Drug screening method for intrathecal chemotherapy of diffuse midline glioma and application

Abstract

The invention provides a drug screening method for intrathecal chemotherapy of diffuse midline glioma, which comprises the following steps: (1) acquiring a diffuse midline glioma sample, and preparing a cell line and an organoid by using the diffuse midline glioma sample; (2) performing drug screening by using the cell line obtained in the step (1); and performing drug screening by using the organoid obtained in the step (1). And (3) verifying the effect of the medicine screened in the step (2) by using high-content imaging. In screening, a specially constructed intrathecal chemotherapy anticancer drug compound library is used, screening is carried out on two levels of a cell line and an organoid, and verification is carried out by using a high-content technology. By means of the method, an innovative treatment scheme of triple intrathecal injection is screened, and a new choice is provided for drug screening of diffuse midline glioma and even drug screening of other central nervous system tumors.

Description

Field of the Invention

[0001] The present application belongs to the field of tumor treatment. Specifically, the present application provides a drug screening method and application for intrathecal chemotherapy of diffuse midline glioma. Background Art

[0002] Brainstem gliomas are the leading cause of brain tumors in children, particularly diffuse midline gliomas with H3K27M mutations, which carry an extremely poor prognosis. Currently, there is a lack of effective treatments for these tumors, and no effective chemotherapy drugs have entered clinical use. A strong and intact blood-brain barrier prevents drugs from reaching the primary tumor site. Furthermore, some patients with diffuse midline gliomas develop central nervous system metastases as their disease progresses. These two factors pose challenges to most in vivo drug studies, thus limiting the use of traditional chemotherapy drugs.

[0003] The blood-brain barrier (BBB) refers to a natural barrier formed by the walls of brain capillaries and glial cells. This highly tight and almost impenetrable cell layer protects the brain from harmful factors such as toxins or bacteria in the blood. The blood-brain barrier only allows a very limited number of small molecules, such as nutrients, to pass through. However, this protective layer makes it difficult for scientists to develop new drugs that can cross the blood-brain barrier and kill central nervous system tumors in situ. Diffuse midline gliomas usually show an intact and denser blood-brain barrier, which is confirmed by the lack of enhancement in the corresponding areas on nuclear magnetic resonance. In in vitro experiments, diffuse midline glioma cells have been shown to lack the ability to destroy the integrity of the blood-brain barrier. Therefore, this poses a greater challenge to drug delivery for the treatment of diffuse midline gliomas.

[0004] Intrathecal drug delivery (IT) bypasses the blood-brain barrier (BBB) by injecting drugs directly into the cerebrospinal fluid (CSF) circulation system, delivering drugs deep into the central nervous system (CNS) while minimizing peripheral systemic toxicity. In diffuse midline gliomas, this technology is primarily implemented through two routes: intraventricular administration via the Ommaya capsule, where the Ommaya capsule is surgically implanted and the drug is injected directly into the lateral ventricle, allowing the drug to diffuse to the tumor area through the CSF circulation; and intrathecal injection via lumbar puncture, where the drug is injected into the subarachnoid space via lumbar puncture and relies on CSF flow for drug distribution. Intrathecal drug delivery was first used in the oncology field for pediatric CNS leukemia. For example, triple intrathecal injection has become a standard regimen for preventing CNS leukemia metastasis. Some chemotherapy drugs are also used to bypass the BBB for glioblastoma, medulloblastoma, and CNS metastasis of lung cancer.

[0005] In the past, there were very few studies on chemotherapy for brainstem gliomas / diffuse midline gliomas using intrathecal administration. Chemotherapy drugs that have been widely used in hematology for central metastasis and drugs that have been tested on a small scale in other primary tumors of the central nervous system have not been proven to be effective in brainstem gliomas. However, these drugs that can be used for clinical intrathecal administration have broad prospects for clinical translation. If the efficacy can be confirmed and potentially effective intrathecal chemotherapy drugs can be screened out, patients with diffuse midline gliomas can be treated quickly and conveniently in the clinic and cross the blood-brain barrier. Therefore, there is an urgent need to develop and innovate drug screening methods for clinical intrathecal chemotherapy of diffuse midline gliomas. Summary of the Invention

[0006] In one aspect, the present application provides a method for drug screening for intrathecal chemotherapy of diffuse midline glioma, the method comprising:

[0007] (1) Obtain diffuse midline glioma samples and use them to prepare cell lines and organoids;

[0008] (2) Using the cell line obtained in step (1) for drug screening; using the organoid obtained in step (1) for drug screening.

