A method for constructing a lung metastasis model of a human melanoma A375-M1 cell line

Abstract

This invention relates to the field of animal model construction, specifically a method for constructing a lung metastasis model of the human melanoma A375-M1 cell line. The human melanoma A375-M1 cell line lung metastasis model was constructed using tail vein injection, and in vivo identification was performed using PET-CT and MRI, while in vitro identification was performed using Bouin's and H&E assays. The human melanoma lung metastasis model constructed by this invention has the advantages of short construction time, simple method, and 100% modeling success rate. This metastasis model simulates the development process and pathological characteristics of melanoma lung metastasis, solving the problem of the lack of ideal animal models for human melanoma lung metastasis. It contributes to the exploration and screening of anti-lung metastasis drugs for human melanoma and plays a positive role in promoting new strategies for combating melanoma lung metastasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal model construction, in particular to a method for constructing a lung metastasis model of human melanoma A375-M1 cell line. BACKGROUND

[0002] Malignant melanoma is a highly invasive and easily metastatic malignant tumor, which has become one of the fastest growing tumors in terms of incidence in recent years, and its mortality rate ranks first among skin malignant tumors. Melanoma is highly malignant and easily metastasizes. About 50-80% of patients with advanced melanoma will develop liver metastasis, and 8-46% of melanoma patients will develop brain or lung metastasis.

[0003] In tumor research, model construction is crucial. Among them, the metastatic tumor model is to artificially inoculate human or murine cells into experimental mice, and the tumor cells spread to various parts of the body through various routes in the experimental mice, and the tumor cells colonize in non-primary organs or tissues to form metastatic foci, which is similar to the process of human tumor metastasis in vivo.

[0004] At present, the preclinical study of advanced metastatic melanoma is mainly based on the murine B16F10 metastatic tumor model, but this model has the problem that the expression of murine cell-related proteins and antibodies is different from that of human cells. In addition, the spontaneous metastasis of human malignant melanoma A375 cell line subcutaneous tumor is also used for preclinical study of advanced metastatic melanoma, but the success rate of this cell line in constructing melanoma metastatic tumor model is low, the modeling time is long, and the metastatic location is random, which is not suitable for the study of specific metastatic organs of melanoma. Therefore, finding a simple and stable method to establish a human melanoma metastasis model plays a key role in the study of melanoma. SUMMARY

[0005] The purpose of the present application is to provide a method for constructing a lung metastasis model of human melanoma A375-M1 cell line.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following scheme:

[0007] (1) Select 3-4 week old male NOD SCID mice, each weighing 17-20g, trypsinize the logarithmic phase A375-M1 cells, resuspend them with pre-cooled PBS, adjust the concentration to 2x10 7 million / mL, inject 100μL into the tail vein of each mouse, and slowly push it in 0.5-1min. Raising in SPF animal laboratory.

[0008] (2) Patients were sacrificed and dissected 7, 14, 21, and 28 days after injection. The lungs were completely removed and stained with three times their volume of Bouin's fixative for 3 hours. After staining and fixation, the number of surface metastases was counted. During the first week, almost no metastases appeared on the lung surface. From the second week onwards, a small number of metastases appeared on the lung surface, and their growth was rapid. By the fourth week, some lung surfaces were completely covered with metastases, consistent with the clinical growth pattern of melanoma metastases. (See results below) Figures 5-6 ).

[0009] (3) The stained and fixed lung tissue was embedded in paraffin, sectioned, stained with H&E, and observed under a microscope. The metastatic lesions within the lung organ exhibited typical focal or nodular patterns characteristic of clinical melanoma lung metastases, and over time, new metastatic lesions continuously increased, rather than simply increasing in tumor volume as in situ tumors. This model effectively simulates the process of tumor cell invasion and proliferation via blood circulation during tumor metastasis. (See...) Figure 7 ).

