A therapeutic microglial cell subpopulation for glioma and a method of inducing the same

Abstract

This invention belongs to the medical field and establishes a clinically relevant mouse glioblastoma treatment model, obtaining "cured" mice. When these "cured" mice were re-challenged with tumors, the tumors spontaneously regressed, and the animals achieved long-term survival. This indicates that the "cured" mice acquired immunity to the tumor, and these mice are named "cured-immune" mice. Intracranial inoculation of tumor cells into the "cured-immune" mice specifically induced a microglia subset exhibiting high expression of the purinergic receptor P2ry12 gene. High ) and immune-boosting functional characteristics, P2ry12 infusion Hi Small glial subsets significantly prolonged the survival time of glioma-bearing mice. Compared with the control group, P2ry12 in "cured-immune" (LTS) mice was significantly reduced. High Ccl12 low Differentially expressed genes in microglial cell subsets are enriched in signaling pathways that promote immune function, hence P2ry12 High Ccl12 low Microglial cell subsets have therapeutic effects on gliomas.

Description

Technical Field

[0001] This invention belongs to the medical field and relates to a microglial cell subset, specifically a therapeutic microglial cell subset for glioma, namely, one with P2ry12. High Ccl12 low The invention relates to a characteristic microglia subpopulation, a method for inducing this subpopulation, and a method for constructing an animal model for inducing a therapeutic microglia subpopulation. Background Technology

[0002] Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival of about 12 months and a 5-year survival rate of 9.8%. Currently, the most effective treatment remains surgery followed by concurrent chemoradiotherapy with temozolomide (TMZ) and adjuvant chemoradiotherapy with temozolomide. However, the tumor recurs rapidly after treatment, and the average survival is only extended by about 2-3 months. [1] Prolonging the survival of glioma patients has become an urgent clinical need. Although numerous drugs, including immunotherapy, have shown promising results in some preclinical studies, they have all failed in clinical trials. [2,3] This strongly suggests that current preclinical animal models for evaluating glioma efficacy do not match clinical practice and cannot accurately reflect the pathophysiological processes and treatment response of GBM. Clearly, using reliable animal models that closely resemble the pathological characteristics and treatment response of clinical GBM is essential to maximizing the translational efficiency of preclinical research findings.

[0003] The development, progression, and treatment response of tumors are closely related to their microenvironment. Gliomas have a unique microenvironment: they possess the blood-brain barrier, have dense brain tissue, and a distinctive brain cell composition. Normal brains lack blood-derived or bone marrow-derived immune cells, thus they are considered "immune-immune organs." Microglia are innate immune cells in the brain, residing there from early embryonic development, primarily performing phagocytic and immune surveillance functions. [4] In pathological states, including tumors, various peripheral immune cells can enter brain tissue and, together with microglia, regulate the immune response. Microglia, stimulated by various pathological factors, undergo strong activation or polarization reactions, exhibiting diverse morphology and functions. They possess a dual role of pro-inflammatory or anti-inflammatory (cytotoxic or protective) action in different diseases or pathological processes. [5] Previously, it was believed that microglia in the adult brain under pathological conditions mainly originated from hematopoietic mononuclear cells; however, the latest lineage tracing combined with single-cell sequencing (scRNA-seq) has corrected this view, confirming that microglia originate from self-renewal and are different from cells (mainly macrophages) transformed from hematopoietic mononuclear cells. [6] Recent single-cell sequencing has confirmed the simultaneous presence of microglia and hematogenous mononuclear / macrophages in glioma tissue. [7]Clearly, the previous concept of conflating intracranial hematogenous mononuclear / macrophage cells with microglia cannot truly explain the function of microglia in gliomas. [6] Single-cell sequencing confirmed that small glial cells are the main immune cells in glioma tissue, but they exhibit high heterogeneity and can be divided into more than ten cell subsets. [7] Currently, the markers for small glial subsets in glioma tissue have not been identified, and their functions are unknown. [5] It is known that T-cell infiltration in GBM tissues is sparse and that single immunotherapy (including PD-1 antibodies, PD-1: programmed death receptor 1, also known as CD279) is ineffective against GBM, suggesting a strong immunosuppressive environment in GBM tissues. [2，3] As the main immune cells in GBM tissues, microglia may primarily play immunosuppressive and pro-cancer roles. Therefore, conventional research methods struggle to identify and determine therapeutically active microglia subsets during the development and progression of GBM.