[0009] (3) Use high-content imaging to verify the effects of the drugs screened in step (2).

[0010] Furthermore, the drug screening in step (2) and step (3) is carried out using an intrathecal anticancer drug compound library, wherein the intrathecal anticancer drug compound library comprises BMT_090605hydrochloride, Ketorolac, Levobupivacaine hydrochloride, Pemetrexed disodium, Pemetrexed, 6-Mercaptopurinehydrate, 6-Mercaptopurine, Methotrexate disodium, Methotrexate, Cytarabine hydrochloride, Cytarabine, Etoposide, Flupirtine Maleate, CCG_50014, Navarixin, UNC3230, AA147, CFM_2, LHVS, GSK 650394, TG003, Decursin, LP_922761, 5-Azacytidine, Etoposide phosphate, Topotecan Hydrochloride, Pemetrexed disodium hemipentahydrate, Genistein, Thalidomide, Gemcitabine, Ifosfamide, Decitabine, Gemcitabinehydrochloride, Busulfan, Thio_TEPA, FdUrd, Diaziquone, 5-Fluorouracil, ACNU, MCNU.

[0011] The names of drugs / compounds in this application may use various Chinese and English names or abbreviations. Within the common sense in the field, these names have the same meaning.

[0012] Furthermore, the intrathecal anticancer drug compound library includes six drugs including methotrexate, cytarabine, etoposide, topotecan, gemcitabine, and pemetrexed, as well as a drug combination of methotrexate, cytarabine, and dexamethasone.

[0013] Furthermore, the concentration of the drug used in step (2) is 1 μmol / L.

[0014] The cerebrospinal fluid concentrations of the drugs calculated from the drug doses in the actual clinical intrathecal injection regimen (150 mL of human cerebrospinal fluid: MTX 220 μM, Ara-C 1.37 mM, ETO 5.66 μM, TOP 6.33 μM, GEM 5.07 mM, PEM 424 μM) are much higher than the in vitro screening concentration (1 μM), so the positive results of this screening method are of guiding significance.

[0015] Furthermore, in step (2), drug screening using the cell line obtained in step (1) includes detecting changes in cell viability after drug application and the IC50 curve of the drug.

[0016] Furthermore, the drug screening using the organoids obtained in step (1) in step (2) includes detecting changes in the size and morphology of the organoids after application of the drug.

[0017] Preferably, the in vitro screening scheme simulates the clinical medication scheme, and the drug is added to the culture medium on the first day of each week. The organoid morphology is photographed and recorded on the 0th, 3rd, 7th, and 14th days of administration. Two weeks after administration, the two-dimensional area of the solid part of the organoid is outlined and calculated using Image J software, and the changes in the morphological size of the organoid after administration are characterized and calculated based on this.

[0018] Furthermore, in step (3), the cell lines and / or organoids obtained in step (1) are subjected to Hoechest / Mitotracker / Calcein-AM / PI fluorescence staining and then scanned using a high-content imaging device to perform fluorescence ratio analysis and / or depth of field extension reconstruction.

[0019] On the other hand, the present application provides a use of a drug combination of methotrexate, cytarabine and dexamethasone in the preparation of a drug for treating diffuse midline glioma, wherein the diffuse midline glioma contains an H3K27M mutation.

[0020] Furthermore, the mass ratio of methotrexate, cytarabine and dexamethasone in the drug is 10-15:35-50:5.

[0021] Furthermore, the drug is an intrathecal drug.

[0022] Furthermore, the drug is an Ommaya capsule-administered drug or a lumbar puncture intrathecal injection drug.

[0023] Combination therapy with methotrexate, cytarabine, and dexamethasone: Single-drug doses of methotrexate are 10-15 mg, cytarabine 35-50 mg, and dexamethasone 5 mg.

