A small molecule combination that efficiently and rapidly induces malignant glioma cells to differentiate into neurons and its application

Abstract

The present invention relates to a small molecule combination that can efficiently and rapidly induce the differentiation of malignant glioma cells into neurons and its application, and belongs to the field of biomedicine technology. The present invention is aimed at the current situation that differentiation therapy drugs have low therapeutic effects on glioblastoma. Through a large number of studies, it is found that a small molecule composition YFSS can effectively and rapidly induce GBM cells to differentiate into mature neuron-like cells. The composition YFSS includes Y27632, Forskolin, SB431542 and SP600125. At the same time, the present invention further develops a method for treating glioblastoma based on the small molecule composition, which continuously releases cerebrospinal fluid containing the composition YFSS to the tumor site, which can effectively treat GBM and improve its prognosis. The present invention provides a new way for the treatment of GBM.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule combination for efficiently and rapidly inducing malignant glioma cells to differentiate into neurons and applications thereof. Background Art

[0002] Glioblastoma (GBM) is a complex and aggressive brain tumor that often has a limited response to traditional surgery, radiotherapy, and chemotherapy, resulting in poor survival outcomes. Currently, a promising approach to treating glioblastoma is to induce terminal differentiation of these cells, leading to an irreversible postmitotic arrest, thereby halting proliferation and subsequently attenuating tumor expansion and spread while minimizing deleterious effects on healthy tissue. This approach can be either biological or chemical and does not target the abnormal gene, but can reverse malignant behaviors such as proliferation and invasion. This strategy was first demonstrated to be effective in the treatment of leukemias, where drugs such as all-trans retinoic acid (ATRA) and arsenic trioxide successfully induced cell differentiation and arrested malignancy. However, these differentiation-inducing agents are effective in hematological cancers but have lower success rates in GBM. A major problem is their limited ability to fully differentiate GBM cells, leading to poor prognosis and recurrence. Therefore, it is crucial to find a differentiation protocol that can not only effectively induce GBM differentiation but also inhibit tumor growth.

[0003] The primary focus of GBM differentiation therapy research is on inducing differentiation into astrocytes or neurons, typically achieved through the use of chemical inducers, protein kinase inhibitors, transcription factors, or miRNA transgenes. Compared to the use of transcription factors or miRNAs, small molecule-induced reprogramming offers several advantages. It avoids the need for genomic insertion of DNA sequences, is cell-permeable and non-immunogenic, is easy to synthesize, store, standardize, and manipulate, is low-cost, exhibits rapid biological effects, and allows for precise control through varying concentrations and combinations. Evidence suggests that a combination of forskolin and CHIR99021 can effectively differentiate rat C6 glioma cells into neuron-like cells and inhibit spinal cord tumor growth. Furthermore, by adding ISX9, I-BET151, and DAPT to this approach, human GBM cells can be reprogrammed into immature neuron-like cells. In animal models, epigenetic reprogramming using histone deacetylase inhibitors enhances cAMP-induced GBM differentiation, thereby inhibiting tumor growth and prolonging survival. Furthermore, the simultaneous use of kinase inhibitors targeting ROCK and mTOR can reprogram human GBM cells into neurons and effectively inhibit GBM growth in vivo. Furthermore, inhibition of the MAPKs / JNK or mTOR and ROCK signaling pathways has been shown to induce differentiation into neuron-like and astrocyte-like cells. However, the ability of these small molecules or small molecule combinations to induce differentiation remains limited. Furthermore, in GBM treatment, neuronal differentiation is more advantageous than astrocyte differentiation because neurons do not proliferate, thus facilitating cell cycle arrest. This led to the present invention. Summary of the Invention

[0004] To solve the above technical problems, the present invention screened different small molecules and their combinations that have effects on neuronal differentiation and found an optimal combination. After culturing with this combination for 2 hours, GBM cells can be promoted to differentiate into neurons, and the differentiation efficiency is as high as over 90%, which is much higher than the effect of existing differentiation methods.

[0005] The first object of the present invention is to provide a method for inducing malignant glioma cells to differentiate into neurons in vitro, comprising the following steps:

[0006] During the culturing of malignant glioma cells, adding the small molecule composition to the neuronal induction medium and continuing the culturing for at least 2 hours;

[0007] The small molecule composition comprises Y27632, forskolin, SB431542 and SP600125, and the added molar ratio is 5-15:50-150:2-6:10-30.

