Application of small molecule medicine composition in transdifferentiation of glioma cells

Abstract

The invention discloses an application of a small molecular medicine composition in transdifferentiation of glioma cells. The small molecular medicine composition comprises LiCl, Pirfenidone, Forskolin and RO4929097, and the small molecular medicine composition comprises LiCl, Pirfenidone, Forskolin and RO4929097. The invention further discloses application of the small molecule medicine composition in treating glioma, preparing a medicine for treating glioma and preparing a medicine for inducing glioma cells to be transdifferentiated into neuron-like cells. According to the present invention, with the application of the small molecule drug combination, the glioblastoma line U251 can be induced into the neuron-like cells, the tumor proliferation and the subcutaneous tumor formation ability can be inhibited, and the median lifetime of the xenotransplantation brain glioma model mouse can be prolonged.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of a small molecule drug combination in glioma cell transdifferentiation. Background Art

[0002] Glioma is a common malignant brain tumor that is difficult to treat, has a poor prognosis, and a high recurrence rate. Therefore, it has always been a hot topic in the study of neurological diseases. Glioma usually originates from a variety of glial cells, including astrocytes, oligodendrocytes, and ependymal cells, and develops into astrocytomas, oligodendrogliomas, ependymomas, etc. According to the classification standards of the World Health Organization (WHO), gliomas are mainly divided into four grades. Among them, grade I and grade II gliomas grow slowly and are low-grade gliomas, while grade III and IV gliomas grow rapidly, are highly invasive, and are highly malignant, and are high-grade gliomas. The median survival time of patients with grade III gliomas is approximately 3 years, while the median survival time of patients with grade IV gliomas is only 15 months. The most common grade IV glioma is glioblastoma (GBM).

[0003] Despite the continuous advancement of medical technology, the treatment of gliomas still faces many difficulties, and the reasons for the difficulty in treatment involve multiple levels. First, gliomas have a very complex tumor microenvironment, which includes various immune cells and non-immune cells, such as microglia, macrophages, T cells, B cells, NK cells, etc. They, together with other non-immune components such as endothelial cells, astrocytes, neurons, etc., constitute the tumor microenvironment (TME) of gliomas. The TME promotes clonal evolution and drug resistance, resulting in extremely high tumor heterogeneity in gliomas. The phenotype and genetic characteristics of tumor cells in different tumor sites of the same patient are significantly different, resulting in different responses of tumor cells to treatment. Therefore, it is difficult for a single therapy to completely eliminate all tumor cells, and it will promote angiogenesis to provide nutrients to the hypoxic internal environment of the tumor. In addition, gliomas are highly invasive, and there is no clear boundary between the tumor and the surrounding brain parenchyma. Tumor cells infiltrate the brain parenchyma along blood vessels, fiber bundles, and ependyma, making it difficult to perform complete surgical resection. Within a few months after surgical resection, recurrent tumors will form near the resection area.

[0004] Traditional treatments include surgical resection, radiotherapy, and chemotherapy. Surgical resection remains the preferred treatment for gliomas. Using techniques such as computed tomography (CT), magnetic resonance imaging (MRI), and fluorescent labeling, the tumor boundaries are precisely identified, aiming to remove as much tumor tissue as possible while maximally preserving neurological function, alleviating symptoms, and prolonging survival. However, due to the highly invasive nature of gliomas, which grow infiltratingly and invade normal brain tissue, complete surgical removal of all tumor cells is difficult. Therefore, further treatment with chemotherapy and radiotherapy is often performed after surgery. Temozolomide (TMZ) is a commonly used chemotherapy agent. TMZ is an oral alkylating drug commonly used to treat malignant gliomas. It can cross the blood-brain barrier and has good penetration into central nervous system tumors. However, tumor cells may develop resistance to temozolomide by counteracting the toxic effects of temozolomide through repairing DNA damage, altering methylation patterns, or upregulating antioxidant enzymes.

[0005] Currently, there are many new treatments for gliomas, including Tumor Therapeutic Fields (TTF), immunotherapy, and targeted therapy. Tumor Therapeutic Fields (TTF) applies electric fields of a specific frequency to the tumor area to disrupt tumor cell division. Immunotherapy and targeted therapy are also increasingly being used in the treatment of gliomas. Immune checkpoint inhibitors are one of the most well-researched and widely used immunotherapies, showing significant efficacy in most tumors. Common immune checkpoints in gliomas include PD-1 and PD-L1. These antibodies bind to PD-1 or PD-L1, blocking the inhibitory effect of tumor cells on immune cells, thereby activating the immune system's anti-tumor activity. Although many cancer treatments have been developed, few have been approved by the FDA for the treatment of gliomas. This is primarily due to the presence of the blood-brain barrier (BBB) in the central nervous system. This barrier, composed of endothelial cells, capillaries, and the basement membrane, serves as a natural barrier protecting the central nervous system from external pathogens and toxins. However, it also makes it difficult for many chemotherapeutic and immunotherapeutic drugs to cross the BBB and reach the tumor site, limiting their efficacy and posing a challenge to the development of glioma treatments. Therefore, there are still many challenges and problems to be solved in the clinical treatment of gliomas, and more effective and safe treatment methods need to be explored.

[0006] Cell reprogramming is the process of converting differentiated cells into another cell type or restoring their pluripotency. Scientists have developed direct reprogramming technology, which uses small molecules, transcription factors, or microRNA to directly convert one lineage into another without going through the pluripotent cell stage. Overexpression of Myod in fibroblasts to reprogram them into myoblasts demonstrated for the first time that transcription factors specific to a single cell type can change cell fate. Central nervous system diseases are often accompanied by a massive loss of neurons, and the ability to generate new neurons in the adult mammalian brain is very limited. Only a small number of neural stem cells in the lateral ventricle and the dentate gyrus of the hippocampus can generate new neurons. Therefore, many scientists are committed to exploring the regeneration of neurons through direct reprogramming. Direct cell reprogramming shows broad application prospects in the field of neural regeneration. Astrocytes are a type of cell with a relatively high proportion in the central nervous system, responsible for supporting and protecting neurons, maintaining the blood-brain barrier, etc. Because astrocytes have the ability to divide and are derived from the same progenitor cells as neurons, they can transform into reactive astrocytes under conditions of neural injury or disease, exhibiting the characteristics of proliferation and surrounding necrotic lesions, also known as glial scars. This makes them ideal targets for reprogramming research.

