenic microenvironment. The patient coverage is also broader.

[0052] 2. By systematically controlling the concentration, duration of administration, and observation period after drug withdrawal, this model can continuously present the complete disease progression chain of UTUC from early mucosal abnormalities, epithelial hyperplasia, focal carcinoma in situ to aggressive high-grade tumors. BBN, as a classic urothelial carcinogen, has had its dose-related carcinogenicity verified by numerous bladder cancer models. This invention utilizes its high affinity for the upper urothelium and successfully reproduces the multi-stage progression pattern of UTUC by precisely controlling the exposure intensity. This model is the first to capture the "temporal dimension" of upper urinary tract tumors, providing a reproducible experimental system for studying tumorigenesis, early driving events, progression mechanisms, and prevention strategies.

[0053] 3. The established model is highly consistent with human UTUC in terms of gross morphology, histopathological features, and expression of immunohistochemical markers, demonstrating good clinical comparability. The induced renal pelvis or ureteral tumors exhibit typical papillary or solid masses, with histological morphology consistent with human UTUC, showing epithelial atypia, layer disorder, and depth of invasion. This fully demonstrates that the model possesses cross-dimensional (macro-micro-molecular) parallelism with clinical UTUC, making it an ideal platform for studying the biological basis of UTUC, drug screening, and treatment response prediction. Attached Figure Description

[0054] Figure 1 From left to right, the figures show the development of renal pelvis cancer in mouse models induced by two-stage sequential BBN for 3, 4, and 5 months. As the induction time increases, the renal pelvis cancer gradually progresses. Figure 2 The images, from left to right, show the development of ureteral cancer in mouse models induced by sequential BBN for 4 and 5 months in two stages. As the induction time increases, the ureteral cancer gradually progresses. Figure 3 In vivo ultrasound findings of renal pelvis cancer in a mouse model induced by two-stage sequential BBN for 4 months; Figure 4 In vivo ultrasound findings of renal pelvis cancer in a mouse model induced by two-stage sequential BBN for 5 months; Figure 5 HE and histochemical staining results of renal pelvic carcinoma tissue in a mouse model induced by two-stage sequential BBN for 3 months; Figure 6 HE and histochemical staining results of renal pelvic carcinoma tissue in a mouse model induced by two-stage sequential BBN for 4 months; Figure 7 HE and histochemical staining results of renal pelvic carcinoma tissue in a mouse model induced by two-stage sequential BBN for 5 months; Figure 8 HE and histochemical staining results of ureteral carcinoma tissue in a mouse model induced by two-stage sequential BBN for 4 months. The red arrows show that UTUC has broken through the muscle layer and infiltrated the growth. Figure 9 HE and histochemical staining results of ureteral cancer tissue in a mouse model induced by two-stage sequential BBN for 5 months. Detailed Implementation

[0055] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0056] Example 1: Drug Induction and Animal Modeling 1.1 Preparation and administration of BBN solution A 0.05% BBN solution is prepared from a 90% BBN stock solution (e.g., add 277.7 μL of 90% BBN stock solution to 500 mL of tap water and mix thoroughly). A 0.025% BBN solution is prepared by diluting a 0.05% BBN solution by one-fold.

[0057] A 0.05% BBN solution was provided as drinking water for mice, allowing them free access to the water; a light-proof water bottle was required (to prevent BBN decomposition). The BBN (N-butyl-N-(4-hydroxybutyl) nitrosamine) used in the experiment was from Selleck, catalog number E0199.

[0058] 1.2 Selection and Grouping of Experimental Animals Female BALB / c mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were used, and drug induction began at 6 weeks of age. Mice were divided into an experimental group (receiving BBN treatment, further subdivided into six subgroups based on the halving of the drug dose: 0 months, 1 month, 2 months, 3 months, 4 months, and 5 months; where 0 months represented the initial BBN induction dose halved to 0.025%, 1 month represented the first month's BBN induction dose being 0.05%, and so on until the end of the 5th month) and a control group (only drinking tap water, with the same treatment duration as the experimental group). Both groups were housed in a constant temperature (23±2℃), constant humidity (50-60%), 12-hour light-dark cycle, and free-range standard feed environment to ensure stable physiological conditions.

