Kit for constructing tumor-bearing atherosclerosis mouse model

Abstract

The invention discloses a kit for constructing a tumor-bearing atherosclerosis mouse model. The kit at least comprises MC38 mouse colon cancer cells, matrigel and high-fat feed / high-cholesterol feed. By adopting the kit, the tumor-bearing atherosclerosis mouse model conforming to the typical clinical characteristics of tumor-associated atherosclerotic cardiovascular diseases can be constructed, and the kit is simple and easy to operate, time-saving, high in success rate and low in production cost.

Description

Technical Field

[0001] The invention belongs to the field of experimental medicine, and particularly relates to a kit for constructing a tumor-bearing atherosclerosis mouse model and uses thereof. Background Art

[0002] In the past 20 years, many innovative treatments have emerged in the field of malignant tumor treatment. These treatments also bring the risk of cardiovascular toxicity, including adverse reactions such as decreased cardiac function, hypertension, thrombosis or embolism. The 2022 European Society of Cardiology Guidelines for Oncology Cardiology first proposed the definition of cancer therapy-related cardiovascular toxicity (CTR-CVT) and provided systematic guidance and recommendations for the diagnosis, treatment, prevention and management of CTR-CVT. It is a milestone event in international oncology cardiology. In addition, as the survival of cancer patients continues to increase, the impact of anti-tumor therapy on some chronic cardiovascular diseases has gradually received attention.

[0003] In recent years, with the continuous improvement of living conditions and the aging of the population in my country, 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 risk of coronary heart disease. Anti-microtubule drugs and small molecule targeted drugs have a chance of causing cardiac ischemia. Recent clinical studies have confirmed that the risk of cardiovascular events in patients treated with ICIs for two years is three times higher than other treatments. As the two major killers of human health, patients with tumors and ASCVD should pay more attention to the evaluation and prognosis of ASCVD disease progression during anti-tumor treatment.

[0004] Because atherosclerosis progresses slowly, ApoE - / - or Ldlr - / - Mice were fed a high-fat diet for more than 3 months to form an atherosclerotic mouse model. For tumor-bearing mouse models, subcutaneous injection of tumor cells grew rapidly, and the tumor volume often reached the ethical upper limit of 2000mm within 1 month. 3 Therefore, it is urgent to construct an animal model of tumor-bearing atherosclerosis to evaluate the effects of anti-tumor therapy on the progression of atherosclerosis and explore its mechanism. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings of the animal model of tumor-bearing atherosclerosis in the prior art, this research group has provided a new method for constructing a tumor-bearing atherosclerosis mouse model through multiple experiments, which significantly improves the success rate of modeling, and the constructed mouse model has representative clinical characteristics, which greatly saves scientific research costs. Specifically, the present invention includes the following technical solutions.

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

[0007] Furthermore, the above-mentioned kit also includes: 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 saline), HE staining reagent, MASSON staining reagent and / or Oil O Red staining reagent.

[0008] Furthermore, the above-mentioned kit also includes: experimental equipment, including cell culture bottles, centrifuge tubes, and surgical instruments.

[0009] In one embodiment, the target of the above kit is ApoE - / - Mouse or Ldlr - / - Mouse, preferably ApoE - / - Mouse.

[0010] Preferably, the above-mentioned ApoE - / - Mice are ApoE on a C57BL / 6J background - / - Mouse.

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

[0012] The above-mentioned matrix glue is a commonly used matrix glue in animal model construction. It is prepared from basement membrane components extracted from mouse tumor tissue, and contains laminin, type IV collagen, heparin sulfate glycoprotein, etc., and can be selected from the following groups: ① Corning Incorporated, Catalog No. 356234 / 354234; ② Bio-Tech Co., Ltd., Catalog No. C0386 / C0387.

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

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

[0015] Compared with the subcutaneous injection of MC38 colorectal cancer cells alone, the advantage of using MC38 colorectal cancer cells mixed with matrix gel is that the matrix gel is prepared from basement membrane components extracted from mouse tumor tissues, and its main components include laminin, type IV collagen, heparin sulfate glycoprotein, etc. These components can provide the support and signals required for cell adhesion, differentiation and proliferation, and can also simulate the characteristics of the basement membrane in the physiological environment, thereby reducing the number of tumor cells implanted and increasing the success rate of tumor implantation. The tumor volume of mice bearing tumors (subcutaneous injection of MC38 colorectal cancer cells) was controlled at 2000mm after 8 weeks. 3 The final constructed TB-AS model can meet the typical clinical characteristics of patients with tumors and atherosclerotic cardiovascular disease (ASCVD), avoiding the excessive proliferation of tumor cells and the premature tumor volume reaching the ethical upper limit of 2000mm after the injection of tumor cells MC38 cells alone. 3 As a result, the patient does not meet the typical clinical features of tumor combined with atherosclerotic cardiovascular disease, which means that the patient is not representative and loses clinical research value.

