Kit for constructing a tumor-bearing atherosclerotic mouse model

Abstract

The application discloses a kit for constructing a tumor-bearing atherosclerotic mouse model, and at least comprises MC38 mouse colon cancer cells, a Matrigel and high-fat feed / high-cholesterol feed. The kit can be used to construct a tumor-bearing atherosclerotic mouse model which is in line with typical clinical features of tumor combined with atherosclerotic cardiovascular diseases, and has the advantages of simple operation, time saving, high success rate and low production cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of experimental medicine, and particularly relates to a kit for constructing a tumor-bearing atherosclerotic mouse model and use thereof. BACKGROUND

[0002] In the past 20 years, a number of innovative treatment methods have emerged in the field of malignant tumor treatment, and these treatment methods have also brought the risk of cardiovascular toxicity, including decreased cardiac function, hypertension, thrombosis or embolism, and other adverse reactions. The 2022 European Society of Cardiology Guidelines for Oncocardiology first proposed the definition of cancer therapy-related cardiovascular toxicity (CTR-CVT), and provided systematic guidance and recommendations for the diagnosis, treatment and prevention of CTR-CVT and management, which is a landmark event in international oncocardiology. In addition, with the continuous prolongation of the survival period of tumor patients, the influence of anti-tumor treatment on some chronic cardiovascular diseases has also gradually been paid attention to.

[0003] In recent years, with the continuous improvement of living conditions and the aging process of the population in China, atherosclerotic cardiovascular disease (ASCVD), including ischemic heart disease and ischemic stroke, has gradually become the most common cause of death. Previous studies have shown that 5-FU and capecitabine can induce exertional angina. Ischemia caused by platinum-containing chemotherapy usually occurs after one of the first three cycles and occurs in patients with potential coronary heart disease risk. Anti-microtubule drugs and small molecule targeted drugs have a chance to cause cardiac ischemia, and recent clinical studies have confirmed that patients treated with ICIs for two years have a three-fold higher risk of cardiovascular events than other therapies. As two major killers of human health, patients with tumors combined with ASCVD should pay more attention to the assessment and prognosis of ASCVD disease progression during anti-tumor treatment.

[0004] Due to the slow course of atherosclerosis, ApoE - / - or Ldlr - / - mice were commonly used in the past, and were fed with high-fat diet for more than 3 months to form atherosclerotic mouse model. However, for tumor-bearing mouse models, subcutaneous injection of tumor cells grows faster, and the tumor volume will reach the ethical limit, i.e. 2000mm 3 , within 1 month. Therefore, it is urgent to construct a tumor-bearing atherosclerotic animal model for evaluating the influence of anti-tumor treatment on the progression of atherosclerosis and exploring the mechanism. SUMMARY

[0005] In order to overcome the above-mentioned deficiencies of the prior art tumor-bearing atherosclerotic animal model, through repeated experiments, the present application provides a new method for constructing a tumor-bearing atherosclerotic mouse model, which significantly improves the success rate of modeling, and the constructed mouse model has clinical feature representation and greatly saves research cost. Specifically, the present application comprises the following technical solutions.

[0006] A kit for constructing a tumor-bearing atherosclerotic mouse model (TB-AS model) comprises at least the following components: MC38 intestinal cancer cells for subcutaneous tumor formation in mice, i.e. MC38 mouse colon cancer cells, matrigel for use in combination with MC38 intestinal cancer cells, and high-fat feed / high-cholesterol feed (high-fat diet) for constructing an atherosclerotic mouse model.

[0007] Further, the above-mentioned kit further comprises biochemical reagents, including 1640 culture medium for culturing and preserving MC38 mouse colon cancer cells, FBS (fetal bovine serum), P / S (penicillin / streptomycin mixed solution, i.e. P / S double antibody), PBS (balanced salt solution PBS, phosphate balanced physiological saline), HE staining reagent, MASSON staining reagent and / or Oil O Red staining reagent.

[0008] Still further, the above-mentioned kit further comprises experimental equipment, including cell culture bottles, centrifuge tubes, and surgical instruments.

[0009] In one embodiment, the above-mentioned kit is implemented for ApoE - / - mice or Ldlr - / - mice, preferably ApoE - / - mice.

