Application of IGFBP2 in diagnosis and immunotherapy of glioblastoma

Abstract

The invention belongs to the technical field of brain glioma markers, and particularly relates to application of IGFBP2 in diagnosis and immunotherapy of glioblastoma. According to the invention, single cell sequencing data analysis reveals that CD47 in GBM is closely associated with an anoxic microenvironment, and meanwhile, it is proved that combined application of the anti-IGFBP2 and anti-CD47 antibodies can significantly improve the curative effect of CD47 targeted therapy. According to the research, the IGFBP2 is determined as a potential biomarker for predicting the anti-CD47 treatment response of the GBM patient for the first time, and a new thought is provided for overcoming the anti-CD47 drug resistance through a combined treatment strategy.

Description

Technical Field

[0001] The present invention belongs to the technical field of brain glioma markers, and particularly relates to the application of IGFBP2 in the diagnosis and immunotherapy of glioblastoma. Background Art

[0002] Glioblastoma (GBM) is the most common and most malignant tumor of the central nervous system, with a median survival of only 15-21 months. The hypoxic microenvironment created by rapid tumor proliferation drives GBM immune escape, a key factor contributing to its high malignancy and treatment resistance. Despite the presence of immune cell infiltration in hypoxic areas, GBM cells can evade phagocytic clearance through various mechanisms, including a "don't eat me" signal mediated by the CD47-SIRPα interaction.

[0003] As a transmembrane protein, CD47 binds to signal-regulatory protein α (SIRPα) on the surface of macrophages to transmit the "don't eat me" signal, thereby inhibiting phagocytosis. Immunotherapeutic strategies targeting CD47 (such as monoclonal antibodies or small molecule inhibitors) have shown promising efficacy in GBM clinical studies. However, the hypoxic microenvironment of GBM may affect the responsiveness of CD47-targeted therapy by upregulating CD47 expression or shaping an immunosuppressive microenvironment. These mechanisms significantly limit the potential application of CD47 monotherapy in solid tumors. In addition, CD47 antibody therapy may induce hematologic toxicity (such as anemia), which is closely related to its widespread expression on the red blood cell surface. Therefore, the heterogeneity of GBM and the potential for immune resistance suggest the need for the development of combination therapy to fully tap the clinical therapeutic potential of CD47.

[0004] Currently, there is no treatment strategy for CD47 immunotherapy resistance in GBM. Therefore, further research on the mechanism of action of CD47 immunotherapy in GBM and the development of potential biomarkers for response to anti-CD47 therapy in GBM patients are of great significance for overcoming GBM immunotherapy resistance and achieving precise diagnosis and treatment of the disease. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention aims to provide an application of IGFBP2 in the diagnosis and immunotherapy of glioblastoma.

[0006] Based on the above objectives, the technical solutions adopted by the present invention are as follows:

[0007] In a first aspect, the present invention provides any of the following uses of biomarkers and / or substances for detecting said biomarkers:

[0008] (A1) Application in the preparation of a product for predicting or assisting in predicting the efficacy of monotherapy for brain glioma;

[0009] (A2) Use in the preparation of products for evaluating or assisting in evaluating the efficacy of monotherapy for brain glioma;

[0010] (A3) Use in the preparation of a product for predicting or assisting in predicting the progression-free survival time of a single immunotherapy for brain glioma;

[0011] The biomarker is IGFBP2.

[0012] Furthermore, the IGFBP2 includes IGFBP2 protein or IGFBP2 gene.

[0013] Furthermore, the monotherapy for glioma is anti-CD47 immunotherapy.

[0014] Furthermore, the products include but are not limited to reagents, kits, chips or test strips.

[0015] Said preparation includes development and / or screening.

[0016] The IGFBP2 gene (Gene ID: 3485) is located on human chromosome 2, NC_000002.12 (216632828..216664436). The amino acid sequence of the IGFBP2 protein is SEQ ID NO.1.

[0017] MLPRVGCPALPLPPPPLLPLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVENHVD STMNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ(SEQ ID NO.1)

[0018] Furthermore, the substance for detecting the biomarker may include a substance for detecting the biomarker by immunohistochemistry (IHC) technology.

[0019] Furthermore, the substance for detecting the biomarker may be a substance for detecting the expression level of IGFBP2 in the exosomes of the patient with brain glioma to be tested.

[0020] In the above application, the substance for detecting the biomarker may include a reagent for detecting the expression level of IGFBP2 protein or the content of IGFBP2 protein.

[0021] In the above applications, the reagent may include an antibody, polypeptide, protein or nucleic acid molecule that binds to the IGFBP2 protein.

[0022] The antibodies binding to IGFBP2 protein described herein include anti-IGFBP2 protein antibodies or functional fragments thereof (such as antibody variable region Fv, single-chain antibody ScFv, antigen-binding fragment Fab or Fab', F(ab)'2, Fab'-SH and other antibody fragments).

[0023] In a second aspect, the present invention further provides a kit, which may include any of the substances for detecting the biomarkers described herein, and the kit may have at least one of the following uses:

[0024] (B1) Predict or assist in predicting the efficacy of monotherapy for glioma;

[0025] (B2) To evaluate or assist in evaluating the efficacy of single-agent immunotherapy for glioma;

[0026] (B3) Predict or assist in predicting the progression-free survival (PFS) of glioma patients treated with monotherapy with immunotherapy.

[0027] The kit may be a therapeutic efficacy prediction kit, a therapeutic efficacy evaluation kit or a companion diagnostic kit.

[0028] The test sample of the kit can be a blood sample or a tissue sample.

[0029] Furthermore, the kit may include IGFBP2 monoclonal antibody or IGFBP2 polyclonal antibody, and the kit may also include immunohistochemistry reagents.

[0030] The third aspect of the present invention further provides any of the following applications of the biomarker as a target:

[0031] (C1) Application in the preparation of a product for improving the efficacy of monotherapy for brain glioma;

[0032] (C2) Use in the preparation of products for the treatment or adjuvant treatment of brain glioma;

[0033] (C3) Use in the preparation of products for use in combination with monotherapy of brain glioma immunotherapy.

[0034] The fourth aspect of the present invention further provides any of the following uses of an IGFBP2 inhibitor:

[0035] (D1) Application in the preparation of a product for improving the efficacy of monotherapy for brain glioma;

[0036] (D2) Application in the preparation of products for the treatment or adjuvant treatment of brain glioma;

[0037] (D3) Use in the preparation of products for inhibiting the occurrence and / or development of brain glioma.

[0038] Furthermore, the product may be a reagent or a medicine.

[0039] The IGFBP2 inhibitor may have at least any one of the following effects:

[0040] (E1) inhibiting or reducing the expression or activity of the IGFBP2 gene;

[0041] (E2) inhibits or reduces the transcription of IGFBP2 gene into mRNA;

[0042] (E3) inhibiting or reducing the translation of IGFBP2 gene into protein;

[0043] (E4) Inhibit or reduce the activity or function of IGFBP2 protein.

[0044] In the above applications, the IGFBP2 inhibitor may include a substance that reduces the expression level of IGFBP2 protein or the content of IGFBP2 protein, or a substance that inhibits the expression of the IGFBP2 gene. The substance may include one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses.

