KR20250052523A - Use of cxcr-4 in diagnosis, prevention or treatment of recurrent brain tumors - Google Patents

Abstract

The present invention relates to the use of CXCR-4 in the diagnosis, prevention or treatment of recurrent brain tumors. Since the use of a CXCR4 antagonist reduces the number of oligodendrocyte-progenitor cell (OPC) lineages migrating into the resection cavity (RC) after surgical resection, and reduces tumor recurrence and mortality, it is expected that the recurrence of glioblastoma (GBM) can be prevented by blocking the CXCL12/CXCR4 pathway.

Description

{USE OF CXCR-4 IN DIAGNOSIS, PREVENTION OR TREATMENT OF RECURRENT BRAIN TUMORS}

The present invention relates to the use of CXCR-4 in the diagnosis, prevention or treatment of recurrent brain tumors, and more particularly, to a pharmaceutical composition for the prevention or treatment of recurrent brain tumors comprising a CXCR-4 inhibitor or a pharmaceutically acceptable salt thereof, a composition for the diagnosis or prognosis of brain tumors comprising an agent for measuring the expression or activity level of CXCR-4, and a method for providing information for the diagnosis or prognosis of brain tumors comprising a step of measuring the mRNA expression of a CXCR-4 gene or the protein activity level thereof.

Cancer is one of the most common causes of death worldwide. Approximately 10 million new cases occur each year, accounting for approximately 12% of all deaths, making it the third most common cause of death. Among various cancers, brain tumors in particular occur regardless of age, and have a higher incidence in children than other cancers. Brain tumors are a general term for primary brain tumors that occur in brain tissue and the meninges surrounding the brain, and secondary brain tumors that metastasize from cancers that occur in the skull or other parts of the body. These brain tumors are different from cancers that occur in other organs in many ways. First, cancers that occur in the lungs, stomach, and breasts are limited to one or two types per organ, and their characteristics are similar and similar. However, a wide variety of cancers occur in the brain. For example, glioblastoma multiforme (GBM), glioma, malignant glioma, lymphadenoma, germ cell tumor, metastatic tumors, etc.

The above glioma is a tumor that accounts for 60% of primary brain tumors. It is a malignant tumor with a high incidence and difficult treatment, and there is currently no special treatment other than radiation therapy.

In the case of glioblastoma, which is classified as the most malignant among gliomas, the resistance to radiation and chemotherapy is very high compared to other cancers, so once diagnosed, the survival period is only 1 year, so proper diagnosis and understanding of the origin and process of each patient's occurrence are important. In particular, glioblastoma that occurs in the brain is the most frequently occurring tumor among adult malignant brain tumors, and even with the best treatment, the median survival period is 12 to 18 months, so the cure rate is extremely low.

Glioblastoma multiforme (GBM) is a highly lethal tumor that mainly relapses locally near the resection cavity (RC) after surgical resection of the primary tumor. Neural stem cells (NSCs) in the subventricular zone (SVZ) harboring oncogenic mutations have recently been reported as the source cells of human GBM.

Glioblastoma is the most aggressive diffuse glioma, accounting for 54% of all gliomas and 16% of all primary brain tumors. Patients with GBM have a poor prognosis, with a median survival of approximately 14–20 months after diagnosis. The current standard of care for GBM includes surgical resection with concurrent radiotherapy and temozolomide. However, these patients have a nearly 100% recurrence rate even with conventional treatment and surgical resection. In approximately 80% of aggressively treated patients, disease progression/recurrence occurs within the resection cavity (RC). After continued efforts to treat the tumor itself, the origin of GBM has emerged as a next-generation target.

In addition to surgical treatment, radiation therapy and chemotherapy are also performed for the treatment of brain tumors, but there is no perfect cure due to the occurrence of resistant mutations and recurrence by tumor stem cells. Therefore, early diagnosis and understanding of the origin of occurrence and development of new treatment methods based on such are required.

Republic of Korea Publication Patent No. 10-2022-0080199 (Published on June 14, 2022)

The present inventors have made extensive research efforts to develop a method for preventing or treating recurrent brain tumors, particularly glioblastoma (GBM). As a result, by using a mouse model established by the present inventors - introducing oncogenic mutations into neural stem cells (NSCs) of the subventricular zone (SVZ) - we have discovered that residual NSCs harboring oncogenic mutations in the SVZ can induce local recurrence after surgical resection of GBM, and that blockade of CXCR-4 can prevent differentiation into oligodendrocyte-progenitor cells (OPCs), a major pathway for tumor recurrence, thereby completing the present invention.

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating recurrent brain tumors, comprising a CXCR-4 (C-X-C chemokine receptor type 4) activity inhibitor or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a composition for diagnosing or predicting the prognosis of a brain tumor, comprising a preparation measuring the expression or activity level of CXCR-4.

Another object of the present invention is to provide a method for providing information for diagnosing or predicting the prognosis of a brain tumor, comprising the step of measuring the mRNA expression level of a CXCR-4 gene or its protein activity level from a biological sample.

The problems to be solved by the present invention are not limited to those mentioned above, and other problems to be solved that are not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

The present inventors have made extensive research efforts to develop a method for preventing or treating recurrent brain tumors, especially glioblastoma (GBM). As a result, using a mouse model established by the present inventors - introducing oncogenic mutations into neural stem cells (NSCs) in the subventricular zone (SVZ) - we have revealed that residual NSCs harboring oncogenic mutations in the SVZ can induce local recurrence after surgical resection of GBM, and that blockade of CXCR-4 can prevent differentiation into oligodendrocyte-progenitor cells (OPCs), a major pathway for tumor recurrence.

The present invention relates to a pharmaceutical composition for preventing or treating recurrent brain tumors, comprising a CXCR-4 inhibitor or a pharmaceutically acceptable salt thereof, a composition for diagnosing or predicting the prognosis of brain tumors, comprising an agent for measuring the expression or activity level of CXCR-4, and a method for providing information for diagnosing or predicting the prognosis of brain tumors, comprising a step of measuring the mRNA expression of a CXCR-4 gene or the protein activity level thereof.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention relates to a pharmaceutical composition for preventing or treating recurrent brain tumor, comprising a CXCR-4 (C-X-C chemokine receptor type 4) activity inhibitor or a pharmaceutically acceptable salt thereof.