[0010] Preferably, the above-mentioned ApoE - / - mice are ApoE - / - mice of C57BL / 6J background.

[0011] Alternatively, the above-mentioned high-fat feed / high-cholesterol feed is D12108C (fat energy supply ratio of 40%, containing 1.25% cholesterol) purchased from Research Diets, which is referred to herein as "high-fat diet".

[0012] The above-mentioned matrigel is commonly used in animal model construction and is prepared from basement membrane components extracted from mouse tumor tissues, and contains laminin, type IV collagen, heparin sulfate glycoprotein, etc., and can be selected from the following group: ① Corning Corporation, item number 356234 / 354234; ② Biyun Tian Co., Ltd., item number C0386 / C0387.

[0013] In one embodiment, when using the above-mentioned kit to construct a tumor-bearing atherosclerotic mouse model, first, the mice are fed with high-fat / high-cholesterol feed to induce the establishment of an atherosclerotic model, i.e., after being fed for 3-6 weeks, preferably 4-5 weeks, for example, 4 weeks, when there is a tendency of lipid metabolism disorder and atherosclerosis in the mice, the MC38 intestinal cancer cells are mixed with Matrigel at a volume ratio of 1:0.3-2.0, preferably 1:0.5-1.8, preferably 1:0.8-1.5, preferably 1:0.9-1.2, more preferably, for example, about 1:1, and then subcutaneously injected into the mice, and the mice are continued to be fed with high-fat / high-cholesterol feed for 6-10 weeks, preferably 7-9 weeks, for example, about 8 weeks, until a successful TB-AS model is constructed.

[0014] It should be understood that, when expressing numerical characteristics herein, the term "about" or "approximately" means that the indicated number can have an error range or floating range of ±10%, ±8%, ±6%, ±4%, or ±2%.

[0015] Wherein, compared with subcutaneously injecting MC38 intestinal cancer cells alone, the advantage of using MC38 intestinal cancer cells mixed with Matrigel is that Matrigel is prepared from basement membrane components extracted from mouse tumor tissue, and contains main components such as laminin, collagen IV, heparin sulfate glycoprotein, etc. These components can provide support and signals required for cell adhesion, differentiation and proliferation, and at the same time can simulate the characteristics of basement membrane in the physiological environment, reduce the number of tumor cell implantation, and improve the success rate of tumor implantation. The tumor volume of the mice after being implanted with tumor (subcutaneously injected with MC38 intestinal cancer cells) for 8 weeks is controlled within 2000mm 3 , which can make the finally constructed TB-AS model meet the typical clinical characteristics of tumor patients combined with atherosclerotic cardiovascular disease (ASCVD), and avoid the problem that the tumor cells proliferate and grow too fast after being injected with tumor cells MC38 cells alone, and the tumor volume reaches the ethical limit of 2000mm 3 too early, which leads to not meeting the typical clinical characteristics of tumor patients combined with atherosclerotic cardiovascular disease, i.e., not having representativeness and losing clinical research value.

[0016] As a specific use mode of the above-mentioned kit, the injection amount of the MC38 intestinal cancer cells is 1×10 4-6 cells per mouse, for example, about 1×10 5 cells per mouse.

[0017] Preferably, the tumor volume of the tumor-bearing atherosclerotic mouse model constructed using the above-mentioned kit is controlled within 2000mm 3 .

[0018] Compared with the prior art mouse model construction method, by selecting the MC38 mouse intestinal cancer cells with slow tumor growth cycle when the mouse is tumor-bearing, and reducing the number of initial planting cells (the number of initial planting cells in the prior art is generally 5x10 5-7 cells per mouse) when subcutaneous injection, the tumor volume of the mouse after 8 weeks of tumor-bearing can be controlled within 2000mm 3 .

[0019] Alternatively, the tumor-bearing atherosclerosis mouse model (TB-AS model) constructed using the above-mentioned kit meets the typical clinical characteristics of tumor-merging atherosclerotic cardiovascular disease (ASCVD) patients, and therefore can be used for anti-tumor drug screening, anti-tumor drug development and clinical treatment research such as PD-1 antibody treatment research, accordingly, in this case, the kit also includes an anti-tumor drug such as a PD-1 antibody.

