Csmd3-modified microglia cells and their anti-tumor applications

Abstract

The application relates to the technical field of tumor immunotherapy, and discloses a Csmd3 modified microglial cell and anti-tumor application thereof. The microglial cell has a lentivirus vector for expressing a nuclear localization activator, a lentivirus vector for expressing a coactivator and a lentivirus vector carrying a single-guide RNA sequence for targeting Csmd3, so that stable overexpression of Csmd3 protein is realized; the engineered cell releases a chemotactic factor to promote peripheral lymphocytes to infiltrate into the parenchyma of a brain solid tumor, promote the development of a local tertiary lymphatic structure and effectively reverse an immunosuppressive microenvironment in situ of the tumor; the cell preparation is used for in-situ micro-injection in the solid tumor, and is used in cooperation with an anti-programmed death receptor 1 monoclonal antibody for treatment on the seventh day after administration. The sequential combination strategy of local chemotaxis and antibody blocking of the pathway maintains the activity of effector cells, and improves the synergistic anti-tumor treatment effect.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, specifically to a Csmd3-modified microglia and its anti-tumor applications. Background Technology

[0002] Glioblastoma is a common primary malignant tumor of the central nervous system. Although tumor immunotherapy has made progress in various solid tumors in recent years, its clinical efficacy against glioblastoma remains limited. This poor prognosis is mainly due to the inherent immunosuppressive state of the brain solid tumor microenvironment. In the typical glioblastoma microenvironment, the tumor lacks effective chemotaxis mechanisms, making it difficult for peripheral blood lymphocytes to cross physiological barriers and infiltrate into the tumor parenchyma. Due to the lack of lymphocyte colonization at the lesion site and the lack of development of tertiary lymphoid structures, it is difficult for the tumor to establish and initiate a specific anti-tumor immune response network in situ.

[0003] To reshape this inhibitory microenvironment, functional modification of cells using genetic engineering has emerged as a potential intervention approach. However, current cell modification technologies face challenges when introducing multiple gene expression elements into chassis cells to construct complex activation systems, leading to promoter competition and gene silencing between different expression cassettes. Conventional transduction and screening methods struggle to eliminate hybrid or wild-type cells that have not fully integrated exogenous sequences, resulting in low purity and poor phenotypic uniformity in the final engineered cell population, and insufficient long-term operational stability of the exogenous transcriptional activation system. This technological bottleneck makes it difficult for modified cells to meet the requirements for large-scale preparation of standardized cell drugs.

[0004] Regarding drug delivery routes and combination therapy strategies, conventional systemic drug administration for glioblastoma is often blocked by the blood-brain barrier, making it difficult for living cells or drug molecules to establish effective local concentrations at the tumor center. Furthermore, even if some effector lymphocytes enter the tumor microenvironment, under long-term, complex inhibitory signal stimulation, newly infiltrated effector cells often upregulate inhibitory receptors, leading to rapid functional exhaustion. Current combination therapy regimens mostly fail to set reasonable intervention times based on the physiological time window for local lymphocyte chemotactic aggregation, lack a sequential mechanism combining in situ drug delivery and antibody blockade, and cannot effectively maintain the killing activity of immune cells after tumor entry, making it difficult to achieve a synergistic anti-tumor effect of relieving immunosuppression and local chemotactic colonization. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides Csmd3-modified microglia and their anti-tumor applications, solving the problems of immunosuppression caused by the lack of immune cell colonization in the glioblastoma microenvironment, insufficient purity and expression stability of multi-gene modified cell populations, and the problems of conventional drug administration being hindered by the blood-brain barrier and the rapid depletion of infiltrating effector cells.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a Csmd3-modified microglia, employing the following technical solution: A Csmd3-modified microglia, wherein the microglia are transfected with a lentiviral vector expressing a nuclear localization activator, a lentiviral vector expressing a co-activator, and a lentiviral vector carrying a single guide RNA sequence targeting Csmd3. The microglia overexpress Csmd3 protein and possess anti-glioblastoma function.

[0007] By employing the aforementioned technical approach and introducing this gene activation system, microglia, through the synergistic transduction of three lentiviral vectors, simultaneously express an inactivated nuclease protein fused with a nuclear localization sequence, a co-activation complex containing a transcriptional activation domain, and transcribe a single-guide RNA molecule with a hairpin aptamer structure within the cell nucleus. Based on the principle of sequence complementarity, the single-guide RNA binds to the promoter target region of the Csmd3 gene, causing the inactivated nuclease protein to localize to the front end of the Csmd3 gene locus. Here, the hairpin aptamer on the single-guide RNA specifically binds to the free co-activation complex, leading to the enrichment of transcription activators in the Csmd3 promoter region. This enrichment then recruits endogenous ribonucleic acid polymerase to initiate transcription, maintaining the sustained high expression of Csmd3 protein in microglia. Overexpression of Csmd3 protein alters the secretory profile of microglia, promoting the infiltration of peripheral circulating lymphocytes into the brain solid tumor parenchyma through chemotaxis. With the aggregation of lymphocytes in the local tumor microenvironment and the development of tertiary lymphoid structures, specific anti-tumor immune killing effects are effectively activated.

[0008] Preferably, the lentiviral vector expressing the nuclear localization activator is VSVG-LENTAI-EF1a.core-dCas9-VP64-2A-BSD-WPRE-pA; The lentiviral vector expressing the coactivator is VSVG-LENTAI-hEF1a-MPH-MS2-N55K-p65-HSF1-HygroR-WPRE-pA; The lentiviral vector carrying the single guide RNA sequence targeting Csmd3 is VSVG-LENTAI-hU6-sgRNA.sp-wMS2-Csmd3-esEF1a-MataGFP-IRES-PuroR-WPRE-pA.

[0009] By adopting the above technical solution, the selected lentiviral backbone is configured with independent promoters and resistance selection tags to prevent promoter competition and gene silencing after the three sets of expression elements are integrated into the same cell genome, ensuring that each element can be translated and expressed independently, thereby maintaining the long-term stability of the transcriptional activation system in microglia.

[0010] Preferably, the microglia are engineered from the mouse microglia line BV2.

[0011] By adopting the above technical solution and selecting a stable microglial cell line that has undergone immortalization treatment as the modification chassis, the engineered cells maintain a relatively uniform growth rate and stable phenotype during in vitro expansion, which is beneficial to the batch stability of subsequent standardized cell preparations.

[0012] Preferably, the method for preparing microglia includes the following steps: Wild-type microglia were seeded into a culture medium and the lentiviral vector expressing the nuclear localization activator and polybrene were added. After the first round of infection, blast fungicides were used for resistance screening and amplification. Lentiviral vector expressing the coactivator and polybrene were added to cells that had completed the first round of infection screening and amplification. After completing the second round of infection, hygromycin B was used for resistance screening and amplification. The lentiviral vector carrying the single guide RNA sequence targeting Csmd3 and polybrene were added into the cells after the second round of infection screening and amplification. After the third round of infection, puromycin was used for resistance screening and amplification. Furthermore, the multiplicity of infection (MNI) in the first round of infection is controlled to be between 10 and 30, and the screening concentration of blastomycin is between 3 μg / mL and 8 μg / mL; the MNI in the second round of infection is controlled to be between 10 and 30, and the screening concentration of hygromycin B is between 150 μg / mL and 250 μg / mL; the MNI in the third round of infection is controlled to be between 10 and 30, and the screening concentration of puromycin is between 1 μg / mL and 3 μg / mL; the final concentration of the polybrene added in the first, second, and third rounds of infection is 10 μg / mL.

[0013] By employing the above-mentioned technical solution and using a sequential multi-round infection combined with a multi-antibiotic gradient screening strategy, wild-type and heterozygous cells that did not fully integrate exogenous fragments were effectively eliminated. Limiting the multiplicity of infection in each round to the aforementioned range ensured the efficiency of exogenous gene introduction while controlling cytotoxicity caused by viral particles. Combined with the set screening drug concentrations, the final cell population possessed complete sets of three exogenous gene sequences, improving the overall purity of the engineered cells.

[0014] Preferably, the microglia are dispersed in phosphate buffer to form a cell suspension for microinjection, and the concentration of the microglia in the cell suspension is defined as 10,000 to 50,000 per microliter.

[0015] By employing the above technical solution, a phosphate buffer system was used to maintain appropriate physiological osmotic pressure and cell viability. The set cell concentration range was adapted to the flow tube diameter of the stereotactic microinjector for brain treatment, preventing excessive concentration from causing needle tract obstruction or local nutrient competitive necrosis, while ensuring the effective number of engrafted cells entering the lesion, thus meeting the physical feasibility of in situ drug delivery and the cell scale required for local treatment.

[0016] Secondly, this invention provides an application of Csmd3-modified microglia in the preparation of drugs for treating glioblastoma, employing the following technical solution: The application of Csmd3-modified microglia in the preparation of drugs for treating glioblastoma involves using the aforementioned Csmd3-modified microglia as the active ingredient to prepare drugs that exert a therapeutic effect against glioblastoma.

[0017] By employing the aforementioned technical approach, engineered microglia, after colonization of the lesion, regulate the local tumor microenvironment through continuous expression of the Csmd3 protein, altering the immune escape state caused by the scarcity of immune cells within glioblastoma. These cells release chemokines, promoting the penetration of peripheral circulating lymphocytes through physiological barriers and their infiltration and aggregation into the solid tumor parenchyma. The enrichment of local lymphocytes promotes the development of tertiary lymphoid structures. This pathological remodeling provides the body's immune system with a microenvironment for in situ antigen presentation and effector cell proliferation, thereby initiating a specific killing response against solid tumors.