[0007] Small-molecule drug-induced reprogramming has widespread application in regenerative medicine. Small-molecule drugs can efficiently regulate cell fate by targeting epigenetic modifications, signaling pathways, and metabolic pathways. Traditional transcription factor-based cell reprogramming studies have shown that this process invariably involves epigenetic modifications and the activation and inhibition of related signaling pathways. This suggests the possibility of inducing lineage conversion by manipulating signaling pathways and epigenetic regulators. Therefore, chemically induced reprogramming could be achieved through small molecules that modulate these pathways. Summary of the Invention

[0008] The purpose of the present invention is to provide a solution that can effectively treat glioma in response to the above technical problems.

[0009] In order to achieve the above object of the invention, the present invention provides an application of a small molecule drug combination in glioma cell transdifferentiation, wherein the small molecule drug combination comprises LiCl, Pirfenidone, Forskolin and RO4929097.

[0010] On the other hand, the present invention also provides a small molecule drug combination comprising LiCl, Pirfenidone, Forskolin and RO4929097.

[0011] On the other hand, the present invention also provides the use of the small molecule drug combination in the treatment of glioma.

[0012] On the other hand, the present invention also provides the use of the small molecule drug combination in the preparation of drugs for treating gliomas.

[0013] On the other hand, the present invention also provides the use of the small molecule drug combination in inducing the transdifferentiation of glioma cells into neuron-like cells.

[0014] On the other hand, the present invention also provides the use of the small molecule drug combination in the preparation of a drug for inducing the transdifferentiation of glioma cells into neuron-like cells.

[0015] On the other hand, the present invention also provides a method for inducing glioma cells to transdifferentiate into neuron-like cells, comprising administering the small molecule drug combination.

[0016] The present invention utilizes a combination of four small molecule drugs: GSK-3β inhibitor LiCl, TGF-β inhibitor Pirfenidone, cAMP agonist Forskolin, and Notch pathway inhibitor RO4929097. This combination can induce the glioblastoma line U251 into neuron-like cells, inhibit tumor proliferation and subcutaneous tumor formation, and prolong the median survival of xenograft brain glioma model mice, demonstrating a certain therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The experimental technical route in the embodiment of the present invention is shown.

[0018] Figure 2 Protein expression in the glioblastoma cell line U251 is shown. (A) Immunofluorescence staining of glial cell-associated proteins. U251 cells express the glial cell markers GFAP and SOX9. (B) Immunofluorescence staining of proliferation-associated proteins. U251 cells express the proliferation-associated protein KI67, the tumor stemness-associated protein SOX2, and the human nuclear protein marker Human Nuclei (HuNu). (C) Immunofluorescence staining of tumor-associated proteins. U251 cells express the malignancy-associated proteins nestin and vimentin. (D) Immunofluorescence staining of neural-associated proteins. U251 cells do not express the immature neuronal marker DCX and weakly express Tuj1 and MAP2 (scale bar, 50 μm).

[0019] Figure 3Results of preliminary drug combination screening are shown. (A) Schematic diagram of the drug combination screening process in the glioblastoma cell line U251. Two days after plating, when the cells reached 80% confluency, the medium was replaced with drug induction medium, with half the volume exchanged every two days. After nine days, the medium was replaced with neural differentiation medium to allow the induced cells to mature. After several days of differentiation, the cells were fixed and immunofluorescence stained to count the number of DCX+ cells. (B) Immunofluorescence staining results for representative drug combination concentrations used in the preliminary screening. After nine days of drug induction, the number of DCX-positive cells reached the highest level when the lithium chloride concentration was increased to 12 mM, the pirfenidone concentration to 10 μM, and the forskolin concentration to 50 μM (scale bar 50 μm). (C) Statistical results of the number of DCX+ cells for different drug combinations and concentrations used in the preliminary screening. Increasing the lithium chloride and pirfenidone concentrations, as well as extending the drug induction time, significantly improved transformation efficiency. (***P < 0.001, one-way ANOVA with Tukey's multiple comparison, n = 3).

[0020] Figure 4 The optimal drug combination is determined. (A) Immunofluorescence staining results comparing LPF with DAPT (LPFD) added to the initial screening drug combination. Tumor stemness-associated protein SOX2 co-labeled with tumor proliferation-associated protein KI67, while DCX-positive cells did not co-label with either SOX2 or KI67. (B) Immunofluorescence staining results comparing glioma cells induced with the drug combination LPFD versus the control group. Vimentin expression in glioma cells was attenuated after LPFD induction. (C) Immunofluorescence staining results comparing LPFR with RO4929097 replacing DAPT versus the control group. LPFR-induced cells expressed DCX, while DCX-positive cells did not co-label with KI67. Tuj1 expression was enhanced compared with the control group. (Scale bar, 50 μm). (D) Statistical analysis of the conversion efficiency of the LPF and LPFD combinations (***P < 0.001, Student's t-test, n = 3). (E) Statistical analysis of transformation efficiency of LPFD and LPFR combinations (***P<0.001, Student's t-test, n=3).

[0021] Figure 5Figure 3 shows the process of morphological changes in glioma cells induced by LPFR under bright field. (A) Under drug induction, cell morphology rapidly changes, with protruding protrusions and becoming rounded and translucent (scale bar, 100 μm). (B) Immunofluorescence staining results on days 4, 9, 14, and 19 of drug induction. DCX expression continues, and the morphology gradually matures. MAP2 expression is not significant on days 4 and 9, but expression increases on days 14 and 19. The number of KI67-positive cells gradually decreases with prolonged induction (scale bar, 50 μm). (C) Statistical analysis of the proportion of DCX-positive cells on days 9, 14, and 19 of drug induction. The proportion of DCX-positive cells is highest on day 9, while DCX expression is absent in the control group at all time points. (D) Statistical analysis of the proportion of KI67-positive cells on days 9, 14, and 19 of drug induction. The proportion of KI67+ cells is lowest in the LPFR group on day 14 (***P < 0.001, Student's t-test, n = 3).