[0010] (4) Multiple modal imaging methods, such as MRI and PET-CT, were used to observe the occurrence and development of lung metastases in model mice and control mice. The imaging characteristics of lung metastases at different developmental stages were also observed. After successful model construction, MRI of the lungs in the model group showed multiple abnormal signals with slightly uneven signal distribution and unclear lesion boundaries, while the control group showed no abnormal signals. PET-CT revealed hyperbacterial activity in the lungs with uneven distribution. These differential diagnostic results are helpful in evaluating the preventive effect and efficacy of drugs and other treatments on metastases.

[0011] (5) Detection of weight changes and survival period of blank control group and model group.

[0012] Based on the above technical solution, in a first aspect, the present invention provides a method for constructing a lung metastasis model of human melanoma A375-M1 cell line, comprising the following steps:

[0013] Log-phase A375-M1 cells in PBS suspension, adjusted to a concentration of 2 × 10⁻⁶. 7 100 μL / mL was injected into the tail vein of 3-4 week old male SCID mice, and a lung metastasis model of human melanoma A375-M1 cell line was obtained after 2-3 weeks.

[0014] The present invention uses the A375-M1 cell line, which is derived from the low metastatic melanoma cell population A375 (female, 54 years old, ATCC CRL-1619) cells. The A375-M1 cell line (ATCC CRL-3222) was obtained by intravenously injecting A375 cells into nude mice.

[0015] The tail vein injection method for constructing a lung metastasis model can simulate the metastasis process of tumor cells in the human body. After being injected via the tail vein, tumor cells enter the arterial blood circulation system through the vascular network, potentially causing multiple metastatic lesions throughout the body. However, because tumor cells are relatively viscous and prone to clumping, they become trapped in the microvessels of the mouse lungs, primarily leading to lung metastases.

[0016] Furthermore, after culturing A375-M1 melanoma cells to the logarithmic growth phase, they were digested with trypsin, and the cell suspension was centrifuged and resuspended in PBS.

[0017] Furthermore, when administering the injection via the tail vein, the injection should be administered slowly, completing the injection within 0.5-1 minute.

[0018] Furthermore, the mice were examined weekly using MRI, PET-CT, H&E sections, and Bouin's fixation staining to assess lung metastasis. The results showed that a human melanoma mouse lung metastasis model was obtained after 2-3 weeks.

[0019] Furthermore, male SCID mice aged 3-4 weeks with a weight of (17±3)g were selected, and each mouse was injected via the tail vein at a concentration of 2×10⁻⁶ g. 7 100 μL of PBS suspension containing 10,000 / mL A375-M1 cells was added. Mice were examined weekly using MRI, PET-CT, H&E sections, and Bouin's fixation staining to assess lung metastasis. Results showed that a human melanoma mouse lung metastasis model was obtained after 2-3 weeks.

[0020] Magnetic Resonance Imaging (MRI) is a medical imaging technique that generates images by utilizing the signals produced by atomic nuclei in living tissue under the influence of strong magnetic fields and radio frequency pulses. MRI uses the interaction between strong magnetic fields and radio frequency pulses and the atomic nuclei in living tissue, generating detailed living images through signal detection, spatial coding, and image reconstruction. This imaging technique is widely used in medical diagnostics, providing non-invasive, high-contrast, and high-resolution structural and functional information.

[0021] Positron emission tomography-computed tomography (PET-CT) is an imaging technique that combines PET and CT. PET provides functional and metabolic information about the tumor, while CT precisely locates the tumor's anatomical position, offering advantages such as sensitivity, accuracy, specificity, and precise localization. PET-CT enables early diagnosis and differential diagnosis of tumors, tumor staging and restaging, which is beneficial for developing tumor treatment plans, evaluating treatment effects, and timely identification of tumor recurrence. 2-Fluoro-2-deoxy-D-glucose (18F-FDG) is a glucose analog that can be actively taken up and aggregated by highly metabolically active solid tumor tissues. It is a radiopharmaceutical commonly used for tumor detection, staging, assessment, and monitoring metabolism, and is currently the most commonly used imaging agent in PET-CT imaging.