[0024] Combination therapy with methotrexate, cytarabine, and dexamethasone is administered intrathecally via lumbar puncture or an Omaya capsule placed in the ventricle;

[0025] The methotrexate, cytarabine, and dexamethasone combination therapy was administered once weekly.

[0026] Those skilled in the art can design intrathecal administration drugs and select suitable excipients therefor according to techniques known in the art, including but not limited to solvents, cosolvents, pH regulators, surfactants, osmotic pressure regulators, antioxidants, preservatives, etc.

[0027] Beneficial effects:

[0028] This study provides the world's first clinically-guided drug screening method specifically for intrathecal chemotherapy, along with a matching ITAC drug screening compound library and a clinical drug screening range for diffuse midline glioma. This is the first reported clinical route-of-administration-guided screening method.

[0029] Screening methods based on multiple preclinical models improve screening robustness. Preclinical models of drug killing in vitro culture media simulate the clinical scenario of subarachnoid drug administration killing tumors in vivo.

[0030] The tumor site of diffuse midline glioma is particularly suitable for intrathecal chemotherapy screening. The tumor is mainly located in the deep midline area of the brain and is surrounded by the lateral ventricles, third ventricles, fourth ventricles and surrounding cerebral cisterns of the subarachnoid space. Surgical resection is difficult, but after intrathecal administration, the drug can diffuse into these areas to kill the tumor. Among them, it is more advantageous for subtypes such as exophytic, aqueductal, and diffuse intrinsic pontine gliomas because these subtypes are closer to the surrounding cerebrospinal fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the process for establishing the drug compound library of the present invention;

[0032] Figure 2 The results of high-throughput screening of the ITAC compound library in vitro in cell lines of the present invention are as follows;

[0033] Figure 3 The in vitro cell line IC50 curve of the intrathecal drugs screened by the present invention;

[0034] Figure 4A This is the drug screening result (ITAC) based on organoid O240724 of the present invention;

[0035] Figure 4B This is the drug screening result (epigenetic and summary results) based on organoid O240724 of the present invention; the word is not big enough, so it is split

[0036] Figure 5 The results of the drug screening based on organoids O241205A and O250102 of the present invention are as follows;

[0037] Figure 6 Screening multiple cases of brainstem glioma organoid triple sheath injection therapy efficacy results for this invention;

[0038] Figure 7 The present invention uses high-content technology to verify the therapeutic effect of intrathecal drug administration based on cell lines;

[0039] Figure 8 The present invention uses high-content technology to verify the therapeutic effect of intrathecal drug administration based on organoids;

[0040] Figure 9 This is a graph showing the fluorescence change trend of animal tumors that demonstrates the efficacy of the triple intrathecal injection therapy screened and verified by in vivo experiments of the present invention;

[0041] Figure 10 This is the animal survival curve for the efficacy of the triple intrathecal injection therapy screened and verified by the in vivo experiment of the present invention;

[0042] Figure 11 This is a schematic diagram of the specific process of drug screening for intrathecal chemotherapy of diffuse midline glioma. DETAILED DESCRIPTION

[0043] Example 1

[0044] The present invention first established an intrathecal anti-cancer compound library (ITAC): by searching the Medline, Web of Science databases and the MCE, TargetMol, and Selleck commercial drug compound libraries, the present invention retrieved and summarized intrathecal injection drugs that have been used in clinical practice, have been conducted / are undergoing clinical trials, and have completed intrathecal drug validation in animals in the preclinical stage using the keywords "intrathecal injection", "Omaya capsule", and "lateral ventricular injection"; then, single concentration (1 μM) high-throughput drug screening was completed on brainstem glioma cell lines with multiple mutation combinations and a brainstem normal stem cell line constructed in vitro.

