Construction method and application of mouse orthotopic osteosarcoma animal model

Abstract

The invention discloses a construction method and application of a mouse in-situ osteosarcoma animal model, and belongs to the technical field of biomedicines.The method adopts a double-step construction mode and comprises the steps that firstly, osteosarcoma cells are inoculated into an immunodeficient nude mouse to preliminarily form tumor tissue; according to the method, the survival rate of tumor cells can be increased, the tumor microenvironment can be more effectively reconstructed, it is ensured that the model is closer to the clinical practical situation in physiology, and the model is more suitable for clinical application. The model effect is obviously better than that of a subcutaneous transplantation tumor model. According to the method disclosed by the invention, a more accurate and reliable mouse in-situ osteosarcoma animal model is successfully constructed by optimizing a modeling process and conditions, and the application prospect in the aspect of screening medicines for preventing or treating osteosarcoma is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for constructing an in situ osteosarcoma animal model in mice and applications thereof. Background Art

[0002] Osteosarcoma (OS) is a highly malignant primary bone tumor that mainly occurs in children and adolescents. As one of the most common malignant bone tumors, osteosarcoma mainly occurs in the epiphyseal ends of long bones, especially in the femur, tibia, and humerus. OS not only invades the surrounding local tissues, but may also further develop into systemic metastasis, affecting the patient's prognosis. The pathogenesis of osteosarcoma has not yet been fully elucidated, but it is known to be related to genetic factors, gene mutations, radioactive radiation, and abnormal cell proliferation during rapid bone growth. Clinically, patients with osteosarcoma often present with local pain, swelling, and limited mobility, and may be accompanied by pathological fractures in the late stage.

[0003] Currently, the main treatments for osteosarcoma include surgical resection and adjuvant chemotherapy. Surgery aims to completely remove tumor tissue, while chemotherapy is used to eliminate potential micrometastatic lesions. Although these treatments have improved patient survival to a certain extent, the therapeutic effects are still unsatisfactory for patients with metastatic or recurrent osteosarcoma. In addition, the toxic side effects and tumor resistance of chemotherapy drugs also limit their clinical application. Therefore, it is particularly important to develop new treatment strategies to reduce the risk of metastasis and recurrence after osteosarcoma treatment.

[0004] In research and clinical trials, it is crucial to construct experimental models that can accurately simulate disease characteristics. Currently, commonly used osteosarcoma models include subcutaneous xenograft models, which simulate the growth process of tumors in vivo by directly implanting tumor cells or tissues under the skin of mice. However, this method has some significant limitations: (1) low tumor formation rate. Not all injected tumor cells can successfully form tumors, which limits the reproducibility and reliability of the model; (2) lack of tumor microenvironment. Subcutaneous xenograft models often fail to simulate the real tumor microenvironment, including cell-cell interactions, angiogenesis, immune response, and osteoclast activation. These factors play a key role in tumor development and metastasis; (3) lack of clinical relevance: because the model is not close to the clinical situation, this may lead to the failure of the therapeutic effects observed in the experiment to be verified in clinical applications.

[0005] Chinese patent publication CN114698595A discloses a method for establishing an in situ tumor animal model and an in situ tumor animal model. This invention uses ultrasound guidance to enable real-time observation of specific parameters of the target organ and surrounding tissues, thereby improving the stability of the orthotopic transplanted tumor model. Chinese patent publication CN118452157A discloses a method for establishing an osteosarcoma PDX model and its application. The PDX model establishment method comprises the following steps: S1: pretreatment of an osteosarcoma patient tumor sample; S2: subcutaneous inoculation of mice. This invention utilizes tumor-bearing models established at multiple sites, maximizing the utilization of limited tumor samples and experimental animals, allowing for rapid expansion of PDX models and improving modeling efficiency and cost. However, these prior art methods still suffer from high technical complexity and sample dependency. Therefore, developing a method for establishing an osteosarcoma model that more closely resembles clinical characteristics, is simple to operate, and is cost-effective is of great significance for in-depth research on the pathogenesis of osteosarcoma, screening for effective therapeutic drugs, and improving patient prognosis. Summary of the Invention

[0006] The present invention provides a method for constructing an in situ osteosarcoma animal model in mice. The in situ osteosarcoma animal model constructed by this method is more convenient, accurate, and reliable, and is physiologically closer to the actual clinical situation.