[0059] 1.3 Animal monitoring and sample processing Mice weight was recorded weekly, and their fur condition, activity level, and response to external stimuli were observed. The presence of upper urinary tract tumors (UTUCs) was confirmed by in vivo ultrasound examination. Mice were sacrificed every month, with all mice sacrificed at the 5-month experimental endpoint. Renal pelvis and ureter were harvested for H&E staining and immunohistochemical staining. Pathological examination confirmed the occurrence of UTUCs. Pathological results are as follows: Figure 1 , Figure 2As shown in Table 1, the tumorigenesis rates of different experimental groups are summarized. It can be seen that after inducing with 0.05% BBN for 2 months, the dosage was changed to 0.025% and induced for another 3 months, resulting in the highest total UTUC tumorigenesis rate, a lower non-tumor mortality rate in mice, and the highest ureteral carcinoma tumorigenesis rate.

[0060] Table 1 Summary of non-tumor mortality and tumor formation rates of various types in different experimental groups

[0061] Example 2: In vivo ultrasound detection of tumor development The ultrasound instrument used in the experiment was a VisualSonics Vevo 2100 high-resolution small animal ultrasound imaging system (probe model: MS-55OD, frequency 21-55MHz, suitable for mice). The results are as follows: Figure 3 , Figure 4 As shown, in mice induced by BBN for 4 and 5 months, in vivo ultrasound showed that the tumors gradually progressed with the extension of induction time.

[0062] Example 3: Pathological staining and histochemical staining HE and histochemical staining were performed on renal pelvis carcinoma and ureter carcinoma tissues from mice at different induction stages. The relevant experimental materials are shown in Table 1. The staining results are as follows: Figures 5-9 As shown, combined with the in vivo ultrasound results in Example 2, HE and Ki-67 staining indicate that renal pelvis carcinoma occurs in the urothelial tract and gradually progresses with increasing induction time. HE and Ki-67 staining also show that ureteral carcinoma further progresses, infiltrating the entire muscle layer and surrounding the ureter. The BBN-induced in situ tumorigenesis model of upper urinary tract urothelial carcinoma in mice constructed in this invention can stably produce tumorigenesis of renal pelvis and ureteral carcinoma at different induction stages, and the tumor development significantly increases with increasing induction time. The gross morphology, histopathology, and histochemical staining results of the constructed tumor model all support a high degree of similarity between this model and clinical patient UTUC in multiple dimensions. All the above examples demonstrate that by modifying the BBN-induced in situ tumorigenesis model of bladder cancer and changing parameters such as mouse strain, sex, drug induction dose, and drug induction time, an in situ tumorigenesis mouse model of UTUC can be constructed.

[0063] Table 2. Experimental materials related to pathological staining and histochemical staining

[0064] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a BBN-induced upper urinary tract urothelial carcinoma animal model, characterized in that, The construction method includes the following steps: administering BBN at a concentration of 0.05% to animals for 0-2 months and at a concentration of 0.025% to animals for 2-5 months to induce an animal model of upper urinary tract urothelial carcinoma; the animals are mice, and the administration is performed by ingesting the drug in solution form through drinking water; the upper urinary tract urothelial carcinoma includes both renal pelvis urothelial carcinoma and ureteral urothelial carcinoma, wherein the tumorigenicity rate of renal pelvis urothelial carcinoma is 66%, the tumorigenicity rate of ureteral urothelial carcinoma is 22%, and the total tumorigenicity rate of upper urinary tract urothelial carcinoma is 88%.

2. The method according to claim 1, characterized in that, The upper urinary tract urothelial carcinoma includes tumor-forming upper urinary tract urothelial carcinoma and non-tumor-forming upper urinary tract urothelial carcinoma.

3. The method according to claim 1, wherein the upper urinary tract urothelial carcinoma is a tumor-forming type of upper urinary tract urothelial carcinoma.

4. The method according to claim 1, characterized in that, The animals were 6-week-old female BALB / c mice.

5. The application of BBN in constructing an animal model of upper urinary tract urothelial carcinoma, characterized in that, Animals were treated with BBN at a concentration of 0.05% via drinking water for the first 0-2 months, and at a concentration of 0.025% via drinking water for the following 2-5 months to induce an upper urinary tract urothelial carcinoma animal model. The animals were mice. The upper urinary tract urothelial carcinoma included both renal pelvis urothelial carcinoma and ureteral urothelial carcinoma, with a tumorigenicity rate of 66% for renal pelvis urothelial carcinoma, 22% for ureteral urothelial carcinoma, and an overall tumorigenicity rate of 88% for upper urinary tract urothelial carcinoma.

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