Method for constructing secondary glioblastoma organoid

Abstract

The invention provides the method for constructing the secondary glioblastoma organ, so that the secondary glioblastoma organ is successfully constructed, and the defect that the secondary glioblastoma organ cannot be cultured by the current brain glioblastoma organ model is overcome; a research tool closer to the original state of glioma is provided for research on secondary glioblastoma of human brain, and a more reliable research tool is provided for tumor microenvironment research, tumor generation mechanism research, drug effect screening, development and application of disease models and the like of secondary glioblastoma.

Description

Technical Field

[0001] The present invention relates to a method for constructing secondary glioblastoma organoids, and relates to the technical field of in vitro culture and detection of organoids. Background Art

[0002] Glioblastoma (GBM) is the most common malignant primary intracranial tumor, including primary glioblastoma that progresses rapidly after a brief clinical history and without evidence of less malignant precursor lesions, and secondary glioblastoma that progresses slowly from low-grade diffuse or anaplastic astrocytomas. The diagnosis of secondary glioblastoma requires histological evidence of a previous low-grade or anaplastic astrocytoma. In the population, only about 5% of cases are classified as secondary glioblastoma. However, it cannot be ruled out that some secondary glioblastomas progress rapidly from less malignant precursor lesions, escape clinical diagnosis, and are misclassified as primary glioblastoma. The distinction between primary and secondary glioblastoma may not affect recent treatment management decisions. However, it is a crucial factor in clinical trials, and these glioblastoma subtypes may also have significant differences in their response to treatment due to their different genetic profiles.

[0003] So far, some glioblastoma organoid models have been established. 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. Since low-grade gliomas are difficult to cure with existing treatments, tumors are often prone to recurrence and grade progression and then progress to secondary glioblastoma. There is currently no good research model for secondary glioblastoma. Secondary glioblastomas are less malignant and have poorer proliferation ability than primary glioblastomas. Currently, there is no method to successfully culture secondary glioblastoma organoids.

[0004] In view of this, the present invention aims to provide a method for constructing secondary glioblastoma organoids, which can successfully construct secondary glioblastoma organoids and provide a research tool that is closer to the original state of glioma for the study of secondary glioblastoma in the human brain. 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 secondary glioblastoma 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 secondary glioblastoma organoids, the culture medium comprising a basal culture medium and additives, wherein the basal culture medium is selected from at least one of DMEM / F12 and Neurobasal; the additives comprise at least one of a supplement, a non-essential amino acid, a growth factor, an antibiotic, and a reducing agent.

[0007] Furthermore, the additives include at least one of sugars and buffers.

[0008] Furthermore, the basal culture medium is a combination of DMEM / F12 and Neurobasal.

[0009] Furthermore, the content of DMEM / F12 and Neurobasal in the basic culture medium is 1:1.

[0010] Furthermore, the final concentration of the DMEM / F12 is 2×.

[0011] Furthermore, the final concentration of Neurobasal is 2×.

[0012] Furthermore, the supplement is selected from one or more of B-27 additives, N2 additives, GlutaMAX additives, NSC-27 additives, and G-5 additives.

[0013] Furthermore, the supplement is a combination of B-27 additive, N2 additive, and GlutaMAX additive.

[0014] Furthermore, the final concentration of the B-27 additive is 50×.

[0015] Furthermore, the final concentration of the N2 additive is 100×.

[0016] Furthermore, the final concentration of the GlutaMAX additive is 100×.

[0017] Furthermore, the non-essential amino acids include one or more of glycine, L-alanine, L-aspartic acid, L-asparagine, L-proline, L-serine, and L-glutamic acid.

[0018] Furthermore, the non-essential amino acids are MEM non-essential amino acid solution.

[0019] Furthermore, the final concentration of the MEM non-essential amino acid solution is 100×.

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

[0021] Furthermore, the growth factor is insulin.

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

[0023] Furthermore, the antibiotic is selected from one or more of penicillin, streptomycin and amphotericin D.

[0024] Furthermore, the antibiotic is penicillin-streptomycin.

[0025] Furthermore, the concentration of penicillin-streptomycin is 10000 U / mL.

[0026] Furthermore, the final concentration of penicillin-streptomycin is 100×.

[0027] Further, the reducing agent is β-mercaptoethanol.

[0028] Furthermore, the final concentration of the β-mercaptoethanol is 1000×.

[0029] Furthermore, the sugar is selected from one or more of heparin and heparin sulfate.