[0007] The specific technical solutions adopted are as follows:

[0008] A method for constructing an orthotopic osteosarcoma model in mice specifically comprises the following steps:

[0009] (1) Cultivate luciferase-labeled osteosarcoma cells to obtain a cell density of 1×10 7 -1×10 8 Cell resuspension at 1000 μg / mL;

[0010] (2) Anesthetizing and fixing nude mice, inoculating the osteosarcoma cells in the cell resuspension solution of step (1) subcutaneously into the nude mice, and then continuing to feed the nude mice, monitoring the size of the subcutaneous tumors in the nude mice, and analyzing the fluorescence intensity;

[0011] (3) When the subcutaneous tumor tissue of nude mice is ≥1000mm 3 , using an in vivo fluorescence imaging device to confirm that the subcutaneous tumor was successfully constructed, and the subcutaneous tumor tissue of the nude mice was removed for later use;

[0012] (4) Anesthetizing and fixing the mouse, making a tibial notch at the proximal end of the tibia of the mouse's lower limb, filling the tibial notch with the subcutaneous tumor tissue from step (3), suturing the surgical incision on the mouse's skin, and raising the mouse to allow the osteosarcoma tissue to grow in the tibial notch, thereby constructing an in situ osteosarcoma animal model in mice.

[0013] The core of the present method lies in a two-step approach using two different animal species: first, osteosarcoma cells are inoculated subcutaneously in immunodeficient nude mice to efficiently establish tumor tissue. Subsequently, the tumor tissue is transferred from the subcutaneous environment to an orthotopic mouse model of osteosarcoma. This process not only improves tumor cell survival but also more effectively recreates the tumor microenvironment.

[0014] Preferably, the osteosarcoma cells labeled with luciferase are K7M2-luc cells.

[0015] Preferably, the nude mice used in step (2) are BALB / c nude mice, and the mice used in step (4) are BALB / c mice.

[0016] Optionally, in step (2) and step (4), the mouse is anesthetized using isoflurane combined with oxygen.

[0017] Preferably, in step (2), the inoculation sites are selected from the armpits on both sides of the nude mouse.

[0018] Preferably, in step (3), after removing the subcutaneous tumor tissue of the nude mouse, the necrotic tissue is removed and the tumor is divided into 0.8-1.2 mm 3 The tissue blocks were soaked in serum-free DMEM culture medium for later use.

[0019] Preferably, in step (4), the depth of the mouse tibial notch is 0.8-1.2 mm. Under the above parameters, it can be ensured that the depth of the notch is 0.8-1.2 mm. 3 The tissue blocks were seeded in the tibial environment.

[0020] The present invention also provides the application of the method for constructing the mouse in situ osteosarcoma animal model in screening drugs for preventing osteosarcoma.

[0021] The present invention also provides the application of the method for constructing the mouse in situ osteosarcoma animal model in screening drugs for treating osteosarcoma.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention first inoculates osteosarcoma cells into immunodeficient nude mice, making full use of the immunodeficiency characteristics of nude mice to achieve rapid tumor formation of tumor cells and quickly transform osteosarcoma cells into tumor tissue; then transfers the tumor tissue from the subcutaneous environment of immunodeficient nude mice to the in situ tibial environment of immune-complete mice to establish an in situ osteosarcoma animal model in mice. This not only helps to improve the survival rate of tumor cells, but also can more effectively reconstruct the tumor microenvironment, ensuring that the model is physiologically closer to the actual clinical situation, and is significantly better than the subcutaneous transplanted tumor, immunodeficient in situ tumor or direct intramedullary injection of cells in mouse in situ tumor model.