In the present invention, "CXCR-4" consists of a base sequence represented by sequence number 1, and specific information can be found in NCBI Reference Sequence ID NG_011587.1.

In one embodiment of the present invention, the CXCR-4 inhibitor may be Plerixafor represented by the following chemical formula 1.

[Chemical Formula 1]

Plerixafor (AMD3100) is a compound represented by the chemical formula 1 and has the chemical name (IUPAC name) of 1,1'-(1,4-phenylenebismethylene)bis(1,4,8,11-tetraazacyclotetradecane)(CAS No. 110078-46-1). The Plerixafor can be easily chemically synthesized and manufactured by a person skilled in the art using a known synthetic method, and used. Alternatively, a commercially manufactured product can be purchased and used.

According to one embodiment of the present invention, it was confirmed that AMD3100, a CXCR4 antagonist, inhibits differentiation from NSCs into OPC lineages in mice and reduces the number of OPC lineages migrating to RCs, and thus prevention or treatment of recurrent brain tumors is possible by using a CXCR-4 activity inhibitor.

The present invention encompasses not only the compound or a pharmaceutically acceptable salt thereof, but also solvates, hydrates, esters and stereoisomers produced therefrom, all of which exhibit the same efficacy, within the scope of the present invention.

In the present invention, the term "pharmaceutically acceptable salt" means a salt formed with any inorganic acid or organic acid or base that does not cause serious irritation to the organism to which it is administered and does not impair the biological activity and physical properties of the compound. As the salt, any salt commonly used in the art, such as an acid addition salt formed with a pharmaceutically acceptable free acid, can be used without limitation.

Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness, or the precipitated salt can be filtered off with suction.

At this time, organic acids and inorganic acids can be used as the free acid, and inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid can be used, and organic acids such as methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid can be used, but are not limited thereto.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. An alkali metal salt or an alkaline earth metal salt can be obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and then evaporating and drying the filtrate. At this time, sodium, potassium, or calcium salts are particularly suitable for preparing the metal salt, but are not limited thereto. In addition, a corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

In the present invention, “prevention” includes all acts of inhibiting or delaying the onset of a recurrent brain tumor by administering the composition of the present invention, and “treatment” includes all acts of improving or beneficially changing the symptoms of a recurrent brain tumor by the composition of the present invention.

In the present invention, "brain tumor" is a tumor that occurs in the brain and central nervous system, and may be used interchangeably with "brain cancer", and may be used interchangeably with "glioma", which is the most representative brain tumor and has a high incidence. Brain tumors can be classified by cell type, morphology, cytogenetics, molecular genetics, immunologic markers, or a combination thereof, and the classification of brain tumors by the World Health Organization classifies central nervous system tumors according to the scale of malignancy based on the histological characteristics of the tumor.

Specifically, the brain tumors include glioblastomas, glioblastoma multiforme, gliosarcoma, anaplastic astrocytomas, oligodendrogliomas, ependymomas, low-grade astrocytomas, medulloblastomas, astrocytic tumors, pilocytic astrocytoma, diffuse astrocytomas, pleomorphic xanthoastrocytomas, subependymal giant cell astrocytomas, anaplastic oligodendrogliomas, oligoastrocytomas, Anaplastic oligoastrocytomas, myxopapillary ependymomas, subependymomas, ependymomas, anaplastic ependymomas, astroblastomas, chordoid gliomas of the third ventricle, gliomatosis cerebris, glangliocytomas, desmoplastic infantile astrocytomas, desmoplastic infantile gangliogliomas, dysembryoplastic neuroepithelial tumors, ependymoblastomas, supratentorial primitive neuroectodermal These tumors may include, but are not limited to, supratentorial primitive neuroectodermal tumors, choroids plexus papilloma, pineal parenchymal tumors of intermediate differentiation, hemangiopericytomas, tumors of the sellar region, craniopharyngioma, capillary hemangioblastoma, and/or primary CNS lymphoma.

More specifically, the brain tumor may be glioblastoma, and the subtype of the glioblastoma may be a proneural subtype, a mesenchymal subtype, a classical subtype, or a neural subtype.

The pharmaceutical composition of the present invention may additionally contain an appropriate carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

In the present invention, a "pharmaceutically acceptable carrier" includes a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the compound to be injected. The type of the carrier usable in the present invention is not particularly limited, and any pharmaceutically acceptable carrier commonly used in the relevant technical field may be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more. In addition, if necessary, other conventional additives such as antioxidants, buffers, and/or bacteriostatic agents may be added and used.

The content of the CXCR-4 inhibitor or a pharmaceutically acceptable salt thereof included in the composition is not particularly limited thereto, but may be included in an amount of 0.01 wt% to 50.0 wt% based on the total weight of the composition, and preferably 0.1 wt% to 10 wt%, but is not limited thereto.

The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, lyophilizers, and suppositories, and may be various oral or parenteral dosage forms. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations may be prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

In addition, preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

Another aspect of the present invention relates to a method for preventing or treating a brain tumor, comprising the step of administering the pharmaceutical composition to a subject suspected of having a brain tumor.

The above pharmaceutical composition can be administered in an amount effective for pharmaceutical purposes to a subject suspected of having a brain tumor for the prevention or treatment of the brain tumor. The above pharmaceutical composition can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, type of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents and can be administered sequentially or simultaneously with conventional therapeutic agents such as temozolomide.

And it can be administered single or multiple times. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors, and it can be easily determined by a person skilled in the art. The preferable dosage of the composition of the present invention varies depending on the condition and weight of the subject, the degree of the disease, the drug form, the route and period of administration, and the appropriate total daily usage amount can be determined by the treating physician within the scope of sound medical judgment, but generally, it can be administered once or several times a day in an amount of 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, more preferably 0.1 to 100 mg/kg.

In the present invention, the subject suspected of having a brain tumor is not particularly limited, and any subject that has developed or can develop a brain tumor can be applied. Specifically, it includes all animals including humans and non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, etc., and by administering a pharmaceutical composition containing the CXCR-4 activity inhibitor of the present invention or a pharmaceutically acceptable salt thereof to a subject suspected of having a brain tumor, the subject can be effectively treated. The brain tumor is as described above.