[0045] Culture of primary brainstem glioma cell lines and normal human brainstem stem cells: Primary cell lines derived from patient tumor tissue (obtained by biopsy or surgical resection) were cultured using brainstem glioma primary cell line culture medium using traditional primary cell line culture methods. Before passaging, the culture flasks were coated with 1:1000 diluted Matrigel (Corning, 356234) at 37°C overnight. The primary culture medium was DMEM high glucose medium (Gibco, 11995073), which also included: 20 ng / mL EGF cytokine (Ciinto SC102), 20 ng / mL bFGF cytokine (Ceintu SC107-10ug), 100U / mL penicillin and 100ug / mL streptomycin (100×, Thermo Fisher Scientific 10378016), 1×N2 (50×, Ceintu S60314017A), 1×B27 (50×, Ceintu S60314015A) and 1×ITS (100×, Ceintu SC25800). The successful establishment of the cell line was confirmed after the 10th passage. The mutation status of the brainstem glioma cell line obtained by whole exome sequencing is shown in the figure below. Figure 2 Heat map legend shown.

[0046] The culture method and culture medium of human normal brainstem stem cells derived from the posterior brain tissue of aborted fetuses are the same as those of primary brainstem glioma cell lines.

[0047] Logarithmically growing cells were plated at 2000 cells / well in 384-well plates and cultured overnight. Drugs were dispensed in vitro using an Echo sonic automated dosing system and added to the cell culture medium to a final concentration of 1 μmol / L for each drug. After 72 h of culture, the cells were titrated using the Cell-titer method (Promega, ) The fluorescence intensity was measured on a microplate reader, and the cell viability was calculated using the formula: relative cell viability (%) = fluorescence intensity of the drug-treated group / fluorescence intensity of the control group × 100%. The above drugs that also meet the following conditions were retained: ① they were previously reported to be associated with anti-tumor effects; ② they have potential to kill diffuse midline gliomas; and ③ they do not kill normal brainstem stem cell lines. A total of 40 drugs (BMT_090605 (hydrochloride), Ketorolac, Levobupivacaine (hydrochloride), Pemetrexed (disodium), Pemetrexed, 6_Mercaptopurine hydrate, 6_Mercaptopurine, Methotrexate (disodium), Methotrexate, Cytarabine (hydrochloride), Cytarabine, Etoposide, Flupirtine (Maleate), CCG_50014, Navarixin, UNC3230, AA147, CFM_2, LHVS, GSK 650394, TG003, Decursin, LP_922761, 5_Azacytidine, Etoposide phosphate, Topotecan (Hydrochloride), Pemetrexed (disodiumhemipenta hydrate), Genistein, Thalidomide, Gemcitabine, Ifosfamide, Decitabine, Gemcitabine (hydrochloride), Busulfan, Thio_TEPA, FdUrd, Diaziquone, 5-Fluorouracil, ACNU, MCNU) to form an ITAC compound library. The process is as follows Figure 1 shown.

[0048] The drug killing results were visualized and analyzed using the Pheatmap package (R v4.2.3) Figure 2) and natural unsupervised clustering of drug-induced cell line killing results to select more effective drugs for diffuse midline glioma. A total of six drugs with high clinical accessibility and previous clinical practice reports were selected: methotrexate (MTX), cytarabine (Ara-C), etoposide (ETO), topotecan (TOP), gemcitabine (GEM), and pemetrexed (PEM). Based on the visualization results, the drugs that are most sensitive to a single cell line can be screened out. For example, MTX is effective on multiple cell lines, Ara-C is effective on 150728, 170720, 190326, and DIPG-17, ETO is more effective on 150728, 170720, and DIPG-17, TOP is effective on 150630, 150714, 170720, and DIPG-17, GEM is more effective on 150630 and 170720, and PEM is more effective on 150728, 170720, and 190326, thus achieving differential screening.

[0049] The range of drugs used for clinical screening also includes combinations of the six drugs mentioned above. Taking into account clinical practice experience, a combination therapy, methotrexate + cytarabine, is included as a supplement to the clinical drug screening range. Because clinical medication requires the addition of dexamethasone to prevent chemical meningitis, this combination therapy actually uses a triple intrathecal injection (TIT) regimen of methotrexate + cytarabine + dexamethasone to simulate a clinical screening scenario (Note: Therefore, all control groups in the schematics of this manual, such as DMSO or NC, are DMSO solutions with added dexamethasone).