[0045] Furthermore, inhibition of IGFBP2 gene expression can be achieved by gene mutation, gene silencing, gene knockout, gene editing or gene knockdown techniques well known to those skilled in the art. For example, RNA interference (RNAi) technology can be used to specifically eliminate or shut down the expression of a specific gene. The tools used for gene editing technology can be CRISPR / Cas9 technology, zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) technology, etc., but are not limited thereto. Hormones that inactivate IGFBP2 gene expression or silence the gene at the post-transcriptional or translational level using gene knockdown (gene knockdown) technology are well known to those skilled in the art. The gene knockdown technology includes RNA interference, Morpholino interference, antisense nucleic acids, ribozymes or dominant negative inhibitory mutations, but are not limited thereto.

[0046] It is well known to those skilled in the art to use shRNA or siRNA expressed by viruses (such as lentivirus, adeno-associated virus) to inhibit gene expression and perform gene silencing.

[0047] The nucleic acid molecule may include shRNA, microRNA, siRNA and / or antisense oligonucleotides.

[0048] Furthermore, the shRNA (short hairpin RNA), microRNA (micro RNA), siRNA (small interfering RNA) and / or antisense oligonucleotides (such as antisense RNA) are used to inhibit the expression of the IGFBP2 gene.

[0049] Furthermore, the shRNA sequence for inhibiting the expression of the IGFBP2 gene is:

[0050] sh-IGFBP2#1: 5'-CCAGTTCTGACACACGTATTT-3'; sh-IGFBP2#2: 5'-ACAGTGCAAGATGTCTCTGAA-3'.

[0051] The IGFBP2 inhibitor may also be an antibody, and the antibody may be an anti-IGFBP2 protein antibody or a functional fragment thereof.

[0052] Herein, the immunotherapy monotherapy may include anti-CD47 therapy.

[0053] Herein, the drug used in the monotherapy of immune therapy may be a CD47 inhibitor.

[0054] The fourth aspect of the present invention further provides a combination drug for preventing or treating glioma, wherein the combination drug may include any of the IGFBP2 inhibitors described herein and a glioma immunotherapy drug.

[0055] Furthermore, the IGFBP2 inhibitor includes IGFBP2 monoclonal antibody or IGFBP2 polyclonal antibody.

[0056] Furthermore, the IGFBP2 inhibitor is anti-IGFBP2.

[0057] Furthermore, the therapeutic dose of the anti-IGFBP2 is 1-20 mg / kg.

[0058] Furthermore, the therapeutic dose of the anti-IGFBP2 is 5-15 mg / kg.

[0059] Furthermore, the therapeutic dose of anti-IGFBP2 can be 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg.

[0060] Furthermore, the therapeutic dose of the anti-IGFBP2 is 10 mg / kg.

[0061] Furthermore, the brain glioma immunotherapy drug may be a CD47 inhibitor.

[0062] Furthermore, the brain glioma immunotherapy drugs include but are not limited to CD47 monoclonal antibodies or CD47 polyclonal antibodies.

[0063] Furthermore, the brain glioma immunotherapy drug is anti-CD47.

[0064] Furthermore, the therapeutic dose of the anti-CD47 is 1-300 μg.

[0065] Furthermore, the therapeutic dose of the anti-CD47 is 100-300 μg.

[0066] Furthermore, the therapeutic dose of anti-CD47 can be 100μg, 110μg, 120μg, 130μg, 140μg, 150μg, 160μg, 170μg, 180μg, 190μg, 200μg, 210μg, 220μg, 230μg, 240μg, 250μg, 260μg, 270μg, 280μg, 290μg, 300μg.

[0067] Furthermore, the therapeutic dose of anti-CD47 is 200 μg.

[0068] The beneficial effects of one or more of the above technical solutions are as follows:

[0069] This study, integrating single-cell RNA sequencing with proteomic analysis, revealed that insulin-like growth factor binding protein 2 (IGFBP2) co-expresses with CD47 in a hypoxia-induced mesenchymal-like GBM subpopulation, synergistically promoting tumor progression and immune evasion. This study reveals the role of the hypoxia-exosomal IGFBP2-CD47 axis in GBM immune evasion, providing a theoretical basis for combined targeted therapy to enhance immune efficacy. Mechanistically, hypoxia induces IGFBP2 expression through HIF-2α-mediated transcriptional activation, which in turn upregulates RAB3A via HIF-1α to increase the secretion of IGFBP2-positive exosomes. IGFBP2 is localized to the exosome surface via integrin α5β1, activating the integrin / FAK / STAT3 signaling pathway to enhance CD47 expression and inhibit macrophage phagocytosis. Calculation of the drug combination index of IGFBP2 and CD47 antibodies confirmed the synergistic effect of combined use of IGFBP2 and CD47 antibodies. This study establishes IGFBP2 as a potential biomarker for predicting response to anti-CD47 therapy in GBM patients, providing new insights into overcoming anti-CD47 resistance through combination therapy. Clinical analysis has shown that combined blockade of IGFBP2 and CD47 synergistically inhibits tumor growth and prolongs survival in an orthotopic GBM model. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Figure 1 is a schematic diagram of the classification of tumor cells into five major subtypes using single-cell sequencing data in an embodiment of the present invention; wherein, A is a schematic diagram of the single-cell RNA sequencing (scRNA) process: tumor tissues of glioblastoma (GBM) patients were sequenced using the 10×Genomics Chromium system, and samples were collected from different anatomical regions (interior: tumor core area; periphery: tumor invasion edge); B is a heat map showing the four major malignant subtypes: neural progenitor-like GBM cells (NPC-GBM), oligodendrocyte precursor-like GBM cells (OPC-GBM), astrocyte-like GBM cells (AC-GBM) and mesenchymal-like GBM cells (MES-GBM), the latter of which are further divided into MES1-GBM and MES2-GBM subpopulations; C is a bar graph showing the proportional distribution of each cell type in scRNA-seq; D is a heat map showing the differences in pathway activity of each malignant subpopulation calculated by the GSVA method.

[0071] Figure 2Figure 1 is a schematic diagram showing that IGFBP2 and CD47 are positively correlated in the hypoxic microenvironment of GBM in the embodiment of the present invention; A is a UMAP and density map showing that the mesenchymal-like (MES-like) GBM subpopulation is enriched in the core area of ​​the tumor, and samples were collected from two areas: the interior (tumor core) and the periphery (tumor invasion edge); B is a UMAP and density map showing that CD47 is highly expressed in the mesenchymal-like GBM subpopulation; C is a gene set enrichment analysis (GSEA) of single-cell RNA sequencing showing that genes positively correlated with CD47 Significantly enriched in the "hypoxia" and "protein secretion" pathways; D is proteomic sequencing confirming that IGFBP2 is significantly upregulated in GBM cells and their exosomes under hypoxic conditions; E is UMAP and density map showing that IGFBP2 and CD47 are co-expressed in the MES1-like GBM subpopulation; F is a bubble map showing that CD47 and IGFBP2 are highly expressed in the MES1-internal subpopulation; G is Monocle2 pseudo-time series analysis showing that the expression of CD47 and IGFBP2 is gradually upregulated with tumor progression; H is Western Blot analysis showed that IGFBP2 expression was upregulated in GBM cells and exosomes under hypoxic conditions; I, Western blot analysis showed that hypoxia upregulated CD47 expression in GBM cells, while knockdown of IGFBP2 inhibited its expression; J, Western blot analysis confirmed that IGFBP2 expression was significantly increased in tumor tissues and plasma exosomes from patients with high-grade glioma; K, flow cytometric analysis of macrophage phagocytosis in GSC20 and GSC267 cells transfected with sh-NC or sh-IGFBP2 (right panel: quantification results, n=3); L, flow cytometric analysis of macrophage phagocytosis in GSC20 cells transfected with ov-NC or ov-IGFBP2 after treatment with IgG or anti-integrin α5β1 blocking antibody (right panel: quantification results, n=3); Data are expressed as mean ± SD, and statistical significance was analyzed by one-way analysis of variance (*P < 0.05; **P < 0.01; ***P < 0.001).