In the present invention, "administration" means introducing the pharmaceutical composition of the present invention into a subject suspected of having a brain tumor by any appropriate method, and the route of administration may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. Specifically, it may be administered by parenteral administration via injection, but is not limited thereto.

Another aspect of the present invention relates to a composition for diagnosing or predicting the prognosis of a brain tumor, comprising a preparation for measuring the expression or activity level of CXCR-4.

In the present invention, "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis means confirming whether a brain tumor has occurred, preferably whether a malignant brain tumor has occurred.

In the present invention, "prognosis" means a prediction of the progression or recovery of a disease, and refers to a prospect or preliminary evaluation. For the purpose of the present invention, prognosis means a prediction of the success of treatment, survival, recurrence, metastasis, etc. in a brain tumor patient after treatment, and preferably means a survival prognosis.

In the present invention, "measuring the mRNA expression level of the CXCR-4 gene" is a process of confirming the presence and expression level of a biomarker gene in a biological sample for the purpose of diagnosing or predicting the prognosis of a brain tumor, and measures the amount of mRNA using an agent used in a method of measuring the level of mRNA transcribed from a target gene. Specifically, the agent for measuring the mRNA level of the CXCR-4 gene in the present invention is preferably an antisense oligonucleotide, a primer pair or a probe, and since the base sequence of the gene encoding the CXCR-4 is registered in the gene bank, a person skilled in the art can design an antisense oligonucleotide, a primer pair or a probe that specifically amplifies a specific region of these genes based on the sequence.

In the present invention, "antisense" refers to an oligomer having a nucleotide base sequence and an intersubunit backbone that can hybridize to a target sequence in RNA by Watson-Crick base pairing to form a heteroduplex, typically with mRNA, within the target sequence. The oligomer can have exact sequence complementarity or near-complementarity to the target sequence.

In the present invention, the "primer" means a short nucleic acid sequence having a short free 3' terminal hydroxyl group, which can form base pairs with a complementary template and functions as a starting point for copying the template. The primer can initiate DNA synthesis in the presence of a reagent for polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer solution and temperature.

In the present invention, the primer means any primer that can be used to amplify the CXCR-4 gene, and the sequence of the primer is not limited as long as it can complementarily bind to the gene and cause amplification.

In the present invention, a "probe" means a nucleic acid fragment such as RNA or DNA, which is short, a few bases, or a long, several hundred bases, and which can specifically bind to mRNA, and is labeled so as to be able to confirm the presence or absence of a specific mRNA. The probe may be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, etc., and the sequence of the probe is not limited as long as it can complementarily bind to the gene.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologues of a natural nucleotide, and modification between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

In the present invention, “measuring CXCR-4 protein activity level” is a process of confirming the presence and expression level of a marker protein in a biological sample for the purpose of diagnosing or predicting the prognosis of a brain tumor, and preferably, the amount of protein can be confirmed using an antibody that specifically binds to CXCR-4 protein.

In the present invention, "antibody" is a term known in the art and means a specific protein molecule directed against an antigenic site. For the purpose of the present invention, an antibody means an antibody that specifically binds to CXCR-4, and such an antibody can be produced by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the gene, and by a conventional method from the obtained protein. This also includes a partial peptide that can be produced from the protein, and the partial peptide of the present invention includes at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or a part thereof that has antigen binding property is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies. The antibody used in the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as a functional fragment of an antibody molecule. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2, and Fv.

Another aspect of the present invention relates to a method for providing information for diagnosing or predicting prognosis of a brain tumor, comprising the step of measuring the mRNA expression level of a CXCR-4 gene or its protein activity level from a biological sample.

In the present invention, the “method for providing information for diagnosis or prognosis prediction” is a preliminary step for diagnosis or prognosis prediction, which provides objective basic information necessary for diagnosis or prognosis prediction of a brain tumor, and excludes a doctor’s clinical judgment or opinion.

In the present invention, a "biological sample" means a direct subject isolated from an individual to measure the expression level of a target gene or protein, and includes samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine. For example, the sample of the present invention is a sample for diagnosing the occurrence of a brain tumor in an individual suspected of having a brain tumor, or a sample used for predicting clinical outcomes after treatment, i.e., the possibility of recurrence and survival prognosis, from an individual who has received surgical or chemical treatment for a brain tumor, and is preferably brain tissue or cells isolated therefrom, but is not limited thereto.

In the present invention, the “analysis method for measuring mRNA expression level” includes, but is not limited to, polymerase chain reaction (PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Realtime RT-PCR), RNase protection assay (RPA), northern blotting, or DNA microarray analysis.

In the present invention, the "analysis method for measuring protein activity level" includes, but is not limited to, western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, fluorescence activated cell sorter (FACS), or protein chip analysis.

The method of the present invention may include a step of comparing the measured level of mRNA expression or protein activity of the CXCR-4 gene with a level measured in a control group.

The above control group includes a negative control group (normal group) or a positive control group (brain tumor patient group).

The positive control group may be an individual known to have a poor prognosis, for example, an individual with a history of metastasis or death after the onset of a brain tumor. In addition, the positive control group may be an individual for which survival information can be confirmed through a known database, and preferably, may be a brain tumor patient with a survival value below average.

In the method of the present invention, if the mRNA expression level of the CXCR-4 gene or its protein activity level is higher than that of the negative control group (normal group), it can be determined that a brain tumor, particularly a malignant brain tumor with a poor prognosis, has already developed or is at a high risk of developing.

In addition, in the method of the present invention, if the level of mRNA expression or protein activity thereof of the CXCR-4 gene is higher than that of the positive control group (brain tumor patient group), it can be judged that the prognosis is relatively poor, and if it is lower than that of the positive control group (brain tumor patient group), it can be judged that the prognosis is relatively good.

The present invention relates to the use of CXCR-4 in the diagnosis, prevention or treatment of recurrent brain tumors. Since the use of a CXCR4 antagonist reduces the number of oligodendrocyte-progenitor cell (OPC) lineages migrating into the resection cavity (RC) after surgical resection, and reduces tumor recurrence and mortality, it is expected that the recurrence of glioblastoma (GBM) can be prevented by blocking the CXCL12/CXCR4 pathway.