[0022] A second aspect of the present invention provides a method for identifying a lung metastasis model of human melanoma A375-M1 cell line constructed using the method described above, comprising the following steps:

[0023] (A) Take control SCID mice and SCID mice that have been modeled for one, two, three and four weeks. After sacrifice, smear the lungs with Bouin's staining and perform H&E sections. Analyze and identify the results.

[0024] (B) Take control SCID mice and SCID mice that have been modeled for three weeks, and inject 100±10 μCi of fluorine

[18] deoxyglucose injection via the tail vein. 18 F-FDG), PET-CT for lung imaging analysis;

[0025] (C) Take control SCID mice and SCID mice that have been modeled for three weeks, and inject 100±10 μCi of fluorine

[18] deoxyglucose injection into the tail vein. 18 F-FDG), MRI for lung imaging analysis.

[0026] The advantages of this invention are:

[0027] 1. This invention provides a new method for constructing an animal model of melanoma lung metastases, which is different from existing human metastatic animal models. It has the advantages of short construction time, simple method and a success rate of up to 100% for metastatic tumors.

[0028] 2. The melanoma lung metastasis animal model constructed by the method described in this invention uses multiple examination methods, including PET-CT, MRI, H&E sections, and Bouin's fixation staining, to detect and identify the formation and development process of the model from multiple aspects and angles.

[0029] 3. This invention simulates the development process and pathological characteristics of lung metastasis in human melanoma, solving the problem of the lack of ideal animal models in the study of the occurrence, development, invasion and metastasis of human melanoma. It provides a good platform for screening drugs and preparations for treating lung metastasis of melanoma and plays a positive role in promoting the exploration of new strategies for treating lung metastasis of melanoma. Attached Figure Description

[0030] Figure 1 H&E sections of the lungs of BALB / C nude mice injected via tail vein for A375 melanoma, scale bar 2 mm.

[0031] Figure 2 H&E sections of the lungs of A375 melanoma mice injected with SCID via the tail vein, scale bar 2 mm.

[0032] Figure 3 H&E sections of the lungs of nude mice with A375-M1 melanoma injected via tail vein, scale bar 2 mm.

[0033] Figure 4 H&E sections of the lungs of A375-M1 melanoma mice injected with SCID via the tail vein, scale bar 2 mm.

[0034] Figure 5 Group A received tail vein injection of A375-M1 cells at a concentration of 1×10⁻⁶. 7 Experimental group, scale bar 1mm. Group B received tail vein injection of A375-M1 cells at a concentration of 2×10⁻⁶. 7 Experimental group, scale bar 1mm.

[0035] Figure 6 These are MRI images of the lung metastasis animal model of this invention. A and B are transverse and coronal MRI images of the chest of the control group SCID mice, respectively; C and D are transverse and coronal MRI images of the chest of the model group SCID mice, respectively. Multiple abnormal signal lesions are visible in C and D. Scale bar: 1 cm.

[0036] Figure 7 These are PET-CT images of the lung metastasis model of the present invention. Figure A, from left to right, shows the transverse, coronal, and sagittal sections of the chest of the control group; Figure B, from left to right, shows the transverse, coronal, and sagittal sections of the chest of the model group; Figure C, upper right, shows the transverse PET-CT image of the control group, and lower left, shows the transverse PET-CT image of the lungs of the model group.

[0037] Figure 8 This is the result of Bouin's fixative staining of the lungs of SCID mice according to the present invention. The yellow spots on the lung surface represent tumor metastases.

[0038] Figure 9This is a statistical chart showing the number of lung metastases per week after SCID mouse modeling in this invention, n=5, *** indicates P<0.001.