[0072] Figure 3Figure 1 is a schematic diagram showing that IGFBP2 is highly expressed in GBM patients and indicates a poor prognosis in the examples of the present invention; A is a gene screening positively correlated with CD47; B is a transmission electron microscopy image showing exosomes isolated from the culture supernatant of glioma cell lines GSC20 and GSC267 (scale bar: 100 nm); C is NanoSight particle tracking analysis of exosome size distribution and number; D is Western Blot analysis of the expression of TSG101, CD81 (exosome marker) and Calnexin (negative control) in N-GDEs and H-GDEs; E is Monocle2 trajectory analysis revealing that the mesenchymal (MES) state gradually evolves with tumor progression; F is immunohistochemical images showing that IGFBP2 expression is significantly increased in tumor tissues of patients with high-grade glioma (scale bar: 100 μm); GH is flow cytometry detection of CD47 expression levels and the proportion of positive cells in GSC20 / GSC267 cells transfected with sh-NC or sh-IGFBP2 (n=3).

[0073] Figure 4 Schematic diagram of exosomal IGFBP2 promoting GBM mesenchymal transition and tumorigenesis in the examples of the present invention; A is a single-cell GSEA analysis showing that IGFBP2-related genes are significantly enriched in the MES1 subpopulation and epithelial-mesenchymal transition (EMT) pathway; B is a Western blot detection of the difference in IGFBP2 protein expression in proneural (PN) and mesenchymal (MES) cancer stem cells (GSCs); C is a quantitative analysis of the diameter of GSC20 / GSC267 cell tumor spheres after treatment with cell supernatant-derived exosomes; D. Western Blot analysis was used to detect the IGFBP2 levels in exosomes of cells transfected with sh-NC or sh-IGFBP2; E is a quantitative comparison of the diameters of tumor spheres in different treatment groups; F is a representative image of H&E staining in each group; G is an immunohistochemical image of Ki67 expression in an orthotopic transplanted tumor model with IGFBP2 knockout (scale bar: 100 μm). Data are expressed as mean ± SD, and statistical significance was determined by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001).

[0074] Figure 5Schematic diagram of IGFBP2 promoting GBM mesenchymal transformation and tumorigenesis in the examples of the present invention; wherein, A is Western blot detection of the expression changes of mesenchymal phenotype markers (CD44, YKL40) and IGFBP2 in GSC20 and GSC267 cells transfected with sh-NC or sh-IGFBP2; B is quantitative analysis of the diameter of tumor spheres of GSC20 and GSC267 cells after sh-IGFBP2 transfection; C is the limiting dilution sphere formation experiment of GSC20 and GSC267 cells after sh-IGFBP2 transfection; DE are in vitro co-culture invasion experiments of GSC20 and GSC267 cells after sh-IGFBP2 transfection (n=3, invasion ability was assessed within 72 hours, scale bar=200μm); F is Western blot Blot analysis of the expression of mesenchymal phenotype markers (CD44, YKL40) in GSC20 and GSC267 cells after treatment with exosomes derived from the cell supernatant; GH is the quantitative analysis of tumor sphere diameter and limiting dilution sphere formation assay after exosome treatment; I is the bioluminescence imaging of the transplanted tumor model constructed by GSC267 cells transfected with sh-IGFBP2 at different time points after surgery (n=5); J is the Kaplan-Meier survival curve of tumor-bearing mice in different groups (n=5); Data are expressed as mean ± SD, and statistical significance was analyzed using one-way analysis of variance and log-rank test (*P<0.05; **P<0.01; ***P<0.001).

[0075] Figure 6 Schematic diagram of HIF-2α upregulating IGFBP2 under hypoxic conditions in an embodiment of the present invention; wherein, A is a cross-analysis of the top 30 genes positively correlated with IGFBP2; B is a heat map showing the transcription factors enriched in NPC-like, OPC-like, AC-like, MES1-like and MES2-like GBM cell subtypes. The data are expressed as mean ± standard deviation, and statistical significance was determined by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001).

[0076] Figure 7Schematic diagram of verification of hypoxia upregulation of IGFBP2 through HIF-2α in the examples of the present invention; A is UMAP and density map showing that IGFBP2 and EPAS1 (HIF-2α encoding gene) are co-expressed in the MES1-like GBM subpopulation; B is Monocle2 pseudo-sequential analysis showing that HIF-2α expression is gradually upregulated with tumor progression; C is UMAP and density map showing that EPAS1 knockout inhibits the state transition trajectory of the MES1-GBM subpopulation; DE is qPCR and Western Blot analysis was performed to detect the expression levels of HIF2α and IGFBP2 in GSC cells treated with si-NC or si-HIF2α. FG showed that the enrichment fold of the anti-HIF-2α antibody group in the promoter region P1 in GSC267 cells was significantly higher than that in the IgG control group. HI showed that the dual-luciferase reporter assay showed that HIF2α overexpression could enhance the promoter activity of the wild-type (WT) but had no effect on the mutant type (Mut1) (HEK-293T cells). Data are expressed as mean ± SD, and statistical significance was analyzed using one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001).

[0077] Figure 8 Schematic diagram of IGFBP2 upregulating CD47 through the integrin α5β1-FAK-STAT3 signaling pathway in an embodiment of the present invention; wherein, A is Coomassie blue staining showing proteins interacting with IGFBP2 identified by immunoprecipitation / mass spectrometry, and arrows indicate IGFBP2 and integrin α5β1 bands; B is a bubble plot showing that ITGA5 and ITGB1 are highly expressed in the MES1-internal subpopulation; C is a UMAP and density map showing the expression localization of ITGA5 / ITGB1 in the MES1-like GBM subpopulation; DE are Western blot detection of FAK-STAT3 pathway proteins and CD47 expression in ov-IGFBP2-transfected GSC20 / GSC267 cells, and observation of the intervention effects of STAT3 inhibitors (Stattic) and FAK inhibitors (Y15); F. Western blot analysis of changes in FAK-STAT3 pathway, mesenchymal markers (CD44 / YKL40) and CD47 expression in ov-IGFBP2-transfected cells after treatment with integrin α5β1 blocking antibody.