The effects of the present invention are not limited to those mentioned above, and other effects not mentioned can be clearly understood by a person having ordinary skill in the art to which the present invention belongs from the following description.

Figure 1 is a diagram illustrating glioma recurrence in the resection cavity (RC) of genome-edited mice carrying oncogenic mutations in neural stem cells (NSCs) of the subventricular zone (SVZ), according to one embodiment of the present invention. Figure 1a, an illustration of the experimental procedure for electroporating plasmids into the SVZ on days post surgical resection (POD) 2, 14, and 28; Figure 1b, coronal sections of mice stained with DAPI on POD 2, 14, and 28 in each group (n=3), where * indicates RC; Figure 1c, an illustration of the strategy for quantifying the number of tdTomato positive cells in different regions, where regions of interest (ROIs) 1-3 in blue, orange, and red are the SVZ, RC, and arbitrary regions of the cortex excluding the SVZ and RC, respectively (scale bar, 500 μm); Fig. 1d, Measurement of tdTomato immunofluorescence intensity at different post-resection time points in ROI 1-3 of SVZ mutant group (*p=0.0423, **p=0.0017, ****p<0.0001); Fig. 1e, RC DAPI staining images on POD 14 day (left, scale bar, 20 μm) and immunostaining images of GFAP, OLIG2 and NeuN positive cells co-localized with tdTomato in RC (right, scale bar, 20 μm); Fig. 1f, Percentage of cells positive for various NSC-derived cell lineage markers (*p=0.0112); Fig. 1g, Schematic of the transplanted tumor model; Fig. 1h, Representative images of sections of tumor-implanted mice on POD 7 day and primary tumors on POD 0, 14 and 28 days (scale bars, 500 μm).
FIG. 2 is a diagram illustrating an example of the present invention, wherein CXCR4 is highly expressed in RC after surgical resection and is associated with the migration of SVZ mutant cells. FIG. 2a, an example of the strategy used to sample mice in SVZ-mutant group at different time points; FIG. 2b, a Venn diagram showing the overlap between the up-regulated DEGs detected through RNA-seq analysis of post-resection samples from POD2, POD14, and recurrent GBM group and control samples (Log2 fold change≥2; adjusted p value≤0.01); FIG. 2c, a Venn diagram showing the overlap between the co-up-regulated DEGs in the cytokine-cytokine receptor interaction pathway; FIG. 2d, a bar graph of KEGG pathway analysis showing the enrichment of up-regulated genes in POD2 group; FIG. 2e, a comparison of cytokine-cytokine receptor interaction pathway enrichment determined through GSEA (NES, normalised enrichment score) from POD2, POD14, and recurrent GBM group; Fig. 2f, Comparison of expression levels of eight genes filtered through panel c among POD2, POD14, and relapsed tumor groups and control samples; Fig. 2g, Measurement of normalized CXCR4 expression levels through time-course transcriptomic profiling; Fig. 2h, Sampling strategy of mouse SVZ, RC, and cortex in SVZ mutation group; Fig. 2i, Images of CXCR4 immunostaining in mouse SVZ without surgical resection, RC at POD14, and normal cortex 11 weeks after surgical resection (Scale bars, 50 μm) (white arrowheads indicate CXCR4-positive cells colocalized with tdTomato-positive cells in SVZ mutation group).
FIG. 3 is a diagram illustrating, according to one embodiment of the present invention, that CXCL12/CXCR4 blockade reduces the number of migrating OPC lineage cells and improves the survival rate of mice. FIG. 3a, images of local tumor development in the AMD3100 treatment group compared with the PBS group on POD28 of surgical resection (n=3) (scale bars, 500 μm); FIG. 3b, Kaplan-Meier survival graph (n=13) (p=0.0356); FIG. 3c, cumulative incidence of tumor development (n=13) (p=0.0256); FIG. 3d, comparison of tumor development rates in the SVZ mutation, AMD3100 treatment, and PBS treatment groups (scale bars, 500 μm); FIG. 3e, images of GFP-positive cells around the RC after migration from the SVZ on POD14 of surgical resection; Fig. 3f, Number of GFP-positive migrating cells around RC after PBS POD14 vs AMD3100 treatment (n=3) (*p<0.05); Fig. 3g, Number of CXCR4-positive cells in tdTomato-positive cells migrating around RC on POD14; Fig. 3h, Number of cells expressing astrocyte marker GFAP, oligodendrocyte lineage cell marker OLIG2 and neuron marker NeuN (n=3); Fig. 3i, Image of undifferentiated tumorspheres; Fig. 3j, Image of differentiated monolayer cell outgrowth after 1 day of incubation; Fig. 3k, Immunostaining images of GFAP, OLIG2 in cells after 7 days of AMD3100 treatment; Fig. 3l, Number of CXCR4-positive cells showing double staining with GFAP, OLIG2 and NeuN in AMD3100 treatment group after 7 days of differentiation.
FIG. 4 is a diagram illustrating, according to one embodiment of the present invention, that CXCR4 expression is increased in recurrent GBM after surgical resection compared to primary tumors in mice and human GBM patients. FIG. 4a, Illustration of primary and recurrent GBM tissues dissected from mice; FIG. 4b, Heatmap plot of DEGs of primary and recurrent tumor samples obtained from in vivo mouse model (n=3); FIG. 4c, Heatmap showing expression levels of 18 signature genes between mouse recurrent tumors and primary tumors (red is overexpressed in recurrent compared to primary samples, green is overexpressed in primary compared to recurrent samples); FIG. 4d, GSEA of CXCR4 pathway enrichment scores in recurrent tumors compared to mouse primary tumors; FIG. 4e, Comparison of normalized CXCR4 read counts in mouse primary and recurrent GBM tissues (**p=0.0094); Fig. 4f, Immunochemical staining images of CXCR4 expression in mouse primary and recurrent GBM tissues; Fig. 4g, Measurement of CXCR4 expression levels in each group (**** p<0.0001); Fig. 4, Illustration of the strategy used to sample from primary and recurrent GBM tissues of human patients; Fig. 4i, GSEA of CXCR4 pathway enrichment scores in recurrent tumors compared to primary tumors of human patients; Fig. 4j, Comparison of CXCR4 expression levels in LR and PD tumor samples (*p=0176); Fig. 4k, Comparison of CXCR4 pathway enrichment scores in LR and PD tumor samples (**p=0063); Fig. 4l, Graph of overall survival rate of GBM recurrent patients in LR group versus PD group according to CXCR4 expression.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Preparation Example 1. Human GBM Biospecimens

A total of 40 pairs of primary and recurrent GBM tissue samples were collected from Seoul National University Hospital with the approval of the Institutional Review Board (IRB) of Seoul National University Hospital and written consent from all participating patients. The tissue samples were collected from patients who underwent surgery for GBM treatment. To ensure the accuracy and validity of the study, the extent of tumor resection was assessed using preoperative and postoperative magnetic resonance imaging (MRI) scans. In addition, the survival period from the first surgery date was calculated to determine the effectiveness of the surgery and evaluate the impact on patient survival.

