Method for constructing IDH mutation IV-level astrocytoma organoid

Abstract

The invention provides a method for constructing the IDH mutation level-IV astrocytoma organ, and provides a culture medium for constructing the IDH mutation level-IV astrocytoma organ, and specific components of the culture medium can realize successful construction of the IDH mutation level-IV astrocytoma organ. And a more reliable research tool is provided for the tumor microenvironment research, the tumor generation mechanism research, the drug effect screening, the development and application of a disease model and the like of the IDH mutation IV-level astrocytoma.

Description

Technical Field

[0001] The invention relates to a method for constructing IDH mutation grade IV astrocytoma organoids, and relates to the technical field of in vitro culture and detection of organoids. Background Art

[0002] Gliomas are the most common primary brain tumors caused by cancerous changes in glial cells in the brain and spinal cord. They are characterized by high recurrence rates, high disability rates, and high mortality rates. The complexity of their molecular characteristics and heterogeneity of their tissue morphology make it difficult to accurately diagnose and successfully treat the tumor. WHO CNS5 divides adult diffuse gliomas into IDH-mutant astrocytomas (WHO grades 2-4), IDH-mutant oligodendrogliomas with 1p / 19q co-deletion (WHO grades 2 and 3), and IDH-wild-type glioblastomas (WHO grade 4). Among them, IDH-mutant astrocytomas are histologically accompanied by necrosis or microvascular proliferation, or with homozygous deletion of CDKN2A / B and are classified as WHO grade 4. They have a poor prognosis, with a median survival of approximately 32 months.

[0003] In order to better study the pathogenic mechanism and treatment of brain glioma, scientists have constructed glioma organoids. Some researchers have used patient tumor tissue fragments and suspension culture to construct patient-derived glioblastoma organoids. Some researchers have used human induced pluripotent stem cells and CRISPR-Cas9 technology to knock out genes such as PTEN and TP53, thereby generating glioblastoma-like organoids. They have a three-dimensional structure, are highly similar to corresponding human tissues, and can reproduce some of the physiological functions of organs in vitro. They are a disease model with great development prospects. At present, there is no method to successfully culture IDH-mutated grade IV astrocytoma organoids.

[0004] In view of this, the present invention aims to provide a method for constructing IDH mutant grade IV astrocytoma organoids, which realizes the construction of an IDH mutant grade IV astrocytoma organoid model. This technology overcomes the shortcoming that the current brain glioma organoid model cannot cultivate IDH mutant grade IV astrocytoma organoids, and is suitable as a new generation of brain glioma organoid model. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a method for constructing IDH mutant grade IV astrocytoma organoids.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A first aspect of the present invention provides a culture medium for culturing IDH-mutated grade IV astrocytoma organoids, the culture medium comprising a basal culture medium and additives.

[0007] Furthermore, the additive includes at least one of epidermal growth factor and fibroblast growth factor.

[0008] Furthermore, the basal culture medium is selected from at least one of DMEM / F12 and Neurobasal.

[0009] Furthermore, the basal culture medium is a combination of DMEM / F12 and Neurobasal.

[0010] Furthermore, the content of DMEM / F12 and Neurobasal in the basic culture medium is 1:1.

[0011] Furthermore, the final concentration of the DMEM / F12 is 2×.

[0012] Furthermore, the final concentration of Neurobasal is 2×.

[0013] Furthermore, the additives also include nutritional supplements.

[0014] Furthermore, the nutritional supplement is selected from one or more of B-27 additives, N2 additives, GlutaMAX additives, NSC-27 additives, and G-5 additives.

[0015] Furthermore, the nutritional supplement is a combination of B-27 additive, N2 additive and GlutaMAX additive.

[0016] Furthermore, the final concentration of the B-27 additive is 50×.

[0017] Furthermore, the final concentration of the N2 additive is 100×.

[0018] Furthermore, the final concentration of the GlutaMAX additive is 100×.

[0019] Furthermore, the additives also include growth factors.