[0039] Figure 10 The images show the weekly H&E staining results of the lungs of SCID mice after modeling. A is the coronal plane, and B is the sagittal plane. Scale bar: 200 μm.

[0040] Figure 11 Four weeks after the SCID mouse model was established, H&E sections of major organs such as heart, liver, spleen, and kidney were collected. Group A was the normal control group; Group B was the SCID mouse model group. Scale bar: 200 μm.

[0041] Figure 12 This is a line graph showing the changes in body weight between the model group and the control group after lung transfer modeling in SCID mice according to the present invention, n=6.

[0042] Figure 13 To investigate the survival curves of the model group and the control group after SCID mouse modeling in this invention, n=6. Detailed Implementation

[0043] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0044] Example 1: Investigation of the melanoma A375 cell line

[0045] (1) Three- to four-week-old male BALB / C nude mice were selected, each weighing (17±3) g. A375 melanoma cells were cultured to the logarithmic growth phase, then digested with trypsin. The cell suspension was centrifuged and resuspended in PBS. Each mouse was inoculated via tail vein at a concentration of 2×10⁻⁶. 7 100 μL of A375 cell suspension (10,000 / mL) was added. One month later, lung tissue was harvested for H&E sections to examine lung metastasis. The section results showed that... Figure 1 The lung connective tissue was intact and clear, with no tumor metastases. A375 melanoma cells injected via tail vein did not induce tumor metastases in the lungs of BALB / c nude mice within one month (n=3).

[0046] (2) Following the method used in (1), nude mice were replaced with 3-4 week old male SCID mice, each weighing (17±3)g, to construct a lung metastasis model. One month after modeling, lung tissue was harvested for H&E sections. The lung connective tissue was intact and clear, with no tumor metastases. Figure 2 A375 melanoma cells, via tail vein induction, failed to form tumor metastases in the lungs of SCID mice within one month (n=3).

[0047] Example 2: Investigation of the melanoma A375-M1 cell line

[0048] (1) Select 3-4 week old male BALB / C nude mice, each weighing (17±3)g. After culturing A375-M1 melanoma cells to the logarithmic growth phase, digest them with trypsin. Centrifuge the cell suspension and resuspend it in PBS. Inoculate each mouse with a tail vein concentration of 2×10⁻⁶ cells. 7 100 μL of A375-M1 cell suspension (10,000 / mL) was administered. One month later, lung tissue was harvested for H&E section examination to assess lung metastasis. The sections showed intact and clear lung connective tissue with no tumor metastases. Figure 3 A375-M1 melanoma cells, via tail vein translocation, failed to form tumor metastases in the lungs of BALB / C nude mice within one month (n=3).

[0049] (2) Following the method used in (1), the nude mice were replaced with 3-4 week old male SCID mice, each weighing (17±3)g, to construct a lung metastasis model. H&E section results showed that multiple dark purple spot-like metastatic lesions appeared in the lungs. Figure 4 A375-M1 melanoma cells were successfully used to establish tumor metastases in the lungs of SCID mice within one month via tail vein induction (n=3).

[0050] Example 3: Investigation of the optimal cell concentration for constructing a melanoma lung metastasis model using the A375-M1 cell line

[0051] Male SCID mice aged 3-4 weeks, each weighing (17±3) g, were selected. A375-M1 melanoma cells were cultured to the logarithmic growth phase, digested with trypsin, and the cell suspension was centrifuged and resuspended in PBS. The mice were randomly divided into 3 groups (n=3), and each group was inoculated via tail vein with a concentration of 1×10⁻⁶ cells / mL. 7 2×10 7 3×10 7 100 μL of A375-M1 cell suspension (3 × 10⁵ / mL). 7 The mice in the 10,000 / mL injection group died within 2-5 minutes, presumably due to excessive cell concentration causing intravascular embolism. One month later, lung tissue was harvested for H&E sections to examine lung metastasis. The section results showed that... Figure 5 In the 10 million / mL group, two slides showed no significant metastatic lesions, while one showed obvious metastatic lesions (2×10⁻⁶). 7 The sections in the 10,000 / mL group showed obvious metastatic foci.