[0022] Figure 6 The effects of drug induction on cell proliferation are shown. (A) Line graph of the OD value at 450 nm of U251 cells measured by CCK8 assay over 7 days of drug treatment. The OD450 values of the control group continued to increase, while the OD450 values of the drug-treated group slowly increased over the first four days and then slowly decreased. (B) Line graph of the OD value at 450 nm of U87-Luci cells measured by CCK8 assay over 4 days of drug treatment. The OD450 values of the control group continued to increase, while the OD450 values of the drug-treated group increased within 48 hours and then decreased (***P < 0.001, Multiple t tests, n = 3).

[0023] Figure 7 The effects of drug induction on the cloning ability of glioma cells are shown. (A) Representative images of the U251 cell cloning assay. The number of clones formed by U251 cells decreased after 9 days of drug treatment. (B) Representative images of the U87-Luci cell cloning assay. The number of clones formed by U87-Luci cells decreased after 6 days of drug treatment. (C) Quantitative analysis of the U251 cell cloning assay. The drug-treated group significantly reduced the number of clones formed by U251 cells. (D) Quantitative analysis of the U87-Luci cell cloning assay. The drug-treated group significantly reduced the number of clones formed by U87-Luci cells (***P<0.001, Student's t test, n=3).

[0024] Figure 8 The effect of drug combination on the tumorigenicity of glioma cells in vivo is shown. (A) Schematic diagram of subcutaneous tumor formation experiment in nude mice. 2×10 6U87-Luci cells treated with 0.35% DMSO or the drug combination LPFR for 6 days were allowed to form subcutaneous tumors, and differences in tumor formation were observed. (B) Injection site. (C) Representative images of in vivo imaging of nude mice on day 14 (left: DMSO-treated group; right: LPFR-treated group). (D) Quantification of fluorescence intensity during in vivo imaging of nude mice over 14 days. On day 2, there was no significant difference in fluorescence intensity between the two groups. Over time, the difference in fluorescence intensity gradually increased, with the DMSO-treated group showing higher fluorescence intensity than the LPFR-treated group. (E) Images of tumor size obtained from samples collected on day 14 (left: DMSO-treated group; right: LPFR-treated group). After 14 days of subcutaneous growth in nude mice, tumors in the control group were significantly larger than those in the drug-treated group. (F) Quantitative analysis of tumor weight obtained on day 14. Tumors in the control group weighed more than those in the drug-treated group (***P < 0.001, Student's t test, n = 6).

[0025] Figure 9 The therapeutic effect of xenograft brain glioma is shown. (A) Schematic diagram of the xenograft brain glioma treatment experiment in nude mice. 3×10 4 U87-luci cells were used to form tumors. On day 4, tumor formation was assessed by intravital imaging and grouping was performed. Starting on day 5, a combination of 10% DMSO + 10% PEG300 or LPFR was injected intravenously retro-orbitally daily. Intravital imaging was performed every 3 days to monitor tumor progression. The end of life of the nude mice treated with either drug or control was observed. (B) Quantitative results of fluorescence intensity from intravital imaging of nude mice over 16 days. Initially, there was no significant difference in tumor fluorescence intensity between the two groups. As time went on, the difference in fluorescence intensity between the two groups gradually increased. (C) Representative images of intravital imaging of nude mice in the control group on day 16. (D) Representative images of intravital imaging of nude mice in the drug group on day 16. (E) Overall survival of nude mice in different treatment groups. The overall survival of mice in the control group (Ctrl, red) and the LPFR-treated group (blue) is shown. The survival difference between the two groups was estimated by the Kaplan-Meier method, and the log-rank test was used to compare the survival difference. The dotted line in the figure represents the median survival rate (50%). Statistical analysis showed that the survival difference between the two groups was statistically significant (n=7, p=0.0066), indicating that LPFR treatment significantly affected the overall survival rate of mice.

[0026] Figure 10 Schematic diagram showing the transdifferentiation process of glioma cells. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the reagents, culture media, laboratory instruments, and biomaterials used in the present invention are all conventional reagents, culture media, laboratory instruments, and biomaterials known to those skilled in the art.

[0028] 1. Experimental reagents

[0029] 1.1 Conventional reagents

[0030] Table 1: List of common reagents

[0031] Reagents company Item No. Tribromoethanol Sigma T48402 tert-Amyl alcohol Sigma 152463 NaOH Sigma 71690 <![CDATA[NaH2PO4]]> Aladin C1525004 <![CDATA[Na2HPO4·12H2O]]> Aladin J1811180 D-Luciferin potassium salt Abmole M8873 PBS powder Boster Biological AR0030 95% saline Jiangxi Kelun Pharmaceutical none Paraformaldehyde (PFA) LEAGENE DF0135 Trizol Thermo Fisher Scientific 15596018 Chloroform Guangzhou Chemical Reagent Factory none Isopropyl alcohol Guangzhou Chemical Reagent Factory none Anhydrous ethanol Guangzhou Chemical Reagent Factory none Triton x-100 Solarbio T8200 Tween-20 Solarbio T8220 BSA powder Sigma WXBC7017V Mounting medium Sigma 10981-100ML Crystal violet Blue Sky C0121 sucrose Guangzhou Chemical Reagent Factory H155 Cell Cycle Staining Kit Lianke Bio CCS012

[0032] 1.2 Glioma cell culture medium

[0033] Table 2: Glioma cell culture medium components and concentrations

[0034] name company Item No. concentration DMEM Medium Gibco C11995500BT - FBS Vazyme F103-01 10％

[0035] 1.3 Small molecule combination induction medium

[0036] Table 3: Small molecule combination induction medium components and concentrations

[0037] name company Item No. concentration DMEM / F12 Medium Gibco C11330500BT - B27supplement Gibco 17504-044 0.4％ N2supplement Gibco 17502-048 0.8％ LiCl Selleck E0153 12mM Pirfenidone Selleck S2907 10 μM Forskolin Selleck S2449 50 μM RO4929097 Selleck S1575 10 μM

[0038] 1.4 Neural Differentiation Medium

[0039] Table 4: Glioma cell culture medium components and concentrations

[0040] name company Item No. concentration DMEM / F12 Medium Gibco C11330500BT - B27supplement Gibco 17504-044 0.4％ N2supplement Gibco 17502-048 0.8％ FBS Vazyme F103-01 0.5％ Penicillin & Streptomycin Gibco 15140122 0.2％ Forskolin Selleck S2449 10 μM Dorsomorphine Cayman chemical 86605643 1 μM Vc Sigma 134032 5 μg / mL FuD Sigma 50919 10 μM Uridine Cayman chemical 58-96-8 10 μM BDNF Peprotech 45002 10 ng / mL GDNF Peprotech 45010 10 ng / mL NT-3 Peprotech 45003 10 ng / mL IGF1 Peprotech 10011 20 ng / mL