[0008] Furthermore, the concentrations of the small molecule composition in the neuron induction medium are: Y276325-15 μM, forskolin 50-150 μM, SB4315422-6 μM, and SP60012510-30 μM.

[0009] Furthermore, the neuronal induction medium is obtained by mixing DMEM / F12 medium and neurobasal medium at a volume ratio of 0.8-1.2:0.8-1.2, and adding components including 0.3-0.7% N2, 0.8-1.2% B27, 18-22ng / ml BDNF, 18-22ng / ml NT3, and 0.8-1.2% GlutaMax.

[0010] Furthermore, antimicrobial agents, such as 0.5-1.5% penicillin / streptomycin, may be added to the neuron induction culture medium.

[0011] The second object of the present invention is to provide a small molecule composition for the preparation of a drug for treating glioblastoma (differentiation), wherein the small molecule composition comprises Y27632, forskolin, SB431542 and SP600125, and the molar ratio thereof is 5-15:50-150:2-6:10-30.

[0012] Furthermore, the small molecule composition is administered to the tumor site during treatment.

[0013] Furthermore, a pharmaceutically acceptable safety background solution containing the small molecule composition is administered to the tumor site.

[0014] Preferably, cerebrospinal fluid containing the small molecule composition is administered to the tumor site.

[0015] Furthermore, the concentrations of the small molecule composition in the cerebrospinal fluid are: Y276325-15 μM, forskolin 50-150 μM, SB4315422-6 μM, and SP60012510-30 μM.

[0016] The third object of the present invention is to provide a drug for treating glioma, which contains a small molecule composition, wherein the small molecule composition is Y27632, forskolin, SB431542 and SP600125, and the molar ratio of Y27632, forskolin, SB431542 and SP600125 is 5-15:50-150:2-6:10-30.

[0017] Furthermore, the drug also contains a background fluid for dispersing the small molecule composition, such as artificial cerebrospinal fluid.

[0018] By means of the above solution, the present invention has at least the following advantages:

[0019] The present invention utilizes the neuron-specific promoter reporter system TUBB3::mcherry to identify a small molecule combination that effectively induces neuronal differentiation of human GBM cells. The combination of Y27632, Forskolin, SB431542 and SP600125 (collectively referred to as YFSS) can effectively and rapidly induce GBM cells to differentiate into mature neuronal-like cells, especially those that are positive for tyrosine hydroxylase. This differentiation process is characterized by a significant reduction in cell proliferation and cell cycle arrest. In addition, the malignant characteristics of GBM cells, such as sphere formation, migration and invasion, are significantly reduced. Among them, YFSS showed high efficacy in differentiating patient-derived GBM cells, and its sustained release in vivo significantly promoted the differentiation of GBM cells. This resulted in a significant reduction in tumor growth in the brains of patient-derived xenograft (PDX) mice and a significant prolongation of their survival.

[0020] The compounds in the small molecule combination YFSS screened in this invention can be easily synthesized and standardized, are cost-effective, can be used in a wide range of clinical applications, and have efficient and rapid bioavailability. This makes small molecule-mediated therapy a practical and potentially effective therapeutic strategy against GBM. Therefore, the differentiation therapy method developed in this invention based on this small molecule combination is very promising in improving GBM prognosis and reducing recurrence.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0023] Figure 1 YFSS mediates the effective neuronal fate change of GBM cells. GBM cells (U87 and U251) were induced to differentiate with YFSS for 3 days and stained with neuronal markers TUBB3 and MAP2 and labeled with Hoechst33258 (A). TUBB3 was quantified. + and MAP2 +Percentage of cells (B), Bar = 50 μm. C, E, qRT-PCR analysis (C) and Western blot analysis (D and E) of relative expression of TUBB3 at different stages of YFSS induction (0, 1, 3, 5, and 7 days), *P < 0.05. F and G, Representative images of GBM cells double-stained for TUBB3 and NEUN, PSD95, or SYN1 treated with YFSS for 7 days, Bar = 50 μm. H, NEUN expression in TUBB3-positive cells + Bar graphs show percentages. I, Representative images of GBM cells treated with YFSS for 7 days loaded with Fluo-4-AM to examine intracellular calcium fluctuations in response to KCl (10 mM). Bar = 100 μm. J, Calcium response calculated as the change in fluorescence (ΔF) relative to the initial fluorescence (F0). K, ΔF / F0 intensity plot showing the response of individual cells to KCl (n = 20).

[0024] Figure 2 The results are from the screening of small molecule compositions.