[0024] (2) The method of the present invention successfully constructed a more accurate and reliable mouse in situ osteosarcoma animal model by optimizing the modeling process and conditions, providing important support for the research on screening drugs for the prevention or treatment of osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the fluorescence image of the subcutaneous tumor in nude mice monitored by the in vivo fluorescence imaging instrument in step (2) of Example 1.

[0026] Figure 2 This is an optical image of a subcutaneous tumor tissue block in a nude mouse.

[0027] Figure 3 This is a diagram of the process of inoculating subcutaneous tumor tissue in step (4) of Example 1.

[0028] Figure 4 This is a diagram of the process of inoculating subcutaneous tumor tissue in Comparative Example 1.

[0029] Figure 5 This is a diagram showing the fluorescence changes of the tumor in the mouse orthotopic osteosarcoma animal model constructed in Example 1 monitored by a living fluorescence imager.

[0030] Figure 6 This is a diagram of the fluorescence changes of the tumor in the subcutaneous osteosarcoma animal model constructed in Comparative Example 1 monitored by a living fluorescence imager.

[0031] Figure 7 The graphs show the changes in tumor volume and mouse weight of the orthotopic osteosarcoma model in mice constructed in Example 1 and the subcutaneous osteosarcoma model in Comparative Example 1, where A represents the tumor volume and B represents the mouse weight.

[0032] Figure 8 Comparison of osteosarcoma specimens obtained from the mouse orthotopic osteosarcoma animal model constructed in Example 1 and the subcutaneous osteosarcoma animal model constructed in Comparative Example 1. DETAILED DESCRIPTION

[0033] In order to make the objects, features and advantages of the present invention more clearly understood, a detailed description is given below using specific embodiments. In the following description, many specific details are set forth to fully understand the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in the various embodiments of the present invention can be combined accordingly without conflicting with each other.

[0034] The procedures for the following examples, in which specific conditions are not specified, generally follow conventional conditions or those recommended by the manufacturer. Any material not described in detail in this specification belongs to the prior art known to those skilled in the art. The experimental materials used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent companies.

[0035] Example 1 Construction of an orthotopic osteosarcoma (OT-OS) animal model

[0036] (1) Resuscitate and culture K7M2-luc cells (purchased from Fenghui Bio). When the K7M2-luc cells in the culture container proliferate to 80%, aspirate the complete culture medium, wash the cells 3 times with sterile PBS, add 2-3 mL of preheated trypsin solution, and place them in a 37°C incubator for digestion. When the cell morphology becomes round under a microscope, add twice the volume of trypsin solution to terminate the digestion. Collect the digested cells in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes to obtain a cell pellet. Aspirate the supernatant and resuspend the cells in a mixture of precooled PBS solution and matrix gel at a ratio of 1:1, and adjust the cell density to 5×10 7 / mL to obtain a cell resuspension.

[0037] (2) Anesthetize and fix the nude mice, and use isoflurane for inhalation anesthesia. Place 6-week-old male BALB / c nude mice in an induction box and use 2% isoflurane combined with oxygen for 5 minutes. Then continue to give 1.5% isoflurane combined with 98.5% oxygen to maintain anesthesia. Inject 100uL of cell resuspension into the right armpit of the BALB / c nude mice, and inoculate K7M2-luc cells subcutaneously into the nude mice. After the injection, gently press the injection site for 30 seconds to prevent the suspension from leaking. Use an alcohol cotton ball to inject the injection point and the surrounding skin. Put the nude mice into the cage for observation. After waking up, put them back in the cage and continue to feed them.