Preparation Example 2. Generation of the Primary and Recurrent GBM RNA-seq Datasets

To further validate our findings, we obtained RNA-seq data for a large cohort of 166 primary GBM patients and 13 recurrent GBM patients from The Cancer Genome Atlas (TCGA), a large, publicly available portal in the United States. All data used in the study, including non-TCGA data, are publicly accessible and can be analyzed through the GlioVis portal. In addition, 40 pairs of primary and recurrent GBM samples were collected and analyzed at Seoul National University Hospital using the Illumina Human HT-12 V4.0 expression Bead chip.

Preparation Example 3. Cre-expressing constructs to model p53, Pten mutations

AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR was provided by Feng Zhang (Addgene plasmid #60229; http://n2t.net/addgene:60229; RRID:Addgene_60229), and pAAV-U6sgp53-U6sgbbSapI-GFAPCre was provided by Sidi Chen (Addgene plasmid #100275; http://n2t.net/addgene:100275; RRID:Addgene_100275). gRNAs targeting p53 (sgP53) and Pten (sgPTEN) were designed as previously described (Lee JH et al. Human glioblastoma arises from subventricular zone cells with low-level driver mutations. Nature. 2018;560(7717):243-247. doi:10.1038/s41586-018-0389-3). The targeting sequences of sgRNAs are as follows: sgP53 5'-TAATAGCTCCTGCATGG-3' (SEQ ID NO: 2), sgPTEN 5'-GGTCAAGATCTTCACAGA-3' (SEQ ID NO: 3). Oligonucleotides containing each sgRNA sequence were synthesized (Cosmogenetech) and annealed in vitro in a thermocycler.

To generate a single vector containing sgRNA targeting p53 , Pten and pGFAP, Cre recombinase, GFAP was amplified from the pAAV-U6sgp53-U6sgbbSapI-GFAPCre plasmid and then converted pCBh to GFAP in the pU6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA plasmid. Next, pU6-sgP53, pU6-sgPTEN were amplified and converted pU6-sgRNA(backbone)_pGFAP-Cre-WPRE-hGHpA to the pU6-sgP53-pU6-sgPTEN_pGFAP-Cre-WPRE-hGHpA plasmid (sgTP-GFAP-cre). Additionally, sgLacz-pGFAP-Cre was generated by inserting the U6-sgRNA(backbone)_pGFAP-Cre-WPRE-hGHpA plasmid.

Example 1. Mouse modeling of SVZ-driven glioma at RC after surgical resection

First, to evaluate the potential of residual NSCs in the SVZ to induce local recurrence of GBM in vivo, we established an in vivo genome-edited mouse model by electroporation of TP53, PTEN, and EGFR mutations into NSCs in the SVZ. As a control, sgRNA targeting the LacZ gene was used instead of TP53 and PTEN. In addition, a resection cavity (RC) was created by cortical resection in a remote cortical area to leave the white matter undisturbed. This model models GBM patients harboring residual mutant NSCs in the SVZ after complete surgical resection of the primary tumor.

1-1. Mouse care and information

All experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital (SNUH-IACUC), and animals were cared for in a facility accredited by AAALAC International (#001169) in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition, NRC (2010). LSL-tdTomato (007914), LSL-Cas9 (25263330), and LSL-EGFRvⅢ (19196966) mice were purchased from the Jackson Laboratory and maintained on C57BL/6 and FVB strain backgrounds. They were housed in isolation cages with free access to food and water in a quiet room until use. Housing rooms were located under specific pathogen-free conditions maintained at 23°C on a split light/dark cycle with lights on at 08:00 and off at 20:00. Routine health checks of the mice were performed by veterinarians and investigators.

Disease-specific survival (DSS) endpoints were determined when mice died or met euthanasia criteria according to the IACUC protocol. Euthanasia criteria included (i) significant body weight loss greater than 20%, (ii) severe neurological impairment including stooped posture with paralysis, seizures, or motor deficits, or (iii) signs of head protrusion.

1-2. In vivo electroporation ( In vivo electroporation)

Postnatal 0-2 day old mouse pups (P0-P2) were anesthetized on ice for more than 5 min. The injection site was set at the center of an imaginary line connecting the lambda and the upper left corner of the eye. An oral-controlled microinjection capillary was assembled and used, and the tip of the capillary was marked with a waterproof permanent marker pen so that it could be easily seen. The volume of the injected solution was measured by a line drawn on the capillary every 1 μl. The capillary was slowly lowered 4 mm into the right lateral ventricle. 1 μl of the plasmid solution supplemented with 1% Fast Green dye was injected through the mouth using a constant and controlled pressure. The capillary was retracted 5 s after the injection was completed. Correct plasmid injection was monitored by visualizing the outline of the dye-filled lateral ventricle through the skull.

After successful injection, 5 mm forceps electrodes (45-0489, BTX-Harvard apparatus) were placed on both sides of the head and five electric pulses (100 V, 50 ms duration, 950 ms interval) were applied using an ECM830 electroporation device (BTX-Harvard apparatus). After electroporation, the animals were placed on a 37°C heating pad until they started to move and then returned to their dams.