[0030] Furthermore, the carbohydrate is heparin sulfate.

[0031] Furthermore, the final concentration of the heparin sulfate is 2 mg / mL.

[0032] Furthermore, the buffer is Hepes.

[0033] Furthermore, the final concentration of Hepes is 10 mM.

[0034] The second aspect of the present invention provides a kit for constructing secondary glioblastoma organoids, the kit comprising the culture medium described in the first aspect of the present invention.

[0035] The third aspect of the present invention provides a method for constructing secondary glioblastoma organoids, the method comprising placing the secondary glioblastoma tissue to be cultured in the culture medium described in the first aspect of the present invention for culture.

[0036] Furthermore, the secondary glioblastoma tissue to be cultured is a pre-treated tissue.

[0037] Furthermore, the pretreatment refers to removing excess tissue from the secondary glioblastoma specimen and then chopping it into pieces to remove residual red blood cells to obtain the secondary glioblastoma tissue to be cultured.

[0038] Furthermore, the redundant tissue refers to vascular tissue and / or necrotic tissue.

[0039] Furthermore, the cutting and mincing refers to cutting the secondary glioblastoma specimen into fragments with a diameter of 0.5-1.0 mm.

[0040] Furthermore, the removal of residual red blood cells is performed using a red blood cell lysing solution.

[0041] Furthermore, the culturing refers to culturing in a culture dish.

[0042] Furthermore, the culture dish is a low-adsorption culture dish.

[0043] Furthermore, the culture conditions are 37° C., 5% CO 2 , and a shaker at 120 rpm / min.

[0044] Further, the specimen or sample refers to a composition obtained from or derived from a target subject, which contains cell entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological characteristics. The sample can be obtained from the subject's blood and other fluid samples of biological origin and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples, biopsy tissues or aspirates; blood or any blood component; body fluids; cells from any time of individual pregnancy or development; or plasma. The term sample includes biological samples that have been processed in any way after they are obtained, such as reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes.

[0045] The fourth aspect of the present invention provides secondary glioblastoma organoids obtained by culturing using the method described in the third aspect of the present invention.

[0046] The fifth aspect of the present invention provides a use of the culture medium according to the first aspect of the present invention, the kit according to the second aspect of the present invention, and / or the organoid according to the fourth aspect of the present invention, wherein the use comprises the following: 1) Application in screening therapeutic drugs for secondary glioblastoma or preparing products for treating secondary glioblastoma; 2) Application in predicting the sensitivity of a subject to a drug for treating secondary glioblastoma or in preparing a product for predicting the sensitivity of a subject to a drug for treating secondary glioblastoma; 3) Use in detecting the responsiveness of a subject to a secondary glioblastoma therapy or in preparing a product for detecting the responsiveness of a subject to a secondary glioblastoma therapy.

[0047] The sixth aspect of the present invention provides a method for screening drugs for the treatment of secondary glioblastoma, the steps of the method comprising: using the organoid described in the fourth aspect of the present invention as a model, detecting the inhibition rate of the substance to be screened on the model, so as to evaluate whether the substance to be screened is a candidate drug for the treatment of secondary glioblastoma.

[0048] In the present invention, screening for therapeutic drugs for secondary glioblastoma refers to treating the secondary glioblastoma organoids provided by the present invention with the substance to be screened. If the activity of the secondary glioblastoma organoids is inhibited or apoptotic, the substance to be screened is a candidate therapeutic drug for secondary glioblastoma; if the activity of the secondary glioblastoma organoids is not inhibited, the substance to be screened is not a candidate therapeutic drug for secondary glioblastoma. The substance to be screened refers to a substance that can be used as a drug, including but not limited to: biological drugs, traditional Chinese medicines, small molecule compounds or pharmaceutically acceptable salts thereof, etc.

[0049] Furthermore, the source of the small molecule compound is selected from: newly synthesized or existing databases; wherein the existing databases include but are not limited to general natural product databases (COCONUT, Super Natural II, NPASS), plant natural product databases (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), Chinese medicine natural product databases (TCM@Taiwan, CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), microbial natural product databases (StreptomeDB, NP Altas, ProCarDB, PAMDB, Lichen Database), marine natural product databases (MNPD, SWMD), natural product databases of different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB), food natural product databases (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet database), toxic natural product databases (Exposome-Explorer, T3DB, Snake Neurotoxin Database, TPPT), natural product industry catalogs (Greenpharma, AnalytiConDiscovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol), databases for deduplicating MS data (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), databases for deduplicating NMR data (NMRShiftDB, NAPROC-13), etc.