[0018] Preferably, the drug for treating glioblastoma is formulated in a form suitable for in situ micro-injection into the solid tumor.

[0019] By employing the above-mentioned technical solution, and addressing the physical limitations of the blood-brain barrier on systemic drug delivery, the cellular drug is formulated as an in-situ micro-injection solution. This allows active cells to bypass the blood-brain barrier and directly colonize the lesion, establishing a local chemotactic signaling source within the tumor parenchyma. Compared to conventional intravenous infusion, this local delivery method enhances the directionality of peripheral lymphocytes' chemotactic infiltration into the tumor.

[0020] Preferably, the drug for treating glioblastoma is configured to be administered in combination with an anti-programmed death receptor 1 monoclonal antibody; and the combination administration specifically involves administering the anti-programmed death receptor 1 monoclonal antibody via intraperitoneal injection starting on the seventh day after the in situ micro-injection of the drug for treating glioblastoma. The initial dose is 400 μg, and the subsequent maintenance dose is 200 μg, administered every other day for six consecutive times.

[0021] By employing the above-mentioned technical approach, inhibitory stimuli in the tumor microenvironment often lead to functional exhaustion of effector cells infiltrating the tumor due to upregulation of inhibitory receptors. The seventh day after microinjection of cells corresponds to the physiological window period of initial development of local tertiary lymphoid structures and massive lymphocyte aggregation. Intervening with an anti-programmed death receptor 1 monoclonal antibody at this time can effectively block the inhibitory pathway, maintain the cytotoxic activity of newly infiltrating effector cells, and delay their exhaustion. The initial dose of 400 μg is designed to rapidly establish an effective blood drug concentration covering the receptor target, while subsequent doses of 200 μg every other day ensure the maintenance of drug activity during the local expansion cycle of effector T cells. This sequential combined strategy of targeted chemotaxis colonization and antibody blockade effectively amplifies the synergistic immune anti-tumor effect.

[0022] This invention provides Csmd3-modified microglia and their anti-tumor applications. It possesses the following beneficial effects: 1. This invention constructs Csmd3-modified microglia that are transferred into a specific gene activation system. By using single-guide RNA to target and specifically recruit transcription activators, stable high expression of Csmd3 protein in the cells is achieved, thereby changing the secretory characteristics of microglia. The chemokines released by microglia cause lymphocytes in the peripheral circulation to cross the physiological barrier and infiltrate into the glioblastoma parenchyma, thereby promoting the development of tertiary lymphoid structures in situ in the tumor and reversing the immunosuppressive state of the brain solid tumor microenvironment lacking immune cell colonization.

[0023] 2. This invention employs three lentiviral backbones equipped with independent promoters and different resistance tags, combined with sequential multi-round infection and multiple antibiotic screening steps to modify stable microglia cell lines. This avoids promoter competition and gene silencing phenomena commonly seen in multi-gene co-expression, eliminates cells with incompletely integrated sequences, ensures the long-term operational stability of the transcriptional activation system in chassis cells, and improves the overall purity and phenotypic uniformity of the engineered cell population, meeting the requirements for standardized cell drug preparation.

[0024] 3. In this invention, the cell drug is formulated as a suspension for in situ micro-injection, and the anti-programmed death receptor 1 monoclonal antibody is sequentially administered starting from the seventh day after injection. The in situ micro-injection allows living cells to bypass the blood-brain barrier and directly colonize at the center of the lesion, establishing a local chemotactic signal source. The antibody drug is introduced at the physiological time window when lymphocytes begin to accumulate in large numbers to block the inhibitory pathway, maintain the killing activity of newly infiltrated effector cells and delay their functional exhaustion, thereby achieving a synergistic anti-tumor effect of local chemotactic aggregation and desuppression. Attached Figure Description

[0025] Figure 1 The figure shows the test results of the CRISPRa system for overexpressing the BV2 microglial cell line Csmd3 and the changes in pro-inflammatory genes after overexpression. Figure 2 This is a diagram showing the results of an in vitro co-culture experiment between engineered microglia overexpressing Csmd3 and glioblastoma cells. Figure 3 This is a diagram showing the results of an in vivo in situ co-implantation experiment of engineered microglia overexpressing Csmd3 and glioblastoma cells. Figure 4 This is a graph showing the test results of engineered microglia inducing the formation of tertiary lymphoid structures in glioblastoma and the efficacy of combined anti-PD-1 immunotherapy in Example 1 of the present invention. Figure 5 This is a diagram showing the test results of the re-challenge experiment on the treatment of mouse tumors with engineered microglia in Example 1 of the present invention. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Preparation Examples 1-5: Preparation Example 1: This preparation example provides a method for preparing mouse microglia overexpressing Csmd3, including the following steps: (1) The mouse microglia cell line BV2 was seeded at a density of 50,000 cells / well in 24-well plates, and 500 μL of high-glucose DMEM complete medium was added. The cells were cultured at 37°C and 5% CO2 for 24 h. The lentiviral vector VSVG-LENTAI-EF1a.core-dCas9-VP64-2A-BSD-WPRE-pA expressing the nuclear localization activator and polybrene at a final concentration of 10 μg / mL were added to the culture wells to control the multiplicity of infection to 20. After 48 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, 5 μg / mL of blastomycin was added for selection for 4 days. Then, the concentration of blastomycin was adjusted to 2.5 μg / mL for maintenance culture for 4 days and the cells were expanded.

[0028] (2) The expanded cells were seeded again at a density of 50,000 cells / well in 24-well plates and cultured for 24 h. Then, the lentiviral vector VSVG-LENTAI-hEF1a-MPH(MS2(N55K)-p65-HSF1)-HygroR-WPRE-pA expressing the co-activator and polybrene at a final concentration of 10 μg / mL were added to control the multiplicity of infection to 20. After 48 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, hygromycin B at a concentration of 200 μg / mL was added for selection for 6 days. Then, the concentration of hygromycin B was adjusted to 100 μg / mL for maintenance culture for 4 days and the cells were expanded.

[0029] (3) The cells that have undergone double transduction and amplification were seeded at a density of 50,000 cells / well in 24-well plates. After culturing for 24 h, the lentiviral vector VSVG-LENTAI-hU6-sgRNA.sp(w / MS2)(Csmd3)-esEF1a-MataGFP-IRES-PuroR-WPRE-pA carrying the single guide RNA sequence number 2# targeting Csmd3 (its nucleotide sequence is shown in SEQ ID NO:7 and SEQ ID NO:8) and polybrene at a final mass concentration of 10 μg / mL were added to control the multiplicity of infection to 20. After 48 h of infection, the cells were digested and seeded into 6-well plates. When the cells grew to 80% confluence, puromycin at a mass concentration of 2 μg / mL was added for selection for 3 days. Then, the mass concentration of puromycin was adjusted to 1 μg / mL for maintenance culture for 4 days. After amplification, engineered mouse microglia overexpressing Csmd3 were finally obtained.

[0030] Preparation Example 2: This preparation example provides a method for preparing mouse microglia overexpressing Csmd3, including the following steps: (1) The mouse microglia cell line BV2 was seeded at a density of 50,000 cells / well in 24-well plates, and 500 μL of high-glucose DMEM complete medium was added. The cells were cultured at 37°C and 5% CO2 for 24 h. The lentiviral vector VSVG-LENTAI-EF1a.core-dCas9-VP64-2A-BSD-WPRE-pA expressing the nuclear localization activator and polybrene at a final concentration of 10 μg / mL were added to the culture wells to control the multiplicity of infection to 20. After 48 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, 5 μg / mL of blastomycin was added for selection for 4 days. Then, the concentration of blastomycin was adjusted to 2.5 μg / mL for maintenance culture for 4 days and the cells were expanded.

[0031] (2) The expanded cells were seeded again at a density of 50,000 cells / well in 24-well plates and cultured for 24 h. Then, the lentiviral vector VSVG-LENTAI-hEF1a-MPH(MS2(N55K)-p65-HSF1)-HygroR-WPRE-pA expressing the co-activator and polybrene at a final concentration of 10 μg / mL were added to control the multiplicity of infection to 20. After 48 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, hygromycin B at a concentration of 200 μg / mL was added for selection for 6 days. Then, the concentration of hygromycin B was adjusted to 100 μg / mL for maintenance culture for 4 days and the cells were expanded.

[0032] (3) The cells that have undergone double transduction and amplification were seeded at a density of 50,000 cells / well in 24-well plates. After culturing for 24 h, the lentiviral vector VSVG-LENTAI-hU6-sgRNA.sp(w / MS2)(Csmd3)-esEF1a-MataGFP-IRES-PuroR-WPRE-pA carrying a single guide RNA sequence targeting Csmd3 (number 3#) and polybrene at a final concentration of 10 μg / mL were added to control the multiplicity of infection to 20. After 48 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, puromycin at a concentration of 2 μg / mL was added for selection for 3 days. Then, the concentration of puromycin was adjusted to 1 μg / mL for maintenance culture for 4 days. After amplification, engineered mouse microglia overexpressing Csmd3 were finally obtained.