[0004] The patent applicant has recently established a new and stable mouse allogeneic glioblastoma orthotopic model (i.e., G422). TN -GBM model) [8] Whole-genome sequencing determined the genotype of this tumor cell line to be IDH1 / 2. 野生型 Chromosome 1 / 19 完整 TERT-promoter 野生型 ATRX 突变 Trp53 突变 It meets the subtype classification criteria for human triple-negative (TN) primary glioblastoma and is therefore named G422. TN -GBM[8]. G422 TN GBM cells are mouse triple-negative primary glioblastoma cells independently induced and established by the applicant of this patent application. They have been deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and the culture name is: Mouse Triple-Negative Primary Glioblastoma G422. TN -GBM; Accession number: CCTCC No: C2020267; Accession date: December 17, 2020. Mice were inoculated in situ with 1×10⁻⁶ g of ... 4 Up to 1×10 5 G422 TN - GBM cells (lethal dose): Tumor tissue exhibits typical human GBM pathological features such as rapid growth, hemorrhage and necrosis, high invasiveness, and T cell deficiency. [8] Mice were inoculated in situ at a dose of 5 × 10⁶. 4 G422 TNGBM cells (optimized stable model): animal survival time was stable at 14-23 days, and median survival was stable at 16-19 days, consistent with the clinical characteristics of short survival in GBM patients. [8] In optimized mouse orthotopic G422 TN In the GBM model, the therapeutic effects of radiotherapy, temozolomide, and temozolomide concurrent chemoradiotherapy at different time windows were similar to those in clinical GBM treatment: i.e., radiotherapy alone was ineffective, surgery and temozolomide monotherapy were effective, and temozolomide concurrent chemoradiotherapy was superior to temozolomide monotherapy; the therapeutic effect of temozolomide monotherapy or concurrent chemoradiotherapy was positively correlated with the earlier the treatment started, i.e., early stage (day 5) was better than mid-stage (day 7) and late stage (day 9); temozolomide monotherapy and / or concurrent chemoradiotherapy were effective but not curative (cure criteria: tumor-bearing mice survived for ≥100 days). [8] These treatment response characteristics reflect the effectiveness and refractory nature of standard clinical treatment for GBM.

[0005] The above-optimized G422 TN In a stable GBM model, we found that temozolomide combined with concurrent chemoradiotherapy and other drugs could achieve a partial cure (cure rate ≤50%). After the tumor-bearing mice were "cured," they were again intracranially inoculated with an equal amount of the same type of G422. TN GBM cells were not treated (i.e., tumor rechallenge trial), and 50% of the "cured" mice survived for ≥100 days (compared to ≤23 days for control tumor-bearing mice), confirming that they had achieved resistance to G422. TN The tumors regressed spontaneously due to immunization with GBM, and these mice were named "cured-immune" mice. These "cured-immune" mice (also known as "long-term survival" or LTS mice) were then re-inoculated with an equal dose of the same type of G422. TN GBM cells were not treated. On day 7 post-inoculation, tumor tissue was collected, and single viable cells were isolated for single-cell RNA sequencing (scRNA-seq). Compared with the control, the tumor tissue of "cured-immune" mice showed a significant increase in only one microglia subpopulation (out of a total of three microglia subpopulations) and T cells, while other subpopulations (especially tumor cells) were significantly reduced. Single-cell RNA sequencing results showed that the increased microglia subpopulation highly expressed the top 5 microglia tag genes: P2ry12 (purinergic receptor), Cx3cr1, Btg2, Hexb, and Cst3; while it expressed low levels of microglia tag genes such as Ccl12 (chemokine (CC motif) ligand 12) (compared to the other two microglia subpopulations). Therefore, this increased microglia subpopulation was named P2ry12. High Ccl12 Low Microglial cell subsets. Signaling pathway functional analysis showed P2ry12 High Ccl12Low The small glial subsets primarily enhance the function of other immune cells (such as T cells) (compared to the control group). This is due to the G422 cells implanted in "cured-immune" mice. TN GBM can spontaneously regress, indicating that its tumor microenvironment is a healing environment, in which increased immune cells play a therapeutic role. Therefore, P2ry12 can be identified. High Ccl12 Low An increase in small colloids is positively correlated with GBM cure. To confirm P2ry12... High Ccl12 Low The therapeutic effects of small colloid subgroups, isolation and purification of P2ry12 High Small glue (representing P2ry12) High Ccl12 Low Small subgroup of colloids), infusion of P2ry12 High Small glues can effectively extend the lifespan of G422. TN GBM tumor-bearing mice survived. Therefore, P2ry12 High Ccl12 LowThe therapeutic effects of small colloid subgroups can be further applied to preclinical studies or clinical trials. References: [1] Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO; European Organization for Research and Treatment of Cancer Brain Tumor andRadiotherapy Groups;National Cancer Institute of Canada Clinical TrialsGroup.Radiotherapy plus concomitant and adjuvant temozolomide forglioblastoma.N Engl J Med.2005Mar 10;352(10):987-9;[2]Shergalis A,BankheadA3rd,Luesakul U,Muangsin N,Neamati N.Current Challenges and Opportunities inTreating Glioblastoma.Pharmacol Rev.2018Jul;70(3):412-445;[3]Chen RQ,Liu F,Qiu XY,Chen XQ.The Prognostic and Therapeutic Value of PD-L1 in Glioma.FrontPharmacol.2019Jan 9;9:1503;[4]Izquierdo P,Attwell D,Madry C.Ion Channels andReceptors as Determinants of Microglial Function.Trends Neurosci.2019Apr;42(4):278-292;[5]Gómez Morillas A,Besson VC,Lerouet D.Microglia andNeuroinflammation:What Place for P2RY12? Int J MolSci.2021Feb 6;22(4):1636;[6]Prinz M,Jung S,Priller J.Microglia Biology:One Century of EvolvingConcepts.Cell.2019Oct 3;179(2):292-311;[7]Klemm F,Maas RR,Bowman RL,KorneteM,Soukup K,Nassiri S,Brouland JP,Iacobuzio-Donahue CA,Brennan C,Tabar V,GutinPH,Daniel RT,Hegi ME,Joyce JA.Interrogation of the MicroenvironmentalLandscape in Brain Tumors Reveals Disease-Specific Alterations of ImmuneCells.Cell.2020Jun 25;181(7):1643-1660.e17;[8]Liu F,Xu X,Li C, Li C, Li Y, YinS, Yu S, Chen XQ. Mannose synergizes with chemoradiotherapy to cure cancer viametabolically targeting HIF-1 in a novel triple-negative glioblastoma mousemodel. Clin Transl Med. 2020Nov; 10(7):e226. Summary of the Invention