[0052] In summary, it was decided to use A375-M1 cells at a concentration of 2×10⁻⁶. 7 A melanoma lung metastasis model was established by injecting 100 μL of the solution into the tail vein of SCID mice at a concentration of 10,000 μL / mL.

[0053] Example 4: Construction of an A375-M1 melanoma lung metastasis model

[0054] Male NOD SCID mice aged 3-4 weeks, each weighing 17-20g, were selected. Logarithmic-phase A375-M1 cells were trypsinized, resuspended in pre-chilled PBS, and the concentration was adjusted to 2×10⁻⁶. 7 100 μL / mL was administered via tail vein injection to each mouse, administered slowly over 0.5–1 minute. Mice were housed in an SPF-grade animal laboratory. Lung metastasis was assessed weekly using MRI, PET-CT, H&E sections, and Bouin's fixation staining. Results showed that a human melanoma mouse lung metastasis model was established after 2–3 weeks.

[0055] Example 5:

[0056] 1. Laboratory animals, materials and equipment

[0057] Experimental animals: blank control NOD SCID mice and NOD SCID mouse model of lung metastasis of A375-M1 melanoma cells for 3 weeks;

[0058] Isoflurane: Hebei Jindafu Pharmaceutical Co., Ltd.; Fluoride

[18] deoxyglucose injection ( 18 F-FDG: Self-made by the Department of Nuclear Medicine, Fudan University Cancer Hospital;

[0059] Experimental equipment: BioSpec 70 / 30USR: Bruker, small animal anesthesia machine: Shenzhen Ruiwode.

[0060] 2. Experimental Methods

[0061] The NOD SCID mouse model of melanoma lung metastasis constructed in this invention underwent lung MRI scans as follows:

[0062] (1) Before the experiment, the mice were fasted for 12 hours. The blank control NOD SCID mice and the NOD SCID mouse model with lung metastasis of A375-M1 melanoma cells for 3 weeks were injected with 18F-FDG 110±10μCi, 100μL, via the tail vein.

[0063] (2) Before imaging, mice were anesthetized with oxygen mixed with 3% (volume fraction) isoflurane on a small animal anesthesia machine. During imaging, anesthesia with oxygen containing 1% (volume fraction) isoflurane was maintained.

[0064] (3) The mice were subjected to transverse and coronal MRI scans using an MRI imaging system. After the scans were completed, the MRI images were transferred to the workstation for measurement.

[0065] 3. Experimental Results

[0066] MRI scans showed multiple abnormal signals in the lungs of the model mice, with the arrows marking obvious metastatic lesions. The lesion boundaries were still clear (see...). Figure 6 ).

[0067] Example 6:

[0068] 1. Laboratory animals, materials and equipment

[0069] Experimental animals: blank control NOD SCID mice and NOD SCID mouse model of lung metastasis of A375-M1 melanoma cells for 3 weeks;

[0070] Isoflurane: Hebei Jindafu Pharmaceutical Co., Ltd.; Fluoride

[18] deoxyglucose injection ( 18 F-FDG: Self-made by the Department of Nuclear Medicine, Fudan University Cancer Hospital;

[0071] Experimental equipment: Siemens Inveon PET / CT, small animal anesthesia machine: Shenzhen Ruiwode.

[0072] 2. Experimental Methods

[0073] The NOD SCID mouse model of melanoma lung metastasis constructed in this invention underwent lung PET-CT scans as follows:

[0074] (1) Before the experiment, the mice were fasted for 12 hours. The blank control NOD SCID mice and the NOD SCID mouse model with lung metastasis of A375-M1 melanoma cells for 3 weeks were injected with the tail vein of each mouse. 18 F-FDG 110±10μCi, 100μL.