[0033] Preparation Example 3: This preparation example provides a method for preparing mouse microglia overexpressing Csmd3, including the following steps: (1) The mouse microglia BV2 cell line was seeded at a density of 30,000 cells / well in 24-well plates, and 500 μL of high-glucose DMEM complete medium was added. The cells were cultured at 37°C and 5% CO2 for 20 h. The lentiviral vector VSVG-LENTAI-EF1a.core-dCas9-VP64-2A-BSD-WPRE-pA expressing the nuclear localization activator and polybrene at a final concentration of 10 μg / mL were added to the culture wells to control the multiplicity of infection to 10. After 36 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, 3 μg / mL of blastomycin was added for selection for 3 days. Then, the concentration of blastomycin was adjusted to 1.5 μg / mL for maintenance culture for 3 days and the cells were expanded.

[0034] (2) The expanded cells were seeded again at a density of 30,000 cells / well in 24-well plates and cultured for 20 h. Then, the lentiviral vector VSVG-LENTAI-hEF1a-MPH(MS2(N55K)-p65-HSF1)-HygroR-WPRE-pA expressing the co-activator and polybrene at a final concentration of 10 μg / mL were added to control the multiplicity of infection to 10. After 36 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, hygromycin B at a concentration of 150 μg / mL was added for selection for 5 days. Then, the concentration of hygromycin B was adjusted to 80 μg / mL for maintenance culture for 3 days and the cells were expanded.

[0035] (3) The cells that have undergone double transduction and amplification were seeded at a density of 30,000 cells / well in 24-well plates. After culturing for 20 h, the lentiviral vector VSVG-LENTAI-hU6-sgRNA.sp(w / MS2)(Csmd3)-esEF1a-MataGFP-IRES-PuroR-WPRE-pA carrying a single-guide RNA sequence targeting Csmd3 (number 2#) and polybrene at a final concentration of 10 μg / mL were added to control the multiplicity of infection to 10. After 36 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, puromycin at a concentration of 1 μg / mL was added for selection for 2 days. Then, the concentration of puromycin was adjusted to 0.5 μg / mL for maintenance culture for 2 days. After amplification, engineered mouse microglia overexpressing Csmd3 were finally obtained.

[0036] Preparation Example 4: This preparation example provides a method for preparing mouse microglia overexpressing Csmd3, including the following steps: (1) The mouse microglia BV2 cell line was seeded at a density of 80,000 cells / well in 24-well plates, and 500 μL of high-glucose DMEM complete medium was added. The cells were cultured at 37°C and 5% CO2 for 28 h. The lentiviral vector VSVG-LENTAI-EF1a.core-dCas9-VP64-2A-BSD-WPRE-pA expressing the nuclear localization activator and polybrene at a final concentration of 10 μg / mL were added to the culture wells to control the multiplicity of infection to 30. After 60 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, 8 μg / mL of blastomycin was added for selection for 5 days. Then, the concentration of blastomycin was adjusted to 3.5 μg / mL for maintenance culture for 5 days and the cells were expanded.

[0037] (2) The expanded cells were seeded again at a density of 80,000 cells / well in 24-well plates and cultured for 28 h. Then, the lentiviral vector VSVG-LENTAI-hEF1a-MPH(MS2(N55K)-p65-HSF1)-HygroR-WPRE-pA expressing the co-activator and polybrene at a final concentration of 10 μg / mL were added to control the multiplicity of infection to 30. After 60 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, hygromycin B at a concentration of 250 μg / mL was added for selection for 7 days. Then, the concentration of hygromycin B was adjusted to 120 μg / mL for maintenance culture for 5 days and the cells were expanded.

[0038] (3) The cells that have undergone double transduction and amplification were seeded at a density of 80,000 cells / well in 24-well plates. After culturing for 28 h, the lentiviral vector VSVG-LENTAI-hU6-sgRNA.sp(w / MS2)(Csmd3)-esEF1a-MataGFP-IRES-PuroR-WPRE-pA carrying a single guide RNA sequence targeting Csmd3 (number 2#) and polybrene at a final concentration of 10 μg / mL were added to control the multiplicity of infection to 30. After 60 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, puromycin at a concentration of 3 μg / mL was added for selection for 4 days. Then, the concentration of puromycin was adjusted to 1.5 μg / mL for maintenance culture for 5 days. After amplification, engineered mouse microglia overexpressing Csmd3 were finally obtained.

[0039] Preparation Example 5: This preparation example provides a method for preparing human microglia overexpressing CSMD3, including the following steps: (1) The mouse microglia cell line BV2 was seeded at a density of 50,000 cells / well in 24-well plates, and 500 μL of high-glucose DMEM complete medium was added. The cells were cultured at 37°C and 5% CO2 for 24 h. The lentiviral vector VSVG-LENTAI-EF1a.core-dCas9-VP64-2A-BSD-WPRE-pA expressing the nuclear localization activator and polybrene at a final concentration of 10 μg / mL were added to the culture wells to control the multiplicity of infection to 20. After 48 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, 5 μg / mL of blastomycin was added for selection for 4 days. Then, the concentration of blastomycin was adjusted to 2.5 μg / mL for maintenance culture for 4 days and the cells were expanded.

[0040] (2) The expanded cells were seeded again at a density of 50,000 cells / well in 24-well plates and cultured for 24 h. Then, the lentiviral vector VSVG-LENTAI-hEF1a-MPH(MS2(N55K)-p65-HSF1)-HygroR-WPRE-pA expressing the co-activator and polybrene at a final concentration of 10 μg / mL were added to control the multiplicity of infection to 20. After 48 h of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, hygromycin B at a concentration of 200 μg / mL was added for selection for 6 days. Then, the concentration of hygromycin B was adjusted to 100 μg / mL for maintenance culture for 4 days and the cells were expanded.

[0041] (3) The cells that have undergone double transduction and expansion were seeded at a density of 50,000 cells / well in 24-well plates. After culturing for 24 hours, a lentiviral vector carrying a single guide RNA sequence targeting CSMD3 and polybrene at a final concentration of 10 μg / mL were added to control the multiplicity of infection to 20. After 48 hours of infection, the cells were digested and seeded into 6-well plates. When the cells reached 80% confluence, puromycin at a concentration of 2 μg / mL was added for selection for 3 days. Then, the concentration of puromycin was adjusted to 1 μg / mL for maintenance culture for 4 days. After expansion, engineered human microglia overexpressing CSMD3 were finally obtained.

[0042] Examples 1-7: Example

[0043] This embodiment provides an anti-tumor application of Csmd3-modified microglia, including the following steps: (1) The engineered mouse microglia overexpressing Csmd3 obtained in Preparation Example 1 were combined with primary mouse glioblastoma cells G422 in logarithmic growth phase. TNGBM cells were digested, collected, and resuspended in phosphate-buffered saline. The two cell types were then mixed at a cell ratio of 2:1 to prepare a cell suspension, in which G422 cells were included. TN The concentration of GBM cells was 25,000 cells / μL, and the concentration of engineered mouse microglia was 50,000 cells / μL.

[0044] (2) Male Kunming rats weighing 20-22g were anesthetized with sodium pentobarbital and fixed on a stereotaxic instrument. A hole was drilled in the right hemisphere of the mouse skull (0.5mm anterior to the anterior fontanelle, 2.0mm to the right of the midline, and a needle depth of 3.5mm). Using a microsyringe, 2μL of the above-mentioned cell suspension was slowly injected into the striatum of the mouse brain at a rate of 1μL / min. At this time, G422 TN The number of GBM cells implanted was 50,000, and the number of engineered microglia implanted was 100,000. After injection, the needle was left in place for 5 minutes and then slowly withdrawn, followed by suturing of the scalp.

[0045] (3) Starting on the 7th day after cell in situ implantation, mice were given anti-programmed death receptor 1 monoclonal antibody via intraperitoneal injection. The initial dose was 400 μg, and the subsequent dose was 200 μg / time, administered every other day, for a total of 6 times during the treatment period. Example

[0046] This embodiment provides an anti-tumor application of Csmd3-modified microglia, including the following steps: (1) The engineered mouse microglia overexpressing Csmd3 obtained in Preparation Example 1 were combined with primary mouse glioblastoma cells G422 in logarithmic growth phase. TN GBM cells were digested, collected, and resuspended in phosphate-buffered saline. The two cell types were then mixed at a 1:1 cell ratio to prepare a cell suspension, in which G422 cells were included. TN The concentration of GBM cells was 10,000 cells / μL, and the concentration of engineered mouse microglia was 10,000 cells / μL.

[0047] (2) Take male Kunming rats weighing 20-22g, anesthetize and perform stereotactic brain injection as in Example 1, and inject 5μL of cell suspension into the striatum of the mouse brain (0.5mm anterior to the anterior fontanelle, 2.0mm to the right of the midline, and 3.5mm deep).

[0048] (3) Starting on the 7th day after cell in situ implantation, mice were given anti-programmed death receptor 1 monoclonal antibody via intraperitoneal injection. The initial dose was 400 μg, and the subsequent dose was 200 μg / time, administered every other day, for a total of 6 times during the treatment period. Example

[0049] This embodiment provides an anti-tumor application of Csmd3-modified microglia, including the following steps: (1) The engineered mouse microglia overexpressing Csmd3 obtained in Preparation Example 1 were combined with primary mouse glioblastoma cells G422 in logarithmic growth phase. TN GBM cells were digested, collected, and resuspended in phosphate-buffered saline. The two cell types were then mixed at a cell ratio of 3:1 to prepare a cell suspension, in which G422 cells were included. TN The concentration of GBM cells was 10,000 cells / μL, and the concentration of engineered mouse microglia was 30,000 cells / μL.