[0006] The objective of this invention is to provide a therapeutic microglia subset for glioma and a method for inducing it, wherein the representative highly expressed gene of this subset is the purinergic receptor P2ry12. High The representative low-expression gene is chemokine (CC motif) ligand 12 (Ccl12). Low Therefore, it is a group of P2ry12 High Ccl12 Low A subset of microglia. P2ry12 High Ccl12 LowMicroglia can be induced to increase in the glioma treatment environment, mainly playing a positive immunomodulatory role and thus having a therapeutic effect on gliomas.

[0007] Another objective of this invention is to provide an induced therapeutic microglial cell subset (P2ry12). High Ccl12 Low The "cure-immunity" animal model (subgroup G422) TN - Effective treatment for GBM (temozolomide concurrent chemoradiotherapy combined with other drugs), this model can also induce increased T cell infiltration in tumor tissue.

[0008] The technical solution for achieving the present invention is: the therapeutic microglia subset (P2ry12) for glioma provided by the present invention. High Clc12 Low The method for inducing microglia (a subset of cells) includes the following steps:

[0009] (a) Lethal doses of G422 were injected into the brains of Kunming mice. TN GBM cells were administered via radiotherapy and chemotherapy combined with immunotherapy on day 7 post-inoculation; the G422 cells mentioned above. TN GBM cells have been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No: C2020267; the culture name is mouse triple-negative primary glioblastoma G422. TN -GBM; Accession number: CCTCC No:C2020267; Accession date: December 17, 2020.

[0010] (b) Mice that achieved a cure in step (a) were subjected to a tumor rechallenge test, i.e., a second inoculation of the striatum of the mouse brain with a lethal dose of G422. TN -GBM cells and no treatment;

[0011] (c) Mice that survived ≥100 days again in step (b) (these mice acquired immunity to the tumor, resulting in spontaneous tumor regression, i.e., "cure-immunity") underwent a second tumor rechallenge test, i.e., a third inoculation of the mice's brain with a lethal dose of G422. TN GBM cells were not treated, and tumor tissue was challenged again on day 7 post-inoculation using P2ry12. High Clc12 Low Microglial subsets and T cells increased significantly, while tumor cells decreased significantly (P2ry12). High Clc12 Low Microglia subset induction was successful.

[0012] The mice that achieve a curative effect as described in step (b) of the induction method of the present invention are mice that have survived for ≥100 days while bearing tumors. According to an article published in Neuro-Oncology in 2018 by Speranza MC et al., mice that survive for ≥100 days (Long-term survivors, LTS) after drug intervention following tumor inoculation are considered to have "cured" tumors in mice; the clinical cure standard for malignant tumors is 5-year survival; 100-day survival of tumor-bearing mice is similar to the 5-year survival of cancer patients; therefore, in the treatment literature of mouse malignant tumor models, survival of ≥100 days in tumor-bearing mice is usually considered "cured".

[0013] The above-described induction method of the present invention involves inoculating a lethal dose of G422 into the mouse brain. TN The specific method for inoculating GBM cells is as follows: 5 × 10⁻⁶ cells are seeded in situ in mice. 4 G422 TN GBM cells. 1×10⁻⁶ cells were seeded into this model. 3 More than one G422 TN - GBM cells resulted in 100% tumor formation and death in mice. To ensure stable animal survival within a narrow range and to guarantee the effectiveness of tumor treatment, we selected a higher tumor cell inoculation rate.

[0014] The aforementioned method of radiotherapy combined with immunotherapy involves concurrent chemoradiotherapy with temozolomide combined with other drugs, including but not limited to PD-1 antibodies, brain globulin transmembrane peptides, long peppermint, TGF-β antibodies, and / or TGF-β inhibitors. A specific regimen for concurrent chemoradiotherapy with temozolomide combined with other drugs may be:

[0015] (a) Temozolomide chemotherapy: Temozolomide was administered to mice by gavage at a dose of 50 mg / kg for a total of 10 doses over 12 days. The administration was repeated for 5 days, followed by a 2-day break, and then another 5 days of administration. In other words, the administration was carried out on the first and last 5 days of the 12-day period, with no administration on the 2-day break in between.