[0025] Figure 3 YFSS inhibits GBM cell proliferation. A and B, Representative images of YFSS-induced GBM cells stained with Ki-67 and labeled with Hoechst 33258 at different stages (0, 1, 3, 5, and 7 days) (A). Quantification of the percentage of Ki-67-positive cells (B). Bar = 250 μm. C, qRT-PCR analysis of Ki-67 expression in YFSS-induced GBM cells at different stages (0, 1, 3, 5, and 7 days). *P < 0.05. D and E, Representative images of GBM cells treated with YFSS for 7 days, labeled with EdU and Hoechst 33258 (D). Calculation of relative EdU incorporation (E). *P < 0.05. Bar = 100 μm.

[0026] Figure 4 YFSS-induced GBM differentiation leads to cell cycle arrest. AD, qRT-PCR analysis of the expression of cell cycle proteins, CDKs, and CKIs in GBM cells at different stages of YFSS induction (0, 1, 3, 5, and 7 days). EH, Western blot analysis of the relative expression of CyclinD1, CDK2, p27KIP1, and p21CIP1 in GBM cells at different stages of YFSS induction (0, 1, 3, 5, and 7 days). I and J, Cell cycle analysis by PI staining and flow cytometry in undifferentiated cells (Control) and cells treated with YFSS for 7 days (YFSS) (I). Quantitative measurement of cell cycle phase (J). *P < 0.05.

[0027] Figure 5YFSS-induced reversal of GBM malignant phenotype. A and B, Representative images of tumor sphere formation assay in GBM cells treated with DMSO (Control) or YFSS (YFSS) for 7 days (A), and statistical analysis of the number and average area of ​​tumor spheres with a diameter greater than 100 μm (B).

[0028] *P < 0.05, Bar = 250 μm. C-F, Representative images of Transwell assays in GBM cells (control) or cells treated with YFSS for 7 days (YFSS) during migration (C and D) and invasion (E and F). *P < 0.05, Bar = 50 μm. qRT-PCR assays were performed to detect the expression of invasion-related proteins MMP1 and MMP3 at different stages (days 0, 1, 3, 5, and 7) in YFSS-induced GBM cells. *P < 0.05.

[0029] Figure 6 Figure 3 shows the changes in proliferation and migration of patient-derived GBM cells after differentiation.

[0030] Figure 7 YFSS mediates efficient neuronal fate conversion in patient-derived GBM cells. A, D, Patient-derived GBM cells treated with YFSS for 3 days with control siRNA (Scramble) or siRNA targeting CEND1 (CEND1-shRNA-1 or CEND1-shRNA-2) were stained with TUBB3 (A) and labeled with EdU (B). The mean fluorescence intensity of each group was quantified (B and D). *P < 0.05 indicates a significant difference compared with the Scramble group; #P < 0.05 indicates a significant difference compared with the Scramble + YFSS group. Bar = 25 μm. E and F, Representative images of double immunofluorescence staining of patient-derived GBM cells treated with YFSS for 7 days for TUBB3 and MAP2, NEUN, PSD95, or SYN1. Bar = 25 μm.

[0031] Figure 8YFSS inhibits tumor development and prolongs mouse survival. A and B, In vivo bioluminescent images (A) and quantification of bioluminescent signals (B) of brain tumors in mice receiving the indicated treatments at 1, 2, 3, and 4 weeks post-implantation. *P < 0.05, indicating a significant difference between the U87 group and the U87 + YFSS group; #P < 0.05, indicating a significant difference between the GBM group and the GBM + YFSS group. C, Kaplan-Meier survival plots of mice transplanted with U87 or patient-derived GBM in the presence or absence of sustained-release YFSS (n = 8 per group). D and E, Representative H&E images (D) and quantification of tumor area (E) of mouse brain sections after 28 days of treatment. *P < 0.05, indicating a significant difference between the U87 group and the U87 + YFSS group; #P < 0.05, indicating a significant difference between the GBM group and the GBM + YFSS group. Bar = 2 mm. F, Representative images of NEUN immunofluorescence staining of brain sections from mice transplanted with U87 or patient-derived GBM for 28 days with or without YFSS sustained release, Hoechst 33258 labeled cell nuclei (left), Bar = 2 mm. The image on the right is a higher magnification of the boxed area on the left, Bar = 200 μm. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0033] The materials and methods involved in the present invention are as follows:

[0034] (1) Human GBM cell culture and neuronal induction:

[0035] Human GBM cell lines (U87 and U251) were originally purchased from the American Type Culture Collection (ATCC) and cultured in Dulbeccos' modified Eagle's medium (DMEM), 10% fetal bovine serum, and 1% penicillin-streptomycin. Three human GBM specimens were obtained from patients who provided written informed consent from the Department of Neurosurgery, Second Affiliated Hospital, Soochow University. The study protocol was approved by the Ethics Committee of Soochow University. Primary GBM cultures were isolated, expanded, and characterized as previously described. For neuronal induction, GBM cells were treated with 5 μM Y27632, 50 μM forskolin, 2 μM SB431542, and 10 μM SP600125 (Selleck) in neuronal induction medium (a 1:1 mixture of DMEM / F12 (Corning) and Neurobasal (Gibico), 0.5% N2, 1% B27, 20 ng / ml BDNF, 20 ng / ml NT3, 1% GlutaMax, and 1% penicillin / streptomycin).

[0036] (2) Lentivirus preparation and transduction

[0037] The TUBB3 (NM_001197181.2) promoter and the fluorescent protein mCherry were linked to a lentiviral vector to indicate TUBB3 transcription and neuronal differentiation efficiency. Lentiviral vectors carrying CEND1 (NM_016564.4)-specific shRNA (CEND1-shRNA-1 or CEND1-shRNA-2) and a negative control (Scramble) were synthesized by GenScript (Nanjing, China). GBM cells were labeled using lentivirus expressing EGFP and luciferase for in vivo tumor growth assays. Preparation of lentivirus and cell infection: Three plasmid expression systems were co-transfected into HEK293T cells to produce lentiviral particles. Purified and concentrated lentivirus was used to infect target cells (U87, U251, and patient-derived GBM cells) at an MOI of 5. After small molecule induction, the efficiency of promoter activation in neuronal-differentiating GBM cells was detected by confocal microscopy (Leica).

[0038] (3) Cell cycle arrest

[0039] The distribution of cell cycle phases was determined by propidium iodide staining and examined by flow cytometry. Briefly, cells (1.0 × 10 6), washed with cold PBS, and fixed with 70% ethanol at -20°C overnight. After centrifugation, the cells were incubated in PBS containing propidium iodide (50 μg / ml) (Sigma-Aldrich) and RNase (1.0 mg / ml) (Sigma-Aldrich) at 37°C in the dark for 30 minutes and sorted by fluorescence-activated cell sorter (FACS; BD). FlowJo software (FlowJo 10.6.2) and ModFit LT software (ModFit LT TM 5.0) to visualize and analyze flow cytometry data.

[0040] (4) Western blot analysis

[0041] GBM cells were lysed and homogenized in RIPA buffer containing protease and phosphatase inhibitors. Protein samples (25 μg) were loaded onto 10% or 15% SDS-PAGE gels. The gels were electrophoresed and transferred to PVDF membranes (Millipore). The membranes were blocked with 5% BSA in TBST buffer for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies against TUBB3 (BioLegend, 1:1000), GAPDH (Cell Signaling Technologies, CST, 1:1000), CyclinD1 (CST, 1:1000), CDK2 (CST, 1:1000), p27KIP1 (CST, 1:1000), p21CIP1 (CST, 1:1000), and CEND1 (Abcam, 1:10000). HRP-conjugated secondary antibodies (anti-mouse: Invitrogen, 1:10,000 or anti-rabbit: Invitrogen, 1:10,000) were incubated at room temperature for 1 hour, and protein bands were visualized using chemiluminescence reagents (Millipore). The relative expression of GAPDH was normalized and quantified by densitometry using ImageJ software.

[0042] (5) Cell proliferation assay

[0043] Cell proliferation was assayed using an EdU detection kit (Beyotime). Briefly, 10 μM EdU was incorporated into cells and incubated at 37°C for 2 hours. Cells were then fixed, permeabilized, and incubated with Click reagent for 30 minutes. Nucleic acids were stained with Hoechst 33258 (Sigma-Aldrich; Catalog #94403), and images were acquired using a confocal microscope (Leica).