[0050] (2) Take male Kunming rats weighing 20-22g, anesthetize and perform stereotactic brain injection as in Example 1, and inject 5μL of cell suspension into the striatum of the mouse brain (0.5mm anterior to the anterior fontanelle, 2.0mm to the right of the midline, and 3.5mm deep).

[0051] (3) Starting on the 7th day after cell in situ implantation, mice were given anti-programmed death receptor 1 monoclonal antibody via intraperitoneal injection. The initial dose was 400 μg, and the subsequent dose was 200 μg / time, administered every other day, for a total of 6 times during the treatment period. Example

[0052] This embodiment provides an anti-tumor application of Csmd3-modified microglia, including the following steps: (1) The engineered mouse microglia overexpressing Csmd3 obtained in Preparation Example 1 were combined with primary mouse glioblastoma cells G422 in logarithmic growth phase. TN GBM cells were digested, collected, and resuspended in phosphate-buffered saline. The two cell types were then mixed at a cell ratio of 2:1 to prepare a cell suspension, in which G422 cells were included. TN The concentration of GBM cells was 10,000 cells / μL, and the concentration of engineered mouse microglia was 20,000 cells / μL.

[0053] (2) Take male Kunming rats weighing 20-22g, anesthetize and perform stereotactic brain injection as in Example 1, and inject 5μL of cell suspension into the striatum of the mouse brain (0.5mm anterior to the anterior fontanelle, 2.0mm to the right of the midline, and 3.5mm deep).

[0054] (3) After the cells were implanted in situ, no anti-programmed death receptor 1 monoclonal antibody or any other drug intervention was given throughout the process. Only an equal volume of phosphate buffer was injected as a control to evaluate the anti-tumor effect of single-drug intervention of engineered cells. Example

[0055] This embodiment provides an anti-tumor application of Csmd3-modified microglia, including the following steps: (1) Primary mouse glioblastoma cells G422 in the logarithmic growth phase TN GBM cells were resuspended separately in phosphate-buffered saline to prepare a tumor cell suspension with a concentration of 10,000 cells / μL. Male Kunming rats weighing 20-22g were anesthetized with sodium pentobarbital and fixed on a stereotaxic apparatus. 5μL of the tumor cell suspension was slowly injected into the striatum of the right hemisphere of the mouse skull (0.5mm anterior to the anterior fontanelle, 2.0mm to the right of the midline, with a needle depth of 3.5mm) to establish an orthotopic solid tumor model of glioblastoma.

[0056] (2) On the 7th day after tumor inoculation, the engineered mouse microglia overexpressing Csmd3 obtained in Preparation Example 1 were resuspended in phosphate buffer to prepare a suspension with a concentration of 20,000 cells / μL. Using a stereotaxic instrument, 5 μL of the microglia suspension (100,000 cells) was slowly injected into the solid tumor at the original injection coordinates.

[0057] (3) Starting on the day of intratumoral injection of microglioma (i.e. 7 days after tumor inoculation), mice were given anti-programmed death receptor 1 monoclonal antibody via intraperitoneal injection. The initial dose was 400 μg, and the subsequent dose was 200 μg / time, administered every other day, for a total of 6 times. Example

[0058] This embodiment provides an anti-tumor application of Csmd3-modified microglia, including the following steps: (1) The engineered mouse microglia overexpressing Csmd3 obtained in Preparation Example 1 were digested and collected with the logarithmically growing mouse glioblastoma cell line GL261 and resuspended in phosphate buffer. The two cell types were mixed at a cell ratio of 2:1 to prepare a cell suspension, wherein the concentration of GL261 cells was 10,000 cells / μL and the concentration of engineered mouse microglia was 20,000 cells / μL.

[0059] (2) Male Kunming rats weighing 20-22g were anesthetized with sodium pentobarbital and fixed on a stereotaxic instrument. A hole was drilled in the right hemisphere of the mouse skull (0.5mm anterior to the anterior fontanelle, 2.0mm to the right of the midline, with a needle depth of 3.5mm). Using a microsyringe, 5μL of the above-mentioned cell suspension was slowly injected into the striatum of the mouse brain at a rate of 1μL / min. At this time, the number of GL261 cells implanted was 50,000, and the number of engineered microglia implanted was 100,000. After the injection, the needle was left in place for 5 minutes and then slowly withdrawn. The scalp was then sutured.

[0060] (3) Starting on the 7th day after cell in situ implantation, mice were given anti-programmed death receptor 1 monoclonal antibody via intraperitoneal injection. The initial dose was 400 μg, and the subsequent dose was 200 μg / time, administered every other day, for a total of 6 times during the treatment period. Example

[0061] This embodiment provides an anti-tumor application of Csmd3-modified microglia, including the following steps: (1) The logarithmic growth phase of the murine glioblastoma cell line GL261 was resuspended in phosphate buffer to prepare a tumor cell suspension with a concentration of 10,000 cells / μL. Male Kunming rats weighing 20-22g were anesthetized with sodium pentobarbital and fixed on a stereotaxic instrument. 5μL of tumor cell suspension was slowly injected into the striatum of the right hemisphere of the mouse skull (0.5mm anterior to the anterior fontanelle, 2.0mm to the right of the midline, and a needle depth of 3.5mm) to establish an orthotopic solid tumor model of glioblastoma.

[0062] (2) On the 7th day after tumor inoculation, the engineered mouse microglia overexpressing Csmd3 obtained in Preparation Example 1 were resuspended in phosphate buffer to prepare a suspension with a concentration of 20,000 cells / μL. Using a stereotaxic instrument, 5 μL of the microglia suspension (100,000 cells) was slowly injected into the solid tumor at the original injection coordinates.

[0063] (3) Starting on the day of intra-glioblastoma injection, mice were given anti-programmed death receptor 1 monoclonal antibody via intraperitoneal injection. The initial dose was 400 μg, followed by 200 μg every other day for a total of 6 doses.

[0064] Comparative Examples 1-2: Comparative Example 1: The difference from Example 1 is that in steps (1) and (2), only the suspension and in-situ injection of G422 are performed. TN -GBM cells (50,000 cells were implanted), without any microglia; in step (3), no anti-programmed death receptor 1 monoclonal antibody was given, only an equal volume of phosphate buffer was injected intraperitoneally, and the rest were the same.

[0065] Comparative Example 2: Compared with Example 1, the difference is that the engineered mouse microglia overexpressing Csmd3 in step (1) are replaced with BV2 microglia transduced with non-targeted negative control sgRNA (NC sgRNA), and the rest are the same.

[0066] Test Examples 1-5: Test Example 1: This test case aims to verify the molecular efficiency of the CRISPRa system guided by different single-guide RNA sequences in overexpressing Csmd3 in microglia, and to confirm the phenotypic transformation of downstream pro-inflammatory molecules triggered by upregulation of the target gene.

[0067] Experimental steps: Negative control microglia transduced with non-targeting sgRNA (MGNC group) obtained from Comparative Example 2, microglia transduced with Csmd3-sgRNA 1#, engineered microglia obtained from Preparation Example 1 (corresponding to Csmd3-sgRNA 2#), and engineered microglia obtained from Preparation Example 2 (corresponding to Csmd3-sgRNA 3#) were collected separately. Cells were washed with pre-chilled phosphate buffer, and lysed thoroughly on ice for 30 min with RIPA lysis buffer containing protease inhibitors. The lysate was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant containing total protein was extracted.

[0068] The protein concentration in the extract supernatant was determined using the BCA method. Sodium dodecyl sulfate loading buffer was added to each sample group for boiling and denaturation. Equal volumes of total protein sample were added to the wells for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0069] The separated proteins were transferred to a PVDF membrane under constant current and blocked with skim milk solution at room temperature for 2 h. Anti-CSMD3 antibody and anti-β-Actin antibody were then added, and the membrane was incubated overnight on a shaker at 4°C. After washing the membrane with Tween-containing elution buffer, horseradish peroxidase-labeled secondary antibody was added and incubated at room temperature for 1 h. ECL chemiluminescent substrate was added, and the luminescence signal was captured using a gel imaging system. The grayscale value of the target band was extracted, and the relative protein expression level of CSMD3 was calculated using β-Actin as an internal control.

[0070] Cell suspensions from the same group in the logarithmic growth phase were collected by centrifugation, and the supernatant was discarded. TRIzol lysis buffer was then added and thoroughly mixed. Chloroform, isopropanol, and anhydrous ethanol were added sequentially to extract and purify total RNA from the cells. The absorbance of the RNA samples was measured using a spectrophotometer to assess nucleic acid purity and concentration.

[0071] A suitable amount of total RNA was used to synthesize cDNA according to the reverse transcription kit instructions. Using the cDNA as a template, specific amplification primers and a real-time quantitative PCR dye were added to prepare the PCR reaction system. In a real-time quantitative PCR instrument, pre-denaturation, denaturation, and annealing extension cycles were sequentially set, and the amplification curves were recorded. The cycle thresholds of the Csmd3 gene and its downstream molecules Il1b (nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2), Tnf (nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4), and Il6 (nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6) were detected. Internal control genes were used for data normalization, and the mRNA expression fold increase of each target gene was calculated using a relative quantification method.