[0020] Furthermore, the growth factor is selected from one or more of insulin, epidermal growth factor, fibroblast growth factor, brain-derived neurotrophic factor, vascular endothelial growth factor, and granulocyte macrophage colony stimulating factor.

[0021] Furthermore, the growth factor is a combination of insulin, epidermal growth factor and fibroblast growth factor.

[0022] Furthermore, the final concentration of the insulin is 2.5 μg / mL.

[0023] Furthermore, the final concentration of the epidermal growth factor is 10 ng / mL.

[0024] Furthermore, the final concentration of the fibroblast growth factor is 10 ng / mL.

[0025] Furthermore, the additive also includes non-essential amino acids.

[0026] Furthermore, the non-essential amino acids include one or more of L-alanine, L-arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, and tyrosine.

[0027] Furthermore, the non-essential amino acids are MEM non-essential amino acid solution.

[0028] Furthermore, the final concentration of the MEM non-essential amino acid solution is 100×.

[0029] Furthermore, the additives also include antioxidants.

[0030] Furthermore, the antioxidant is selected from one or more of β-mercaptoethanol, glutathione, dithiothreitol, and dithioerythritol.

[0031] Furthermore, the antioxidant is β-mercaptoethanol.

[0032] Furthermore, the final concentration of the β-mercaptoethanol is 1000×.

[0033] Furthermore, the additives also include antibiotics.

[0034] Furthermore, the antibiotic is selected from one or more of penicillin, streptomycin, amphotericin B, gentamicin, kanamycin, and penicillin-streptomycin dual antibiotic.

[0035] Furthermore, the antibiotic is penicillin-streptomycin dual antibiotic.

[0036] Furthermore, the concentration of the penicillin-streptomycin dual antibody is 10000 U / mL.

[0037] Furthermore, the final concentration of the penicillin-streptomycin dual antibody is 100×.

[0038] The second aspect of the present invention provides a method for constructing IDH mutant grade IV astrocytoma organoids, the method comprising culturing pre-treated IDH mutant grade IV astrocytoma tissue with the culture medium described in the first aspect of the present invention.

[0039] Furthermore, the culturing refers to culturing in a culture dish.

[0040] Furthermore, the culture dish is a low-adsorption culture dish.

[0041] Furthermore, the pre-treatment step includes removing impurities from the IDH mutant grade IV astrocytoma specimen and then cutting it into pieces.

[0042] Furthermore, the cutting and shredding refers to cutting the specimen into pieces with a diameter of 0.5-1.0 mm.

[0043] Furthermore, the impurities refer to vascular tissue and / or necrotic tissue.

[0044] Furthermore, the culture conditions are 37° C., 5% CO 2 , and a shaker at 120 rpm / min.

[0045] The third aspect of the present invention provides an organoid of an IDH-mutated grade IV astrocytoma cultured by the method described in the second aspect of the present invention.

[0046] The fourth aspect of the present invention provides a model for screening IDH mutation grade IV astrocytoma therapeutic drugs, predicting the sensitivity of a subject to IDH mutation grade IV astrocytoma drug or predicting the responsiveness of a subject to IDH mutation grade IV astrocytoma treatment methods, wherein the model comprises the organoid described in the third aspect of the present invention.

[0047] The fifth aspect of the present invention provides a kit for culturing IDH-mutated grade IV astrocytoma, the kit comprising the culture medium described in the first aspect of the present invention.

[0048] The sixth aspect of the present invention provides the use of the culture medium described in the first aspect of the present invention, the organoid described in the third aspect of the present invention, the model described in the fourth aspect of the present invention and / or the kit described in the fifth aspect of the present invention, wherein the use includes use in preparing a product.

[0049] Furthermore, the products include: products for screening IDH mutation grade IV astrocytoma therapeutic drugs, products for predicting the sensitivity of subjects to IDH mutation grade IV astrocytoma drug, or products for predicting the responsiveness of subjects to IDH mutation grade IV astrocytoma treatment methods.