[0038] (3) Monitor the size of subcutaneous tumors in nude mice and analyze the fluorescence intensity. D-luciferin potassium salt (150 mg / kg) was intraperitoneally injected into BALB / c nude mice. After about 10 minutes, the size of the tumor in the BALB / c nude mice was photographed using an in vivo fluorescence imager (IVIS) and the fluorescence intensity was analyzed using software. Representative images are shown below. Figure 1 As shown, the fluorescent signal was concentrated in the right axillary area of the nude mouse, indicating that the tumor was successfully established;

[0039] When osteosarcoma tissue grows to 1000mm 3 BALB / c nude mice were killed, the skin and subcutaneous fascia were carefully cut, the subcutaneous tumor tissue was completely exposed and removed, the tumor tissue was cut open, the necrotic tissue was carefully scraped off, and the subcutaneous tumor tissue was divided into approximately 1 mm 3 The tissue block ( Figure 2), soaked in serum-free DMEM culture medium for later use.

[0040] (4) After anesthetizing and fixing a 6-week-old male BALB / c mouse (using the same method as step (2)), the skin around the mouse knee joint was disinfected with iodine tincture, and the proximal end of the mouse tibia was exposed. A tibial notch with a depth of about 1 mm was made with the tip of ophthalmic scissors, and the 1 mm obtained in step (3) was cut into the proximal end of the tibia. 3 The osteosarcoma tissue was filled into the tibial gap, and the surgical incision on the mouse skin was sutured and disinfected with iodine 3 times. Figure 3 As shown, mice were fed to allow osteosarcoma tissue to grow at the tibial notch, thereby establishing an in situ osteosarcoma animal model in mice.

[0041] (5) In vivo imaging was performed on the 7th, 14th, and 21st days after the establishment of the mouse orthotopic osteosarcoma animal model to observe the changes in the tumor. The operation method was the same as step (3). The results are as follows: Figure 5 As shown in Figure 2, the tumor continued to grow and progress after the orthotopic osteosarcoma animal model was established, and the enhancement of the fluorescence signal intuitively demonstrated the dynamic changes of the tumor. At the same time, the tumor volume and mouse weight were measured every other day after the orthotopic osteosarcoma animal model was established. The results are shown in Figure 2. Figure 7 As shown in Figure A, by regularly measuring the tumor volume, it was found that the tumor volume increased significantly over time, indicating that the tumor grew rapidly. Figure 7 Figure B shows that the weight and overall health of the mice remained stable throughout the experiment, and no obvious weight loss was observed, indicating that tumor growth did not cause serious systemic effects on the mice.

[0042] (6) The endpoint of the mouse experiment was defined as mouse death or tumor volume greater than 1500 mm 3 The tumor volume is calculated as follows: Volume = (length × width 2 ) / 2, on the 21st day after tumor resection, 5 BALB / c mice in each group were euthanized, and the whole lower limb specimens were removed and quickly fixed in 4% paraformaldehyde. The obtained osteosarcoma specimens are shown in the figure. Figure 8 As shown in the first row, it can be seen that the tumor volume that meets the experimental endpoint definition is greater than 1500mm 3 standards.

[0043] Example 2 Construction of subcutaneous osteosarcoma (SC-OS) animal model

[0044] (1) According to steps (1) to (3) in Example 1, a volume of about 1 mm 3 Subcutaneous tumor tissue;

[0045] (2) After 6-week-old male BALB / c nude mice were anesthetized and fixed (the same method as above), the skin around the knee joint of the nude mice was disinfected with iodine and about 1 mm3 The osteosarcoma tissue was transplanted subcutaneously, and the surgical incision on the skin of the nude mice was sutured and disinfected with iodine for 3 times. Figure 4 As shown, nude mice were bred to allow subcutaneous osteosarcoma tissue to grow, thereby establishing a subcutaneous osteosarcoma animal model.