[0016] (b) Concurrent X-ray radiotherapy: The whole brain of mice was irradiated with X-rays once, 10 Gy; radiotherapy was given on the same day before the first administration of temozolomide, i.e., the temozolomide concurrent chemoradiotherapy regimen.

[0017] (c) PD-1 antibody treatment: Mice were injected intraperitoneally with 200 micrograms of PD-1 antibody, 6 doses / 12 days, starting on the same day after the first administration of temozolomide;

[0018] (d) Treatment with brain globulin-penetrating peptide (TAT-Ngb), or long peppermint, or TGF-β antibody, or TGF-β inhibitor:

[0019] The treatment regimen of the brain globulin transmembrane peptide (TAT-Ngb) can be as follows: On the same day after the first administration of temozolomide, the brain globulin transmembrane peptide is administered intraperitoneally to mice at a dose of 10 mg / kg for a total of 10 doses over 12 days. The administration is repeated for 5 days, followed by a 2-day break, and then another 5 days of administration. In other words, the administration is repeated for the first and last 5 days of the 12-day period, with no administration for the 2 days in between.

[0020] The treatment regimen for long pepperamide can be: intraperitoneal injection, 5 mg / kg, 10 doses / 12 days, administration for 5 days, stop administration for 2 days, and then administration for another 5 days, that is, administration for the first and last 5 days of the 12 days, and no administration for the middle 2 days;

[0021] The TGF-β antibody treatment regimen may be: intraperitoneal injection, 200 micrograms / mouse, 6 doses / 12 days;

[0022] The TGF-β inhibitor (Galunisertib) treatment regimen can be: oral administration via gavage, 50 mg / kg, twice daily, 10 doses / 12 days, with 5 days of administration, 2 days of discontinuation, and then another 5 days of administration, i.e., administration for the first and last 5 days of the 12-day period, with no administration for the 2 days in between.

[0023] The therapeutic microglial cell subset (P2ry12) for glioma provided by this invention High Clc12 Low The microglia subset (P2ry12) was obtained by induction using the aforementioned method followed by isolation and purification. High Clc12 Low A subset of microglia is characterized by high expression of the purinergic receptor P2ry12 and low expression of the chemokine (CC motif) ligand 12 (Clc12). The top five microglia marker genes with high expression are P2ry12, Cx3cr1, Btg2, Hexb, and Cstd. P2ry12 High Clc12 Low Microglial cell subsets can be used to prepare or screen drugs for the treatment of gliomas.

[0024] The present invention provides the induction of P2ry12 High Clc12 Low A method for constructing an in situ glioma “cure-immunity” animal model of microglia subsets (also known as an animal model of induced therapeutic microglia subsets) includes steps (a) to (b) or steps (a) to (c) of the induction method provided by the present invention described above.

[0025] This invention establishes a glioma "cure-immunity" animal model, and stably passages G422 gliomas in an optimized "subcutaneous inoculation → 24-hour in vitro culture → striatal inoculation" model system. TNGBM cells; seeded at an optimized 5×10⁻⁶ cells. 4 G422 TN GBM cells were injected into the right striatum of male Kunming mice. Treatment began on day 7 post-inoculation with a combination of temozolomide concurrent chemoradiotherapy, PD-1 antibody, and TAT-Ngb (Neuroglobin transmembrane peptide; Ngb, Neuroglobin) (until day 18). Survival time of tumor-bearing mice was recorded (inoculation day 0), and survival time was analyzed. Mice surviving more than 100 days were defined as cured (standard mouse tumor cure criteria, "cured" mice). Optimized 5×10⁻⁶ GBM cells were injected into the left striatum of "cured" mice (corresponding to the inoculation site on the right striatum). 4 G422 TN GBM cells (optimization process as before) were used without treatment (i.e., tumor re-challenge), with normal age-matched male Kunming mice inoculated with tumor cells as the control group; the survival time of tumor-bearing mice was recorded; mice that survived more than 100 days after re-challenge were confirmed to have tumor immunity (immunity acquired after cure, i.e., "cure-immune" mice, or "long-term survival" LTS mice); 5×10⁻⁶ GBM cells were then inoculated again at different locations on the left striatum of these "cure-immune" mice. 4 G422 TN GBM cells (optimization process as before) were used without treatment, with normal age-matched male Kunming mice inoculated with tumor cells as the control. On day 7 after inoculation, the animals were sacrificed, and tumor brain tissue was collected. Single live cells were isolated, and the quality of live cells was confirmed to meet the standards. The cells were then sent to Novogene Biotech for single-cell RNA sequencing (10X Genomics).