[0078] Figure 9Schematic diagram of IGFBP2 upregulating CD47 through the integrin α5β1-FAK-STAT3 signaling pathway in an embodiment of the present invention; wherein, A is UMAP and density map showing that IGFBP2 and EPAS1 are co-expressed in the MES1-like GBM subpopulation; B is Co-IP and Western blot verification of the interaction between IGFBP2 and integrin α5β1 (using anti-IGFBP2 antibody); C is GSEA showing that genes positively correlated with IGFBP2 are enriched in the "focal adhesion", "ECM receptor interaction" and "STAT3 signaling" pathways; D is Western blot detection of changes in protein expression of the FAK-STAT3 pathway and CD47 in sh-IGFBP2-transfected GSC cells; E is changes in protein expression of the FAK-STAT3 pathway and CD47 in exosome-treated GSC cells; F is ov-IGFBP2-transfected GSC267 cells after anti-integrin α5β1 After treatment with blocking antibodies, the protein expression of FAK-STAT3 pathway, mesenchymal markers and CD47 changed; GH is the flow cytometric analysis of the proportion of CD47+ cells in GSC cells transfected with ov-IGFBP2 after anti-integrin α5β1 blocking (right panel: quantification results, n=3); IJ is the flow cytometric analysis of the macrophage phagocytosis rate in GSC cells transfected with ov-IGFBP2 after anti-integrin α5β1 blocking (right panel: quantification results, n=3). Data are expressed as mean ± SD, and statistical significance was analyzed by one-way analysis of variance (*P<0.05; **P<0.01; ***P<0.001).

[0079] Figure 10Schematic diagram of IGFBP2 localizing on the surface of exosomes through integrin α5β1 in an embodiment of the present invention; wherein, A is an electron micrograph and immunogold labeling (anti-IGFBP2, ITGA5, ITGB1 and CD81 antibodies, 10 nm gold particle labeling, scale bar = 100 nm) of exosomes derived from GSC20 and GSC267 cells; B is Western blot detection of membrane proteins (IGFBP2, ITGA5, ITGB1, CD81) and intraluminal protein HSP70 after exosomes were treated with proteinase K gradient; C is protein expression of IGFBP2, ITGA5, ITGB1 and HSP70 in exosomes under normoxic and hypoxic conditions; D E are ELISA quantification of IGFBP2 levels in exosomes derived from GSC cells transfected with sh-ITGA5 or sh-ITGB1; F is Western blot analysis of IGFBP2, ITGA5, ITGB1 and HSP70 in exosomes derived from GSC cells transfected with sh-ITGA5 or sh-ITGB1 Blot detection; G is a schematic diagram: IGFBP2 is localized on the surface of exosomes through integrin α5β1 under normoxia and hypoxia; H. Immunogold-labeled electron microscopy images of IGFBP2 in GSC cells under normoxia and hypoxia (right: number of IGFBP2+ particles in a single cell, n=5), data are expressed as mean ± standard deviation, and statistical significance was analyzed by one-way ANOVA (*P<0.05; **P<0.01; ***P<0.001).

[0080] Figure 11 Schematic diagram of hypoxia upregulating RAB3A to promote exosome secretion in the present invention; A is RNA sequencing of the GSE45117 and GSE232725 datasets showing increased RAB3A expression under hypoxia; BC are qPCR and Western blot analysis of RAB3A expression levels in sh-RAB3A-transfected GSC20 / GSC267 cells; D is Western blot analysis of the same cell number (20×10 6 ) of sh-NC / sh-RAB3A-transfected cells. The data are expressed as mean ± SD, and the statistical significance was determined by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001).

[0081] Figure 12Schematic diagram of hypoxia upregulating RAB3A to promote exosome secretion in the embodiment of the present invention; AB is qPCR and Western blot detection of upregulated RAB3A expression under hypoxia; C is Western blot detection of HIF-1α, RAB3A and β-actin protein expression in GSC cells under hypoxia; DE is NTA detection of changes in exosome size and number of sh-RAB3A-transfected GSC cells under hypoxia (2×10 7 cells); FG. Electron microscopy observation of the number of ILVs in MVBs of sh-Rab3A-transfected GSC cells (right panel: number of ILVs in a single MVB, n=5, scale bar=1 μm); HI. Confocal microscopy analysis of the number of CD63-labeled MVBs after Rab3A knockdown (right panel: number of CD63+ particles in a single cell, n=5, scale bar=50 μm); Data are expressed as mean ± SD, and statistical significance was analyzed by one-way ANOVA (*P<0.05; **P<0.01; ***P<0.001).

[0082] Figure 13 Figure 2 shows the combined blocking of IGFBP2 and CD47 in the embodiment of the present invention to synergistically inhibit GBM immune escape and malignant progression; AB are flow cytometric analyses of macrophage phagocytosis rate in GSC cells treated with anti-CD47 and anti-IGFBP2 antibodies (right panel: quantitative results, n=3); C is a schematic diagram of the experimental design for the treatment of orthotopic transplanted tumor models; DE are postoperative bioluminescence imaging and tumor activity quantification of GSC267 transplanted tumor models treated with anti-CD47 and anti-IGFBP2 antibodies (n=5); F is the Kaplan-Meier survival curve of tumor-bearing mice in different treatment groups (n=5); G is a schematic diagram of the mechanism by which the hypoxia-exosomal IGFBP2-CD47 axis regulates GBM immune escape; data are expressed as mean ± standard deviation, and statistical significance was determined using one-way analysis of variance and log-rank test (*P<0.05; **P<0.01; ***P<0.001). DETAILED DESCRIPTION

[0083] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0084] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0085] In the present invention, the IGFBP2 gene (Gene ID: 3485) is located on human chromosome 2, NC_000002.12 (216632828..216664436). The amino acid sequence of the IGFBP2 protein is SEQ ID NO.1.

[0086] This application discovered for the first time that IGFBP2 is highly expressed in cells and exosomes under hypoxic conditions, and is significantly positively correlated with CD47. IGFBP2 is a small molecule glycoprotein of the insulin-like growth factor binding protein (IGFBP) family. As a secreted protein, IGFBP2 regulates key biological processes such as cell adhesion, migration and invasion by interacting with cell surface receptors such as integrins. In addition, IGFBP2 plays a key role in regulating the tumor microenvironment and may enhance the resistance of tumor cells to immune surveillance by promoting immune escape mechanisms. Single-cell sequencing technology revealed the close association between IGFBP2 and CD47 in the GBM hypoxic niche, and further confirmed that exosomal IGFBP2 upregulated CD47 expression through the integrin / FAK / STAT3 signaling pathway, and the combined use of anti-IGFBP2 antibodies and anti-CD47 antibodies can significantly inhibit tumor growth and prolong the survival of tumor-bearing mice.

[0087] Example

[0088] 1. Experimental methods

[0089] cell lines

[0090] The mouse glioblastoma (GBM) cell line GL261 was purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Human GBM stem cells GSC20 and GSC267 were kindly provided by the Bhat laboratory at the MD Anderson Cancer Center and cultured in F123 medium supplemented with B-27 (Gibco, USA), 20 ng / mL recombinant human epidermal growth factor (rhEGF, R&D Systems, USA), and 20 ng / mL recombinant human basic fibroblast growth factor (rhbFGF, R&D Systems, USA) as described. All cell lines were authenticated by short tandem repeat (STR) profiling and confirmed to be free of mycoplasma contamination.