[0041] 1.5 Other reagents required for cell culture

[0042] Table 5: Other reagents required for cell culture

[0043] name company Item No. DPBS Gibco C14190500BT 0.05% Trypsin-EDTA Gibco 25300-054 Matrigel BD 356234 Gelatin from porcine skin Sigma 48722-500G-F

[0044] 1.6 Antibodies

[0045] Table 6: Antibody List

[0046] Antibody Species company Item No. concentration Ki67 Rat Invitrogen 14-5698-82 1:1000 SOX2 Rabbit Millipore Ab5603 1:1000 SOX9 Rabbit Millipore Ab5535 1:1000 HuNu Mouse Millipore MAB1281 1:1000 DCX Rabbit Abcam Ab18723 1:2000 MAP2 Chicken Abcam Ab5392 1:2000 Tuj1 Mouse Sigma T8660 1:1000 GFAP Rat Invitrogen 13-0300 1:1000 Mash1 Rabbit Abcam ab211327 1:1000 Alexa Fluor 647 Goat anti chicken Life A21449 1:1000 Alexa Fluor 647 Donkey anti mouse Life A31571 1:1000 Alexa Fluro 647 Donkey anti Rabbit Invitrogen A31573 1:500 Alexa Fluro 647 Donkey anti guinea pig Jackson 706-605-148 1:1000 Alexa Fluor 488 Donkey anti mouse Life A21202 1:1000 Alexa Fluor 488 Goat anti chicken Life A11039 1:1000 Alexa Fluor 555 Donkey anti rabbit Life A31572 1:1000 Alexa Fluor 546 Goat anti chicken Life A11040 1:1000 DAPI - Roche 70508621 1:1000

[0047] 1.7 PBS buffer (0.01M)

[0048] Pour one packet of PBS powder into a 2L beaker and add 1.9L of ultrapure water. Place the beaker on a magnetic stirrer and stir until the powder is completely dissolved. Adjust the volume to 2L and dispense into individual bottles for later use.

[0049] 1.8 1.25% tribromoethanol solution

[0050] Accurately weigh 1.25g of tribromoethanol powder into a 100ml glass container wrapped in tinfoil. Add 2.5ml of tert-amyl alcohol and 90ml of deionized water. Stir overnight on a magnetic stirrer to completely dissolve the tribromoethanol. The next day, add deionized water to bring the total volume to 100ml. Filter using a 0.22μm pore size Millipore filter. Store in a refrigerator at 4°C.

[0051] 1.9D-luciferase sodium salt

[0052] 500 mg of D-luciferase sodium salt was dissolved in 33 ml of sterile DPBS to prepare a 15 mg / ml solution, which was then stored in a -80°C refrigerator.

[0053] 1.10 0.1% Gelatin

[0054] Weigh 0.2g of gelatin particles into a small beaker, add 200ml of DPBS, and place in an autoclave at 121°C for 30 minutes to swell and sterilize the gelatin. Mix the solution thoroughly, filter it through a 0.22μm filter in a clean bench, transfer it to a sterile bottle, and store it in a refrigerator at 4°C.

[0055] 2. Experimental content

[0056] 2.1 Cell experimental methods

[0057] 2.1.1 Culture of human glioblastoma cell lines

[0058] The U251 glioblastoma cell line was purchased from Wuhan Pronocell Life Science Co., Ltd. and cultured in a high-glucose DMEM medium containing 10% fetal bovine serum.

[0059] When cells reach a density of approximately 80%-90% in the culture dish, passage them. First, remove the old culture medium, then slowly add 1 ml of DPBS along the edge of the dish. Gently shake the dish to remove cell debris and residual culture medium. Next, add 1 ml of 0.05% trypsin solution to the dish and place it in a 37°C incubator for 2 minutes. Once most cells have shrunk and become rounded, the intercellular spaces have increased, and the remaining cells easily fall off when the dish is tapped, quickly add complete culture medium containing 10% fetal bovine serum to terminate the digestion process. Gently pipette the mixture to evenly disperse the cells. Then, transfer the cell suspension to a centrifuge tube and centrifuge at 1200 rpm for 3 minutes. After centrifugation, discard the supernatant, resuspend the cells in 1 ml of fresh culture medium, and passage them at a 1:5 ratio. Finally, continue incubating the cell suspension in a 37°C, CO2-containing incubator.

[0060] 2.1.2 Small molecule drug combination induces glioma cell transdifferentiation

[0061] Sterile round coverslips were placed in a 24-well plate. 300 μL of 0.1% gelatin solution was added to each well for slide coating. The next day, the 0.1% gelatin solution was aspirated and washed three times with sterile ddH2O. 1.6 x 10 4 U251 cells were seeded onto glass slides. When the cell confluence reached 80%, the medium was replaced with either the drug combination medium or the medium containing 0.35% DMSO for 9 days of induction. After drug induction, the medium was replaced with neural differentiation medium to further promote cell differentiation.

[0062] 2.1.3 Cell immunofluorescence staining and imaging

[0063] Remove the cell culture plate from the incubator and discard the culture medium. Gently add an appropriate amount of PBS solution to the wells. Place the plate on a shaker and gently shake at 75 rpm at room temperature for 5 minutes to wash. Then, aspirate the PBS solution. Next, add 300 μL of PFA solution to each well to fix the cells for approximately 15 minutes. After fixation, wash the plate three times with PBS to remove residual PFA. Next, add PBST solution containing 0.1% Triton X-100 to the wells and incubate for 10 minutes to increase cell membrane permeability. After permeabilization, wash the plate three times with PBS. Then, add 3% bovine serum albumin (BSA) solution and block for 30 minutes to reduce nonspecific binding. After the blocking solution is completely absorbed, add the appropriate amount of primary antibody to the 3% BSA solution. The blocking solution is then aspirated, and a solution containing the primary antibody is added. Incubate overnight at 4°C to allow the primary antibody to bind to the target antigen. The next day, take the well plate out of the 4°C refrigerator and let it stand for a while to warm up to room temperature. After warming up, wash the well plate three times with PBST solution containing 0.2% Tween-20 to remove unbound primary antibody. Subsequently, add a solution containing secondary antibody and DAPI (nuclear dye) to the well plate and incubate in the dark for 45 minutes. The secondary antibody can recognize and bind to the primary antibody, while DAPI is used to mark the cell nucleus. After incubation, wash the well plate twice again with PBST, and then wash it twice with PBS. Finally, place the slide upside down on a slide containing an anti-fluorescence quenching mounting medium to complete the cell immunofluorescence staining. The staining results can be imaged using a Zeiss upright fluorescence microscope, or observed using a Zeiss laser confocal microscope for high-magnification imaging.