[0026] CellRanger (version 4.0.0) was used to perform quality control filtering, alignment, and quantification on the raw single-cell RNA sequencing data, ultimately obtaining the gene expression matrix for each cell. Seurat (version 3.0.2) was then used for further cell filtering (UMI > 2000 per cell, number of genes > 500, mitochondrial gene expression percentage < 0.2%), gene filtering (expression in at least 10 cells), standardization, and cell subpopulation classification. The clustering results showed that the tumor tissue could be divided into a total of 17 cell subpopulations (clusters 0-16), including 3 microglia subpopulations (clusters 2, 8, and 9) and 1 T cell subpopulation (cluster 7). Compared with the control, in the "cured-immune" mice (i.e., LTS), only clusters 8 (microglia) and 7 (T cells) showed a significant increase (including absolute numbers and relative ratios), while all tumor cell subpopulations (i.e., clusters 0, 1, 3, 4, and 6) were significantly decreased. Other subpopulations showed no significant decrease or change. The top 5 tag genes for clusters 2, 8, and 9 are as follows: C1qc high C1qa high C1qb high Ccl12 high Cstd high P2ry12 high Cx3cr1 high Btg2 high Hexb high Cstd high Aif1 high C1qc high C1qb high Tmsb4x high Ccl12 high Based on the high expression of purinergic receptor P2ry12 and low expression of chemokine (CC motif) ligand 12 (Ccl12) in the cluster 8 microglia subset; Low Based on the high expression of Ccl12 in the clusters 2 and 9, this subgroup was named P2ry12. High Ccl12 Low Small gel; Metascape database analysis, compared with the control, P2ry12 in the "cure-immunity" group High Ccl12 Low Differentially expressed genes in the microglia subset are primarily enriched in immune response and immune regulatory signaling pathways, including positive regulation of the immune response, cytokine-mediated signaling pathways, acquired immune responses, and leukocyte-mediated immunity. The number of G422 mice that died after inoculation in the "cure-immune" group was [not specified].TN Following GBM cell induction, the tumor regressed spontaneously, and the animals survived long-term. This confirms that the increased immune cells within the tumors of "cured-immune" mice have a therapeutic effect. Clearly, the increased P2ry12 cells... High Ccl12 Low Microglia and T cells with G422 TN - GBM is positively correlated with cure rates.

[0027] To further confirm P2ry12 High Ccl12 Low The therapeutic effects of small gelatin, as described above in G422 TN GBM orthotopic model mice were treated with a combination of temozolomide concurrent chemoradiotherapy, PD-1 antibody, and brain globulin transmembrane peptide (as before). Flow cytometry was used to sort P2ry12 from tumor tissues that responded to the treatment. High Small colloid subsets of live cells (represented by P2ry12) High Ccl12 Low (Small gel); Take 200 P2ry12 gels High Microcell infusion on day 5 at G422 TN -Intratumoral GBM; Results demonstrated the effectiveness of P2ry12 infusion High The survival time of mice treated with small-peptide injections was significantly prolonged (compared to those treated with P2ry12 infusion). Low Compared to the smaller rubber group).

[0028] This invention provides a reliable orthotopic glioma "cure-immunity" mouse model that can be used to induce P2ry12. High Ccl12 Low Therapeutic microglia and T-cell therapy, or preclinical efficacy evaluation and target screening for curative treatment of malignant gliomas. We validated the effect of temozolomide concurrent chemoradiotherapy + PD-1 antibody + brain globulin transmembrane peptide on G422. TN The model demonstrated the curative effect of GBM in situ tumors (treatment initiated 7 days after tumor implantation, with a cure rate of 25%). Further tumor rechallenge experiments confirmed that this combined cure resulted in immunity to the same tumor (immunization rate of 50%), and that the long-term survival of rechallenged animals was a result of the immunotherapeutic environment. Therefore, this model can be used to evaluate the efficacy of immunotherapy for malignant gliomas, providing important evaluation data for preclinical treatment studies. In summary, we established a stable and reliable "cure-immunity" mouse model of malignant gliomas, which can realistically reflect the effectiveness of treatment and the induced tumor-specific immunity, and can be used for preclinical evaluation of novel anti-tumor immunotherapies and therapeutic targets.