[0020] The present application provides a novel tumor-bearing atherosclerosis mouse model construction tool and method, which can be used to construct a tumor-bearing atherosclerosis mouse model that meets the typical clinical characteristics of tumor-merging atherosclerotic cardiovascular disease, not only improves the experimental work efficiency, but also increases the success rate, can greatly reduce the production cost of the tumor-bearing atherosclerosis mouse model, and has popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a flowchart of the present application for constructing a tumor-bearing atherosclerosis mouse model.

[0022] Figure 2 The in vivo tumor growth of the TB-AS model non-treatment group (TB-AS) mice and the model PD-1 antibody aPD-1 treatment group mice (TB-AS+ aPD-1) is shown.

[0023] Figure 3 The typical images of H&E staining, Oil O Red staining and Masson staining of the aortic root plaque of the TB-AS group mice and the aPD-1 treatment group mice (TB-AS+ aPD-1) are shown (scale = 250 μm).

[0024] Figure 4 The aortic root plaque area and collagen deposition statistics of the TB-AS group mice and the aPD-1 treatment group mice (TB-AS+ aPD-1) are shown (*** represents p<0.001). Among them, collagen deposition represents the formation of atherosclerotic fibrous plaque, collagen fibers wrap around the necrotic core to form fibrous plaque, the more fibrous collagen in the plaque, the thicker the fibrous cap, and the more stable the plaque. DETAILED DESCRIPTION

[0025] The tumor-bearing atherosclerotic mice (TB-AS mice) model with typical clinical features of tumor combined with atherosclerotic cardiovascular disease (ASCVD) can be used to relatively accurately evaluate the in vivo experiment of the influence of anti-tumor treatment, such as PD-1 treatment, on the progression of atherosclerosis, and is the basis of the model for the development of biomarkers of the disease, the study of pathogenesis, the screening of anti-tumor drugs, the evaluation of efficacy, and the adverse reactions of atherosclerosis.

[0026] Due to the slow course of atherosclerosis, ApoE - / - or Ldlr - / - mice are generally fed with high-fat diet for more than 3 months to form atherosclerotic mouse model. However, for tumor-bearing mouse model, the tumor cells grow faster after subcutaneous injection, and the tumor volume often reaches the ethical limit, i.e. 2000mm 3 , within 1 month.

[0027] Under this contradiction, the research group tried a new modeling method, determined a reasonable tumor implantation method, tumor implantation time and tumor implantation dose, mixed MC38 intestinal cancer cells with matrigel at a specific ratio, and found that matrigel could effectively prevent MC38 intestinal cancer cells from proliferating too fast, thereby limiting the tumor volume in the mouse model to the expected range, avoiding the tumor volume reaching the ethical limit of 2000mm 3 within 1 month after tumor implantation, resulting in the loss of clinical model value of the constructed mouse due to not meeting the typical clinical features of tumor combined with ASCVD patients. Through multiple experiments, the technical scheme is determined as follows: on the one hand, the ApoE - / - mice are implanted with tumors at the time point when there is lipid metabolism disorder and atherosclerosis tendency after being fed with high-fat diet for 4 weeks, and the final condition meets the clinical features of tumor combined with ASCVD patients; on the other hand, the MC38 intestinal cancer cells with slow tumor growth cycle are selected when implanting tumors, and the number of initial implanted cells is reduced (the number of initial implanted cells in the prior art is generally 5×10 5-7 cells per mouse), so that the tumor volume of the mouse after being implanted with tumors for 8 weeks is controlled within 2000mm 3 , forming a tumor-bearing atherosclerotic mouse model with clinical representative characteristics.

[0028] In a preferred embodiment, the number of MC38 intestinal cancer cells subcutaneously injected when constructing the mouse model can be 1×10 4 to 1×10 6 cells per mouse. If the number of MC38 cells subcutaneously injected is less than 1×10 4The slow tumor implantation rate per mouse (1 × 10⁶ cells / mouse) leads to excessively long modeling time, reducing modeling efficiency and resulting in high mouse model costs. If the number of MC38 cells injected subcutaneously exceeds 1 × 10⁶ cells / mouse, the model will be more expensive. 6 Each individual tumor develops too rapidly, often reaching the ethically mandated upper limit of 2000 mm² within one month. 3 This can easily lead to modeling failure, resulting in mice that do not have clinically representative characteristics.