1-3. Surgical resection

To perform surgical resection, 4-week-old mice were anesthetized by intraperitoneal injection of a ketamine-xylazine mixture. The mice were then placed in a stereotaxic frame (Stoelting) to confirm the precise location of the surgical site. A longitudinal skin incision was made, and a 2-mm diameter craniotomy was made using a hand-held drill centered 2 mm posterior to the bregma and 2 mm lateral to the midline. The corpus callosum was left intact, and the cortex was lesioned using a flat pipette tip attached to a vacuum pump (VACUSIP, INTEGRA Biosciences). After resection, the mice were placed on a 37°C heating pad until fully recovered from anesthesia and then returned to their home cages for postoperative care and monitoring.

1-4. Stereotaxic injection of AAV5 into cortex after surgical resection

To construct adeno-associated virus (AAV) targeting astrocytes, pAAV5 capsid plasmid, viral assembly helper plasmid (pAd deltaF6, UPENN Vector Core), and target plasmid were co-transfected into HEK293T cells using polyethylenimine (1 mg/ml). DMEM (Gibco) containing fetal bovine serum (FBS; Gibco) was replaced with serum-free medium before transfection. Six hours after transfection, the medium was replaced again with DMEM containing FBS. The transfected cells were incubated for 72 hours. The harvested cells were resuspended in 50% fresh DMEM and 0.04% DNase I (Worthington) in nuclease-free water and lysed by three series of freeze-thaw cycles. The cell debris and AAV-containing supernatant were separated by centrifugation, and the supernatant was collected. AAV was collected by polyethylene glycol-mediated purification.

Four-week-old mice were anesthetized with an intraperitoneal injection of a ketamine-xylazine mixture for stereotactic injection and placed in a stereotactic frame (Stoelting). Using the stereotactic apparatus, a Hamilton syringe was inserted through the hole and positioned on the brain surface. A syringe pump (KD Scientific Inc.) was used to inject 100 nl of virus solution at a flow rate of 25 nl/min. The syringe was slowly withdrawn 3 min after injection. Virus was injected into the margin of the resection cavity (RC) immediately after surgical resection or 2 weeks before surgical resection. The coordinates for preoperative injection were 0.8, -1, and -1 (in mm: caudal, lateral, and ventral to bregma). After injection, the mice were placed on a 37°C heating pad until fully recovered and then returned to their cages.

As a result, tdTomato-positive cells were found to have migrated to the RC. In addition, 63.6% (7 out of 11) of the mouse models developed brain tumors around the RC, excluding other sites, 4 weeks after surgery, whereas no tumors were observed in the control mice (Figs. 1b and 1c). These results suggest that residual SVZ NSCs with mutations can migrate to the resection site and induce malignant gliomas around the RC after surgery.

Next, we hypothesized that recurrent GBM tumors in the RC after primary tumor resection could arise from two sources of tumor-initiating cells: residual tumor cells regrowing after incomplete resection of the primary tumor around the RC after a period of dormancy or clonal evolution of residual NSCs in the SVZ to the tumor around the RC, and set up a mouse model consisting of a two-step and dual-color reporting system to investigate which of these is correct. In the first step, the SVZ of LSL-EGFRviii; LSL-Cas9-GFP mice was transduced with plasmids via postnatal electroporation to generate GFP-positive NSCs harboring the mutation in the SVZ. In the second step, tdTomato-positive tumor cells were transplanted into the cortex of mice and the mice were closely monitored for tumor regrowth. Complete resection of the tdTomato-positive primary tumor was attempted by surgery 1 week later ( Fig. 1g ). The tdTomato reporter indicates that recurrent tumors arise from residual tumor cells of the primary tumor, while the GFP reporter highlights the contribution of SVZ mutant NSCs to reconstructing recurrent tumors.

1-5. Orthotopic implantation of GBM tumorshpere cells and surgical removal of primary tumor

tdTomato+ tumorigenic cells derived from LSL-EGFRviii f/+; LSL-tdTomato f/+ mice were stereotactically transplanted into 3-week-old LSL-EGFRviii f/+; LSL-Cas9-GFP f/+ mice (previously injected with sgTP-GFAP-cre plasmid and electroporated on P0-2).

After 3–4 days of culture, tdTomato+ tumorsphere cells were harvested and dissociated into a single-cell suspension. The cells were resuspended in a small volume of regular DMEM/F12 at a concentration of 1 × 10 ⁵ cells/μl, and 1 μl was stereotactically injected into the cortex of LSL-EGFRviii f/+; LSL-Cas9-GFP f/+ mice. After 1 week of growth, bulky primary tumors developed and were removed by surgical resection as described above. Mice were monitored daily for signs of tumor burden. Moribund mice were sacrificed and their brains were processed for histological analysis.

As a result, when the primary tdTomato-positive tumor was removed, 33% of the mice developed GFP-positive recurrent tumors in the RC region (Fig. 1h). These results suggest that residual NSCs harboring oncogenic mutations in the SVZ can reconstitute recurrent GBM tumors after resection of the primary tumor.

Furthermore, to further investigate the temporal progression between surgical intervention and GBM formation in RC, the progression of tumor regrowth over time was observed.

1-6. Image analysis of mouse brain

Mice in each condition were sacrificed and perfused. Brains were then removed, fixed in freshly prepared phosphate-buffered 4% paraformaldehyde (4% PFA), cryoprotected overnight in 30% buffered sucrose, and embedded in OCT compound (3801480, Leica Biosystems)-embedded cryoblocks and stored at -80°C. Cryostat-cut sections (25 μm thick) were collected and mounted on glass slides. Cryostat-cut sections were collected at 4 μm thickness for H&E staining. Slides were then stained with the following antibodies: mouse antibody to Nestin (1:200 dilution; MAB5326, Merck Millipore), rabbit antibody to GFAP (1:500 dilution; Z0334, DAKO), rabbit antibody to oligodendrocyte transcription factor 2 (OLIG2; 1:500 dilution; AB9610, Merck Millipore), rat antibody to platelet-derived growth factor receptor. α (PDGFRα; 1:200 dilution; 14-4321, eBioscience), rabbit antibody to S100β (1:500 dilution; ab52642, Abcam), rat antibody to myelin basic protein (MBP; 1:500 dilution; MAB386, Merck Millipore), rabbit antibody to Ki67 (1:500 dilution; ab15580, Abcam), rabbit antibody to NeuN (1:500 dilution; ab104225, Abcam), mouse antibody to C-X-C chemokine receptor type 4 (CXCR4; 1:500 dilution; sc-53534, Santa Cruz Biotechnology), rabbit polyclonal antibody to CXCL12 (1:500 dilution; ab25117, Abcam). DAPI included in the mounting solution (P36931, ThermoFisher) was used for nuclear staining. Images were acquired using a Zeiss LSM800 confocal microscope with a Z-stack with a step size of 1.5 μm. Fluorescence intensity reflecting the distribution of fluorescent reporter-positive cells was converted to gray value and measured using ImageJ software (http://rsbweb.nih.gov/ij/).