[0029] This invention belongs to the medical field and establishes a clinically relevant mouse glioblastoma (GBM) treatment model, obtaining "cured" mice. When these "cured" mice were re-challenged with tumors, the tumors spontaneously regressed, and the animals achieved long-term survival. This indicates that the "cured" mice acquired immunity to the tumor, and these mice are named "cured-immune" mice. Intracranial inoculation of tumor cells into the "cured-immune" mice specifically induced a microglia (microglia) subset, which is characterized by high expression of the purinergic receptor P2ry12 gene. High ) and immune-boosting functional characteristics, P2ry12 infusion Hi Small glial subsets significantly prolonged the survival time of glioma-bearing mice. We independently established a stable orthotopic model of triple-negative (TN) glioblastoma in mice (named G422). TN G422 tumors (GBM) share similar malignant pathological characteristics and conventional radiotherapy and chemotherapy features with clinical GBM: rapid growth, high invasiveness, T-cell deficiency, and high lethality (survival time <30 days in tumor-bearing mice); surgery combined with temozolomide concurrent chemoradiotherapy or temozolomide concurrent chemoradiotherapy is effective, but not curative (cure criteria: tumor-bearing survival ≥100 days). Temozolomide concurrent chemoradiotherapy combined with brain globulin transmembrane peptide (TAT-Ngb) and PD-1 antibody can reduce the mortality rate of G422 tumors by 25%. TN GBM tumor-bearing mice were cured; these "cured" mice were then re-inoculated with the same amount of the same tumor cells but without treatment (i.e., tumor rechallenge). 50% of these "cured" mice acquired immunity and survived for ≥100 days ("cured-immunized" mice). These "cured-immunized" mice underwent another tumor rechallenge, and brain tumor tissue was harvested on day 7 after tumor cell inoculation for single-cell RNA sequencing. Results showed a significant increase in only one microglia subset, accompanied by a significant increase in T cells and a significant decrease in tumor cells (control group consisted of age-matched untreated tumor-bearing mice). Single-cell sequencing analysis revealed that the top 5 highly expressed characteristic genes in this microglia subset were P2ry12, Cx3cr1, Btg2, Hexb, and Cst3, while the low-expressed characteristic genes mainly included chemokine (CC motif) ligand 12 (Ccl12). low Therefore, this group of small gums is defined as P2ry12. High Ccl12 low Subgroup; P2ry12 in "cure-immune" (i.e., LTS) mice compared to control. High Ccl12 low Differential gene enrichment in small glial subsets occurred in signaling pathways related to immune function. On day 5, G422... TN GBM tumor-bearing mice were infused with P2ry12. High The small colloid subgroup significantly prolonged animal survival time. Therefore, it can be determined that P2ry12...Hi Ccl12 lo Microglial cell subsets have therapeutic effects on gliomas. Attached Figure Description

[0030] Figure 1 Mouse in situ G422 TN - Schematic diagram of the combined treatment schedule (temozolomide concurrent chemoradiotherapy combined with PD-1 antibody and brain globulin transmembrane peptide) for GBM model. Timeline: Time axis; arrows indicate tumor cell inoculation or administration dates. Day 0: Inoculation date; Days 7-18: Radiotherapy or drug therapy period. RT, X-ray irradiation; TMZ, temozolomide, administered orally (po) 4 hours after RT irradiation, once daily (qd), followed by administration at fixed times of 9-10 am, for a total of 10 doses; PD-1 antibody, intraperitoneal injection (ip), once daily, for a total of 6 doses; TAT-Ngb, brain globulin transmembrane peptide, intraperitoneal injection, once daily, for a total of 10 doses; In the multi-drug therapy regimen, administration is in the following order: TMZ - 1-hour interval - PD-1 antibody - 1-hour interval - TAT - brain globulin, with administration dates indicated by arrows. Note: The color intensity of the arrows in the diagram does not represent any additional meaning, but only indicates the administration time points.

[0031] Figure 2 : Left: Survival time and weight change (right): Results of tumor-bearing mice in each combined treatment group. Mice were intracranially inoculated with 5 × 10⁶ mice. 4 G422 TN After GBM cell therapy, patients were randomly assigned to the following 8 groups (n = 8 / group): Control (control group, intraperitoneal injection of equal volume of normal saline), TAT-Ngb, PD-1, TAT-Ngb / PD-1 (combination therapy of brain globulin transmembrane peptide and PD-1 antibody), RT / TMZ (temozolomide concurrent irradiation), RT / TMZ / TAT-Ngb (temozolomide concurrent irradiation combined with TAT-Ngb), RT / TMZ / PD-1 (temozolomide concurrent irradiation combined with PD-1 antibody), and RT / TMZ / PD-1 / TAT-Ngb (temozolomide concurrent irradiation combined with PD-1 antibody and TAT-Ngb). Figure 1 The results of the treatment regimen showed that only the combined treatment of RT / TMZ / PD-1 / TAT-Ngb had a curative effect (survival of tumor cells for >100 days after inoculation, the standard for tumor cure in mice; cure rate 25%, 2 / 8). **P<0.01; ***P<0.001.

[0032] Figure 3Comparison of intracranial tumor size in mice treated with RT / TMZ / PD-1 / TAT-Ngb and RT / TMZ / PD-1 combination therapy (left image shows tumor size results for tumor cells expressing luciferase, right image is a statistical chart of the left image). In the above... Figure 2 In the treatment groups, all tumor-bearing mice in the RT / TMZ / PD-1 / TAT-Ngb and RT / TMZ / PD-1 combined treatment groups underwent in vivo bioluminescent imaging (BLI) on day 20 after tumor inoculation (days 7-18 of the treatment period) to visualize intracranial tumor size. The results showed that the mean relative intracranial tumor size in the RT / TMZ / PD-1 / TAT-Ngb treatment group was significantly lower than that in the RT / TMZ / PD-1 treatment group (tumor cells expressed luciferase). *P<0.05.