[0064] 2.1.4 Cell proliferation ability assay

[0065] U251 or U87-Luci cells were first resuspended in DMEM medium containing 10% fetal bovine serum (FBS), and then the cells were evenly seeded into 96-well plates at a density of 4000 / well, and 100 microliters of culture medium was added to each well. The seeded cells were placed in a constant temperature incubator at 37°C and cultured for 24 hours until the cells were completely attached. After attachment, the medium was replaced with LPFR or 0.35% DMSO culture medium, and 5 replicates were set up for each group. When the detection time arrived, 10 μL of CCK-8 reagent was added to each well, and then the 96-well plate was transferred to a 37°C incubator containing 5% carbon dioxide and incubated for another 40 minutes. The absorbance of each well was measured at 450 nm using an enzyme reader, and the obtained data were plotted and analyzed.

[0066] 2.1.5 Clone formation assay

[0067] U251 or U87-Luci cells were suspended in DMEM culture medium containing 10% fetal bovine serum (FBS) to prepare a single cell suspension. U251 cells were seeded in a 6-well plate at a cell density of 600 cells / well, and U87-Luci cells were seeded in a 6-well plate at a cell density of 400 cells / well, with 3 replicates for each group. The cells were cultured in a 37°C constant temperature incubator for 24 hours to allow the cells to fully adhere to the wall. The next day, the medium was replaced with LPFR or 0.35% DMSO culture medium. U251 was cultured for 9 days and U87-Luci was cultured for 6 days. Visible clones were formed in the well plate under bright field observation. The culture medium was discarded, and the cells were rinsed once with DPBS and fixed with 4% paraformaldehyde solution for 15 minutes. After fixation, the 4% paraformaldehyde solution was discarded and the cells were rinsed twice again with DPBS. Crystal violet solution was added to stain the cells in the dark for 10 minutes. After staining, the crystal violet solution was recovered and the cells were washed three times with pure water to remove excess crystal violet solution. Finally, the wells were air-dried and the formed cell colonies were photographed and counted, and the statistics were plotted.

[0068] 2.2 Animal experimental methods

[0069] 2.2.1 Establishment of nude mouse subcutaneous glioma model

[0070] The experimental animals BALB / c-nu used were purchased from Guangdong Jicui Yaokang. All experimental design principles were approved by the Experimental Animal Ethics Committee of Jinan University and complied with the ethics of animal experiments. The ethical approval number is 20240619-06. First, the U87-Luci cells in the drug-treated group and the control group were digested with trypsin at a concentration of 0.05%, and the digestion time did not exceed 3 minutes. Subsequently, the cells were collected and centrifuged, and the supernatant was removed after centrifugation. The cells were then resuspended in DPBS and the cells were counted. The concentration of the cell suspension was adjusted to an appropriate level with DPBS, and the adjusted cell suspension was finally placed on ice waiting for tumor loading.

[0071] Five-week-old female nude mice were anesthetized with isoflurane, and the inguinal skin of the mice was disinfected with iodine. A 1ml syringe was used to draw up 100μL of the cell suspension. The needle was inserted dorsally, approximately 1cm below the skin, and then slowly bolus injection was performed. A noticeable bulge was observed below the skin after injection, confirming that the cell suspension was injected into the subcutaneous tissue rather than the muscle. After the injection, the needle was slowly withdrawn with a twist to prevent leakage.

[0072] 2.2.2 In vivo fluorescence imaging of nude mice

[0073] Nude mice were intraperitoneally injected with a 150 mg / kg solution of D-luciferase sodium salt. Five minutes after the injection, the nude mice were anesthetized by intraperitoneal injection of 0.25% tribromoethanol solution (20 ml / kg). Three minutes after anesthesia, if there was no leg extension response after pinching the fingers, the nude mice were placed in a small animal in vivo imaging device for imaging. The exposure time for the subcutaneous tumor model was 1 s, and the exposure time for the xenograft brain glioma model was 50 s. In vivo imaging was performed at subsequent time points. The fluorescence intensity of the tumor area was measured using Living Image software, and statistical analysis and graphing were performed.

[0074] 2.2.3 Subcutaneous tumor collection in nude mice

[0075] Anesthetize nude mice by intraperitoneal injection of 0.25% tribromoethanol solution (20 ml / kg). Confirm anesthesia by pinching the fingers for 3 minutes without leg extension reaction, and perform cardiac perfusion. Secure the limbs to the operating table, cut upward along the abdominal cavity, and open the chest cavity to fully expose the heart. Insert the needle into the left atrium, secure the needle, and perfuse normal saline until the limbs and liver turn white, then stop perfusion. Cut the skin in the groin to expose the tumor, carefully separate the tumor, photograph it, and weigh it. Store in a -80℃ refrigerator.

[0076] 2.2.4 Establishment of nude mouse glioma model

[0077] First, digest the U87-Luci cells with 0.05% trypsin for no more than 3 minutes. Then, collect the cells and centrifuge them. After centrifugation, remove the supernatant, resuspend the cells in DPBS, and count the cells. Adjust the cell suspension concentration to 1.5 × 10 4 Finally, the cell suspension was placed on ice and waited for tumor loading.

[0078] Five-week-old female nude mice were injected with 0.25% tribromoethanol solution (20 mL / kg). Anesthesia was confirmed by pinching the fingers for 3 minutes and confirming the absence of a leg extension response. The mice were then placed on an operating table. The head skin was disinfected with iodine, and the scalp was incised with a sterile scalpel blade to expose the skull. The mice were secured using a mouse adapter and leveled under a microscope. After leveling, a hole >1 mm in diameter was drilled at the coordinates A / P -2.1, M / L +1.5, and D / V -1.5, using the bregma as the origin. A glass electrode was then used to aspirate the tumor cell suspension, and 2 μL of the cell suspension was injected into the CA1 region of the hippocampus at a rate of 500 nL / min. After injection, the electrode was slowly withdrawn after 5 minutes. Finally, the scalp of the nude mouse was sutured, the wound disinfected with iodine, and the mice were placed on a heating pad for observation until awakening. The mice were then returned to their cages. In vivo imaging was performed every 4 days to monitor tumor growth.