[0091] Patients and specimens

[0092] Human glioma tissue, plasma, and normal brain tissue (derived from the cortex of patients undergoing decompressive surgery for patients with traumatic brain injury or hypertensive intracerebral hemorrhage) were obtained from inpatients at Qilu Hospital. All participants provided written informed consent, and the study was approved by the Research Ethics Committee of Qilu Hospital of Shandong University (approval number: KYLL-2022(ZM)-439).

[0093] Co-immunoprecipitation (Co-IP) experiments

[0094] The Pierce Direct Magnetic IP / Co-IP kit (88828, Thermo Fisher Scientific) was used according to the manufacturer's instructions. The protocol was as follows: After cell lysis, anti-IGFBP2 antibody (ab188200, Abcam) or an isotype IgG control (2729, CST) was added and incubated with rotation overnight at 4°C. Magnetic beads were then added and incubated at 37°C for 2 hours. Precipitated proteins were boiled in SDS loading buffer and analyzed by mass spectrometry or Western blot.

[0095] Exosome isolation and identification

[0096] Glioma cell culture supernatants or human plasma were collected and centrifuged sequentially (300g for 10 minutes, 2000g for 30 minutes, and 123,000g for 45 minutes). The supernatant was filtered through a 0.22 μm filter and ultracentrifuged (110,000g for 70 minutes). The pellet was washed with PBS and centrifuged again (110,000g for 70 minutes). The exosomes were resuspended in PBS and stored at −80°C. Exosome morphology was observed using transmission electron microscopy (HT-7700, Hitachi), and particle size and concentration were determined using a ZetaView system (Particle Metrix, Germany).

[0097] Western blot

[0098] Cellular or exosome proteins were extracted using RIPA lysis buffer containing protease inhibitors, and protein concentrations were quantified using the BCA assay (Beyotime, China). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Antibody details are shown in Table 1.

[0099] Table 1 Antibody details

[0100] antibody brand article number CD47 Cell Signaling Technology 63000 CD47 BioXcell BE0019 β-actin Proteintech 20536-1-AP IGFBP2 Abcam ab188200 IGFBP2 R&D Systems #AF674 HSP70 Proteintech 10995-1-AP IgG BioXcell #BP0089 CD44 Cell Signaling Technology 37259 YKL40 Cell Signaling Technology 47066 ITGA5 Cell Signaling Technology 98204 ITGB1 Cell Signaling Technology 34971 FAK Cell Signaling Technology 3285 p-FAK Cell Signaling Technology 3283 STAT3 Cell Signaling Technology 9139 p-STAT3 Cell Signaling Technology 9145 HIF-1α Proteintech 20960-1-AP HIF-2α Proteintech 26422-1-AP CD81 Proteintech 66866-1-Ig TSG101 Proteintech 28283-1-AP Calnexin Proteintech 10427-2-AP RAB3A Proteintech 15029-1-AP

[0101] siRNA and plasmid transfection

[0102] siRNA, ov-IGFBP2 plasmid, and negative control (NC) were synthesized by Boshan Biotechnology (Shanghai) Co., Ltd. Transfection was performed using Lipofectamine 3000 reagent (#L3000015, Invitrogen). 5 μL of 20 μM siRNA, 1 μg of control, and ov-IGFBP2 plasmid were added to each well of a six-well plate. Stable cell lines transfected with sh-IGFBP2 and sh-RAB3A were established by puromycin selection. siRNA and shRNA sequences are shown in Table 2.

[0103] Table 2 siRNA and shRNA sequences

[0104] Name Sequence(5’-3’) sh-CTRL / si-NC 5'-UUCUCCGAACGUGUCACGUTT-3' sh-IGFBP2#1 5'-CCAGTTCTGACACACGTATTT-3' sh-IGFBP2#2 5'-ACAGTGCAAGATGTCTCTGAA-3' si-CTRL 5'-UUCUCCGAACGUGUCACGUTT-3' si-HIF1α#1 5'-CAAUCAAGAAGUUGCAUUATT-3' si-HIF1α#2 5'-UCGACUAUCUGCUCCAAGUUCTT-3' si-HIF2α#1 5'-CUCCUCAGUUUGCUCUGAATT-3' si-HIF2α#2 5'-CAGAACUGAUUGGUUACCATT-3' sh-Rab3A#1 5'-GACCATCTATCGCAACGACAA-3' sh-Rab3A#2 5'-CTACATGTTCAAGATTCTCAT-3'

[0105] RNA extraction and real-time quantitative PCR

[0106] Cellular RNA was extracted using RNA-Quick Purification Kit (RN001, ES Science), reverse transcribed using ReverTra Ace qPCR RT Kit (FSQ-101, TOYOBO), and quantitative PCR was performed using TB Green TM Premix ExTaq TM (Takara). β-actin was used as the internal reference. The primer sequences are shown in Table 3.

[0107] Table 3 Primer sequence list

[0108] Name Sequence(5'-3') Homo-IGFBP2-123F ACAATGGCGATGACCACTCA Homo-IGFBP2-123R CCAGCTCCTTCATACCCGAC Homo-RAB3A-93F CGAGTTCTTTGAGGCAAGCG Homo-RAB3A-93R GACTCGGACATCTTCTCGCA Homo-HIF1A-86F GGCAGCAACGACACAGAAAC Homo-HIF1A-86R TTTTCGTTGGGTGAGGGGAG Homo-HIF2A(EPAS1)-79F TCATGGGACTTACACAGGTGG Homo-HIF2A(EPAS1)-79R CGAATCTCCTCATGGTCGCA P1-141F(CHIP) GGATTATTTTAGCGGGCGGAG P1-141R(CHIP) GCCCCGAATGGTCTTAGTGAC P2-91F(CHIP) CTCGCGAACTGAACTGAGAGC P2-91R(CHIP) GAGAATACGGATAACTCGCGG

[0109] Luciferase reporter gene assay

[0110] 293T cells were seeded in 24-well plates for 24 hours and then co-transfected with 0.75 μg of pGL3-basic-IGFBP2 promoter-luciferase reporter vector, 0.25 μg of HIF-2α expression plasmid (or empty vector pENTER), and 0.1 μg of pRL-TK internal control plasmid. 48 hours later, the activity was detected using a dual-luciferase reporter system (Beyotime).

[0111] HIF-2α expression plasmid or empty vector pENTER and pRL-TK internal reference plasmid were purchased from Genema.

[0112] Construction of pGL3-basic-IGFBP2 promoter-luciferase reporter vector:

[0113] The IGFBP2 promoter-WT gene sequence (wild-type gene sequence, SEQ ID NO.2), IGFBP2 promoter-mut1 gene sequence (mutant 1 gene sequence, SEQ ID NO.3), and IGFBP2 promoter-mut2 gene sequence (mutant 2 gene sequence, SEQ ID NO.4) were synthesized respectively. Kpn I and Hind III restriction sites were designed at both ends of the above synthetic sequences, respectively. The above sequences were double-digested with Kpn I and Hind III and then inserted into the Kpn I and Hind III restriction sites of the pGL3-basic plasmid (purchased from Jinan Boshang Biotechnology Co., Ltd.) to obtain pGL3-WT, pGL3-Mut1, and pGL3-Mut2 vectors.