[0050] In summary, the world's first clinical route-guided intrathecal anti-cancer drug compound library (ITAC) was established, and the clinical drug screening range for diffuse midline glioma was determined, including the above-mentioned 6 drugs (MTX, Ara-C, ETO, TOP, GEM, PEM) and 1 combination therapy (TIT). The appropriate clinical drug screening range can improve screening efficiency and screening positivity, accelerate clinical translation, and improve the targeted killing of diffuse midline glioma.

[0051] Example 2

[0052] Logarithmically growing cells were plated at 3000 cells / well in 96-well plates and cultured overnight. 50 mmol / L stock solutions of MTX, Ara-C, ETO, TOP, GEM, and PEM compounds were diluted in vitro in a 1:3 gradient. The drugs were taken and added to the cell culture medium to make the final drug concentrations in each well 50 μmol / L, 16.67 μmmol / L, 5.56 μmmol / L, 1.85 μmmol / L, 0.62 μmmol / L, 0.21 μmmol / L, 0.068 μmmol / L, 0.023 μmmol / L, 0.0076 μmmol / L, and 0 μmmol / L. After further culture for 72 h, the cells were titrated using the Cell-titer method (Promega, ) The fluorescence intensity was measured on a microplate reader and the cell viability was calculated using the formula: relative cell viability (%) = fluorescence intensity of the drug-treated group / fluorescence intensity of the control group × 100%. Graphpad Prism 9 software was used to fit and plot the half-maximal inhibitory concentration (IC50) of each compound for each brainstem glioma cell line. Figure 3 As shown in the figure, MTX is effective against various cell lines, Ara-C is effective against 170720 and 190326, ETO is effective against 150714, 170720, and 190326, and has a lower IC50 against 150714, TOP is effective against 150714 and 170720, GEM is more effective against 150714 and 170720, and PEM is effective against 150714, 170720, and 190326. Based on the IC50 value, GEM or MTX can be selected for 150714, Ara-C or MTX for 170720, and MTX for 190326, achieving differentiated optimal screening.

[0053] Example 3

[0054] The present invention constructs patient-derived brainstem glioma organoids (covering diffuse midline gliomas) in vitro according to the patented technical solution of Example 1 of CN118126952A, and completes the single concentration (1 μM) screening of drugs within the clinical drug screening range in vitro. The addition of drugs to the in vitro culture medium to kill organoids perfectly simulates the clinical scenario of killing tumors by cerebrospinal fluid immersion after intrathecal injection into the subarachnoid space in vivo. The in vitro screening scheme simulates the clinical medication scheme, and the drug is added to the culture medium on the first day of each week. The morphology of the organoids is photographed and recorded on the 0th, 3rd, 7th, and 14th days of administration, corresponding to the 1st / 8th day of each month of the clinical chemotherapy cycle. Two weeks after administration, the two-dimensional area of the solid part of the organoid is outlined and calculated using Image J software, and the changes in the morphological size of the organoids after administration are characterized and calculated based on this. The Pheatmap package is used for visual analysis to screen out the most suitable drugs for different organoids. One of the constructed brainstem glioma organoids (O240724) was derived from a patient undergoing surgery for a grade IV diffuse midline glioma. The tumor originated in the midbrain. The organoids were screened for drugs within the aforementioned ITAC clinical drug screening range and compared with drugs previously reported for epigenetic regulation of H3K27M (PAB: panobinostat, TAZ: tazemetostat; ONC: ONC-201). It was found that intrathecal injection of drugs showed efficacy that was not inferior to epigenetic regulation drugs, and some drugs had even advantages. Among them, TIT therapy had the best effect (Figure 4).

[0055] Another brainstem glioma organoid (O241205A) was derived from a biopsy of a patient with a grade IV diffuse midline glioma originating in the pons. The organoids were screened for drugs within the aforementioned ITAC clinical drug screening range, with TIT therapy showing the best efficacy ( Figure 5 Part A of the .

[0056] Another brainstem glioma organoid (O250102) was derived from a patient who underwent surgery for a grade IV diffuse midline glioma. The tumor involved the entire brainstem. The organoids were screened for drugs within the aforementioned ITAC clinical drug screening range and the epigenetic regulatory drug ONC201. TIT therapy was the most effective ( Figure 5 Part B of the

[0057] In summary, clinical drug screening based on organoid level was achieved.