[0079] 2.2.5 Retroorbital intravenous injection in nude mice

[0080] Use an insulin needle to draw up the drug solution to be injected and deflate air bubbles. Anesthetize nude mice with isoflurane. Confirm anesthesia by monitoring respiratory rate. Secure the mouse with your left hand. Gently protrude the eyeball within its socket. Hold the needle in your right hand, with the needle tip at a 45-60° angle to the medial canthus and the needle tip at a 45° angle to the tip of the mouse's nose. Slowly insert the needle into the medial canthus until there is a noticeable miss. The insertion depth is approximately 3-5 mm. After the bolus injection is complete, slowly withdraw the needle and gently press the eye to stop any bleeding.

[0081] 2.3 Data processing and statistical analysis

[0082] Immunohistochemistry results were analyzed using ZEN 2.3 Blue Edition. Graphpad Prism 8 software was used for statistical analysis. Data are presented as mean or mean ± SD. Analyses were performed using t-tests and one-way ANOVA. Results were considered significant when P < 0.05. N = 3.

[0083] 2.4 Experimental design

[0084] First, a screening experiment was conducted on U251 cells, identifying clinically applicable small molecule drugs by activating or inhibiting relevant signaling pathways. The selected drug combinations were found to be able to induce the emergence of DCX+ cells. After the drug combination was finalized, bulk RNA sequencing and RT-qPCR were used to preliminarily explore the transdifferentiation mechanism. CCK8 assays, colony formation assays, and in vivo tumorigenesis in nude mice were then used to investigate whether the drug combination could inhibit the tumor characteristics of gliomas. Finally, a nude mouse brain glioma model was established, and the selected drug combination was administered via retro-orbital intravenous injection, hoping to inhibit glioma progression in vivo and prolong survival.

[0085] Technical routes such as Figure 1 shown.

[0086] 3. Experimental results

[0087] 3.1 Protein expression in glioma cell lines

[0088] To understand the characteristics of the transdifferentiation of human glioblastoma cell line U251, immunofluorescence staining was performed on U251 cells without drug induction to determine their protein expression.

[0089] First, the glial cell-related proteins of U251 cells were detected to check whether they had glial cell-related characteristics. The results showed that U251 cells expressed glial cell-related proteins GFAP and SOX9 ( Figure 2 , A).

[0090] In order to determine the malignancy and proliferation characteristics of the glioblastoma cell lines used, immunofluorescence staining of proliferation-related proteins KI67 and SOX2 was performed. High expression of Ki67 indicates high proliferation activity of tumor cells, which is often associated with poor prognosis of glioma patients. Clinically, patients with high KI67 often have more aggressive tumors and shorter survival. SOX2 is highly expressed in glioma stem cells (GSCs), maintaining their stem cell characteristics and tumorigenicity, and driving the invasiveness and migration of gliomas. The results showed that U251 highly expressed KI67 and SOX2 ( Figure 2 , B), indicating that the cells have strong proliferation ability and high malignancy. U251 expresses human nuclear marker Human Nuclei ( Figure 2 , B), identified as a human cell line.

[0091] Next, tumor-related proteins that are often highly expressed in gliomas were detected. In gliomas, high expression of Nestin usually indicates that tumor cells have a higher proliferation ability and is related to the malignancy of gliomas. Vimentin is a vimentin protein that is often highly expressed in malignant tumors. Vimentin has a higher expression level in gliomas with high malignancy, and its high expression is associated with high invasiveness and poor prognosis of glioma patients. High expression of Nestin and Vimentin was detected in untreated U251 ( Figure 2 , C), indicating that U251 is highly invasive and malignant.

[0092] At the same time, some neuronal markers were detected. The U251 cell line does not express the immature neuronal marker Doublecortin (DCX), but weakly expresses TUBB3 (Tuj1) and microtubule-associated protein 2 (MAP2) ( Figure 2 , D).

[0093] 3.2 Screening of drug combinations to transdifferentiate glioma cells into neurons

[0094] 3.2.1 Screening of three basic combinations of small molecules

[0095] Drug screening was performed using the U251 cell line. Since U251 cells are astrocyte-derived, express glial fibrillary acidic protein (GFAP), and possess typical astrocyte characteristics, drug combinations that induce glioma transdifferentiation were initially screened (Table 7).

[0096] Table 7: Screened drugs and their pathways of action

[0097] path drug Use concentration WNT pathway agonists LiCl 2mM / 4mM / 6mM / 12mM TGFβ pathway inhibitors Pirfenidone 10nM / 20nM / 30nM / 50nM / 10μM / 20μM cAMP agonists Forskolin 50 μM Notch pathway inhibitors RO4929097 5μM / 10μM

[0098] First, the U251 cell line was induced with a combination of candidate drugs for 4 days. The neural differentiation medium was then replaced and cultured for 4 days to allow the transformed neurons to mature further. The cells were then fixed with PFA and immunofluorescence staining was performed. DCX expression was detected as a criterion for whether neuronal conversion had occurred. DCX is a microtubule-associated protein that plays an important role in the migration and maturation of nerve cells. It is highly expressed in immature neurons and neuroblasts, which can help researchers observe the process of neural development.