[0075] (2) Before imaging, mice were anesthetized with oxygen mixed with 3% (volume fraction) isoflurane on a small animal anesthesia machine. During imaging, anesthesia with oxygen containing 1% (volume fraction) isoflurane was maintained.

[0076] (3) One hour later, the mice were scanned in transverse, coronal and sagittal planes using the Siemens Inveon PET / CT system. The data were analyzed and processed using ASIPro VM software.

[0077] 3. Experimental Results

[0078] Results from a NOD SCID mouse model of melanoma lung metastases showed that, compared to the control group, PET contrast agent produced a cloudy, diffuse, and highly bright image in the lungs, with the surface of the lungs exhibiting higher brightness than the interior. This indicates that the metastatic lesions are diffusely distributed, and that there are relatively more metastatic lesions on the lung surface (see...). Figure 7CT scan results further confirmed that the lungs appeared black and without enhancement. This indicates that the metastatic lesions are small and scattered, consistent with the results of in vitro autopsy (see...). Figure 8 ).

[0079] Example 7:

[0080] 1. Laboratory animals and reagents:

[0081] Blank control NOD SCID mice and NOD SCID model mice with lung metastasis of A375-M1 melanoma cells;

[0082] Reagent: Bouin's fixative staining solution, manufacturer: Nanjing Senbeijia Biotechnology Co., Ltd.

[0083] 3. Experimental Methods

[0084] (1) The mice were euthanized by dislocation of the cervical vertebrae, and the intact lungs were dissected. After washing with physiological saline and draining, the lungs were immediately stained with 3 times the volume of Bouin's fixative for 2 hours. After staining, the lungs were washed 1-2 times with physiological saline (e.g. Figure 8 ).

[0085] (2) The number of metastatic lesions on the fixed and stained lung surface of mice was counted (e.g., Figure 9 ).

[0086] 4. Experimental Results:

[0087] After staining, metastatic lesions appear as yellow fluorescent protrusions, evenly distributed on the costal surface of the lung and within the horizontal and oblique fissures of the lung. Normal lung tissue is pale yellow or brown.

[0088] The cauldron statistics showed that after metastatic lesions were discovered in the second week, they exhibited a geometric growth trend. The number of metastatic lesions in the first, second, third, and fourth weeks were 1±1, 30±5, 212±41, and 420±55, respectively.

[0089] Example 8:

[0090] Reagents: Hematoxylin staining solution: Google Bio; Eosin staining solution: Google Bio

[0091] (1) Sampling: The heart, liver, spleen, lung and kidney of the model group at 1, 2, 3 and 4 weeks were fixed in 4% paraformaldehyde for 24 hours. The heart, liver, spleen, lung and kidney were removed from the fixative and the target organs were trimmed with a scalpel in a fume hood. The trimmed tissue and corresponding labels were placed in a dehydration box.

[0092] (2) Dehydration: Place the dehydration box into the basket and dehydrate it in the dehydrator by sequentially applying alcohol. 75% alcohol 4h - 85% alcohol 2h - 90% alcohol 2h - 95% alcohol 1h - anhydrous ethanol I 30min - anhydrous ethanol II 30min - benzene 5-10min - xylene I 5-10min - xylene II 5-10min - wax I 1h - wax II 1h - wax III 1h.

[0093] (3) Embedding: The paraffin-soaked organs are embedded in an embedding machine. First, the molten paraffin is placed into the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box and placed into the embedding frame according to the embedding surface requirements, and the corresponding label is attached. The tissue is cooled in a -20°C refrigerator. After the paraffin solidifies, the paraffin block is removed from the embedding frame and the paraffin block is trimmed.

[0094] (4) Sectioning: Place the trimmed wax block on a paraffin microtome and section it to a thickness of 4μm. Float the sections on 40℃ warm water in a slide spreader to flatten the tissue. Use a glass slide to lift the tissue and place it in a 60℃ oven to bake.