[0072] Table 1. Data on the relative expression levels of target genes and proteins in microglia of each group

[0073] Figure 1 The figure shows the test results of the CRISPRa system for overexpressing the BV2 microglial cell line Csmd3 and the changes in pro-inflammatory genes after overexpression. in, Figure 1 (A) is a schematic diagram of CRISPRa activating endogenous Csmd3 overexpression in BV2 microglia. "CRISPRa for CSMD3 overexpression" in the diagram represents the CRISPRa system used for overexpressing the CSMD3 protein. "Screening" represents the screening process. 1#, 2#, and 3# represent single-guide RNAs targeting the Csmd3 gene with different sequence numbers (i.e., Csmd3-sgRNA in the diagram). "MG OE−Csmd3,BV2 "This indicates the final BV2 microglia cell line overexpressing Csmd3 obtained through screening; Figure 1 (B) is a representative Western blot image of CSMD3 protein expression in each group of cells. NC corresponds to the non-targeted negative control cells prepared in Example 2. 1#, 2#, and 3# correspond to engineered microglia carrying different sequence numbers of Csmd3-sgRNA, respectively. 2# corresponds to the cells obtained in Example 1, and 3# corresponds to the cells obtained in Example 2. "CRISPRasystem in BV2 cells" indicates that the detection system is the CRISPRa system in the BV2 cell line. "406kD" and "42kD" represent the molecular weight (kilodaltons) of the target protein CSMD3 and the β-actin used as an internal control, respectively. Figure 1(C) is a statistical bar chart of the relative protein expression level of CSMD3 in each experimental group (i.e., the relative protein level shown on the vertical axis); Figure 1 (D) is a statistical bar chart of the relative mRNA expression level of Csmd3 in each experimental group (i.e., the relative mRNA expression shown on the vertical axis); Figure 1 (E) is a statistical bar chart showing the relative mRNA expression levels of three pro-inflammatory factors—interleukin-1β (Il1b), tumor necrosis factor (TnF), and interleukin-6 (Il6)—in cells with different phenotypes. The gray "MG" in the legend... NC,BV2 "Represents negative control microglia of the infected control sequence, the red "MG" OE−Csmd3,BV2 "This represents engineered microglia overexpressing Csmd3 (corresponding to preparation example 1); for Figure 1 (C) Figure 1 (D) and Figure 1 The statistical data in (E) are presented with error bars at the top of the bar chart representing the mean plus or minus the standard error of the data. Asterisks are used to indicate statistically significant differences between groups, where * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and "ns" indicates no significant difference.

[0074] in conclusion: Based on the data in Table 1 and the appendix Figure 1 The CRISPRa system, used in vitro, was applied to engineer microglia, effectively increasing the transcriptional levels of target genes. Combined with... Figure 1 (A) System design diagram and Figure 1 Transcriptional analysis (D) revealed that the effective activation of the Csmd3 gene transcription process within the cell depends on the target site selection of the guide RNA sequence. Specifically, referring to... Figure 1 (B) protein blot staining bands and Figure 1 (C) Quantitative statistics show that the single-guide RNA carrying sequence number 2# exhibits higher transcriptional activation efficiency after binding to the promoter region of the target gene. In Preparation Example 1, both the abundance of Csmd3 messenger RNA and the final expression level of CSMD3 protein showed an upregulation trend, significantly better than the other control groups, including Comparative Example 2, thus verifying the expression regulation effect of this system at the cellular level.

[0075] Microglia, as innate immune cells resident in the central nervous system, directly influence the immune microenvironment of local brain tissues through molecular phenotypic transformation. Upregulation of Csmd3 expression not only manifests as an increase in the abundance of this target but also alters the expression status of some intracellular inflammation-related molecules. (Refer to...) Figure 1 The downstream gene quantification results of (E) are compared with the corresponding data in Table 1. The engineered cells obtained in Preparation Example 1 showed increased transcription levels of pro-inflammatory factors such as Il1b, Tnf, and Il6 under resting culture conditions without intervention from exogenous inflammatory stimuli in vitro.

[0076] The changes in this expression profile indicate that the gene-editing strategy developed in this invention can effectively promote the polarization of microglia towards a pro-inflammatory phenotype. The synthesis and release of these pro-inflammatory factors help improve the local immunosuppressive microenvironment in tumors, providing in vitro experimental data to support subsequent in vivo evaluation of the anti-tumor ability of these engineered cells.

[0077] Test Example 2: This test case aims to verify the inhibitory effect of engineered microglia on glioblastoma cells and their responsiveness to environmental inflammatory signals through direct and indirect co-culture systems in vitro.

[0078] Experimental steps: Prepare luciferase-labeled GL261 glioblastoma cells in logarithmic growth phase (hereinafter referred to as GL261-Luc cells), and prepare engineered microglia overexpressing Csmd3 obtained in Example 1. OE-Csmd3,BV2 ) and negative control microglia (MG) prepared in Comparative Example 2 NC,BV2 ).

[0079] GL261-Luc cells were seeded in multi-well culture plates, and equal amounts of engineered microglia or negative control microglia were added to establish direct co-culture systems. Wells seeded only with GL261-Luc cells were retained as blank control groups. Each culture system was divided into an untreated group and a lipopolysaccharide (LPS)-treated group. A specific concentration of LPS solution was added to the culture medium in the LPS-treated group.

[0080] At the start of culture (0 h) and after 24 h of continued culture, D-luciferin potassium substrate was added to each well. In vitro luciferase luminescence images were acquired using an in vivo optical imaging system. Regions of interest were delineated in each well using image analysis software, and fluorescence intensity values ​​were extracted. The fluorescence intensity ratio between the engineered microglia group and the negative control group was calculated at different time points and under different treatment conditions.

[0081] An indirect co-culture system was established using Transwell chambers equipped with microporous membranes. GL261 cells were seeded in the lower culture plate, and engineered microglia or negative control microglia were seeded in the upper chamber. A control group with no cells in the upper chamber was included. After 24 hours of culture, the upper chamber containing microglia was removed.

[0082] GL261 cells were collected from the lower culture wells, and CCK-8 assay working solution was added and incubated in the dark. The absorbance of each culture well at 450 nm was measured using a microplate reader, and the relative viability of tumor cells was calculated.

[0083] Table 2. Fluorescence intensity and relative cell viability data of tumor cells in each co-culture group

[0084] Note: The "-" in the table indicates that the number of duplicate samples set for the test items differs (ROI intensity test group is set to 3 duplicate samples and 4 duplicate experiments, CCK-8 method group is set to 3 duplicate samples and 6 duplicate experiments), and the corresponding test item does not have duplicate sample data with that number.

[0085] Figure 2 This is a diagram showing the results of an in vitro co-culture experiment between engineered microglia overexpressing Csmd3 and glioblastoma cells. in, Figure 2 (A) is a schematic diagram of an in vitro co-culture experiment. In the diagram, "In vitro co-culture experiment" indicates an in vitro co-culture experiment; "Direct" represents direct co-culture; "Indirect" represents indirect co-culture using a layer-separated structure; "Luciferase imaging" indicates luciferase imaging detection; "LPS" represents lipopolysaccharide treatment; "CCK-8" indicates detection using the Cell Counting Kit-8 method; and "MG..." NC,BV2 "This indicates the negative control microglia in response ratio 2," "MG" OE-Csmd3,BV2 "" represents the engineered microglia obtained in Preparation Example 1, and "GL261-GBM" represents glioblastoma cells; Figure 2(B) shows in vitro luciferase luminescence images of GL261-GBM cells cultured alone (labeled Blank, i.e., blank control) and in direct co-culture systems with two types of microglia. In the figure, "In vitro Luciferase imaging of GL261-GBM cells" indicates luciferase imaging of tumor cells, "Untreated" represents no treatment, and the red "0h" and "24h" represent the initial culture state and the 24-hour time point, respectively. The "p / s / cm2 / sr" next to the color bar on the right is the unit of luminescence radiance (photons / second / square centimeter / steredegrees). Figure 2 (C) is a statistical graph of the regions of interest (ROI values) extracted from co-culture systems under different conditions. The vertical axis “ROI(p / s / cm2 / sr,104)” represents the fluorescence intensity value after conversion according to the order of magnitude. Figure 2 (D) is a line graph showing the ratio of fluorescence intensity between the engineered microglia group and the negative control group under untreated and LPS-treated conditions when directly exposed to tumor cells. The vertical axis “ROI ratio of GL261-Luc” represents the ratio of signal intensity between the two groups. Figure 2 (E) is a bar chart of the relative cell viability of tumor cells collected from the lower layer after indirect co-culture. In the figure, "CCK-8, GL261-GBM" indicates that the CCK-8 method was used to detect GL261-GBM cells, and "Relative cell viability" indicates relative cell viability. Regarding the above Figure 2 (C) Figure 2 (D) and Figure 2 The data in (E) is presented in the graph. The error bars in the bars and lines represent the mean plus or minus the standard error of the data. The asterisks are used to indicate statistically significant differences between groups, where * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and "ns" indicates no significant difference.