[0099] First, a combination of lower concentrations of LiCl (lithium chloride, Lithium Chloride, CAS No. 7447-41-8), Pirfenidone (Pirfenidone, CAS No. 53179-13-8), and Forskolin (Forskolin, CAS No. 66575-29-9) was used to induce the cells after the cell density reached 60%-70%. After four days of drug treatment, the medium was changed to neural differentiation medium to make the induced neurons more mature ( Figure 3 , A). However, under the induction of low concentrations of this drug combination, no transdifferentiation and tumor suppression were observed ( Figure 3 , B). The drug concentrations were then gradually increased, and it was found that when the pirfenidone concentration was increased to 30nM and the LiCl concentration was increased to 6mM, the cells underwent morphological changes and a very small number of DCX+ cells were induced. Only about 30 DCX+ cells could be observed on a 12mm diameter slide. When the drug concentrations were continuously increased to 10μM pirfenidone, 12mM LiCl, and 50μM forskolin, the number of DCX+ cells increased to 4 per square millimeter. In addition to increasing the drug concentrations, the drug induction time was extended from 4 days to 9 days, and it was found that the number of DCX+ cells per square millimeter increased to 4 per square millimeter ( Figure 3 , C), and DCX+ cells do not express KI67 ( Figure 3 , B). Therefore, the basic drug combination was initially determined to be LPF (12 mM LiCl, 10 μM Pirfenidone, 50 μM Forskolin), and LPF was induced for 9 days.

[0100] 3.2.2 Determination of the optimal drug combination

[0101] Since the number of cells transformed under the basic combination of LPF induction is still small, it is hoped to further improve the transformation efficiency. When 5μM DAPT (a common Notch signaling inhibitor that inhibits the Notch pathway by inhibiting γ-secretase, CAS No. 208255-80-5) was added to the LPF combination (LPFD), it was found that the number of DCX+ cells increased significantly, reaching 18 per square millimeter ( Figure 4 , D). In addition, the expression of KI67, a protein related to cell proliferation, was evaluated and it was found that the number of KI67+ cells in the DCX+ cell area decreased ( Figure 4 , A), and the expression of intermediate filament protein Vimentin in cells treated with LPFD was weakened, and the originally dense fibrous structure was disassembled ( Figure 4 , B). Vimentin expression levels reflect the mesenchymal characteristics of cells, and high expression is associated with the migration and invasion abilities of tumor cells. Therefore, from the perspective of tumor characteristics, LPFD treatment can reduce the migration and invasion abilities of U251 cells. This suggests that inhibition of the Notch signaling pathway can promote transdifferentiation and tumor suppression in U251 cells. DAPT should be replaced with safer clinical agents.

[0102] The γ-secretase inhibitor RO4929097 (CAS No. 847925-91-1) can effectively inhibit the Notch signaling pathway and also shows an inhibitory effect on the ERK1 / 2 signaling pathway, thereby having an anti-EMT (epithelial-mesenchymal transition) effect, thereby inhibiting the growth of tumor cells such as melanoma. When RO4929097 was added to the combined LPF instead of DAPT, a more significant transdifferentiation phenomenon was observed, reaching approximately 55 DCX+ cells per square millimeter ( Figure 4 , E), indicating that RO4929097 has a good effect in replacing DAPT, so the optimal drug combination is determined to be LPFR (12mM LiCl, 10μM Pirfenidone, 50μM Forskolin, 10μM RO4929097) ( Figure 4 , C). Figure 10 A schematic diagram of the transdifferentiation process of glioma cells is shown. Glioma cells are induced into neuron-like cells under the combined action of a combination of four LPFR small molecules (L, LiCl; P, Pirfenidone; F, Forskolin; R, RO4929097).

[0103] 3.2.3 Time course analysis of drug-mediated glioma cell reprogramming

[0104] The drug-induced transdifferentiation process of glioma cells was observed under a microscope. Under bright field, it was found that the cells in the control group always showed a typical glial morphology, with flat and expanded cells and a significant increase in cell number; while the cells in the drug-induced group began to show morphological changes on the first day, indicating that the cells began to respond to drug treatment; on the fifth day of drug treatment, cell protrusions became more obvious, the cytoskeleton contracted and elongated, and the cell body became round and translucent ( Figure 5 , A), therefore, from the morphological point of view, the U251 cells after drug induction are obviously different from the glioma cell morphology and are closer to the neuronal morphology.

[0105] Morphologically, the cells responded quickly to the drug, undergoing morphological changes. Monitoring was performed on days 4, 9, 14, and 19 after drug exposure, analyzing the expression of neuronal markers DCX and MAP2, as well as the tumor cell proliferation marker KI67, following LPFR treatment.

[0106] Immunofluorescence staining results showed that a small number of DCX-positive cells appeared from the fourth day during drug induction, and the highest proportion of DCX-positive cells appeared at the end of drug induction on the ninth day, about 3%. After switching to neural differentiation medium for subsequent culture, the proportion of DCX+ cells decreased compared with the ninth day, but at the same time, MAP2 expression was enhanced, and the morphology of DCX+ cells showed a more mature neuronal morphology ( Figure 5 , B; Figure 5 , C). On the fourth and ninth days, the proportion of KI67+ cells in the control group was 7%, while that in the treatment group was 6%, with no significant difference. After switching to neural differentiation medium for subsequent culture, the proportion of KI67+ cells in the drug-treated group was significantly lower than that in the control group on the fourteenth day, with the control group being about 10% and the treatment group being only 2% ( Figure 5 , D), suggesting that some immature neurons may have matured or died after drug induction was removed. The decreased proportion of KI67+ cells in the control group on day 19 compared to day 14 suggests that prolonged culture may have reduced cell proliferation and led to partial contact inhibition.

[0107] 3.3 Inhibitory effects of drug combinations on tumor characteristics of glioma cells

[0108] 3.3.1 Inhibition of glioma cell proliferation by drug combination

[0109] The CCK-8 assay was used to evaluate the effect of the drug combination on the proliferation ability of two glioma cell lines, U251 and U87-Luci.

[0110] The results showed that in the U251 cell line, the cell number within 24 hours of drug treatment was comparable to that of the control group. However, the cell number began to decrease after 24 hours, and then there was a slight growth trend within 4 days. However, after 4 days, the cell number continued to decrease, which was significantly different from the control group ( Figure 6 , A). This indicates that the combination has a significant cell proliferation inhibitory effect on the U251 cell line.

[0111] Since the cells in the U87-Luci control group grow rapidly and form a large number of spheres after four days, and the tumor spheres are easily detached during the medium change process, the changes in cell proliferation ability within 4 days of drug treatment were only measured in the U87-Luci cell line. The experimental results showed that the OD values of the control group and the drug-treated group continued to increase within 48 hours, and the cells showed a trend of continuous proliferation. However, the OD value of the drug-treated group continued to decrease in the later period, indicating that the number of cells gradually decreased ( Figure 6 , B), reflecting that drug treatment also has a significant inhibitory effect on the proliferation of U87-Luci.