[0050] 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.

[0051] The seventh aspect of the present invention provides a method for predicting the sensitivity of a subject to a drug for treating secondary glioblastoma, the method comprising: using the organoid described in the fourth aspect of the present invention as a model, detecting the inhibition rate of the drug for treating secondary glioblastoma on the model, so as to evaluate the sensitivity of the subject to the drug for treating secondary glioblastoma.

[0052] Furthermore, predicting the sensitivity of the subject to the therapeutic drug for secondary glioblastoma refers to treating the secondary glioblastoma organoids derived from the subject with the therapeutic drug for secondary glioblastoma. If the activity of the secondary glioblastoma organoids is inhibited or apoptotic, the subject is sensitive to the therapeutic drug; if the activity of the secondary glioblastoma organoids is not inhibited, the subject is insensitive to the therapeutic drug.

[0053] Furthermore, the secondary glioblastoma treatment drugs are drugs known to those skilled in the art that can be used to treat secondary glioblastoma, including imidazole tetrazinones, nitrosoureas, procarbazine, platinums, vinblastines and camptothecins, as well as small molecule targeted tumor drugs, immunotherapy drugs and / or macromolecular antibody drugs.

[0054] In the present invention, sensitivity refers to the responsiveness of the subject or the secondary glioblastoma organoids derived from the subject to the drug. The data that can reflect the sensitivity or responsiveness may be obtained in an in vitro environment or in an in vivo environment, and the data can be quantified. Any data that can reflect the degree of drug sensitivity can be used in the present invention.

[0055] The eighth aspect of the present invention provides a method for detecting the responsiveness of a subject to secondary glioblastoma therapy, the steps of the method comprising: using the organoid described in the fourth aspect of the present invention as a model, detecting the inhibition rate of the secondary glioblastoma therapy on the model to evaluate the responsiveness of the subject to the secondary glioblastoma therapy.

[0056] In the present invention, detecting the responsiveness of a subject to a secondary glioblastoma therapy refers to treating the secondary glioblastoma organoids derived from the subject with a therapy method. If the activity of the secondary glioblastoma organoids is inhibited or apoptotic, the subject has a good responsiveness to the therapy; if the activity of the secondary glioblastoma organoids is not inhibited, the subject has a poor responsiveness to the therapy. Therapeutic methods include radiotherapy, chemotherapy, tumor treatment electric fields, proton beam therapy, surgical therapy, and immunotherapy.

[0057] The subject or experimenter described in the present invention refers to any animal, including humans and non-human animals. The term non-human animals includes all vertebrates, for example, mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a specific embodiment of the present invention, the subject or experimenter is preferably a human.

[0058] Advantages and beneficial effects of the present invention: The organoid culture method provided by the present invention realizes the construction of secondary glioblastoma organoids, overcomes the shortcoming that the current brain glioma organoid model cannot culture secondary glioblastoma organoids, and provides a research tool that is closer to the original state of glioma for the study of secondary glioblastoma in the human brain, and provides a more reliable research tool for the tumor microenvironment research, tumor occurrence mechanism research, drug efficacy screening, disease model development and application of secondary glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] 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

[0060] 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.

[0061] Example 1 Construction of secondary glioblastoma organoids 1. Culture medium configuration 1. Basic culture medium: DMEM / F12:Neurobasal = 1:1.

[0062] 2. Additives: B27 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×, heparin sulfate 2 mg / mL, Hepes 10 mM.

[0063] 2. Cultivation Methods 1. Fresh ex vivo secondary glioblastoma specimens obtained through craniotomy (the patient was undergoing the second surgery, the pathological result of the first surgery was a low-grade glioma, intraoperative frozen pathology suggested a high possibility of glioma, and postoperative pathology confirmed secondary glioblastoma) 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 of relevant personnel had been obtained for specimen acquisition.

[0064] 2. Pour the specimen into a large dish on a clean bench under sterile operation.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Table 1. Comparative Example Three Culture Medium Compositions

[0073] 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.

[0074] 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, Group A 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.

[0075] Table 2. Cell activity comparison table

[0076] 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 secondary glioblastoma organoids, characterized in that: The culture medium comprises a basic culture medium and additives, wherein: The basal culture medium is selected from at least one of DMEM / F12 and Neurobasal; The additive comprises at least one of a supplement, a non-essential amino acid, a growth factor, an antibiotic, and a reducing agent; Preferably, the additive further comprises at least one of a sugar and a buffer.