[0033] Figure 4 The image shows the results of tumor re-challenge in cured mice and intracranial tumor imaging (left) and survival analysis (right) in control mice. Figure 2 Tumor-cured mice (RT / TMZ / PD-1 / TAT-Ngb group, n=2) underwent a tumor rechallenge experiment (day 110). 5 × 10⁶ cells were injected into the left striatum of both cured mice and age-matched normal control mice (control, n=4). 4 G422 TN GBM cells were injected without any treatment. On day 10 post-inoculation, in vivo BLI imaging was performed on the animals to show tumor size (left image). Tumor rechallenge and survival time results of control tumor-bearing mice are shown in the right image. The results demonstrate that only one cured mouse survived for >100 days after tumor inoculation (i.e., successful rechallenge, "cured-immune"). In this model, 5 × 10⁻⁶ GBM cells were injected. 4 G422 TN After GBM cells were applied, all untreated tumor-bearing mice survived for less than 23 days. Therefore, "cured-immune" mice can only mean that the mice have acquired immunity to the tumor, with immune cells that kill tumor cells in the tumor microenvironment, preventing tumor growth and causing it to regress automatically, thus achieving long-term survival again.

[0034] Figure 5 Schematic diagram of the experimental procedure for single-cell RNA sequencing of tumor tissue in "cured-immune" mice undergoing secondary re-challenge. Day D. Mice in situ G422. TN For combination therapy regimens for GBM models, please refer to [link / reference]. Figure 1 For the results of the combination therapy (i.e., the RT / TMZ / PD-1 / TAT-Ngb group), please refer to [link to relevant documentation]. Figure 2 The results of tumor rechallenge in "cured" mice (n=2) can be found in [link to relevant documentation]. Figure 4One of the tumor-challenged mice achieved long-term survival, becoming a "cure-immune" mouse. This "cure-immune" mouse (LTS group, n=1, day 220) received a second intracranial injection of 5 × 10⁵ cells / mL into the left striatum (at a different location than the previous injection). 4 G422 TN GBM cells were used as controls in age-matched normal mice (n=2, with equal amounts of tumor cells inoculated at the same location). On day 7 after tumor cell implantation, the tumor tissue was perfused with pre-cooled PBS, minced, and immediately digested (collagenase IV + DNase I). Single cells were isolated using Percoll in a 70% / 37% / 30% (mixed cell layer) solution (800g, 4℃, 30min) to prepare live cells. Once the live cells met the quality standards, they were sent to Novogene Biotech for single-cell RNA sequencing (scRNA-seq).

[0035] Figure 6 : A diagram showing cell subpopulations based on single-cell RNA sequencing results. CellRanger (version 4.0.0) and Seurat (version 3.0.2) were used to sequence G422 cells. TNThe raw data from single-cell sequencing of GBM tumor tissue were organized and classified to determine the total number of effective sequencing cells: control mice, 7547 cells; LTS (“cured-immune” mice), 4465 cells. Cell clustering results showed that tumor tissue cells could be divided into 17 cell subpopulations (clusterID, 0-16), with the following cell counts for each subpopulation: 3302 (cluster 0), 2935 (cluster 1), 1149 (cluster 2), 965 (cluster 3), 956 (cluster 4), 675 (cluster 5), 553 (cluster 6), 471 (cluster 7), 232 (cluster 8), 214 (cluster 9), 204 (cluster 10), 103 (cluster 11), 82 (cluster 12), 61 (cluster 13), 53 (cluster 14), 29 (cluster 15), and 28 (cluster 16). The SingleR package was used for annotation of major cell types. Cell groups 0-16 were annotated as follows: Group 0: Fibroblasts; Group 1: Fibroblasts; Group 2: Microglia; Group 3: Fibroblasts; Group 4: Fibroblasts; Group 5: Monocytes; Group 6: Erythrocytes; Group 7: T cells; Group 8: Microglia; Group 9: Microglia; Group 10: Endothelial cells; Group 11: Macrophages; Group 12: B cells. The cell types included: 13 endothelial cells, 14 oligodendrocytes, 15 monocytes, and 16 monocytes. Three microglia subpopulations were identified: clusters 2, 8, and 9 (Cluster IDs: 2, 8, 9). Since the reference dataset only contained normal mouse cell types, tumor cells were not shown in the annotation results. However, fibroblasts (Clusters 0, 1, 3, 4) and erythrocytes (Cluster 6, washed and removed, and without transcriptional function), which are absent in brain tissue, were abundant. Therefore, Clusters 0, 1, 3, 4, and 6 are presumed to be G422. TN -GBM cell subset, which corresponds to the largest number of tumor cells in tumor tissue. Figure 7Results of chromosome copy number variation analysis to determine tumor cell subsets. A core characteristic of tumor cells is their genomic alteration; therefore, chromosome copy number variations (inferCNV) can be used to identify tumor cell subsets. Cluster 2 (microglia) and Cluster 5 (monocytes) were used as reference cells. InferCNV analysis was performed on Cluster 0, 1, 3, 4, and 6 cell subsets. The results showed that Cluster 0, 1, 3, 4, and 6 (B) cell subsets had significant copy number deletions on chromosomes 1, 2, 14, and 17, and significant copy number increases on chromosomes 3 and 8 (arrows indicate high CNV regions). Therefore, Cluster 0, 1, 3, 4, and 6 can be identified as G422. TN -GBM cell subset. Figure 8 The relative expression of the top 5 differentially expressed genes in each subpopulation across all cell subpopulations is shown. Specifically, the top 5 differentially expressed genes in the Cluster 2 subpopulation are: C1qc, C1qa, C1qb, Ccl12, and Ctsd; in the Cluster 8 subpopulation: P2ry12, Cx3cr1, Btg2, Hexb, and Cst3; and in the Cluster 9 subpopulation: Aif1, C1qc, C1qb, Tmsb4x, and Ccl12. The high expression of P2ry12 and low expression of Ccl12 in the Cluster 8 subpopulation are more prominent than in the other two subpopulations; therefore, the Cluster 8 subpopulation is named P2ry12. High Ccl12 Low Subgroup.