[0016] As a specific use of the above kit, the injection volume of MC38 colon cancer cells is 1×10 4-6 cells / cell, for example, about 1×10 5 Cells / piece.

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

[0018] Compared with the existing mouse model construction method, the MC38 mouse intestinal cancer cells with a slower tumor growth cycle were selected when the mice were tumor-bearing, and the number of initial implanted cells was reduced during subcutaneous injection (the number of initial implanted cells in the existing technology is generally 5×10 5-7 cells / mouse), which can keep the tumor volume of mice within 2000mm after 8 weeks of tumor bearing. 3 Within.

[0019] Optionally, the tumor-bearing atherosclerosis mouse model (TB-AS model) constructed using the above kit meets the typical clinical characteristics of patients with tumors and atherosclerotic cardiovascular disease (ASCVD), and can therefore 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 anti-tumor drugs such as PD-1 antibodies.

[0020] The present invention provides novel tools and methods for constructing a tumor-bearing atherosclerosis mouse model, which can be used to construct a tumor-bearing atherosclerosis mouse model that meets the typical clinical characteristics of tumor-combined atherosclerotic cardiovascular disease, which not only improves the experimental work efficiency, but also increases the success rate, and can significantly reduce the production cost of the tumor-bearing atherosclerosis mouse model, and has promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the process of constructing a tumor-bearing atherosclerosis mouse model according to the present invention.

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

[0023] Figure 3 Typical images of H&E staining, Oil O Red staining, and Masson staining of aortic root plaques in TB-AS group mice and αPD-1 treatment group mice (TB-AS+αPD-1) are shown (scale bar = 250 μm).

[0024] Figure 4 The plaque area and collagen deposition statistics of the aorta root of mice in the TB-AS group and mice treated with αPD-1 (TB-AS+αPD-1) are shown (*** represents p<0.001). Among them, collagen deposition represents the formation of atherosclerotic fibrous plaques. Collagen fibers wrap around the necrotic core to form fibrous plaques. 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, which has typical clinical features of tumor-combined atherosclerotic cardiovascular disease, can be used for in vivo experiments to relatively accurately evaluate the effects of anti-tumor therapies, such as PD-1 therapy, on the progression of atherosclerosis. It is the basis for the development of biomarkers for the disease, the study of pathogenesis, the screening of anti-tumor drugs, the evaluation of efficacy, and the model for the adverse reactions of atherosclerosis.

[0026] Because atherosclerosis progresses slowly, ApoE - / - or Ldlr - / - Mice were fed a high-fat diet for more than 3 months to form an atherosclerotic mouse model. For tumor-bearing mouse models, subcutaneous injection of tumor cells grew rapidly, and the tumor volume often reached the ethical upper limit of 2000mm within 1 month. 3 .

[0027] Faced with this contradiction, our research group tried a new modeling method, determined the appropriate tumor implantation method, time and dose, mixed MC38 colorectal cancer cells with matrigel in a specific ratio and used them together. It was unexpectedly found that matrigel could effectively prevent MC38 colorectal 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 upper limit of 2000mm within 1 month after implantation. 3 , which resulted in the constructed mice losing their clinical model value because they did not meet the typical clinical characteristics of patients with tumors and ASCVD. Through multiple experiments, the technical solution determined that ApoE - / - When mice were fed a high-fat diet for 4 weeks, they were given tumors at a time point when lipid metabolism disorders and atherosclerosis tended to occur in the body. The final condition was consistent with the clinical characteristics of patients with tumors and ASCVD. On the other hand, MC38 colorectal cancer cells of mice with a slower tumor growth cycle were selected for tumor bearing, and the number of initial implanted cells was reduced during subcutaneous injection (the number of initial implanted cells in the prior art is generally 5×10 5-7 cells / mouse), so that the tumor volume of mice was controlled at 2000mm after 8 weeks of tumor bearing. 3 Within 100 μg / mL, a tumor-bearing atherosclerosis mouse model with clinically representative characteristics was formed.