[0086] in conclusion: Based on the data in Table 2 and the appendix Figure 2 In vitro co-culture systems can be used to evaluate the inhibitory effect of microglia on the growth of glioblastoma cells. Figure 2 (A) Experimental Design and Figure 2 (B) Figure 2Imaging results (C) show that under standard culture conditions without the addition of exogenous stimuli, the fluorescence signal of tumor cells in the blank group increased over time. The fluorescence intensity of tumor cells in the engineered microglia group was significantly lower than that in the negative control group. Upregulation of Csmd3 enhanced the ability of microglia to inhibit the in vitro proliferation of tumor cells.

[0087] After treatment with added lipopolysaccharide, Figure 2 The fluorescence intensity ratio curve of (D) shows a decreasing trend. Engineered microglia exhibit better tumor-suppressive effects under lipopolysaccharide stimulation than under untreated conditions. Overexpression of Csmd3 in microglia increases the sensitivity of target cells to pro-inflammatory signals, making them exhibit more pronounced cytotoxic effects when subjected to pro-inflammatory stimuli.

[0088] Indirect co-culture systems are primarily used to analyze the paracrine effects of soluble cell factors. Based on... Figure 2 (E) and the relative cell viability data in Table 2 show that the survival rate of tumor cells in the lower layer of the engineered microglia group decreased after direct cell contact was blocked using a microporous membrane. The engineered cells created a microenvironment unfavorable to tumor cell survival in the culture medium by secreting cytokines. In vitro experimental results validated the positive regulatory effect of Csmd3 overexpression on the antitumor activity of microglia through both direct contact and paracrine pathways.

[0089] Test Example 3: This test case aims to evaluate the intervention effect of engineered microglia on glioblastoma growth and its impact on animal survival in the complex intracranial microenvironment through an in vivo orthotopic co-implantation mouse model.

[0090] Experimental steps: GL261-GBM cells and G422 cells in logarithmic growth phase were collected separately. TN GBM cells, and negative control microglia (MG) cells prepared in Comparative Example 2. NC,BV2 ) and engineered microglia (MG) cells prepared in Example 1 OE-Csmd3,BV2 The concentration of each cell suspension was adjusted, and glioblastoma cells and microglia were mixed at a ratio of 1:2, so that each injection dose contained 5 × 10⁻⁶ cells. 4 One tumor cell and 1×10 5 Small glial cells. Only 5 × 10⁻⁶ cells were retained. 4 A suspension of glioblastoma cells was used as a blank control.

[0091] Healthy, age-appropriate experimental mice were selected and subjected to intraperitoneal anesthesia. The anesthetized mice were then fixed onto a stereotaxic apparatus, and the scalp was routinely prepared and disinfected. The scalp was incised along the midline of the skull to expose the skull, and a micro-drill was used to create a hole at a specific coordinate location. Using a micro-syringe, the prepared cell suspension was slowly injected into the striatum region of the mouse's right brain. After injection, the needle was left in place for several minutes to prevent reflux. The needle was then slowly withdrawn, and the scalp was sutured, completing the establishment of the intracranial in situ co-implantation model.

[0092] On day 7 after cell inoculation, a subset of mice from each group were randomly selected and euthanized. Brain tissue was harvested intact after cardiac perfusion. The brain tissue was fixed in paraformaldehyde solution, dehydrated, cleared, and then embedded in paraffin. Serial sections were prepared using a microtome, and histological staining was performed using hematoxylin and eosin (H&E) staining. Panoramic images of the sections were acquired using a digital slide scanner, and tumor boundaries were delineated using image processing software. The tumor burden area of ​​the largest cross-section was measured.

[0093] For a mouse model inoculated with luciferase-expressing GL261-GBM cells, D-luciferin potassium salt substrate was injected intraperitoneally on day 13 after inoculation. Once the substrate was evenly distributed in vivo, the mice were placed in a dark chamber of an in vivo optical imaging system, and bioluminescent signals from the brain were acquired under isoflurane-maintained anesthesia. The tumor location was selected as the region of interest using accompanying software, and photon emissivity values ​​were extracted and quantified.

[0094] The remaining tumor-bearing mice in each group were retained for survival observation. The mice's mental state, weight changes, and survival status were monitored and recorded daily. The time of natural death or reaching the humane endpoint was used as the endpoint for survival recording. The data of each group were summarized and survival distribution curves were plotted.

[0095] Table 3. Tumor burden and luminescence intensity test data of the in vivo orthotopic co-implanted model

[0096] Note: The "-" in the table indicates that the number of replicate samples for the test item is different (5 or 6 replicates for some tissue section determinations and 7 replicates for in vivo imaging), and there is no replicate sample data with that number set in the corresponding test item.

[0097] Figure 3 This is a diagram showing the results of an in vivo in situ co-implantation experiment of engineered microglia overexpressing Csmd3 and glioblastoma cells. in, Figure 3(A) is a schematic diagram of in vivo co-implantation experiments. In the diagram, "In vivo co-implantation experiments" indicates in vivo co-implantation experiments, and "GL261-GBM" and "G422" are also mentioned. TN "-GBM" represents two glioblastoma cell lines, with the number of cells implanted labeled as "5×10". 4 “MG” NC,BV2 "This indicates the negative control microglia in response ratio 2," "MG" OE-Csmd3,BV2 "This represents the engineered microglia obtained in Preparation Example 1, with the number of cells implanted labeled as "1×10". 5 "2:1" indicates the ratio of microglia to tumor cells injected; Figure 3 (B) is a representative H&E staining and quantitative statistical graph of tumor burden in the brain tissue of mice in each group 7 days after co-implantation of the GL261-GBM model. "Blank" represents the blank control group injected with only tumor cells, and "Tumor burden" represents the tumor burden calculated by area. The vertical axis is in square millimeters (mm). 2 (1mm) indicates the scale of the pathological slide image; Figure 3 (C) is an in vivo bioluminescence imaging image of an intracranial tumor in a mouse with the GL261-GBM model on day 13 post-implantation. The image shows "In vivo Luciferase imaging of GL261-GBM model," indicating in vivo luciferase imaging of the GL261-GBM model. "Day 13 pi" represents day 13 post-implantation (pi is an abbreviation for post-implantation). The color bar at the bottom indicates "0.00-4.0×10". 5 "This represents the color gradation range of luminous intensity; Figure 3 (D) is a statistical graph of the region of interest (ROI) extracted from the above GL261-GBM live imaging. The vertical axis is "ROI(photons / sec, 10 5 ")" indicates the number of photons emitted per second multiplied by 10. 5 ; Figure 3 (E) is the survival curve of GL261-GBM model mice. The vertical axis “Percent survival” represents the survival percentage of mice, and the horizontal axis “Days” represents the number of days. Figure 3 (F) is G422 TN -Representative H&E staining maps and quantitative statistical graphs of tumor burden area for each group after co-implantation of the GBM model on day 7; Figure 3 (G) is G422 TN - Survival curve of GBM model mice; For the data presented above, the error bars in the graph represent the mean plus or minus the standard error of the data. The asterisks in the graph indicate statistically significant differences between groups, where * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and "ns" indicates no significant difference.

[0098] Test Example 4: This test case aims to verify the mechanism by which engineered microglia induce the formation of tertiary lymphoid structures in the glioblastoma microenvironment through clinical data analysis and in vivo animal experiments, and to evaluate its therapeutic efficacy when used in combination with anti-PD-1 immunotherapy.

[0099] Experimental steps: Transcriptome sequencing data and related clinical follow-up information of glioma patients were obtained from public databases. Bioinformatics algorithms were used to extract expression matrices of characteristic gene sets from Csmd3-positive microglia and tertiary lymphoid structures, and Pearson correlation coefficients between the two scoring features were calculated. Patients were grouped according to their scoring levels, and Kaplan-Meier curves were plotted for overall survival. The activation distribution of intercellular communication signaling in the single-cell transcriptome dataset was analyzed.

[0100] Collect G422 from test case 3 TN -Intact brain tissue from GBM orthotopic co-implantation model mice. After fixation and paraffin embedding, serial tissue sections were prepared. Immunohistochemical staining was used to label the sections with anti-CD3 and anti-CD20 antibodies, respectively. The sections were observed under an optical microscope, and aggregated lymphocyte clumps with overlapping CD3-positive T-cell and CD20-positive B-cell regions in serial sections were defined as tertiary lymphoid structures. The number and area of ​​tertiary lymphoid structures, as well as the density of lymphocytes in non-tertiary lymphoid structure regions, were quantitatively analyzed under a microscope in different experimental groups.

[0101] Stripping G422 TN Fresh brain tumor tissue samples were collected from mice in the GBM orthotopic co-implantation model (Comparative Example 1, Comparative Example 2, and Example 1). Total RNA was extracted from the tissues and cDNA templates were synthesized. The relative transcriptional expression level of the Pdcd1 gene (its nucleotide sequence is shown in SEQ ID NO:9 and SEQ ID NO:10) was detected using specific primers in a real-time quantitative PCR system. The fold change in expression was calculated using an internal reference gene after data normalization.