[0044] (6) Immunofluorescence

[0045] GBM cells or sections were fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.2) for 30 minutes and permeabilized with PHT solution (3% BSA and 0.1% Triton X-100 in PBS) for 1 hour at room temperature. Primary antibodies against TUBB3 (BioLegend, 1:500), MAP2 (CST, 1:500), SYN1 (CST, 1:200), Ki-67 (CST, 1:400), NEUN (Abcam, 1:200), and tyrosine hydroxylase (TH) were incubated overnight at 4°C. Alexa Fluor TM Incubation was performed with 594 / 488 goat anti-mouse IgG (H+L) (Invitrogen, 1:500) or anti-rabbit IgG (H+L) (Invitrogen, 1:500) at 37°C for 1 hour. Cell nuclei were labeled with Hoechst 33258 (Sigma-Aldrich). Fluorescence signals were detected using confocal laser scanning (Leica).

[0046] (7) Calcium imaging

[0047] Calcium imaging and data analysis were performed by Leica Microsystems LAS AF (Leica AF6000 microscope). GBM cells (U87 and U251) differentiated by YFSS for 7 days were loaded with 2 μM Fluo-4AM (Beyotime) in a 5% CO2 solution at 37°C for 30 minutes. Fluo-4AM was added to Tyrode solution (128mM NaCl, 2mM KCl, 2mM CaCl2, 2mM MgCl2, 30mM glucose and 25mM HEPES). Calcium flux was monitored for 90 seconds with an exposure time of 200 milliseconds and an exposure interval of 2 seconds. For KCl stimulation experiments, a final concentration of 25mM was added to confirm the neural properties of the responding cells. The calcium response was calculated as the value of the fluorescence change (ΔF) relative to the initial fluorescence (F0) quantified using ImageJ software.

[0048] (8) Tumor sphere formation assay

[0049] To assess the clonality of GBM cells after neural induction, 100 cells were seeded per well in neural progenitor cell culture medium (Neurobasal, 1% N2, 1% B27, 10 ng / ml EGF, and 20 ng / ml bFGF). The number and area of ​​tumor spheres were counted and scored using ImageJ.

[0050] (9) qRT-PCR and RNA-seq

[0051] Total RNA was isolated using Trizol reagent (Invitrogen) according to the user guide for qRT-PCR and RNA-seq analysis. For qRT-PCR, total RNA was reverse transcribed using the MaximaFirst Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific) according to the supplier's recommended procedures. qRT-PCR detection was performed using Premix ExTaqTM (Takara Bio). All samples were performed in triplicate. The average value was used for measurement, and the results were normalized to the expression of GAPDH. For RNA-seq, total RNA was extracted and eukaryotic mRNA was enriched by oligonucleotide (dT) magnetic beads. The enriched mRNA was then fragmented into short fragments using fragment buffer and reverse transcribed into cDNA using NEBNext UltraRNALibrary Prep Kit for Illumina (New England Biolabs, NEB). The purified double-stranded cDNA fragments were end-repaired, A bases were added and connected to Illumina sequencing adapters. The ligation reaction was purified with AMPure XP microbeads and amplified by PCR. The resulting cDNA library was sequenced by Gene Denovo Biotechnology Co. (Guangzhou, China) using Illumina Novasek6000. The RNA-Seq data were then used for gene set enrichment analysis (GSEA) and analyzed by weighted gene co-expression network analysis (WGCNA). For differentially expressed genes in RNA-seq data, Gene Ontology (GO) and KEGG pathway analysis were performed using DAVID (https: / / david.ncifcrf.gov).

[0052] (10) Migration and invasion assays

[0053] The migration and invasion of GBM cells or neurally differentiated GBM cells were studied using Transwell chambers. Cells were plated at 5 × 10 cells per 100 μl. 4Cells were seeded at a density of 100 cells in neuronal induction medium with or without small molecules and plated onto 24-well Transwell plates with 8 μm pores (Corning) for migration assays or onto the upper chamber of a Transwell coated with Matrigel (BD Biosciences) for invasion assays, and the lower chamber was filled with the same medium as the upper chamber. After incubation for 24 hours, GBM cells that did not migrate or invade were scraped off. Cells on the bottom surface were fixed with 4% paraformaldehyde for 10 minutes and stained with 0.4% crystal violet for 30 minutes. The number of migrating and invading cells was counted from five randomly selected fields using ImageJ.