As a result, we observed that cells positive for tdTomato migrated directly from the SVZ to the surgical bed (Fig. 1b). In mice with TP53, PTEN, and EGFR mutations, mutant NSCs that migrated from the SVZ were found only around the RC of the cortex. In addition, we observed that the number of tdTomato-positive cells increased markedly over time around the RC, far from the mutant SVZ. We found that oligodendrocyte progenitor cells (OPCs) were the major cell subtype that migrated from the SVZ to the RC (Fig. 1e and 1f). These results suggest that mutant NSCs in the SVZ exhibit preferential migration behavior and lead to the development of high-grade malignant gliomas in the RC after surgery through abnormal growth of the OPC lineage.

Example 2. Transcriptional profiling of mouse brain tissue

2-1. RNA sequencing

Frozen brain tissue samples from primary and recurrent GBM patients were rapidly frozen in liquid nitrogen to maintain integrity and stored at -80°C. RNA extraction from tissue samples was performed using the micro RNeasy kit from QIAGEN, which has been recognized for its high efficiency and quality.

To facilitate sequencing and analysis of RNA-seq data, experienced Macrogen technicians performed cDNA synthesis, library preparation, and sequencing. The Novoseq 6000 instrument manufactured by Illumina was used, and 80 million reads (40 million in each direction) were obtained for each sample. The paired-end reads were aligned to the UCSC mm10 reference using HISAT2 (v.2.2.0), and the gencode gtf annotation file was used for annotation.

Quantification of gene expression was computed using featureCounts from Subread (v1.6.4) on each genome-mapped reads file. Cell count files were merged into a matrix using a homemade python script (Python 3.6), and raw read counts were used for further analysis. Data were filtered for genes with at least 10 counts in all samples.

Then, DESeq2 (v.1.22.2) was used to compute the size coefficients of each sample and perform variance stabilization transformation. Batch correction was performed to incorporate technical and biological relevant features into the model. The variance stabilization transformed bulk RNA-seq data was used as input for clustering.

Differentially expressed genes (DEGs) analysis was performed using DESeq2 (ref. 74), and batch status was incorporated as a covariate in the expression model. Upregulated genes were defined as genes with a Log2Fold Change greater than 1 and an FDR less than 0.01. Gene set enrichment analysis (GSEA) (v. 3.0) was performed on genes ranked by differential expression, and the pathways used in GSEA were obtained from the Molecular Signatures Database (MSigDB). Pathways were filtered for pathways with a minimum size of 15 and a maximum size of 500.

The R package "clusterProfiler" (v3.10.1) was used to perform GSEA of Gene Ontology (GO) to compare biological processes (BPs), cellular components (CCs), and molecular functions (MFs) of differentially expressed genes (DEGs). The results showed that the five most important biological processes were identified, with FDR less than 0.05 and log2FC less than or equal to 1. In addition, the same package was used to perform KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of DEGs. The results were visualized using a bar graph of the top 20 pathways.

As a result, there was a significant overlap of up-regulated DEGs in the POD2, POD14, and recurrent GBM groups after resection (Fig. 2b). The expression level of CXCR4 was also found to be significantly increased in the post-resection group compared to the control group, and its expression level in RC also showed a continuous increase over time, suggesting a correlation with the migration of SVZ mutant cells (Fig. 2g).

To further determine the role of CXCR4 in the development of recurrent tumors, we performed transcriptional profiling analyses of three primary murine tumors derived from the SVZ without resection and three recurrent tumors after surgical resection.

As a result, there were 576 up-regulated and 531 down-regulated differentially expressed genes (DEGs) in the recurrent tumor samples (Fig. 4b). Compared with the primary murine glioma, the recurrent tumors showed relatively higher expression of CXCR4 (Fig. 4e). The gene set of the CXCR4 signaling pathway showed higher enrichment scores in the recurrent tumor samples (Fig. 4c). Immunostaining was performed to confirm that CXCR4 was significantly highly expressed in the recurrent tumors (Fig. 4f-g). These results suggest that CXCR4 is a potential driving force for the recurrence of glioma after surgery.

Furthermore, to determine the expression and role of the CXCR4 axis in local recurrence around the RC in human patients, we performed transcriptional profiling analysis on 56 primary and recurrent tumor samples from GBM patients. Here, we compared gene expression profiles between recurrent tumor samples from LR and PD patients, distinguishing between local recurrence (LR) around the RC after complete resection with no evidence of residual tumor and other recurrence patterns (PD), including progression of residual tumor and distant intracranial metastases after incomplete resection.

As a result, there were 446 differentially expressed genes (DEGs), including 352 genes significantly upregulated in recurrent GBM and 94 genes specific to primary GBM (an adjusted p-value < 0.05 and a 1-fold change). The CXCR4 signaling pathway gene set showed a higher enrichment score (Fig. 4i). In addition, the expression level of CXCR4 was higher in the recurrent tumor samples of LR patients than in the recurrent tumor samples of PD patients (Fig. 4j). The CXCR4 molecular pathway had a higher enrichment fraction in the LR patient group, suggesting that CXCR4 signaling may also play a role in promoting LR of glioblastoma (Fig. 4k). Meanwhile, patients with high CXCR4 expression showed longer overall survival (OS) (P=0.018) (Fig. 4l), implying the prognostic effect of distinct recurrence patterns. These results suggest that targeting CXCR4 signaling may be a promising therapeutic approach to prevent or reduce LR in glioblastoma, and that differential approaches to relapse may be needed depending on the cellular source and mechanism of relapse.

Example 3. Blockade of the CXCL12/CXCR4 axis in vivo and in vitro in vivo and in vitro )

Based on the results of the above examples, a specific CXCR4 blocker, Plerixafor (AMD3100, Sigma, A5602), was used to investigate the role of the CXCL12/CXCR4 chemokine axis in glioblastoma recurrence.