[0112] Therefore, even though the drug combination could not induce transdifferentiation of U87-Luci cells due to the heterogeneity of tumor cells, the results showed that it had the ability to inhibit the proliferation of both cell lines.

[0113] 3.3.2 Inhibition of glioma cell clonogenicity by drug combination

[0114] The clone-forming ability reflects the dependence and proliferation ability of the cell population and can, to a certain extent, reflect the tumor-forming ability of cells in vivo.

[0115] In this experiment, U251 and U87-Luci cells were seeded at a density of 500 cells per well in 6-well plates. After 24 hours of adherence, the cells were replaced with drug culture medium or control culture medium. U251 cells were cultured for 9 days, and U87-Luci cells were cultured for 6 days. Statistics showed that after LPFR treatment, only 4 colonies were formed per well in U251 cells, while the average number of colonies per well in the control group was 102 ( Figure 7 , A, C). In U87-Luci cells, the average number of clones per well in the control group was approximately 53, while no visible clones were formed in the treatment group ( Figure 7 , B, D). Therefore, LPFR has an inhibitory effect on the colony formation of both U251 and U87-Luci.

[0116] 3.4 Evaluation of the subcutaneous tumorigenicity of gliomas induced by drug combinations

[0117] Due to the different characteristics of the two cell lines, U251 could not rapidly form solid tumors in nude mice. In order to evaluate the tumorigenicity of drug-induced tumor cells in vivo, 2×10 5 U87-Luci cells were injected into the subcutaneous tissue of the left and right groin of nude mice to form tumors ( Figure 8Because U87-Luci has been genetically modified to express the firefly luciferase gene, it can emit bioluminescence when injected intraperitoneally with a specific substrate (such as D-luciferin). The number of photons produced is positively correlated with the concentration of luciferase. Therefore, the activity of tumors in nude mice can be determined by measuring the fluorescence intensity using a small animal in vivo imaging device.

[0118] On the second day after cell transplantation, live imaging was performed and no significant difference in the fluorescence intensity of the tumors on both sides was observed, indicating that the number of tumor-bearing cells on both sides was roughly the same. Subsequently, live imaging was performed on the 4th, 7th, 10th, and 14th days after tumor implantation to observe the changes in tumor fluorescence intensity. It was found that the difference in fluorescence intensity between the two groups became larger and larger as time went on. By the 14th day, the average fluorescence intensity of the tumors in the control group was 2.96E+10 [p / s], while the average fluorescence intensity of the tumors in the treatment group was 6.9E+09 [p / s], which was significantly lower than that in the control group ( Figure 8 , C, D). Samples were collected on the 14th day. The average tumor weight of the control group was 0.4 g, while the average tumor weight of the treatment group was only 0.05 g, which was significantly lower than that of the control group ( Figure 8 , E, F). Therefore, the tumorigenicity of glioma cells in nude mice was reduced after drug treatment.

[0119] 3.5 Therapeutic effect of drug combination on brain glioma

[0120] To explore whether LPFR can be effective in treating gliomas in situ in the brain, we first attempted systemic administration via retro-orbital intravenous injection, as intravenous injection allows for adjustment of drug concentrations and multiple dosing.

[0121] 3×10 4 U87-luci cells were injected into the CA1 region of the hippocampus of nude mice to form tumors. In vivo imaging was performed on the fourth day after cell transplantation, and the fluorescence intensity of the tumors in the mice was roughly the same. The mice in the treatment group and the control group were injected with 100 μL of the drug into the retroorbital vein twice a day for 6 consecutive days ( Figure 9 , A). The treatment group was injected with the drug combination LPFR, and the control group was injected with the solvent 10% DMSO and 10% PEG300 (Table 8). In order to observe the growth trend of the tumor during drug treatment, in vivo imaging was performed every 4 days. It was observed that on the 8th and 12th days, there was no significant difference in the fluorescence intensity of the tumors in the control group and the treatment group. Both groups of tumors were rapidly proliferating, reflecting that the treatment group had no obvious trend of inhibiting the tumor. However, from the 12th day to the 16th day, the tumors in the drug-treated mice tended to progress slowly. On the 16th day after tumor loading, the average fluorescence intensity of the tumors in the control group was 4.50E+08 [p / s], and the average fluorescence intensity of the tumors in the drug-treated group was 1.50E+08 [p / s] ( Figure 9, BD). In experiments on glioma model mice, the end-of-life criteria usually follow the humanitarian end-of-life principle. When the mice show a significant decrease in activity and their weight continues to drop to 20% of their original weight, they are considered to have reached the end of life. The median survival of mice after drug treatment was also prolonged. The median survival of mice treated with the control group was 21.5 days, while the median survival of mice treated with the drug was 24 days ( Figure 9 , E). The survival difference between the two groups was statistically significant. However, the tumors continued to grow, especially in the later stages, and the mice died quickly in the latter stages of the experiment.

[0122] Table 8: Injection of drugs in treatment group and control group

[0123] control group LPFR group DMSO 10％ DMSO 10％ PEG300 10％ PEG300 10％ LiCl 0.2M Pirfenidone 0.3mM Forskolin 0.5mM RO4929097 0.6mM

[0124] It can be seen that the present invention has screened out a combination of four small molecule drugs LPFR, which can induce human glioma cells U251 into neuron-like cells, inhibit tumor proliferation and subcutaneous tumor formation, and prolong the median survival time of xenograft brain glioma model mice, showing a certain therapeutic effect.

Claims

1. Application of a small molecule drug combination in glioma cell transdifferentiation, characterized in that: The small molecule drug combination includes LiCl, Pirfenidone, Forskolin and RO4929097.

2. A small molecule drug combination comprising LiCl, Pirfenidone, Forskolin and RO4929097.

3. Use of the small molecule drug combination according to claim 2 in the treatment of glioma.

4. Use of the small molecule drug combination according to claim 2 in the preparation of a drug for treating glioma.

5. Use of the small molecule drug combination according to claim 2 in inducing transdifferentiation of glioma cells into neuron-like cells.

6. Use of the small molecule drug combination according to claim 2 in the preparation of a drug for inducing transdifferentiation of glioma cells into neuron-like cells.

7. A method for inducing transdifferentiation of glioma cells into neuron-like cells, comprising administering the small molecule drug combination according to claim 2.

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