[0102] Preparation of G422 TNA single-cell suspension of GBM cells mixed with negative control microglia or engineered microglia in a specific ratio was used. An intracranial co-implantation model was established in mice using stereotactic brain imaging. Mice in each co-implantation model were randomly assigned to either a control group or an anti-PD-1 treatment group. Starting from day 7 post-implantation, mice in the treatment group were intraperitoneally injected with anti-PD-1 monoclonal antibody every other day, with an initial dose of 400 μg and a maintenance dose of 200 μg for the next five injections. Mice in the control group were injected with an equal volume of physiological buffer. The survival status of the mice was monitored daily until the experimental endpoint was reached, and survival time data were recorded to plot survival distribution curves.

[0103] Table 4. In vivo TLS quantification and Pdcd1 gene expression test data

[0104] Note: The presence of a "-" in the table indicates that due to differences in the replication settings of the test items (e.g., RT-qPCR analysis is set to 4 replicates, and histological sections are set to 8 replicates), no corresponding replicate sample number was set for that item.

[0105] Figure 4 This is a graph showing the test results of engineered microglia inducing the formation of tertiary lymphoid structures (TLS) in glioblastoma and the efficacy of combined anti-PD-1 immunotherapy in Example 1 of the present invention. in, Figure 4 (A) is a correlation plot of glioma samples in the CGGA (left) and TCGA (right) public datasets, with the vertical axis showing "Csmd3". + "microglia signature score" indicates Csmd3 + Microglial cell characteristic score, the horizontal axis "TLSsignature score" represents the TLS characteristic score, "R" in the figure is the Pearson correlation coefficient (0.67 and 0.59 respectively), "p" is the statistical significance value (both less than 2.2e-16). Figure 4 (B) shows the survival curves of patients with different signature scores in the TCGA dataset. The vertical axis "Percent Survival" represents the survival percentage, and the horizontal axis "Months" represents the number of months. "P < 0.0001" in the figure indicates that the survival difference is statistically significant; "CMG:Csmd3 + "microglia" indicates that CMG is Csmd3. +The abbreviation for microglia is "CMGhigh TLShigh", which represents a group with high expression of both traits; "CMGlow TLSlow", which represents a group with low expression of both traits; "CMGlow TLShigh", which represents a group with low expression of CMG and high expression of TLS; and "CMGhigh TLSlow", which represents a group with high expression of CMG and low expression of TLS. Figure 4 (C) is a bar chart of relative information flow between cells. The blue bars represent the control group, and the red bars represent the model group (2°-LTS(ICu)). The horizontal axis lists various signaling pathways and molecules (including GAS, SELPLG, APP, CCL, CD86, MHC-II, THY1, PECAM1, SELL, CADM, SEMA4, GRN, TGFb, JAM, CD45, CD22, ITGAL-ITGB2, CXCL, MIF, GALECTIN, CD52, LCK, MHC-I). The green arrows in the figure specifically point to the CCL and CXCL chemokine signaling pathways. Figure 4 (D) is a representative image of a series of brain slices, “TLS:CD3”. + CD20 + G422 TN "-GBM" indicates G422 TN -CD3 and CD20 double-positive grade tertiary lymphoid structures in the GBM tumor model, with the left longitudinal section marking the comparative groups 2 (MG). NC,BV2 ) and Example 1 Group (MG OE-Csmd3,BV2 The top horizontal markers indicate the T-cell marker CD3 and the B-cell marker CD20, and "50μm" is the image scale bar. Figure 4 (E) is a quantitative statistical chart of TLS in the slice. The left vertical axis "TLS number / section" represents the number of TLS in each slice (i.e., No.TLS), and the right vertical axis "TLS area (×10)" represents the number of TLS in each slice. 4 μm 2 ") / section" represents the TLS area of ​​each slice (i.e., Area.TLS); Figure 4 (F) shows the immunohistochemical staining images of the non-TLS regions in each group, "Non-TLS region, G422". TN "-GBM" indicates G422 TN - The non-TLS region of the GBM tumor model is horizontally labeled with the blank control group (Blank, corresponding to comparison 1) and comparison 2 group (MG). NC,BV2) and Example 1 Group (MG OE-Csmd3,BV2 The vertical axis indicates the staining indices CD3 and CD20, and "50μm" is the image scale bar. Figure 4 (G) is a quantitative bar chart for the aforementioned non-TLS regions, with the left graph's vertical axis showing "CD3". + T cells / mm 2 "This represents the number of CD3-positive T cells per square millimeter. The vertical axis of the right graph shows "CD20". + B cells / mm 2 "This indicates the number of CD20-positive B cells per square millimeter;" Figure 4 (H) is a scatter plot of correlations in the TCGA dataset, with the vertical axis "PDCD1" representing the expression level of the programmed death receptor 1 gene, and the horizontal axis "Csmd3" representing the expression level of the programmed death receptor 1 gene. + "microglia" indicates Csmd3 + Microglial cell characteristics, "R=0.51, p<2.2e-16" shows the correlation coefficient and significance value; Figure 4 (I) is a quantitative PCR analysis diagram of mouse tumor tissue. "Pdcd-1" represents the target gene being detected, and the vertical axis "Relative mRNA expression" represents the relative messenger RNA expression level. Figure 4 (J) is a bar chart of αPD-1 responsiveness to PD-1 antibody therapy. The vertical axis "Percent" represents the percentage of patients, and the horizontal axes "Low" and "High" represent Csmd3, respectively. + Microglial cell characteristics (Csmd3) + The diagram shows the low and high expression groups of the microglia signature. In the legend, blue squares represent response treatment and gray squares represent non-response. Figure 4 (K) is a schematic diagram of the in vivo co-implantation and drug delivery experimental design, in which tumor cells (G422) are marked. TN The number of GBM implants is 5x10 4 "Comparative Example 2 cells (MG)" NC,BV2 ) or Example 1 cells (MG OE-Csmd3,BV2 The number of implants is 1x10 5The two types of cells were mixed in a 2:1 ratio and implanted into mice. The horizontal axis “Days post-implantation (pi)” represents the number of days after implantation. The axis is marked with specific day nodes “0, 7, 9, 11, 13, 15, 17”. The green arrows corresponding to the dates and “αPD-1” below indicate that anti-PD-1 antibody injections were administered on days 7, 9, 11, 13, 15, and 17. Figure 4 (L) is "G422" containing the ingredients of Example 1. TN -GBM+MG OE-Csmd3,BV2 "The co-implantation model survival curves show the percentage of survival on the vertical axis and the number of days on the horizontal axis. In the legend, the black solid line "Control" represents the control subgroup that did not receive antibody injection, and the pink solid line "αPD-1" represents the subgroup that received antibody treatment. "1 / 13" and "2 / 14" represent the results of 1 out of 13 mice in the control group and 2 out of 14 mice in the antibody treatment group, respectively." Figure 4 (M) is "G422" containing the components of Comparative Example 2. TN -GBM+MG NC,BV2 "Survival curves of co-implantation model, where "Control" is the control subgroup, "αPD-1" is the antibody treatment subgroup, and "ns" indicates no significant difference between the two groups.

[0106] for Figure 4 (E) Figure 4 (G) Figure 4 The data in (I) is presented with bar chart error bars representing the mean plus or minus the standard error. Asterisks in the chart indicate statistically significant differences between groups, where * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and "ns" indicates no significant difference.

[0107] in conclusion: Based on the data in Table 4 and the appendix Figure 4 Bioinformatics analysis of clinical samples showed that the abundance of Csmd3-positive microglia infiltrating gliomas was positively correlated with the formation of tertiary lymphoid structures, and the co-expression of these two histological features was closely associated with prolonged overall patient survival. Further signaling pathway analysis indicated that specific activation of related chemokine pathways constitutes the molecular basis for mediating intercellular communication and lymphocyte recruitment.

[0108] Histological examination of animals further validated the immune remodeling function of these engineered cells in vivo. (Reference) Figure 4 (D) Figure 4(E) and the quantitative statistical results in Table 4 show that the engineered microglia corresponding to the Example 1 group were at G422. TN GBM tumor microenvironment promotes local aggregation of T cells and B cells, thereby forming a tertiary lymphoid structure. Compared with Comparative Group 2, the number and distribution area of ​​tertiary lymphoid structures in the pathological sections of Example 1 group were significantly increased. Figure 4 (F) and Figure 4 Data from (G) show that even in scattered tumor areas where complete lymphoid structures were not formed, the infiltration density of CD3-positive T cells and CD20-positive B cells was significantly increased. The increased focal lymphocyte abundance within the tumor indicates that engineered microglia have the ability to improve the immunosuppressive microenvironment of solid tumors.

[0109] The chemotactic recruitment of immune cells in the local microenvironment is accompanied by changes in the sensitivity of tumor tissue to immune checkpoint blockade. Figure 4 (H) Figure 4 (I) and the quantitative analysis in Table 4 showed that, with the increase in the number of infiltrating immune cells, the transcriptional level of the programmed death receptor 1 gene in the tumor parenchyma of the Example 1 group was significantly upregulated. Combined with the analysis of public clinical cohorts, this expression pattern can be confirmed to generally predict a higher objective response rate to drug therapy targeting immune checkpoints in such patients.

[0110] In vivo combination therapy studies provide a preclinical efficacy evaluation of this treatment strategy. (Refer to...) Figure 4 (M), the comparative model 2, lacking effective immune cell infiltration, exhibited primary resistance to single-drug anti-PD-1 antibody, and combination therapy failed to improve disease progression in the animals. However, in animal models implanted with engineered microglia, such as... Figure 4 As shown in (L), the intervention of anti-PD-1 antibody further prolonged the survival of experimental mice. The cells prepared in Example 1 provided a histological basis for subsequent anti-tumor immune responses by inducing the generation of tertiary lymphoid structures around the tumor and promoting the activation of effector lymphocytes. This mechanism transforms glioblastoma, which is originally immune cell-deficient, into a phenotype sensitive to immune checkpoint inhibitors, providing definite evidence of survival benefit for combination therapy in a living environment.