[0029] When the number of MC38 colon cancer cells injected subcutaneously was approximately 1 × 10⁻⁶ 5 When the tumor size is 1 cell / cell, the preferred volume ratio of MC38 colon cancer cells to Matrigel is 1:0.3-2.0. If the volume ratio of MC38 colon cancer cells to Matrigel is higher than 1:0.3, the Matrigel, due to its small dosage, is insufficient to exert its limiting effect as a "cell proliferation decelerator," leading to excessively rapid tumor formation. Often, the tumor volume will reach the ethically mandated upper limit of 2000 mm² within one month. 3 This can easily lead to modeling failure, resulting in mice that lack clinical representativeness. If the volume ratio of MC38 colon cancer cells to matrix gel is less than 1:2.0, the excessive amount of matrix gel may inhibit the proliferation of MC38 cells in mice, resulting in slower tumor implantation, longer modeling time, reduced modeling efficiency, and higher mouse model costs.

[0030] This invention determines the optimal time and dosage for tumor implantation of MC38 colon cancer cells / stromal gel mixtures, thereby effectively improving the success rate of modeling. Furthermore, the reagents used in this modeling method are all non-toxic and will not cause additional adverse drug reactions. It also more closely reflects the clinical characteristics of patients with cancer complicated by ASCVD.

[0031] The tumor-bearing atherosclerotic mouse model constructed using the method of this invention can be widely used for research on the mechanism of action of anti-tumor and / or atherosclerotic drugs, new drug development, and pre-clinical drug trials, and is an important and commonly used experimental animal model.

[0032] In this article, the term "tumor-bearing atherosclerotic mouse model" may sometimes also be referred to as "tumor-bearing atherosclerotic model" or "tumor-bearing atherosclerotic mouse," etc. For the sake of simplicity, it can be abbreviated as "mouse model" or "model mouse," which have the same meaning and can be used interchangeably.

[0033] In the modeling method of this invention, routine operations such as subcutaneous injection of MC38 cells, in vivo imaging, observation of tumor growth, and staining of tissue sample sections are well known to those skilled in the art.

[0034] In a specific embodiment, the kit for constructing a tumor-bearing atherosclerotic mouse model provided by the present application further comprises at least one of the following items in addition to the aforementioned MC38 cell strain, biochemical reagents, and experimental apparatus: a carrying tool box, the space of which is divided into a defined space capable of accommodating one or more containers, such as medicine bottles, test tubes, and the like, each of which contains a separate component for the method of the present application; an instruction manual, which can be written on the bottles, test tubes, and the like, or on a separate piece of paper, or on the outside or inside of the containers, such as a paper with an operation demonstration video APP download window, such as a two-dimensional code, and the instruction manual can also be in the form of multimedia, such as a U disk, a network disk, and the like.

[0035] The present application will be further described in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present application and are not intended to limit the scope of the present application.

[0036] Examples

[0037] In this paper, the addition amount, content, and concentration of various substances are mentioned, and the percentage content mentioned herein refers to the mass percentage (wt%) or volume / weight percentage (w / v%) unless otherwise specified.

[0038] In the examples herein, if no specific temperature is specified for the operation, the temperature generally refers to room temperature (10-35°C).

[0039] Main materials and instruments:

[0040] Animals: 6-week-old, 18-20 g weight ApoE - / - Male mice were purchased from Jisui Yaoke.

[0041] Cell strain: MC38 intestinal cancer cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0042] Matrigel: Corning Corporation, item number 356234 / 354234; Biyun Tian Co., Ltd., item number C0386 / C0387.

[0043] Experimental reagents and apparatus: 1640 medium, FBS, P / S, PBS, HE staining reagent, MASSON staining reagent, Oil O Red staining reagent, culture bottle, centrifuge tube.

[0044] Main instruments: CO2 incubator, sterilization pot, refrigerator, syringe, clean bench, centrifuge, small animal live imaging system, fluorescence inverted phase contrast microscope.