[0054] (11) In vivo xenograft tumor model

[0055] In vivo evaluation of GBM differentiation therapy was performed using an intracranial GBM xenograft model. Prior to implantation, mice were anesthetized with an intraperitoneal (ip) injection of ketamine (100 mg / kg). Luciferase-expressing U87 or human primary GBM cells (2 × 10 cells per mouse) were transfected into the 40-well plate. 5 Cells (cells) were suspended in 5 μl PBS and implanted into the brains of 8-week-old immunodeficient NOD-SCID mice using a stereotactic apparatus (Kopf Instruments) (coordinates: 2 mm posterior, 2 mm lateral to the anterior mammary gland, 2.5 mm deep from the dura mater). Seven days after implantation, 100 μl of artificial cerebrospinal fluid (purchased from Solarbio) containing small molecules (15 μM Y27632, 150 μM forskolin, 6 μM SB431542, and 30 μM SP600125) was continuously released into the orthotopic tumor tissue at a rate of 0.25 μl / h using a sustained-release pump for 14 days (RWD Life Science, China). Bioluminescence was detected using a PerkinElmer IVIS Spectrum, and luciferase activity was measured to monitor tumor volume weekly. All mice were euthanized when they reached a moribund state. Their brains were removed, embedded, cut into 20 μm thick sections on a cryostat, and stained with H&E or immunofluorescence. Survival data of xenograft tumor models were compared using the Kaplan-Meier method with the log-rank test (n=8).All experiments were approved by the Institutional Animal Care and Use Committee.

[0056] (12) Statistical analysis

[0057] Data are presented as mean ± SEM. Statistical analysis was performed using Student's t-test or one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. *, # P < 0.05 was considered statistically significant. All experiments were repeated at least three times.

[0058] Example 1: Effective induction of neuronal differentiation of GBM cells using a small molecule mixture

[0059] In our previous studies, we successfully used small molecules to reprogram fibroblasts into neurons, showing promise for spinal cord injury repair. To identify small molecules that could effectively differentiate human GBM cells into neurons, we developed a TUBB3 promoter-reporter gene system and established the U87-TUBB3::mCherry cell line, enabling monitoring of the neuronal marker TUBB3. Using U87-TUBB3::mCherry cells, we screened various small molecules known to play a role in neuronal transdifferentiation, including Y27632, SB431542, CHIR99021, LDN193189, SP600125, DAPT, SU5402, VPA, forskolin, ISX9, and I-BET151. We found that the mixture YFSS (Y27632, forskolin, SB431542, and SP600125) performed best in inducing neuronal differentiation in U87 cells, with an efficiency of 92.4 ± 0.9%, as indicated by mCherry expression. Moreover, omitting any YFSS component significantly reduced differentiation efficiency ( Figure 2 A and B). In vitro differentiation of U87 and U251 cells using neuronal induction medium supplemented with YFSS resulted in significant morphological changes within a day, and even as early as 2 hours ( Figure 2 ), including the loss of polymorphic features and the development of elongated neuron-like structures. These morphological changes further evolved into complex neuron-like processes ( Figure 2 C), and strong TUBB3 expression ( Figure 2 A).

[0060] In our study, TUBB3 in U87 and U251 cell lines + The expression of MAP2 in U87 and U251 cells increased significantly to 85.1±1.9% and 82.5±1.9% respectively 3 days after induction. In addition, MAP2 was expressed in both cell lines, with the ratios of MAP2 in U87 and U251 being 64.7±1.6% and 65.9±3.7% respectively. Figure 1 As shown in A and B. During differentiation, the expression of TUBB3 at the mRNA and protein levels increased steadily and significantly increased after three days ( Figure 1 CE). Similarly, MAP2 expression began to increase on the first and third days of differentiation in U87 and U251, respectively, and remained at a high level thereafter ( Figure 2 D). In addition, the expression of NEUN in U87 and U251 cells increased significantly on days 5 and 3 of differentiation, indicating maturation ( Figure 2E). After 7 days of YFSS induction, cells showed obvious NEUN staining, with U87 and U251 showing 93.4±0.4% and 94.4±0.2% positive, respectively. In addition, synaptic proteins PSD95 and SYN1 also showed positive staining ( Figure 1 FH).

[0061] To assess the functional properties of the neuron-like cells generated by differentiation, we performed calcium imaging after 7 days of differentiation. When treated with potassium chloride (KCl), both differentiated U87 and U251 cells displayed a characteristic neuronal calcium transient response. This response is characterized by a rapid and significant increase in intracellular calcium levels, such as Figure 1 These findings indicate that the differentiated cells have acquired the physiological functions of neurons. Therefore, these results show that the YFSS small molecule mixture can effectively induce the differentiation of human GBM cells into functional neurons.