[0050] The seventh aspect of the present invention provides a method for screening IDH mutation grade IV astrocytoma therapeutic drugs, predicting the sensitivity of a subject to IDH mutation grade IV astrocytoma drug, or predicting the responsiveness of a subject to IDH mutation grade IV astrocytoma treatment methods, the method comprising any of the following: 1) Treating the model described in the fourth aspect of the present invention with the substance to be screened. If the activity of the model is inhibited or apoptosis occurs, the substance to be screened is a candidate therapeutic drug; if the activity of the model is not inhibited, the substance to be screened is not a candidate therapeutic drug.

[0051] Furthermore, the substances to be screened include biological drugs, traditional Chinese medicines, small molecule compounds or pharmaceutically acceptable salts thereof.

[0052] Furthermore, the small molecule compound can be selected from any compound database, including a newly synthesized database or an existing database.

[0053] Further, the pharmaceutically acceptable salt refers to a small molecule compound prepared by reacting with a suitable organic or inorganic acid or acid derivative. Pharmaceutically acceptable salts include, but are not limited to, hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, benzenesulfonate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate, nitrate, sodium salt, calcium salt, potassium salt, zinc salt and meglumine salt.

[0054] In some embodiments, the method further includes: further testing the candidate therapeutic drug obtained in the above steps, wherein the test includes testing its effect in treating secondary glioblastoma. If the tested candidate therapeutic drug has a significant inhibitory effect on secondary glioblastoma, it means that the candidate therapeutic drug is a candidate therapeutic drug for secondary glioblastoma.

[0055] 2) The model described in the fourth aspect of the present invention is treated with a therapeutic drug for IDH-mutated grade IV astrocytoma. If the activity of the model is inhibited or apoptosis occurs, the subject is sensitive to the drug; if the activity of the model is not inhibited, the subject is insensitive to the drug.

[0056] Furthermore, the therapeutic drugs for IDH-mutated grade IV astrocytoma are drugs known to those skilled in the art that can be used to treat IDH-mutated grade IV astrocytoma, including but not limited to small molecule targeted tumor drugs, immunotherapy drugs and / or macromolecular antibody drugs.

[0057] 3) Treating the model described in the fourth aspect of the present invention with the IDH-mutated grade IV astrocytoma treatment method; if the activity of the model is inhibited or apoptosis occurs, the subject is sensitive to the treatment method; if the activity of the model is not inhibited, the subject is insensitive to the treatment method.

[0058] Furthermore, the treatment methods include radiotherapy, chemotherapy, tumor treating electric fields, proton beam therapy, surgical therapy, and immunotherapy.

[0059] Advantages and beneficial effects of the present invention: The organoid culture medium and culture method provided by the present invention can successfully construct organoids of IDH mutant grade IV astrocytoma, providing a more reliable research tool that is widely used in many aspects such as tumor microenvironment research, tumor occurrence mechanism research, drug efficacy screening, development and application of disease models, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a microscopic view of an organoid after 10 days of culture in the medium provided by the present invention; Figure 2 This is a statistical graph of organoid activity after 10 days of culture in the culture medium provided by the present invention; Figure 3 This is a microscope view of the organoids after 10 days of culture in the three culture media in the comparative example. Figure 3 A in the figure is the culture result of comparative culture medium A. Figure 3 B in the figure is the culture result of comparative culture medium B. Figure 3 C in the figure is the culture result of comparative culture medium C; Figure 4 This is a statistical chart of organoid activity after 10 days of culture in group A culture medium in the comparative example. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0062] In the present invention, IDH refers to isocitrate dehydrogenase. IDH mutation means that the tumor cells of astrocytoma contain an altered or "mutated" IDH gene, so that the tumor cells do not produce IDH protein. IDH mutations are present in low-grade gliomas, secondary malignant gliomas, melanomas, angioimmunoblastic T-cell lymphomas, myeloproliferative tumors, myelodysplastic syndromes (MDS), and acute myelocytic leukemia (AML). All central nervous system tumors are given grades I to IV based on the degree of similarity between the appearance and behavior of the tumor cells and the cells commonly found in the central nervous system. The grading system used by most pathologists is called the WHO grade. Detecting the presence of IDH mutations in the tumor can help distinguish astrocytomas from other brain and spinal cord tumors and help decide on subsequent treatment strategies.