3-1. Processing of AMD3100

After surgical resection, mice were injected intraperitoneally with AMD3100 (1.25 mg/kg) or PBS twice daily at 6-h intervals. After the final injection, mice were sacrificed and brains were removed.

3-2. Spheroid culture and in vitro differentiation analysis in vitro analysis of differentiation)

GBM tumorspheres (tdTomato tumorspheres) were generated from LSL-EGFRviii f/+ induced tumors; LSL-tdTomato f/+ mice were generated using the pU6-sgP53-pU6-sgPTEN_CBh-Cas9-P2A-Cre plasmid and grown as tumorspheres in neural stem cell medium: DMEM/F12 (Corning, 0-090-CV) supplemented with B27 (50X, Gibco, 17504044), penicillin-streptomycin (Gibco, 15140122), recombinant human EGF (Novoprotein, C046) and bFGF (Novoprotein, C029) at a final concentration of 20 ng/ml each. To maintain the cell line, tumor cells were dissociated with Accutase (Gibco, A1110501), plated at a density of 2 x 10 ⁵ /ml, and passaged every 4-5 days.

SVZ mutant tumorspheres (mNSC+ TS) were generated from LSL-EGFRviii f/+ mutant SVZ derived from LSL-Cas9-GFP f/+ mice using the pU6-sgP53-pU6-sgPTEN_pGFAP-Cre-WPRE-hGHpA plasmid and grown as tumorspheres in neural stem cell medium. They were maintained identically to the GBM tumorspheres.

For mNSC+ TS differentiation assays, cells were detached with Accutase and plated at a density of 40,000 cells per plate (pre-coated with 20 μg/mL Poly-L-ornithine and 10 μg/mL Laminin, Sigma L2020 and P4957) in neural stem cell medium containing reduced concentration of fetal bovine serum (FBS, 1%). AMD3100 (25 μM) or PBS was supplemented to cells every 72 h (in triplicate). After 7 days of treatment, cells were immunostained and processed for lineage differentiation analysis.

As a result, we found that blocking the CXCL12/CXCR4 axis reduced the number of migrating oligodendrocyte progenitor cells (OPCs) and improved tumor control and survival (Fig. 3a–e). Meanwhile, while OPCs were the predominant differentiated subtype in the control group, treatment with AMD3100 reduced the number of OPCs and caused their preferential differentiation into GFAP-positive astrocytes (Fig. 3f–g). These results suggest that CXCR4 blockade can affect the differentiation of NSCs and prevent OPC differentiation, a major pathway for tumor reconstruction. These results suggest that blocking CXCL12/CXCR4 may be a potential therapeutic strategy to prevent local recurrence due to residual NSCs after surgical resection of primary tumors.

Statistical analyses

Data are presented as mean ± SEM. Results were analyzed by t-test, Manny-Whitney U test, or Fisher's exact test, as appropriate, using GraphPad Prism version 9.4.1 (Graph-Pad Software, Inc.). Cumulative tumor incidence and survival data for mice were analyzed by Kaplan-Meier analysis. All P values less than 0.05 were considered statistically significant. All experiments involving the use of animals or cell sources were randomized. Sample size was determined a priori based on variability found in pilot and similar experiments. The investigators were not blinded to allocation during experiments and analysis of results.

The present invention relates to a pharmaceutical composition for preventing or treating recurrent brain tumors comprising a CXCR-4 inhibitor or a pharmaceutically acceptable salt thereof, a composition for diagnosing or predicting the prognosis of brain tumors comprising an agent for measuring the expression or activity level of CXCR-4, and a method for providing information for diagnosing or predicting the prognosis of brain tumors comprising a step of measuring the mRNA expression of a CXCR-4 gene or the protein activity level thereof, which can be used in the medical industry related to cancer treatment.

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Claims (9)

A pharmaceutical composition for preventing or treating recurrent brain tumors, comprising a CXCR-4 (CXC chemokine receptor type 4) inhibitor or a pharmaceutically acceptable salt thereof.
In the first paragraph, the pharmaceutical composition wherein the CXCR-4 inhibitor is Plerixafor represented by the following chemical formula 1:
[Chemical Formula 1]
.
In claim 1, the brain tumor is glioblastomas, glioblastoma multiforme, gliosarcoma, anaplastic astrocytomas, oligodendrogliomas, ependymomas, low-grade astrocytomas, medulloblastomas, astrocytic tumors, pilocytic astrocytoma, diffuse astrocytomas, pleomorphic xanthoastrocytomas, subependymal giant cell astrocytomas, anaplastic oligodendrogliomas, Oligoastrocytomas, anaplastic oligoastrocytomas, myxopapillary ependymomas, subependymomas, ependymomas, anaplastic ependymomas, astroblastomas, chordoid gliomas of the third ventricle, gliomatosis cerebris, glangliocytomas, desmoplastic infantile astrocytomas, desmoplastic infantile gangliogliomas, dysembryoplastic neuroepithelial tumors, A pharmaceutical composition characterized in that the tumor is selected from the group consisting of ependymoblastomas, supratentorial primitive neuroectodermal tumors, choroids plexus papilloma, pineal parenchymal tumors of intermediate differentiation, hemangiopericytomas, tumors of the sellar region, craniopharyngioma, capillary hemangioblastoma, and primary CNS lymphoma.
A pharmaceutical composition according to claim 1, wherein the brain tumor is glioblastoma.
A pharmaceutical composition in claim 4, wherein the subtype of the glioblastoma is a proneural subtype, a mesenchymal subtype, a classical subtype, or a neural subtype.
A composition for diagnosing or predicting the prognosis of a brain tumor, comprising a preparation measuring the expression or activity level of CXCR-4.
A composition for diagnosing or predicting the prognosis of a brain tumor, wherein the preparation in claim 6 is a primer pair or probe that specifically binds to the CXCR-4 gene.
A composition for diagnosing or predicting the prognosis of a brain tumor, wherein the agent in claim 6 is an antibody that specifically binds to the CXCR-4 protein.
A method for providing information for diagnosing or predicting the prognosis of a brain tumor, comprising: a step of measuring the mRNA expression level of a CXCR-4 gene or its protein activity level from a biological sample.