[0095] (5) Dewaxing paraffin sections to water: Place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then wash with distilled water.

[0096] (6) Hematoxylin staining of cell nuclei: Slices are stained with Harris hematoxylin for 3-8 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water.

[0097] (7) Eosin staining of cytoplasm: Immerse the section in eosin staining solution for 1-3 min.

[0098] (8) Dehydration and mounting: Immerse the sections sequentially in 95% ethanol I for 5 min, 95% ethanol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to dehydrate and clear the sections. Remove the sections from the xylene and allow them to dry slightly. Mount with neutral resin (e.g., Figure 10 , 11 ).

[0099] Cross-sectional and coronal H&E staining results of the lung metastasis model showed that the metastatic lesions gradually formed starting two weeks after modeling, exhibiting typical focal shapes and rapid spread, mostly occurring on the lung surface and near intrapulmonary blood vessels.

[0100] H&E section examination and post-dissection observation revealed no tumor foci in organs other than the lungs. The H&E section examination results further demonstrate that this method can successfully construct a human melanoma lung metastasis model within 2 weeks.

[0101] Example 9:

[0102] After modeling, the body weight of blank control NOD SCID mice and A375-M1 melanoma cell lung metastasis NOD SCID model mice was measured every 2 days (e.g. Figure 12 ).

[0103] The results showed that there was no significant difference in body weight between the control group and the model group during the first four weeks after modeling.

[0104] Example 10:

[0105] Blank control NOD SCID mice and A375-M1 melanoma cell lung metastasis NOD SCID model mice were housed under SPF conditions, and mortality was recorded (e.g., Figure 13 ).

[0106] The results showed that the model mice began to die on day 39 after tumor inoculation, and all mice in the model group died by day 51.

[0107] In summary, we successfully constructed a nude mouse lung metastasis model of melanoma and systematically investigated its characteristics. We determined the method for constructing a SCID mouse lung metastasis model by injecting A375-M1 melanoma cell suspension via tail vein, and established identification methods using in vivo MRI, PET-CT, and in vitro H&E and Bouin's assays.

[0108] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for constructing a lung metastasis model of human melanoma A375-M1 cell line, characterized in that, Includes the following steps: Male SCID mice aged 3-4 weeks with a weight of (17±3) g were selected. A375-M1 melanoma cells were cultured to the logarithmic growth phase, digested with trypsin, and the cell suspension was centrifuged and resuspended in PBS. The cells were then injected via the tail vein at a concentration of 2×10⁻⁶ cells per mouse. 7 100 μL of PBS suspension containing 10,000 / mL A375-M1 cells was added; a human melanoma mouse lung metastasis model was obtained after 2-3 weeks. The A375-M1 cells were derived from the low-metastatic melanoma cell population A375 cells, and the highly metastatic cell line A375-M1 was obtained by intravenously injecting A375 cells into nude mice.

2. The method for constructing a lung metastasis model of human melanoma A375-M1 cell line according to claim 1, characterized in that, Mice were examined for lung metastasis weekly using MRI, PET-CT, H&E sections, and Bouin's fixation staining.

3. A method for identifying a lung metastasis model of human melanoma A375-M1 cell line constructed using the construction method described in claim 1 or 2, characterized in that, Includes the following steps: (A) Control SCID mice and SCID mice modeled for one, two, three, and four weeks were taken, sacrificed, and their lungs were stained with Bouin's stain and H&E sections were prepared. The results were analyzed and identified. (B) Take control SCID mice and SCID mice that have been modeled for three weeks, inject 100±10 μCi of fluorine[18] deoxyglucose injection into the tail vein, and perform PET-CT imaging analysis of the lungs; (C) Take control SCID mice and model SCID mice three weeks after birth, inject 100±10 μCi fluoride[18] deoxyglucose injection into the tail vein, and analyze the lung imaging with MRI.

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