[0058] Example 4

[0059] The present invention screens out the use of triple intrathecal injection therapy (MTX+Ara-C+DEX) for treating diffuse midline glioma.

[0060] Twelve brainstem glioma organoids (from patients with different histopathological types, different pathological grades, and diffuse midline gliomas / non-diffuse midline gliomas) were screened for drugs within the scope of clinical drug screening in ITAC. The screening results were visualized using Pheatmap and unsupervised clustering was performed based on the source of the organoids. It can be seen that among the different diffuse midline glioma organoids, TIT therapy was the best in the screening, and natural clustering showed that the organoid groups sensitive to TIT were all grade IV diffuse midline gliomas with H3K27M mutations ( Figure 6 H3 wild-type gliomas are insensitive to TIT. Organoids are more closely aligned with real-life patient conditions than cell lines or animal models. In summary, triple intrathecal therapy (MTX+Ara-C+DEX) has significant potential for treating diffuse midline gliomas.

[0061] Example 5

[0062] The present invention also provides a method for screening drugs for clinical drug screening range of intrathecal chemotherapy of diffuse midline glioma based on high content technology:

[0063] Brainstem glioma cells in logarithmic growth were plated into 96-well plates at 5,000 cells / well and cultured overnight. After intervention with a single concentration of each drug (small dose for a short time, specifically 10 nM for 24 hours), the cells were stained with Hoechest / Mitotracker / Caspase and Hoechest / Calcein-AM / PI double sequence fluorescence (Hoechest: #C1027, Beyotime; Mitotracker: #A66442, Thermo Fisher; Caspase: #C10432, Thermo Fisher; Calcein-AM / PI: #C1371, Beyotime). After scanning with a high-content imaging device (OperaPhenix / Harmony), fluorescence proportion analysis was performed, and the drug that was most sensitive to the cells was selected to complete the screening. The proportion of each indicator was as follows: Figure 7 As shown in parts AD, TIT significantly changed the cell phenotype, as shown in the fluorescence staining images. Figure 7 As shown in the EF section.

[0064] In addition, after applying a single concentration of drug intervention (short-term, specifically 1 μM for 72 hours) to a patient-derived brainstem glioma organoid constructed in vitro (O241216, from a patient undergoing surgery for grade IV diffuse midline glioma, the tumor originated in the thalamus), the cells were stained with Hoechest / Mitotracker / Calcein-AM / PI fluorescence and scanned with a high-content imaging device for extended depth of field reconstruction. The most effective drug for killing the tumor was selected for screening, and TIT therapy was most effective for this patient ( Figure 8), this method can be used to complete the screening more realistically.

[0065] Example 6

[0066] The experiment used 6-8 week-old female BALB / c nude mice to establish an orthotopic brainstem tumor model of H3K27M mutant brainstem glioma (cell line 190326, which harbors the H3K27M mutation and also TP53 and PIK3CA mutations). The specific method is as follows: a primary brainstem glioma cell suspension (100,000 cells / 5 μL) was prepared and stereotactically injected into the mouse brainstem over 2 minutes, maintaining a slow and uniform injection rate. The injection site was approximately 1 mm posterior to the lambdoid suture and 1 mm lateral to the midline of the mouse skull. One week after inoculation, the mice were observed for general condition, and the fluorescence signal intensity of the orthotopic brainstem tumor was measured using an in vivo fluorescence imager (IVIS Lumina Series III). Subsequently, the tumor-bearing mice were randomly divided into two groups based on the fluorescence signal intensity: a control group and a triple intrathecal injection group to ensure balanced tumor burden between the two groups.

[0067] Triple intrathecal therapy (TIT) treatment group: First, a TIT mixed solution was prepared: DEX compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of DEX compound was adjusted to 4 μmol / L; MTX compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of MTX compound was adjusted to 10 μmol / L; Ara-C compound was dissolved in 0.9% (v / w) sodium chloride solution, and the concentration of Ara-C compound was adjusted to 60 μmol / L. Stereotactic injection was performed into the lateral ventricle of mice every 4 days to simulate intrathecal injection, and a total of 5 μL of the freshly prepared mixed solution was injected each time (drug concentrations in cerebrospinal fluid: MTX 1 μmol / L, Ara-C 6 μmol / L, DEX 0.4 μmol / L, the same ratio as the clinical medication regimen).