[0063] In the present invention, the compound database includes a newly synthesized database and an existing database, wherein the existing database includes but is not limited to a general natural product database (COCONUT, Super Natural II, NPASS), a plant natural product database (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), a Chinese medicine natural product database (TCM@Taiwan, CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), a microbial natural product database (StreptomeDB, NP Altas, ProCarDB, PAMDB, Lichen Database), a marine natural product database (MNPD, SWMD), a natural product database of different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB), a food natural product database (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet database), a toxic natural product database (Exposome-Explorer, T3DB, Snake Neurotoxin Database, TPPT), natural product industry catalogs (Greenpharma, AnalytiConDiscovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol), databases that use MS data for deduplication (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), databases that use NMR data for deduplication (NMRShiftDB, NAPROC-13), etc.

[0064] In the present invention, "therapeutic drugs" and "therapeutic methods" as used herein include alleviating, inhibiting or improving the symptoms or conditions of a disease; inhibiting the occurrence of complications; improving or preventing potential metabolic syndrome; inhibiting the occurrence of a disease or symptom, such as controlling the development of a disease or condition; alleviating a disease or symptom; reducing a disease or symptom; alleviating complications caused by a disease or symptom, or preventing or treating signs caused by a disease or symptom. As used herein, a compound or pharmaceutical composition, after administration, can improve a disease, symptom or condition, especially improve its severity, delay the onset, slow the progression of the disease, or reduce the duration of the disease. Whether fixed or temporary administration, continuous administration or intermittent administration, can be attributed to or related to the administration.

[0065] Example 1 Construction of IDH-mutated grade IV astrocytoma organoids 1. Culture medium configuration 1. Basic culture medium: DMEM / F12:Neurobasal = 1:1.

[0066] 2. Additives: B-27 additive 50×, N2 additive 100×, MEM non-essential amino acid solution 100×, GlutaMAX additive 100×, penicillin-streptomycin (10,000 U / mL) 100×, insulin 2.5 μg / mL, β-mercaptoethanol 1000×, EGF 10 ng / mL, FGF 10 ng / mL.

[0067] 2. Cultivation Methods 1. Fresh ex vivo IDH-mutated grade IV astrocytoma specimens obtained through craniotomy (intraoperative frozen pathology suggests a high possibility of glioma, and postoperative pathology confirms IDH-mutated grade IV astrocytoma) were placed in pre-cooled brain glioma preservation solution and transported to the laboratory in an ice bath and incubator box for no more than 4 hours. Informed consent has been obtained from relevant personnel for specimen acquisition.

[0068] 2. Pour the specimen into a large dish on a clean bench under sterile operation.

[0069] 3. Use surgical blades, high-pressure steam sterilized microscissors and microtweezers to remove the red vascular tissue of the sample and the black necrotic tissue produced by surgical electrocoagulation. Carefully cut the specimen into fragments with a diameter of about 0.5-1.0 mm. Transfer the fragments to a 15 mL centrifuge tube, add 10 mL DPBS to mix thoroughly, let it stand for 1 minute, remove the less active tissue and impurities in the supernatant, and repeat the washing 3 times.

[0070] 4. After removing the supernatant, add 5 mL of 1× RBC lysis buffer to remove residual red blood cells. During the removal process, place the centrifuge tube on a shaker in a 37°C, 5% CO2 incubator with the shaker speed set to 120 rpm / min for 10 minutes.

[0071] 5. After the cleaning is completed, let the centrifuge tube stand for about 1 minute. The chopped tissue fragments will settle at the bottom. Aspirate the supernatant, add 10 mL of DPBS to mix thoroughly, let it stand to remove the supernatant, repeat the washing twice, add 10 mL of culture medium, let it stand for 1 minute, and remove the supernatant.