[0111] Test Example 5: This test case aims to evaluate, through a tumor rechallenge experiment, whether mice that had previously been treated with the protocol in Example 1 and survived for a long period of time had developed a systemic and persistent anti-tumor immune memory against glioblastoma.

[0112] Experimental steps: Cured mice (i.e., LTS mice containing the components of Example 1) that survived for more than 100 days in the previous in situ co-implantation experiment without any signs of tumor recurrence were selected as the experimental group. Healthy wild-type mice of the same age and sex were introduced as the control group (i.e., blank controls inoculated only with tumor cells). G422 mice in the logarithmic growth phase labeled with luciferase were prepared. TN GBM tumor cells were collected and the concentration of the cell suspension was adjusted.

[0113] Two groups of mice were intraperitoneally anesthetized and fixed in a stereotaxic apparatus. At corresponding coordinate locations in the cerebral hemispheres of both groups of mice, a small amount of 5 × 10⁻⁶ natriuretic peptide (Na₂O₃) was slowly injected using a microinjector. 4 G422 TN -GBM cell suspension. After inoculation, the scalp was routinely sutured, and the mice were returned to their rearing cages to recover.

[0114] Seven days after tumor re-inoculation, mice in both groups were injected intraperitoneally with D-fluorescein potassium substrate solution. After the substrate had fully circulated and distributed, the anesthetized mice were transferred into the dark chamber of the in vivo optical imaging system. Bioluminescence signals from the mouse heads were acquired, and the fluorescence intensity of the region of interest (ROI) in the luminescent area was quantified using system software to assess the early proliferation and colonization of tumors within the brain.

[0115] After optical imaging monitoring was discontinued, mice were kept under standard feeding conditions, and their physiological status and survival were recorded daily for both groups. The tumor rechallenge inoculation diary was recorded as day 0, and observation continued for 100 days. The observation cutoff point was defined as the mice reaching a humanitarian endpoint, such as natural death or weight loss. Survival days were recorded, and survival rate curves were plotted.

[0116] Table 5. Luminescence intensity and survival data of tumor re-challenge mice on day 7.

[0117] Figure 5 This is a graph showing the test results of the re-challenge experiment on the treatment of mouse tumors with engineered microglia in Example 1 of the present invention; in, Figure 5 (A) is a flowchart illustrating the tumor rechallenge experiment in cured mice. The flowchart shows "Tumor rechallenge of LTS-MG". OE-Csmd3,BV2 "mice" indicates tumor rechallenge in long-term survival (LTS) mice containing the components of Example 1, "5x10 4 G422 TN"GBM cells" indicates the number of glioblastoma cells re-implanted; the syringe and mouse head graphic represent the intracranial stereotactic injection operation; "Cured mice" are cured mice; "Days pi" represents the number of days after the initial implantation; node "100" is the start time of the re-challenge (marked as day 100); the "BLI" and mouse head fluorescence image below "107" indicate that in vivo bioluminescence imaging was performed on day 7 after the re-challenge (day 107 of the overall process); "200" and "Survival" indicate that the survival period was continuously observed until day 200 of the overall process. Figure 5 (B) is an in vivo bioluminescence imaging image of intracranial tumors in two groups of mice 7 days after tumor rechallenge vaccination. "Control" represents age-matched and sex-matched mice using the Comparative Example 1 protocol. OE-Csmd3,BV2 "Represents the cured mice in Example 1; "2.8 × 10 5 "and 9.5×10" 5 "Indicates the lower and upper reference values ​​of luminescence radiance in the imaging image; Figure 5 (C) shows the survival curves of the two groups of mice after tumor rechallenge. The vertical axis "Percent survival" represents the survival percentage, and the horizontal axis "Days" represents the number of observation days calculated from the date of rechallenge vaccination. In the legend, the black solid line "Control" corresponds to group 1, and the pink solid line "LTS-MG" corresponds to group 1. OE-Csmd3,BV2 "Corresponding to the cured group in Example 1, "100% (3 / 3)" means that all 3 mice in the cured group in Example 1 survived the entire rechallenge observation period.

[0118] in conclusion: Based on the data in Table 5 and the appendix Figure 5 In the previous example, the therapeutic intervention using engineered microglia (Example 1) enabled the establishment of a long-lasting and specific anti-tumor immune memory in the host. Combined with... Figure 5 (B) In vivo imaging and the quantitative data in Table 5 show that mice in the control group 1, lacking pre-existing immunity, were vaccinated with G422. TN Following GBM cell inoculation, tumor bioluminescence signals were detected in the brain on day 7, indicating successful colonization and proliferation of exogenous tumor cells. In contrast, LTS mice that received cell therapy in Example 1 and survived for more than 100 days showed lower fluorescence intensity in their brains, remaining close to baseline background levels. This result demonstrates that the immune system in cured mice can effectively recognize and eliminate re-inoculated homologous tumor cells, inhibiting their tumorigenesis in the brain.

[0119] The early clearance effect of lesions ultimately manifested in the differences in animal survival. Figure 5Survival data (C) objectively show that all mice in Comparative Example 1 died shortly after inoculation due to the continuous growth of intracranial tumors. In contrast, the mice in the cured group of Example 1, after receiving the same dose of tumor cells, achieved a 100% survival rate and showed no signs of recurrence throughout the 100-day observation window. The effector memory lymphocytes formed in the host effectively weakened the inherent immune tolerance of the glioblastoma microenvironment. In this protocol, microglia overexpressing Csmd3 not only played a role in recruiting lymphocytes and establishing a tertiary lymphoid structure in the early stages, but also promoted the transformation of local antigen presentation into systemic long-term immune protection, thereby blocking the tumor recurrence pathway.

Claims

1. A Csmd3-modified microglia, characterized in that, The microglia were transfected with lentiviral vectors expressing nuclear localization activators, lentiviral vectors expressing coactivators, and lentiviral vectors carrying single guide RNA sequences targeting Csmd3. The microglia overexpress Csmd3 protein and possess anti-glioblastoma function.

2. The Csmd3-modified microglia according to claim 1, characterized in that, The lentiviral vector expressing the nuclear localization activator is VSVG-LENTAI-EF1a.core-dCas9-VP64-2A-BSD-WPRE-pA; The lentiviral vector expressing the coactivator is VSVG-LENTAI-hEF1a-MPH(MS2(N55K)-p65-HSF1)-HygroR-WPRE-pA; The lentiviral vector carrying the single guide RNA sequence targeting Csmd3 is VSVG-LENTAI-hU6-sgRNA.sp(w / MS2)(Csmd3)-esEF1a-MataGFP-IRES-PuroR-WPRE-pA.

3. The Csmd3-modified microglia according to claim 1, characterized in that, The microglia were engineered from the mouse microglia line BV2.

4. The Csmd3-modified microglia according to claim 1, characterized in that, The method for preparing the microglia includes the following steps: Wild-type microglia were seeded into a culture medium and the lentiviral vector expressing the nuclear localization activator and polybrene were added. After the first round of infection, blast fungicides were used for resistance screening and amplification. Lentiviral vector expressing the coactivator and polybrene were added to cells that had completed the first round of infection screening and amplification. After completing the second round of infection, hygromycin B was used for resistance screening and amplification. The lentiviral vector carrying the single guide RNA sequence targeting Csmd3 and polyglobulin were added into the cells after the second round of infection screening and amplification. After the third round of infection, puromycin was used for resistance screening and amplification.

5. The Csmd3-modified microglia according to claim 4, characterized in that, The multiplicity of infection in the first round of infection was controlled at 10-30, and the screening concentration of the blast fungicide was 3-8 μg / mL. The multiplicity of infection during the second round of infection was controlled at 10-30, and the screening concentration of hygromycin B was 150-250 μg / mL. The infection multiple was controlled to be 10-30 during the third round of infection, and the puromycin screening concentration was 1-3 μg / mL. In the first, second, and third rounds of infection, the final mass concentration of the added polybrene was 10 μg / mL.

6. The Csmd3-modified microglia according to claim 1, characterized in that, The microglia are dispersed in phosphate buffer to form a cell suspension for microinjection, and the concentration of the microglia in the cell suspension is defined as 10,000-50,000 per microliter.

7. The use of Csmd3-modified microglia according to any one of claims 1 to 6 in the preparation of drugs for treating glioblastoma.

8. The application according to claim 7, characterized in that, The drug for treating glioblastoma is formulated in a form suitable for in situ micro-injection into the solid tumor.

9. The application according to claim 7, characterized in that, The drug for treating glioblastoma is formulated to be administered in combination with an anti-programmed death receptor 1 monoclonal antibody.

10. The application according to claim 9, characterized in that, The specific method of combined drug administration is as follows: The anti-programmed death receptor 1 monoclonal antibody was administered via intraperitoneal injection starting on the seventh day after the in situ microinjection of the drug for treating glioblastoma. The initial dose was 400 μg, and the subsequent maintenance dose was 200 μg, administered every other day for six consecutive times.

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