2. The culture medium according to claim 1, characterized in that The basal culture 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×; Preferably, the supplement is selected from one or more of B-27 additive, N2 additive, GlutaMAX additive, NSC-27 additive, G-5 additive; Preferably, the supplement is a combination of B-27 supplement, N2 supplement, and GlutaMAX supplement; 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 non-essential amino acids include one or more of glycine, L-alanine, L-aspartic acid, L-asparagine, L-proline, L-serine, and L-glutamic acid; 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 growth factor comprises one or more of insulin, epidermal growth factor, basic fibroblast growth factor, brain-derived neurotrophic factor, vascular endothelial growth factor, and granulocyte macrophage colony stimulating factor; Preferably, the growth factor is insulin; Preferably, the final concentration of the insulin is 2.5 μg / mL; Preferably, the antibiotic is selected from one or more of penicillin, streptomycin, and amphotericin D; Preferably, the antibiotic is penicillin-streptomycin; Preferably, the concentration of penicillin-streptomycin is 10000 U / mL; Preferably, the final concentration of penicillin-streptomycin is 100×; Preferably, the reducing agent is β-mercaptoethanol; Preferably, the final concentration of the β-mercaptoethanol is 1000×; Preferably, the carbohydrate is selected from one or more of heparin and heparin sulfate; Preferably, the carbohydrate is heparin sulfate; Preferably, the final concentration of the heparin sulfate is 2 mg / mL; Preferably, the buffer is Hepes; Preferably, the final concentration of Hepes is 10 mM.

3. A kit for constructing secondary glioblastoma organoids, characterized in that: The kit comprises the culture medium according to any one of claims 1 to 2.

4. A method for constructing secondary glioblastoma organoids, characterized in that: The method comprises placing the secondary glioblastoma tissue to be cultured in the culture medium according to any one of claims 1-2 for culture.

5. The method according to claim 4, characterized in that The secondary glioblastoma tissue to be cultured is a pre-treated tissue; Preferably, the pretreatment refers to removing excess tissue from the secondary glioblastoma specimen and then chopping it into pieces to remove residual red blood cells, so as to obtain the secondary glioblastoma tissue to be cultured; Preferably, the redundant tissue refers to vascular tissue and / or necrotic tissue; Preferably, the cutting and shredding refers to cutting the secondary glioblastoma specimen into fragments with a diameter of 0.5-1.0 mm; Preferably, the residual red blood cells are removed by using a red blood cell lysing solution. Preferably, the culturing refers to culturing in a culture dish; Preferably, the culture dish is a low-adsorption culture dish; Preferably, the culture conditions are 37° C., 5% CO 2 , and a shaker at 120 rpm / min.

6. Secondary glioblastoma organoids obtained by culturing using the method described in any one of claims 4-5.

7. Use of the culture medium according to any one of claims 1 to 2, the kit according to claim 3 and / or the organoid according to claim 6, characterized in that: The applications include the following: 1) Application in screening therapeutic drugs for secondary glioblastoma or preparing products for treating secondary glioblastoma; 2) Application in predicting the sensitivity of a subject to a drug for treating secondary glioblastoma or in preparing a product for predicting the sensitivity of a subject to a drug for treating secondary glioblastoma; 3) Use in detecting the responsiveness of a subject to a secondary glioblastoma therapy or in preparing a product for detecting the responsiveness of a subject to a secondary glioblastoma therapy.

8. A method for screening a drug for treating secondary glioblastoma, the method comprising the steps of: Using the organoid described in claim 6 as a model, the inhibition rate of the substance to be screened on the model is detected to evaluate whether the substance to be screened is a candidate therapeutic drug for secondary glioblastoma.

9. A method for predicting the sensitivity of a subject to a drug for treating secondary glioblastoma, the method comprising the steps of: Using the organoid described in claim 6 as a model, the inhibition rate of the secondary glioblastoma treatment drug on the model is detected to evaluate the sensitivity of the subject to the secondary glioblastoma treatment drug.

10. A method for detecting the responsiveness of a subject to a secondary glioblastoma therapy, the method comprising: Using the organoid described in claim 6 as a model, the inhibition rate of the model by secondary glioblastoma therapy is detected to evaluate the responsiveness of the subject to the secondary glioblastoma therapy.

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