[0072] 6. Transfer the above organoids to low-adsorption six-well plates, with about 50-60 organoids per six-well plate, and add 2 mL of culture medium to each six-well plate.

[0073] 7. Place the low-adsorption six-well plate containing organoids and culture medium on a shaker in a 37°C, 5% CO2 incubator. Set the shaker speed to 120 rpm / min. Change the culture medium every 2 to 3 days according to the color change of the culture medium. Replace 2 / 3 of the culture medium each time. Observe the growth status of the organoids every day, take photos to record the growth rate and morphological changes.

[0074] 8. Place the culture plate at 2-8°C or on ice, incubate for 30-90 minutes, and shake gently. Add PBS to wash 2-3 times, place in a 15 ml centrifuge tube and let stand for 1-2 minutes. After the organoids have settled, add an appropriate amount of trypsin to the 15 ml centrifuge tube and digest at 37°C for 10 minutes. After the organoids are digested into single cells, use a 70 μm sieve to remove incompletely digested organoids, leaving the cell suspension, centrifuge at 1000 r for 5 min, remove the supernatant; add 2 ml PBS to resuspend, take 20 μl of the cell suspension, add an equal amount of AOPI dye solution and mix well, count and analyze the cell activity using a cell counter.

[0075] 3. Cultivation Results 1. According to the comparative example and the culture method provided by the present invention, the organoids were cultured for 10 days. The culture medium used in the comparative example is shown in Table 1.

[0076] Table 1. Comparative Example Three Culture Medium Compositions

[0077] 2. Growth status comparison results are as follows Figure 1 , Figure 3 As shown, it can be seen that the organoids cultured using the culture medium and method described in the embodiments of the present invention are round in shape, and the surrounding proliferating organoid tissue tentacles are more numerous and dense, indicating that they are in good growth condition.

[0078] 3. Cell activity comparison results are as follows Figure 2 , Figure 4 As shown in Table 2, since the culture results of the three culture media in the comparative example are the same or similar, one of them is selected for comparison. Figure 2 It can be seen that the organoids cultured by the method described in the embodiment of the present invention have good activity and uniform distribution. From Table 2, it can be seen that the cell viability of the organoids cultured by the method described in the embodiment of the present invention is much higher than that of the organoids cultured by the method described in the comparative example, and the roundness and agglomeration rate are also higher than those of the comparative example.

[0079] Table 2. Cell activity comparison table

[0080] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A culture medium for culturing IDH-mutated grade IV astrocytoma organoids, characterized in that: The culture medium comprises a basic culture medium and additives; Preferably, the additive includes at least one of epidermal growth factor and fibroblast growth factor.

2. The culture medium according to claim 1, characterized in that The basal culture medium is selected from at least one of DMEM / F12 and Neurobasal; Preferably, the basal medium is a combination of DMEM / F12 and Neurobasal; Preferably, the content of DMEM / F12 and Neurobasal in the basal medium is 1:1; Preferably, the final concentration of the DMEM / F12 is 2×; Preferably, the final concentration of Neurobasal is 2×.