[0046] (3) In vivo imaging was performed on days 7, 14, and 21 after the establishment of the subcutaneous osteosarcoma animal model to observe the changes in the tumor. The operation was the same as above. The results are shown in Figure 2. Figure 6 As shown in Figure 2, the tumor growth of the subcutaneous osteosarcoma animal model was slow and the fluorescence signal was weak, reflecting the differences in tumor microenvironment and biological behavior between the subcutaneous osteosarcoma animal model and the orthotopic osteosarcoma animal model in mice. At the same time, the tumor volume and mouse body weight were measured every other day after the subcutaneous osteosarcoma animal model was established. The results are shown in Figure 2. Figure 7 As shown in Figure A, by regularly measuring tumor volume, we found that the tumor growth rate of the subcutaneous osteosarcoma animal model was significantly slower than that of the orthotopic osteosarcoma animal model in mice. Figure 7 As shown in Figure 2B, the weight and overall health of the mice remained stable throughout the experiment, and no obvious weight loss was observed, indicating that tumor growth did not cause serious systemic effects on the mice.

[0047] (4) On the 21st day after tumor resection, 5 BALB / c mice in each group were euthanized, and the entire lower limb specimens were removed and quickly fixed in 4% paraformaldehyde. The obtained osteosarcoma specimens are shown in the following figure. Figure 8 As shown in the second row of Figure 2, although the tumor volumes of both models reached the threshold defined by the experimental endpoint (>1500mm 3 ), but the tumor specimen volume of the subcutaneous osteosarcoma animal model was significantly smaller than that of the orthotopic osteosarcoma animal model.

[0048] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing an orthotopic osteosarcoma model in mice, characterized in that: The specific steps include: (1) Cultivate luciferase-labeled osteosarcoma cells to obtain a cell density of 1×10 7 -1×10 8 Cell resuspension at 1000 cells / mL; (2) Anesthetizing and fixing nude mice, inoculating the osteosarcoma cells in the cell resuspension solution of step (1) subcutaneously into the nude mice, and then continuing to feed the nude mice, monitoring the size of the subcutaneous tumors in the nude mice, and analyzing the fluorescence intensity; (3) When the subcutaneous tumor tissue of nude mice is ≥1000mm 3 , using an in vivo fluorescence imaging device to confirm that the subcutaneous tumor was successfully constructed, and the subcutaneous tumor tissue of the nude mice was removed for later use; (4) Anesthetizing and fixing the mouse, making a tibial notch at the proximal end of the tibia of the mouse's lower limb, filling the tibial notch with the subcutaneous tumor tissue from step (3), suturing the surgical incision on the mouse's skin, and raising the mouse to allow the osteosarcoma tissue to grow in the tibial notch, thereby constructing an in situ osteosarcoma animal model in mice.

2. The method for constructing an orthotopic osteosarcoma animal model in mice according to claim 1, characterized in that: The osteosarcoma cells labeled with luciferase are K7M2-luc cells.

3. The method for constructing an orthotopic osteosarcoma animal model in mice according to claim 1, wherein: The nude mice used in step (2) are BALB / c nude mice, and the mice used in step (4) are BALB / c mice.

4. The method for constructing an orthotopic osteosarcoma animal model in mice according to claim 3, characterized in that: In step (2), the inoculation sites were selected from the armpits on both sides of the nude mice.

5. The method for constructing an orthotopic osteosarcoma animal model in mice according to claim 1, wherein: In step (3), after removing the subcutaneous tumor tissue of the nude mouse, the necrotic tissue was removed and the tumor was divided into 0.8-1.2 mm 3 The tissue blocks were soaked in serum-free DMEM culture medium for later use.

6. The method for constructing an orthotopic osteosarcoma animal model in mice according to claim 1, wherein: In step (4), the depth of the notch in the mouse tibia is 0.8-1.2 mm.

7. Use of the method for constructing an orthotopic osteosarcoma model in mice according to any one of claims 1 to 6 in screening drugs for preventing osteosarcoma.

8. Use of the method for constructing an orthotopic osteosarcoma model in mice according to any one of claims 1 to 6 in screening drugs for treating osteosarcoma.

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