[0045] The experimental methods include cell culture, mouse feeding, animal dissection, tissue sample staining, etc. The experimental scheme is approved by the Animal Management and Ethics Committee of Fudan University.

[0046] Example 1: MC38 colon cancer cell culture

[0047] MC38 colon cancer cells were taken out from the liquid nitrogen tank; the cells were resuscitated in a water bath at 37°C; centrifuged, the freeze storage solution was discarded, and an equal volume of complete culture medium (RPMI 1640 culture medium 89%, FBS 10%, P / S 1%) was added and mixed, and then centrifuged. Then add complete culture medium to resuspend and place in a CO2 incubator for 24h, when the cell density reaches about 5×10 5 / ml, start subculture.

[0048] The subculture process of suspension cells is strictly in accordance with sterile operation, and the steps are as follows:

[0049] 1. Resuspend and mix the cells in the culture bottle;

[0050] 2. Take 2 / 3 or half of the mixed cell suspension to a new culture bottle;

[0051] 3. Add an equal amount or twice the amount of fresh culture medium to the original bottle and the new divided bottle, respectively, to keep the cell density at about 5×10 5 / ml;

[0052] 4. Pay attention to the change of pH value of the culture medium and the cell density, and change the medium regularly (2-3 times a week), and when the cell density is greater than 2×10 6 / ml, repeat step 1 or freeze storage.

[0053] Collect MC38 cell culture solution, centrifuge, discard the supernatant, wash with PBS twice, and resuspend with PBS. Count under a microscope, adjust the cell concentration to 2×10 6 / ml, mix with an equal volume of Matrigel, and use this mixture for subcutaneous injection of mice.

[0054] Example 2: Construction of tumor-bearing atherosclerotic mouse model

[0055] The process of constructing a tumor-bearing atherosclerotic mouse model and PD-1 antibody treatment is shown in Figure 1 .

[0056] Take 6-week-old ApoE - / - male mice weighing 18-20g, feed them with high-fat diet to establish atherosclerotic mouse model (AS model), and after 4 weeks of high-fat diet feeding, the AS model is completed.

[0057] Each AS model mouse was subcutaneously injected with 0.1 ml of the above cell / matrix glue mixture, and continued to be fed with a high-fat diet for 8 weeks. After the start of modeling, the body weight of the mice was measured every day, and the activity, hair, and body size of the mice were observed. After tumor implantation, the two largest perpendicular axes (L and W) were measured and recorded every 3 days with a vernier caliper to monitor tumor growth.

[0058] After 8 weeks of high-fat diet feeding, the MC38 intestinal cancer cell tumor was successfully implanted, and the atherosclerotic mouse model with tumor, i.e., TB-AS model, was constructed.

[0059] Example 3: PD-1 antibody treatment in mouse model

[0060] One day after tumor implantation in TB-AS mice, the mice were injected intraperitoneally with PD-1 antibody (aPD-1, 10 mg / kg, 3 times a week for 8 weeks) to simulate clinical PD-1 antibody treatment (referred to as TB-AS + aPD-1 model), and the mice were divided into the following 2 groups using isotype control IgG as a negative control: TB-AS group, TB-AS + aPD-1 group. After the start of modeling, the body weight of the mice was measured every day, and the activity, hair, and body size of the mice were observed. After tumor implantation, the two largest perpendicular axes (L and W) were measured and recorded every 3 days with a vernier caliper to monitor tumor growth.

[0061] The in vivo tumor volume changes of TB-AS model non-treatment group (TB-AS) mice and model PD-1 antibody aPD-1 treatment group mice (TB-AS + aPD-1) are shown in Figure 2 The curve clearly shows that aPD-1 has a tumor inhibiting effect.

[0062] Example 4: Detection in mouse model

[0063] After modeling was completed, the mice in each group were euthanized, and the mouse aortic root was taken for frozen section, which was subjected to HE staining, MASSON staining, and oil red staining to evaluate the plaque phenotype of the mice. The results are shown in Figure 3 .