[0062] Example 2: YFSS-mediated neuronal conversion of GBM cells leads to reduced cell proliferation and prolonged cell cycle

[0063] To evaluate the proliferation of GBM cells (U87 and U251) during neuronal differentiation, we examined the expression of Ki-67 at different stages (days 0, 1, 3, 5, and 7). Immunofluorescence staining showed that Ki-67-positive cells were significantly reduced starting from day 3 of differentiation ( Figure 3 A and B), a significant decrease in Ki-67 mRNA levels was observed starting from day 1 ( Figure 3 C). This indicates that there is a significant obstacle to cell proliferation during differentiation. Further analysis using EdU incorporation within 7 days showed that proliferation activity decreased significantly after YFSS treatment, with the EdU positive rate of U87 decreasing from 33.4±3.4% to 4.4±0.5% and the positive rate of U251 decreasing from 45.4±1.2% to 16.4±1.6% ( Figure 3 D and E). These results clearly show that when GBM cells differentiate into neurons, their proliferation is significantly reduced.

[0064] To understand the role of cell cycle regulation in GBM cell proliferation during differentiation, we analyzed U87 cells at different differentiation stages (days 0, 1, 3, 5, and 7). Transcriptome microarray analysis revealed a consistent decrease in the expression of cyclins and cyclin-dependent kinases (CDKs), including CyclinA2, B1, B2, D1, E1, E2, and CDK1, 2, 4, 6, 7, and 8. In contrast, cyclin-dependent kinase inhibitors (CKIs), such as p21CIP1 and p27KIP1, increased throughout differentiation. KEGG analysis using GSEA confirmed a significant downregulation of cell cycle and DNA replication pathways in differentiated cells.

[0065] qRT-PCR analysis confirmed these findings, showing that the expression of cell cycle proteins and CDKs decreased significantly from the onset of differentiation, while the expression of CKIs p21CIP1 and p27KIP1 increased ( Figure 4 AD). CyclinD1 protein levels mirrored mRNA levels, initially decreasing in U87 cells before returning to baseline after three days, while remaining consistently low in U251 cells. CDK2 expression patterns differed temporally, with an initial decrease followed by fluctuations in both cell lines. Expression levels of p21CIP1 and p27KIP1 at the protein level showed a continuous increase throughout differentiation in U87 cells. In U251 cells, expression of p21CIP1 and p27KIP1 also increased, with the former reaching a maximum level on day 3 and the latter on day 5 ( Figure 4 EH).

[0066] Flow cytometry analysis showed that after 7 days of YFSS treatment, the G1 phase of U87 and U251 cells was significantly increased, and the G2 phase was reduced. It is worth noting that the S phase of U87 cells was also increased ( Figure 4 These findings strongly suggest that YFSS treatment impedes cell proliferation and induces cell cycle arrest in the G1 and S phases during the differentiation of GBM cells into neurons.

[0067] Example 3: YFSS inhibits tumor spheroid formation and reduces the migration and invasion ability of GBM cells

[0068] The decreased proliferation and cell cycle exit observed in GBM cells undergoing neuronal differentiation by YFSS led us to hypothesize that tumor sphere formation, migration, and invasion might be similarly affected. When U87 and U251 cells were cultured in neurosphere medium for 7 days, YFSS significantly inhibited tumor sphere formation, as evidenced by a decrease in sphere size and a reduction in spheres with diameters exceeding 100 μm ( Figure 5 A and B).

[0069] Migration was assessed using a Transwell assay, which showed that the number of cells migrating into the lower chamber was significantly reduced after 7 days of differentiation compared to undifferentiated cells ( Figure 5 C and D). The invasive capacity of both cell lines, measured by Matrigel invasion assay, was also significantly reduced after differentiation ( Figure 5 E and F). The expression levels of invasion-related genes MMP1 and MMP3 decreased from the first day of differentiation, indicating a significant reduction in invasive potential ( Figure 5G, H). The expression of other invasion-related genes, vascular endothelial growth factor C (VEGFC) and ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), also showed a similar decreased trend, whereas the expression of tissue inhibitor of metalloproteinases (TIMP) family genes, namely TIMP1, TIMP2, and TIMP4, known as inhibitors of invasion, increased.

[0070] These observations collectively indicate that YFSS not only inhibits tumorsphere formation but also significantly reduces the migration and invasion of GBM cells during neuronal differentiation, contributing to the reversal of GBM malignant phenotype.