3. The culture medium according to claim 1, characterized in that The additives also include nutritional supplements; Preferably, the nutritional supplement is selected from one or more of B-27 additive, N2 additive, GlutaMAX additive, NSC-27 additive, G-5 additive; Preferably, the nutritional supplement is a combination of B-27 additive, N2 additive, and GlutaMAX additive; Preferably, the final concentration of the B-27 additive is 50×; Preferably, the final concentration of the N2 additive is 100×; Preferably, the final concentration of the GlutaMAX additive is 100×; Preferably, the additive further comprises a growth factor; Preferably, the growth factor is selected from one or more of insulin, epidermal growth factor, fibroblast growth factor, brain-derived neurotrophic factor, vascular endothelial growth factor, and granulocyte macrophage colony stimulating factor; Preferably, the growth factor is a combination of insulin, epidermal growth factor, and fibroblast growth factor; Preferably, the final concentration of the insulin is 2.5 μg / mL; Preferably, the final concentration of the epidermal growth factor is 10 ng / mL; Preferably, the final concentration of the fibroblast growth factor is 10 ng / mL; Preferably, the additive further comprises non-essential amino acids; Preferably, the non-essential amino acids include one or more of L-alanine, L-arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, and tyrosine; Preferably, the non-essential amino acids are MEM non-essential amino acid solution; Preferably, the final concentration of the MEM non-essential amino acid solution is 100×; Preferably, the additive further comprises an antioxidant; Preferably, the antioxidant is selected from one or more of β-mercaptoethanol, glutathione, dithiothreitol, and dithioerythritol; Preferably, the antioxidant is β-mercaptoethanol; Preferably, the final concentration of β-mercaptoethanol is 1000×; Preferably, the additive further comprises antibiotics; Preferably, the antibiotic is selected from one or more of penicillin, streptomycin, amphotericin B, gentamicin, kanamycin, and penicillin-streptomycin dual antibiotic; Preferably, the antibiotic is penicillin-streptomycin dual antibiotic; Preferably, the concentration of the penicillin-streptomycin dual antibody is 10000 U / mL; Preferably, the final concentration of the penicillin-streptomycin dual antibody is 100×.

4. A method for constructing IDH mutant grade IV astrocytoma organoids, characterized in that: The method comprises culturing the pre-treated IDH-mutated grade IV astrocytoma tissue with the culture medium according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The culturing refers to culturing in a culture dish; Preferably, the culture dish is a low-adsorption culture dish; Preferably, the pre-treatment step comprises removing impurities from the IDH mutation grade IV astrocytoma specimen and then cutting it into pieces; Preferably, the cutting is to cut the specimen into pieces with a diameter of 0.5-1.0 mm; Preferably, the impurities are vascular tissue and / or necrotic tissue; Preferably, the culture conditions are 37° C., 5% CO 2 , and a shaker at 120 rpm / min.

6. Organoids of IDH-mutated grade IV astrocytoma cultured according to the method of claim 4 or 5.

7. A model for screening IDH mutation grade IV astrocytoma therapeutic drugs, predicting the sensitivity of subjects to IDH mutation grade IV astrocytoma drugs, or predicting the responsiveness of subjects to IDH mutation grade IV astrocytoma treatment methods, characterized in that: The model comprises the organoid of claim 6.

8. A kit for culturing IDH-mutated grade IV astrocytoma, characterized in that: The kit comprises the culture medium according to any one of claims 1 to 3.

9. Use of the culture medium according to any one of claims 1 to 3, the organoid according to claim 6, the model according to claim 7 and / or the kit according to claim 8, characterized in that: The application includes application in preparing products; Preferably, the products include: products for screening therapeutic drugs for IDH-mutated grade IV astrocytoma, products for predicting the sensitivity of a subject to a drug for IDH-mutated grade IV astrocytoma, or products for predicting the responsiveness of a subject to a therapeutic method for IDH-mutated grade IV astrocytoma.

10. A method for screening therapeutic drugs for IDH-mutated grade IV astrocytoma, predicting the drug sensitivity of a subject to IDH-mutated grade IV astrocytoma, or predicting the responsiveness of a subject to a therapeutic method for IDH-mutated grade IV astrocytoma, characterized in that: The method includes any of the following: 1) treating the model described in claim 7 with the substance to be screened, and if the activity of the model is inhibited or apoptosis occurs, the substance to be screened is a candidate therapeutic drug; if the activity of the model is not inhibited, the substance to be screened is not a candidate therapeutic drug; 2) treating the model described in claim 7 with a therapeutic drug for IDH-mutated grade IV astrocytoma; if the activity of the model is inhibited or apoptosis occurs, the subject is sensitive to the drug; if the activity of the model is not inhibited, the subject is insensitive to the drug; 3) Treating the model described in claim 7 with the treatment method for IDH-mutated grade IV astrocytoma; if the activity of the model is inhibited or apoptosis occurs, the subject is sensitive to the treatment method; if the activity of the model is not inhibited, the subject is insensitive to the treatment method.

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