[0064] The plaque area, lipid accumulation area, and collagen deposition ratio were calculated using Image Pro Plus 6.0. As can be seen from the images shown in Figure 3 It can be seen from the images shown in that the atherosclerotic phenotype was successfully constructed, and at the same time, HE staining and oil red staining showed that the plaque area of the aortic root of the TB-AS + aPD-1 group mice was significantly increased, and the lipid accumulation was significantly increased compared with the TB-AS group; MASSON staining showed that the collagen deposition in the plaque of the aortic root of the TB-AS + aPD-1 group mice was reduced compared with the TB-AS group, indicating that aPD-1 significantly promoted plaque progression and inhibited plaque stability.

[0065] Statistical analysis: All data are expressed as mean ± standard deviation (SD). Analysis between groups was performed using one-way ANOVA, and Turkey's test was used for statistical analysis using Graphpad Prism 8.0 software, and *p<0.05 was set as having statistical difference. Results are shown in Figure 4 The plaque area of the TB-AS mouse model was lower than that of the TB-AS + aPD-1 treated mouse model, and the collage content of the TB-AS mouse model was higher than that of the TB-AS + aPD-1 treated mouse model. It is shown that aPD-1 significantly promotes plaque progression while inhibiting tumor cell growth, and inhibits the stability of plaques.

[0066] The experimental results show that the TB-AS mouse model is successfully constructed by using the method of the present application, which provides a basic animal model for studying the influence of anti-tumor treatment on the occurrence and development of atherosclerotic plaques and the stability of plaques in patients with tumor combined with ASCVD. On the basis of this model, firstly, disease biomarkers can be developed to provide cardiovascular risk assessment for patients with tumor combined with ASCVD during anti-tumor treatment in clinic. Secondly, the mechanism of anti-tumor treatment promoting plaque development can be further studied through this model to provide new targets for drug development. In addition, as an economical, convenient and highly feasible model, it provides a convenient model platform for future drug development and drug screening.

[0067] The above examples are for the convenience of those skilled in the art to understand and use the present application. It is obvious for those skilled in the art to make modifications to the examples and apply the general principles described herein to other examples without creative labor. Therefore, the present application is not limited to the above examples. Improvements and modifications made by those skilled in the art according to the principles of the present application without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A kit for constructing a tumor-bearing atherosclerotic mouse model, namely the TB-AS model, characterized in that, It shall include at least the following components: MC38 colon cancer cells for subcutaneous tumor formation in mice, matrix gel for use in combination with MC38 colon cancer cells, and a high-fat / high-cholesterol diet for constructing a mouse model of atherosclerosis. When the kit is used to construct a mouse model of tumor-bearing atherosclerosis, mice are first fed a high-fat / high-cholesterol diet to induce the establishment of the atherosclerosis model. When the mice show lipid metabolism disorder and atherosclerosis tendency, MC38 colon cancer cells are mixed with matrix gel at a volume ratio of 1:0.3-2.0 and injected subcutaneously into the mice. The mice are then fed a high-fat / high-cholesterol diet for 6-10 weeks until the TB-AS model is successfully constructed.

2. The kit according to claim 1, characterized in that, Also includes: Biochemical reagents, including 1640 medium, FBS, P / S, PBS, HE staining reagent, MASSON staining reagent and / or Oil Red staining reagent.

3. The kit according to claim 1, characterized in that, Also includes: Experimental equipment includes cell culture flasks, centrifuge tubes, and surgical instruments.

4. The kit according to claim 1, characterized in that, The kit is designed for ApoE. - / - Mouse or Ldlr - / - Mice.

5. The kit according to claim 4, characterized in that, The kit is used to treat ApoE in the C57BL / 6J background. - / - Mice.

6. The kit according to claim 1, characterized in that, The volume ratio of MC38 colon cancer cells to stromal colloid is 1:0.9-1.

2.

7. The kit according to claim 1, characterized in that, The injection dose of the MC38 colon cancer cells was 1×10⁻⁶. 4-6 Cell / animal 8. The kit according to claim 1, characterized in that, The tumor volume of the constructed tumor-bearing atherosclerotic mouse model was controlled at 2000 mm. 3 Within.

9. The kit according to claim 1, characterized in that, The tumor-bearing atherosclerotic mouse model is used for anti-tumor drug screening, anti-tumor drug development, and clinical treatment research.

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