[0068] Control group: Stereotactic injection was performed into the lateral ventricle of mice every 4 days to simulate intrathecal injection, with a total of 5 μL of freshly prepared DEX solution injected each time (drug concentration in cerebrospinal fluid: DEX 0.4 μmol / L).

[0069] There was no significant weight loss in mice during the drug administration period, and no mouse deaths due to the drug occurred. During the drug administration period, the fluorescence value of brainstem in situ tumors was measured once a week, and the fluorescence intensity was recorded. The overall survival of the two groups of mice was tracked.

[0070] Fluorescence intensity changes as Figure 9 The survival curve is shown as Figure 10 As shown, triple intrathecal injection therapy significantly inhibited tumor growth of diffuse midline glioma in vivo and prolonged survival, with the median survival of animals almost doubling.

Claims

1. A method for drug screening for intrathecal chemotherapy of diffuse midline glioma, characterized in that: The method comprises: (1) Obtain diffuse midline glioma samples and use them to prepare cell lines and organoids; (2) Using the cell line obtained in step (1) for drug screening; using the organoid obtained in step (1) for drug screening. (3) Use high-content imaging to verify the effects of the drugs screened in step (2).

2. The method according to claim 1, wherein the drug screening in step (2) is performed using an intrathecal anticancer drug compound library, wherein the intrathecal anticancer drug compound library comprises BMT_090605hydrochloride, Ketorolac, Levobupivacaine hydrochloride, Pemetrexed disodium, Pemetrexed, 6-Mercaptopurinehydrate, 6-Mercaptopurine, Methotrexate disodium, Methotrexate, Cytarabinehydrochloride, Cytarabine, Etoposide, Flupirtine Maleate, CCG_50014, Navarixin, UNC3230, AA147, CFM-2, LHVS, GSK 650394, TG003, Decursin, LP_922761, 5-Azacytidine, Etoposide phosphate, Topotecan Hydrochloride, Pemetrexed disodium hemipentahydrate, Genistein, Thalidomide, Gemcitabine, Ifosfamide, Decitabine, Gemcitabinehydrochloride, Busulfan, Thio_TEPA, FdUrd, Diaziquone, 5-Fluorouracil, ACNU, MCNU.

3. The method according to claim 2, wherein the intrathecal anticancer drug compound library comprises six drugs including methotrexate, cytarabine, etoposide, topotecan, gemcitabine, and pemetrexed, as well as a drug combination of methotrexate, cytarabine, and dexamethasone.

4. The method according to any one of claims 1 to 3, wherein the concentration of the drug used in steps (2) and (3) is 1 μmol / L.

5. The method according to any one of claims 1 to 4, wherein the drug screening using the cell line obtained in step (1) in step (2) comprises detecting changes in cell viability and the IC50 curve of the drug after drug application; and the drug screening using the organoid obtained in step (1) in step (2) comprises detecting changes in size and morphology of the organoid after drug application.

6. The method according to any one of claims 1 to 5, wherein in step (3), the cell lines and / or organoids obtained in step (1) are subjected to Hoechest / Mitotracker / Calcein-AM / PI fluorescence staining and then scanned using a high-content imaging device to perform fluorescence ratio analysis and / or extended depth of field reconstruction.

7. Use of a drug combination of methotrexate, cytarabine and dexamethasone in the preparation of a medicament for treating diffuse midline glioma, wherein the diffuse midline glioma contains an H3K27M mutation.

8. The use according to claim 7, wherein the mass ratio of methotrexate, cytarabine and dexamethasone in the drug is 10-15:35-50:

5.

9. The use according to claim 7 or 8, wherein the drug is an intrathecal drug.

10. The use according to claim 9, wherein the drug is an Ommaya capsule-administered drug or a lumbar puncture intrathecal injection drug.

Images