[0028] In a preferred embodiment, the number of MC38 intestinal cancer cells injected subcutaneously when constructing a mouse model can be 1×10 4 to 1×10 6 If the number of MC38 cells injected subcutaneously is less than 1×10 4The tumor implantation speed is slow, which leads to a long modeling time, reduces the modeling efficiency, and causes the cost of mouse models to be too high. If the number of MC38 cells injected subcutaneously is higher than 1×10 6 The tumor growth rate is too fast, and the tumor volume often reaches the ethical upper limit of 2000mm within 1 month. 3 , which can easily lead to modeling failure and the resulting mice do not have clinically representative characteristics.

[0029] When the number of MC38 colorectal cancer cells injected subcutaneously was approximately 1×10 5 When the volume ratio of MC38 colorectal cancer cells to matrigel is 1:0.3-2.0, the optimal mixing ratio is 1:0.3-2.0. If the volume ratio of MC38 colorectal cancer cells to matrigel is higher than 1:0.3, the matrigel will not be able to play the limiting role of "cell proliferation decelerator" due to the small amount of matrigel used, resulting in too fast tumor formation. The tumor volume will often reach the ethical upper limit of 2000mm within 1 month. 3 , which can easily lead to modeling failure and the resulting mice do not have clinically representative characteristics. If the volume ratio of MC38 colorectal cancer cells to matrix gel is lower than 1:2.0, the matrix gel may tend to inhibit the proliferation of MC38 cells in mice due to excessive dosage, resulting in slower tumor implantation, too long modeling time, reduced modeling efficiency, and excessively high mouse model costs.

[0030] The present invention determines a reasonable implantation time and dosage of the MC38 colon cancer cell / matrigel mixture, thereby effectively improving the success rate of modeling. In addition, the reagents involved in the modeling method are not significantly toxic and will not cause additional adverse drug reactions. At the same time, it has clinical characteristics that are closer to patients with tumors and ASCVD.

[0031] The tumor-bearing atherosclerosis mouse model constructed by the method of the present invention can be widely used in the study of the drug action mechanism of anti-tumor and / or atherosclerosis therapeutic drugs, new drug development and pre-clinical drug trials, and is an important and commonly used experimental animal model.

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

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

[0034] In a specific embodiment, the kit for constructing a tumor-bearing atherosclerosis mouse model provided by the present invention may include, in addition to the aforementioned MC38 cell line, biochemical reagents, and experimental equipment, at least one of the following items: a carrying tool box, the space of which is divided into a limited space that can accommodate one or more containers and operating instruments, such as medicine bottles, test tubes, and the like, each of which contains a separate component for the method of the present invention; instructions, which can be written on bottles, test tubes and the like, or on a separate piece of paper, or on the outside or inside of the container, such as a paper with an operation demonstration video APP download window such as a QR code, and the instructions can also be in the form of multimedia, such as a USB flash drive, a network disk, etc.

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

[0036] Example

[0037] This article involves the addition amount, content and concentration of various substances, and the percentage content mentioned here refers to mass percentage (wt%) or volume / weight percentage (w / v%) unless otherwise specified.

[0038] In the embodiments herein, if no specific description is given for the operating temperature, the temperature generally refers to room temperature (10-35° C.).

[0039] Main materials and instruments:

[0040] Animals: 6-week-old, 18-20 g ApoE-positive mice on a C57BL / 6J background - / - Male mice were purchased from Jicui Yaokang.

[0041] Cell line: MC38 colorectal cancer cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0042] Matrigel: Corning Incorporated, catalog number 356234 / 354234; Bio-Tech Limited, catalog number C0386 / C0387.

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

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

[0045] The experimental methods including cell culture, mouse husbandry, animal dissection, and tissue sample staining were performed using routine operating procedures, and the experimental protocol was approved by the Animal Care and Ethics Committee of Fudan University.

[0046] Example 1: MC38 colon cancer cell culture

[0047] Take out MC38 colon cancer cells from the liquid nitrogen tank; resuscitate the cells in a water bath at 37°C; centrifuge, discard the cryopreservation solution, add an equal volume of complete culture medium (RPMI 1640 culture medium 89%, FBS 10%, P / S 1%), mix well, and centrifuge. Then add complete culture medium to resuspend, place in a CO2 incubator and culture for 24 hours. When the cell density reaches 5×10 5 / ml, and start subculturing.