[0114] Chromatin immunoprecipitation (ChIP)

[0115] The binding site of HIF-2α in the promoter region of PDIA3P1 was predicted by JASPAR database, and the experiment was performed by MagnaChIP. TM Kit (17-10086, Millipore). Brief process: Formaldehyde cross-linking of proteins and DNA → Cell lysis → Chromatin sonication → Immunoprecipitation with anti-HIF-2α antibody (CST, D6T8V) → Reversal of cross-linking → DNA purification → qPCR analysis (primers see Table 3).

[0116] Immunofluorescence

[0117] Cells were seeded on confocal microplates, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% BSA. The cells were then incubated with primary antibodies overnight at 4°C and Alexa Fluor 594-conjugated secondary antibodies (Yeasen, 1:200) for 1 hour at room temperature in the dark. The cytoskeleton was labeled with FITC-phalloidin (Yeasen, 1:200), and nuclei were stained with DAPI (Yeasen, 1:5000). Images were acquired using a Leica SP8 confocal microscope.

[0118] Flow cytometry

[0119] Phagocytosis assay: THP1 cells were induced to differentiate into macrophages with 100 nM PMA for 48 h. CFSE-labeled GBM cells GSC20 and GSC267 were treated with isotype control antibody IgG (5 μg / mL, BioXcell, #BP0089), IGFBP2 antibody (5 μg / mL, R&D Systems, #AF674), CD47 antibody (10 μg / mL BE0019, BioXcell), IGFBP2 antibody (5 μg / mL, R&D Systems, #AF674), and CD47 antibody (10 μg / mL BE0019, BioXcell), respectively, for 24 h. The above GBM cells (GSC20 and GSC267) (1×10 6 ) were co-cultured with macrophages for 24 hours, and the phagocytic rate was detected by flow cytometry after labeling with APC-CD11b antibody (BioLegend).

[0120] In GSC20 cells, the CD47 antibody and IGFBP2 antibody were diluted in series according to the above concentrations, and the cells were treated with different concentration gradients of CD47 antibody and IGFBP2 antibody to calculate the drug combination index of the two.

[0121] CD47 detection: GSC cells were incubated with anti-CD47 eFluor 710 antibody (eBioscience) at room temperature for 30 minutes and analyzed by BD Accuri C6 flow cytometer. The data were processed using FlowJo V10.

[0122] Tumor sphere formation experiment GSCs were seeded at 1000 cells / well in 6-well plates. After culturing for 1-2 weeks, the diameter of the tumor spheres was measured under a Leica DMi8 microscope.

[0123] Limiting dilution assay (ELDA)

[0124] GSCs were seeded into ultra-low-adhesion 96-well plates at gradient densities (1-128 cells / well, 10 replicates). The sphere formation rate was counted after 7 days and analyzed using ELDA software (http: / / bioinf.wehi.edu.au / software / elda / ).

[0125] GBM-brain organoid co-culture invasion model

[0126] According to the literature (Bjerkvig R, et al. Cancer Res. 1986), 18-day-old rat fetal brain organoids were co-cultured with GFP-labeled GBM cell spheres for 72 hours, and the invasion was observed using a Leica TCS SP8 confocal microscope.

[0127] Animal experiments

[0128] GBM model: BALB / c nude mice (GemPharmatech) aged 4-6 weeks were intracranially inoculated with GSC267-luc cells (5×10 5 Tumor progression (days 4, 7, 14, and 28) was monitored using the IVIS Spectrum in vivo imaging system (PerkinElmer), and survival was recorded.

[0129] Combined therapy experiment: C57BL / 6 mice were intracranially inoculated with GL261-luc cells (inoculation volume was 2×10 5) were then divided into four groups: those injected via the tail vein with an anti-CD47 antibody (BE0019, BioXcell), those injected with an anti-IGFBP2 antibody (#AF674, R&D Systems), those injected with both an anti-CD47 antibody (BE0019, BioXcell) and an anti-IGFBP2 antibody (#AF674, R&D Systems), and those injected with an isotype control antibody, IgG (#BP0089, BioXcell). The CD47 antibody was injected at a dose of 10 mg / kg, the IGFBP2 antibody at a dose of 200 μg, and the IgG at a dose of 200 μg three times a week. Tumor volume was assessed by in vivo imaging. The experiment was approved by the Animal Ethics Committee of Qilu Hospital of Shandong University (DWLL-2023-169).

[0130] Single-cell RNA sequencing

[0131] Sample processing: The tissue was washed with PBS and minced, digested by enzyme → filtered → lysed red blood cells → resuspended in PBS → counted using CountStar (300-600 viable cells / μL).

[0132] Library construction: Single-cell GEMs were generated using the 10x Genomics Chromium system and sequenced using the Illumina Novaseq6000 (PE150, ≥100,000 reads / cell).

[0133] Data analysis: Cell Ranger 3.0 alignment to the GRCh38 reference genome → Seurat V2.0 normalization → PCA dimensionality reduction → UMAP clustering → Monocle2 pseudo-series analysis → GSEA functional enrichment.

[0134] Statistical analysis

[0135] Data were analyzed using GraphPad Prism 8 and R Studio (4.3.1). Survival analysis was performed using the log-rank test, and intergroup comparisons were performed using the t-test, Wilcoxon test, or one-way ANOVA. P < 0.05 was considered significant.

[0136] 2. Results

[0137] Positive correlation between IGFBP2 and CD47 in MES-GBM subpopulations in the hypoxic microenvironment

[0138] To explore the association between hypoxic microenvironment and immune escape in glioblastoma (GBM), this study collected tissue samples from the tumor core (Inner) and invasive edge (Periphery) of GBM patients and performed single-cell transcriptome sequencing using the 10× Genomics Chromium system ( Figure 1 A). According to previous annotations, tumor cells are divided into four malignant subtypes: neural precursor-like (NPC-GBM), oligodendrocyte precursor-like (OPC-GBM), astrocyte-like (AC-GBM), and mesenchymal-like (MES-GBM). MES-GBM is further subdivided into MES1-GBM and MES2-GBM subtypes ( Figure 1 B, Figure 2 A). Spatial distribution analysis showed that the proportion of MES-GBM in the tumor core was significantly higher than that in the peripheral area ( Figure 1 C, Figure 2 A). GSVA functional enrichment analysis showed that the MES1-GBM subpopulation exhibited highly complex functional characteristics, with epithelial-mesenchymal transition (EMT), angiogenesis, and hypoxia-related signaling pathways significantly activated in the core region ( Figure 1 D), suggesting that the GBM core region contains a hypoxia-driven immunosuppressive microenvironment dominated by MES1-GBM. Single-cell analysis showed that CD47 was specifically highly expressed in the MES1-GBM subpopulation ( Figure 2 B). GSEA enrichment analysis revealed that CD47 positively correlated genes were significantly enriched in hypoxia response and protein secretion pathways ( Figure 2 C, Figure 3 A), and these pathways are more activated in core MES1-GBM ( Figure 1 D). To analyze the effect of hypoxia-regulated proteins on CD47, we isolated exosomes secreted by glioma stem cells (GSCs) under hypoxia stimulation (morphology, size and concentration characteristics are shown in Figure 3 BD), and analyzed the protein composition of GBM cells and exosomes under hypoxic conditions by proteomic sequencing. The results showed that IGFBP2 ( Figure 3 A) was significantly upregulated in hypoxic GBM cells and exosomes ( Figure 2 D). Single cell co-expression density analysis further confirmed that IGFBP2 and CD47 specifically co-localized in the core MES1-GBM subpopulation ( Figure 2 EF). Monocle2-based pseudo-temporal trajectory analysis showed that the expression of CD47 and IGFBP2 gradually increased with tumor progression and reached a peak in the hypoxic core area ( Figure 2 G, Figure 3 E). Western blotting experiments confirmed that hypoxia can induce the expression of CD47 and IGFBP2, while knockdown of IGFBP2 can inhibit the expression of CD47 ( Figure 2HI, Figure 3 GH). Functional experiments showed that IGFBP2 knockdown cells were more easily phagocytosed by macrophages ( Figure 2 K), while overexpression of IGFBP2 promotes immune escape, an effect that can be reversed by anti-CD47 antibody (aCD47) ( Figure 2 L). By analyzing GBM patient tumor tissue and plasma exosomes, Western blotting and immunohistochemistry confirmed that IGFBP2 was highly expressed in GBM tissue and exosomes, and the expression level increased with the increase of pathological grade ( Figure 2 J, Figure 3 F), suggesting that it may serve as a potential biomarker for glioma diagnosis and grading. In summary, this study suggests that the coordinated expression of CD47 and IGFBP2 in the hypoxia-driven MES1-GBM subpopulation promotes tumor progression and exosome-mediated signaling, and that IGFBP2, as a hypoxia-inducible marker, is closely associated with CD47-mediated immune escape.