[0071] Example 4: YFSS effectively promotes neuronal differentiation of patient-derived GBM cells

[0072] Given the heterogeneity and mutational diversity of GBM, patient-derived samples are closer to its genetic and phenotypic characteristics than traditional cell lines. We isolated cells from GBM tissue resected from three patients and cultured them in neurosphere medium. Once adhered to PLL-coated substrates, these cells were subjected to YFSS induction in neuronal induction medium for 7 days. Compared with the control group (Scramble+SFM), patient-derived GBM cells formed spheres and underwent neuronal differentiation, as shown by increased TUBB3 fluorescence intensity and decreased EdU incorporation ( Figure 6 A, Figure 7 AD). This differentiation was marked by the colocalization of TUBB3 with mature neuronal markers Map2, NEUN, SYN1, and PSD95, and was partially reversed by CEND1 knockdown ( Figure 7 AB and 7E-F).

[0073] In addition, the migration of patient-derived GBM cells was examined. Without treatment, GBM spheroids expanded over time, but YFSS treatment significantly inhibited this proliferation. CEND1 downregulation mitigated this effect to some extent, highlighting the role of CEND1 in reducing GBM cell proliferation and migration during differentiation. Figure 6 BC).

[0074] These results indicate that YFSS not only induces patient-derived GBM cells to differentiate into mature neurons but also implies that CEND1 is a key regulator in this conversion process.

[0075] Example 5: YFSS-induced reprogramming hinders GBM tumor growth in vivo

[0076] To evaluate the clinical potential of YFSS in vivo, we utilized the luciferase-GFP-expressing U87 GBM cell line and patient-derived GBM cells to track tumor growth. We established a patient-derived xenograft (PDX) model and administered YFSS directly to the tumor site via a slow-release micro-osmotic pump. This approach allowed for controlled release and promoted the differentiation of GBM cells into neurons in vivo. Tumor progression was monitored weekly using bioluminescence imaging on a PerkinElmer IVIS Spectrum.

[0077] The results showed that the tumors in the control group (U87 and GBM) grew rapidly, while the tumors in the YFSS-treated group (U87+YFSS and GBM+YFSS) progressed significantly slower ( Figure 8 A and B). Survival analysis after cell transplantation showed that YFSS treatment significantly improved the survival time of the mouse models: approximately 42 days for U87+YFSS and approximately 37.5 days for GBM+YFSS, compared with 30 days and 26.5 days for the respective control groups ( Figure 8 C).

[0078] Histological analysis at 28 dpi confirmed that in the YFSS-treated group, tumor volume was reduced and the expression of the neuronal marker NEUN was increased, indicating that tumor cells successfully differentiated into neurons ( Figure 8 DF). These findings highlight the potential of YFSS in orthotopic GBM treatment, significantly reducing tumor growth and prolonging survival in the NOD mouse model.

[0079] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for inducing malignant glioma cells to differentiate into neurons in vitro, characterized in that: The following steps are involved: During the culturing of malignant glioma cells, adding the small molecule composition to the neuronal induction medium and continuing the culturing for at least 2 hours; The small molecule composition consists of Y27632, forskolin, SB431542 and SP600125, and the concentrations of the small molecule composition in the neuron induction medium are: Y27632 5 μM, forskolin 50 μM, SB431542 2 μM, SP600125 10 μM; The neuron induction medium is prepared by mixing DMEM / F12 medium and neurobasal medium at a volume ratio of 1:1, and adding components including 0.5% N2, 1% B27, 20 ng / ml BDNF, 20 ng / ml NT3, and 1% GlutaMax.

2. The method according to claim 1, characterized in that The neuron induction culture medium also contains an antibacterial agent.

3. Use of a small molecule composition in the preparation of a drug for treating glioblastoma, characterized in that: The small molecule composition comprises Y27632, forskolin, SB431542, and SP600125, and the molar ratio thereof is 5:50:2:

10.

4. The use according to claim 3, characterized in that The small molecule composition is administered to the tumor site for treatment.

5. The use according to claim 3, characterized in that A pharmaceutically acceptable safety background solution containing the small molecule composition is administered to the tumor site.

6. The use according to claim 5, characterized in that The cerebrospinal fluid containing the small molecule composition is administered to the tumor site.

7. The use according to claim 5 or 6, characterized in that The concentrations of the small molecule composition in the background solution were: Y27632 5 μM, forskolin 50 μM, SB431542 2 μM, and SP600125 10 μM.

8. A drug for treating glioma, characterized in that: The medicine contains a small molecule composition, which is Y27632, forskolin, SB431542 and SP600125, and the molar ratio of Y27632, forskolin, SB431542 and SP600125 is 5:50:2:10.

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