[0048] The routine culture and subculture process of suspension cells strictly follows aseptic operation, and the steps are as follows:

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

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

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

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

[0053] The MC38 cell culture medium was collected and centrifuged, the supernatant was discarded, the cells were washed twice with PBS and resuspended in PBS. The cells were counted under a microscope and the cell concentration was adjusted to 2×10 6 / ml, mixed with an equal volume of Matrigel, and the mixture was used for subcutaneous injection into mice.

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

[0055] The process of establishing a tumor-bearing atherosclerosis mouse model and PD-1 antibody treatment is as follows Figure 1 shown.

[0056] Take ApoE from 6-week-old mice weighing 18-20g - / - Male mice were fed a high-fat diet to establish an atherosclerosis mouse model (AS model). After 4 weeks of high-fat diet feeding, the AS model was completed.

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

[0058] After 8 weeks of high-fat diet feeding, MC38 colorectal cancer cells were successfully implanted, and a tumor-bearing atherosclerosis mouse model, namely the TB-AS model, was constructed.

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

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

[0061] The changes in tumor volume in the TB-AS model non-treatment group (TB-AS) and the PD-1 antibody αPD-1 treatment group (TB-AS+αPD-1) are shown in Figure 2. Figure 2 The curve clearly shows that αPD-1 has an inhibitory effect on tumors.

[0062] Example 4: Mouse model testing

[0063] After modeling, mice in each group were euthanized, and the hearts of mice were taken for frozen sections of the aortic root. HE staining, MASSON staining, and oil red staining were performed to evaluate the phenotype of mouse plaques. The results are shown in Figure 3 middle.

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

[0065] Statistical analysis: All data are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for multiple group analysis, and Turkey test statistical analysis was performed using Graphpad Prism 8.0 software. *p < 0.05 was set as statistically significant. The results are shown in Figure 4 The plaque area of ​​the TB-AS mouse model was lower than that of the TB-AS+αPD-1 treated mouse model, while the collagen content of the TB-AS mouse model was higher than that of the TB-AS+αPD-1 treated mouse model. This indicates that αPD-1 significantly promotes plaque progression and inhibits plaque stability while inhibiting tumor cell growth.

[0066] The experimental results show that the TB-AS mouse model was successfully constructed using the method of the present invention, which provides a basic animal model for studying the effects of anti-tumor therapy on the development and stability of atherosclerotic plaques in patients with tumors and ASCVD. On the basis of this model, firstly, disease biomarkers can be developed to provide cardiovascular risk assessment for clinical patients with tumors and ASCVD during anti-tumor therapy. Secondly, this model can be used to further study the mechanism by which anti-tumor therapy promotes plaque development, providing 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 embodiments are intended to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make modifications to the embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A kit for constructing a tumor-bearing atherosclerosis mouse model, namely a TB-AS model, characterized in that: The invention at least comprises the following components: MC38 intestinal cancer cells for forming tumors under the skin of mice, matrix gel for use in combination with MC38 intestinal cancer cells, and high-fat feed / high-cholesterol feed for constructing an atherosclerosis mouse model.

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

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

4. The kit according to claim 1, characterized in that The object of the kit is ApoE - / - Mouse or Ldlr - / - Mouse, preferably ApoE - / - Mouse.

5. The kit according to claim 1, characterized in that The object of the kit is ApoE in C57BL / 6J background - / - Mouse.

6. The kit according to claim 1, characterized in that The high fat / high cholesterol diet was D12108C from ResearchDiets.

7. The kit according to claim 1, characterized in that When used to construct a tumor-bearing atherosclerosis mouse model, the mice are first fed with a high-fat diet / high-cholesterol diet to induce the establishment of an atherosclerosis model. When there is lipid metabolism disorder and atherosclerosis tendency in the mice, MC38 colorectal cancer cells and matrix gel are mixed at a volume ratio of 1:0.3-2.0, preferably a volume ratio of 1:0.5-1.8, preferably a volume ratio of 1:0.8-1.5, preferably a volume ratio of 1:0.9-1.2, and more preferably, for example, a volume ratio of about 1:1, and the mice are subcutaneously injected. The high-fat diet / high-cholesterol diet is continued for 6-10 weeks, preferably 7-9 weeks, for example, about 8 weeks, until the TB-AS model is successfully constructed.

8. The kit according to claim 1, wherein The injection volume of MC38 colorectal cancer cells was 1×10 4-6 cells / cell, for example, about 1×10 5 Cells / piece.

9. The kit according to claim 1, characterized in that The tumor volume of the constructed tumor-bearing atherosclerosis mouse model was controlled at 2000mm 3 Within.

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

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