[0139] 2. IGFBP2 drives a dual-modal mechanism of GBM mesenchymal transition and tumorigenesis

[0140] To explore the regulatory role of IGFBP2 on GBM mesenchymal transition (MES transition), single-cell GSEA analysis showed that IGFBP2 positively correlated genes were significantly enriched in the MES1 subpopulation and epithelial-mesenchymal transition (EMT) pathway ( Figure 4 A). Western blotting experiments confirmed that knockdown of IGFBP2 could inhibit the expression of MES-GBM markers (CD44, YKL40) ( Figure 5 A). Through neurosphere formation experiments and limiting dilution experiments, we found that IGFBP2 significantly enhanced the self-renewal ability of MES-type glioma stem cells (GSC20, GSC267) ( Figure 4 C, Figure 5 BC). Based on the high invasive characteristics of MES-GBM, this study established a GBM tumor spheroid-rat brain organoid co-culture invasion model to simulate the physiological invasion microenvironment. The experiment showed that the invasion ability of GBM spheroids with IGFBP2 knockdown to brain tissue was significantly weakened ( Figure 5 DE). Further exosome intervention experiments showed that sh-IGFBP2 exosomes inhibited GBM mesenchymal transition ( Figure 4 DE, Figure 5 GH), suggesting that IGFBP2 drives the malignant phenotype through exosome-mediated intercellular communication. GSC267 was orthotopically transplanted into the brain of 4-week-old nude mice to establish a xenograft tumor model. The results showed that IGFBP2 knockdown significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice ( Figure 4 FG, Figure 5In summary, IGFBP2 promotes GBM-mesenchymal transition and tumorigenesis through dual mechanisms: cell-autonomous signaling (regulating MES markers) and exosome-dependent signaling (mediating microenvironment remodeling).

[0141] 3. Molecular mechanism of hypoxia-induced transcriptional activation of IGFBP2 via HIF-2α

[0142] To analyze the regulatory mechanism of IGFBP2-specific high expression in MES1-GBM subgroup, this study screened the top 30 genes positively correlated with IGFBP2 by intersection analysis ( Figure 6 A) and the top 10 transcription factors enriched in MES1-GBM ( Figure 6 B) found that hypoxia-inducible factor HIF-2α (encoding gene EPAS1) is a core co-expression regulator of IGFBP2 ( Figure 7 A). Pseudo-sequential analysis based on monocle2 showed that EPAS1 expression gradually increased with tumor progression and was significantly enriched in the core hypoxic area ( Figure 7 B). Using CellOracle (32) to simulate EPAS1 gene knockout, it was found that its deletion could inhibit the state transformation trajectory of the MES1-GBM subpopulation ( Figure 7 C). RT-qPCR and Western blotting experiments confirmed that knockdown of HIF-2α under 1% hypoxia could block the expression of IGFBP2 mRNA and protein ( Figure 7 DE). According to the JASPAR database, HIF-2α has two potential binding sites (P1 and P2) in the IGFBP2 promoter region ( Figure 7 F). ChIP-qPCR experiments confirmed that HIF-2α specifically binds to the P1 site of the IGFBP2 promoter ( Figure 7 G). Dual luciferase reporter system showed that overexpression of HIF-2α significantly enhanced IGFBP2 promoter activity, while mutation of the P1 site completely abolished this effect ( Figure 7 In summary, hypoxia directly induces IGFBP2 expression through a HIF-2α-dependent transcriptional mechanism.

[0143] 4. IGFBP2 drives CD47-mediated immune escape via the integrin / FAK / STAT3 signaling axis

[0144] This study focused on the molecular mechanism by which IGFBP2 regulates CD47 expression. Previous reports have shown that IGFBP2 promotes tumor malignancy by binding to integrin receptors. Our co-immunoprecipitation (Co-IP) experiments and mass spectrometry sequencing data showed that integrin α5β1 (ITGA5 / ITGB1) is a potential binding protein of IGFBP2 ( Figure 8A, Table S4). Single-cell RNA analysis further revealed that ITGB1 and ITGA5 were specifically co-expressed in the MES1-GBM subpopulation and were mainly enriched in the inner cell region of this subpopulation ( Figure 9 A, Figure 8 BC). Co-IP experiments confirmed that IGFBP2 can directly bind to integrin α5β1 in GSC20 and GSC267 cells ( Figure 9 B) It is known that integrins regulate the downstream STAT3 signaling pathway by activating FAK, and STAT3 can upregulate CD47 expression. Single-cell sequencing GSEA analysis showed that IGFBP2 positively correlated genes were significantly enriched in "focal adhesion formation", "extracellular matrix receptor interaction" and "STAT3 signaling pathway" ( Figure 9 C), suggesting that IGFBP2 may regulate CD47 through the integrin-FAK-STAT3 signaling axis. Experiments have shown that knocking down IGFBP2 can inhibit the activity of the FAK-STAT3 pathway and downregulate CD47 expression through cell-autonomous and exosome-dependent signals ( Figure 9 DE); overexpression of IGFBP2 activated this pathway and upregulated CD47, an effect that could be reversed by STAT3 inhibitors (Stattic, 5 μM) and FAK inhibitors (Y15, 10 μM) ( Figure 8 DE). Functional experiments confirmed that glioma stem cells overexpressing IGFBP2 escaped macrophage phagocytosis by activating the FAK-STAT3 pathway, and ITGB1 / ITGA5 neutralizing antibodies could effectively block this immune escape phenotype ( Figure 9 FH, Figure 8 F). In summary, IGFBP2 activates the FAK-STAT3 signaling axis by binding to integrin α5β1, thereby promoting CD47-mediated immune escape, providing a new strategy for targeting immune checkpoints.

[0145] 5. Hypoxia enhances the localization of IGFBP2 on the exosome surface through integrin α5β1-mediated binding

[0146] To explore the distribution characteristics of IGFBP2 in exosomes in the tumor microenvironment, we first confirmed that IGFBP2 and integrin α5β1 co-localized on the surface of exosome membranes by immunogold labeling experiments ( Figure 10 A). Proteinase K was further used to degrade exosome surface proteins (such as CD81), while the intracellular protein HSP70 was not degraded. The results showed that IGFBP2 and integrin α5β1 were located on the surface of exosomes, and hypoxia significantly upregulated the level of IGFBP2 in exosomes ( Figure 10BC). To verify the mechanism by which integrin α5β1 mediates the localization of IGFBP2 on the exosome membrane, we knocked down integrin α5β1 in glioma stem cells (GSCs) and found that the level of IGFBP2 on the exosome surface was significantly reduced ( Figure 10 DF). Combined with the above results, we propose that IGFBP2 is localized on the surface of the exosome membrane by interacting with integrin α5β1, and its binding efficiency is enhanced under hypoxic conditions ( Figure 10 G). Given the key role of the endosomal system in the formation of multivesicular endosomes (MVBs) and exosome secretion, immunoelectron microscopy experiments showed that the enrichment of IGFBP2 in endosomes increased significantly under hypoxic conditions ( Figure 10 H), suggesting that hypoxia promotes the internalization of more IGFBP2 into cells and its subsequent secretion onto the surface of exosomes via the MVB pathway. In summary, hypoxia enhances the localization of IGFBP2 on the surface of exosomes through integrin α5β1-mediated binding, providing a structural basis for its intercellular communication mediated by exosomes.

[0147] 6. Hypoxia promotes the secretion of IGFBP2-positive exosomes by upregulating RAB3A

[0148] To understand the mechanism by which hypoxia promotes exosome secretion, we used RNA sequencing data from GSE45117 and GSE232725 and found that RAB3A, a key regulator of exosome production, was significantly upregulated under hypoxic conditions ( Figure 11 A). RAB3A belongs to the RabGTPase family (including the RAB3A / RAB27A system) and is a core molecule that regulates exosome secretion and vesicle formation. RT-qPCR and Western blot experiments confirmed that hypoxia upregulates RAB3A expression ( Figure 12 AB), while knockdown of HIF-1α inhibited its expression ( Figure 11 BD), suggesting that hypoxia activates RAB3A through HIF-1α-dependent transcription. To clarify the regulatory role of RAB3A on exosome secretion, we knocked down RAB3A and measured the exosome secretion of glioma stem cells (GSCs). Nanoparticle tracking analysis (NTA) and exosomal IGFBP2 protein detection showed that RAB3A knockdown under hypoxic conditions significantly inhibited exosome secretion ( Figure 12 CE). Further electron microscopic observation revealed that RAB3A knockdown led to an abnormal increase in the number of intraluminal vesicles (ILVs) in multivesicular endosomes (MVBs) ( Figure 12 FG), while immunofluorescence (CD63 labeled MVBs) showed that the number of MVBs accumulated due to the blockage of exosome secretion ( Figure 12HI), indicating that RAB3A deficiency inhibits exosome release by hindering the fusion of MVBs with the plasma membrane, leading to the retention of ILVs within MVBs. In summary, hypoxia upregulates IGFBP2 expression through HIF-2α-dependent transcriptional activation and enhances the secretion of IGFBP2-positive exosomes in a HIF-1α-RAB3A-dependent manner. This mechanism reveals a novel pathway by which hypoxia propagates IGFBP2 signaling through exosomes, driving GBM immune evasion.

[0149] 7. Combined blockade of IGFBP2 and CD47 synergistically inhibits GBM malignant progression

[0150] To evaluate the synergistic effect of targeting IGFBP2 on anti-CD47 therapy, we used anti-IGFBP2 antibodies in combination with anti-CD47 antibodies to treat glioma stem cells (GSCs). In vitro experiments showed that the dual antibody combination significantly enhanced the phagocytic ability of macrophages on GSCs ( Figure 13 AB). The combination of drugs showed that the two had a synergistic effect. In addition, by inoculating GL261 cells into the brain of C57 mice to construct an orthotopic transplant tumor model, in vivo experiments further confirmed that the tumor growth in the combined treatment group was significantly inhibited, and the survival of tumor-bearing mice was significantly prolonged ( Figure 13 CF).

[0151] The above results indicate that dual blockade of IGFBP2 and CD47 can inhibit GBM immune escape and malignant progression through synergistic effects, providing a new strategy for combined immunotherapy of GBM.

[0152] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. The biomarker and / or the substance for detecting the biomarker are used in any of the following applications: (A1) Application in the preparation of a product for predicting or assisting in predicting the efficacy of monotherapy for brain glioma; (A2) Use in the preparation of products for evaluating or assisting in evaluating the efficacy of monotherapy for brain glioma; (A3) Use in the preparation of a product for predicting or assisting in predicting the progression-free survival time of a single immunotherapy for brain glioma; The biomarker is IGFBP2.

2. The use according to claim 1, characterized in that The substance for detecting biomarkers includes a reagent for detecting the expression level or content of IGFBP2 protein.

3. The use according to claim 2, characterized in that The reagents include antibodies, polypeptides, proteins or nucleic acid molecules that bind to IGFBP2 protein.

4. A kit, characterized in that The kit comprises the substance for detecting the biomarker according to any one of claims 1 to 3, and the kit has at least one of the following uses: (B1) Predict or assist in predicting the efficacy of monotherapy for glioma; (B2) To evaluate or assist in evaluating the efficacy of single-agent immunotherapy for glioma; (B3) Predict or assist in predicting the progression-free survival (PFS) of glioma patients treated with monotherapy with immunotherapy.

5. Any of the following uses of the biomarker according to claim 1 as a target: (C1) Application in the preparation of a product for improving the efficacy of monotherapy for brain glioma; (C2) Use in the preparation of products for the treatment or adjuvant treatment of brain glioma; (C3) Use in the preparation of products for use in combination with monotherapy of brain glioma immunotherapy.

6. Any of the following uses of an IGFBP2 inhibitor: (D1) Application in the preparation of a product for improving the efficacy of monotherapy for brain glioma; (D2) Application in the preparation of products for the treatment or adjuvant treatment of brain glioma; (D3) Use in the preparation of products for inhibiting the occurrence and / or development of brain glioma.

7. The use according to claim 6, characterized in that The IGFBP2 inhibitor includes a substance that reduces the expression level of IGFBP2 protein or the content of IGFBP2 protein, or a substance that inhibits the expression of the IGFBP2 gene, and the substance includes one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses.

8. The use according to claim 7, characterized in that The antibodies include antibodies against IGFBP2 protein or functional fragments thereof.

9. The use according to any one of claims 1-3 or 5-8, or the kit according to claim 4, characterized in that: The monotherapy is anti-CD47 therapy.

10. A combined drug for preventing or treating brain glioma, characterized in that: The combined drug comprises the IGFBP2 inhibitor according to any one of claims 6 to 8 and a glioma immunotherapy drug.

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