[0036] Figure 9 Results of a comparison of major differentially expressed genes between the cluster2 and cluster8 microglia subsets in tumor tissues of "cured-immune" mice. Results showed that P2ry12 expression was significantly higher in the cluster8 microglia subset than in the cluster2 subset. (LTS, "cured-immune" mice)

[0037] Figure 10 Results of a comparison of major differentially expressed genes between the cluster9 and cluster8 microglia subpopulations in tumor tissues of "cured-immune" mice. The results showed that P2ry12 was also a prominent highly expressed gene in the cluster8 microglia subpopulation.

[0038] Figure 11Differences in GAS and IL-2 cell communication signaling pathways between Cluster 8 glial subsets and Cluster 2 and Cluster 9 glial subsets. Results showed that Cluster 8 glial subsets primarily communicate with other cell subsets via the IL-2 signaling pathway, while Cluster 2 and 9 glial subsets primarily communicate with other cell subsets via the GAS signaling pathway.

[0039] Figure 12 Results of a comparison of major differentially expressed genes in the Cluster 8 glial subset in tumor tissues of "cured-immune" mice and control mice. Results show representative differentially expressed genes in the Cluster 8 glial subset between "cured-immune" and control mice.

[0040] Figure 13 The results showed that the differentially expressed GO signaling pathway of the Cluster 8 microglia subset in tumor tissues of "cured-immune" mice and control mice were enriched. The results indicated that the differentially expressed GO gene enrichment was mainly concentrated in immune-related signaling pathways, suggesting that in GBM tissues of "cured-immune" mice, the Cluster 8 microglia subset is associated with a positively regulatory environment for anti-tumor immunity and is positively correlated with T cell proliferation. Therefore, it can be determined that the Cluster 8 microglia subset has an immunopromoting effect in tumor tissues of "cured-immune" mice.

[0041] Figure 14 Enrichment results of KEGG signaling pathway in differentially expressed genes in the Cluster 8 microglia subset of tumor tissues from "cured-immune" mice and control mice. Metascape enrichment analysis showed that the differentially expressed KEGG genes were mainly enriched in immune-related signaling pathways.

[0042] Figure 15 Enrichment of KEGG signaling pathway in differentially expressed genes of the Cluster 9 microglia subset in tumor tissues of "cured-immune" mice and control mice. Results showed that KEGG enrichment in the Cluster 9 microglia subset was mainly concentrated in non-immune-related signaling pathways.

[0043] Figure 16 Enrichment of KEGG signaling pathway in differentially expressed genes in the Cluster 2 microglia subset of tumor tissues from "cured-immune" mice and control mice. The results showed that the KEGG enrichment of differentially expressed genes was mainly concentrated in non-immune-related signaling pathways.

[0044] Figure 17 P2ry12 High Small colloid subsets treated in situ G422 mice TN Results of GBM. Mice were intracranially inoculated with 5 × 10⁻⁶ g of [unspecified substance]. 4 G422TN After GBM cell inoculation, patients were randomly divided into two groups: a control group (n=8) and a combination therapy group (n=20). Combination therapy (temozolomide concurrent chemoradiotherapy + PD-1 antibody + brain globulin transmembrane peptide) was administered from day 5 to 16 post-inoculation, with the same dosing regimen as above. Figure 1 On day 17, tumor tissue was collected, and tumor tissue that was effective in treatment (50%) was selected. Single viable cells were isolated and co-incubated with P2ry12 fluorescent antibody. Effective P2ry12 was then sorted by flow cytometry. High and P2ry12 Low Live cells. 200 P2ry12 cells High Or P2ry12 Low Live cells were injected into mice in situ on day 5 of G422 cells. TN -GBM tissue (n=7 / group). Results show that P2ry12 infusion... High Small-particle mice with tumors showed significantly prolonged survival time (***P<0.001, compared with P2ry12). Low (Group comparison).

[0045] Figure 18 : A composite diagram generated to provide abstract figures. Detailed Implementation

[0046] Example 1

[0047] The mouse orthotopic G422 provided by this invention TN The GBM glioma "cure-immunity" model can induce a therapeutic microglia subset that is characterized by high P2ry12 expression and low Ccl12 expression. High Ccl12 Low Small adhesive. The P2ry12 provided by this invention. High Ccl12 Low Small glial subsets primarily possess immunomodulatory functions, promoting T-cell infiltration into tumor tissue and exhibiting immunotherapeutic effects against gliomas. The mouse orthotopic G422 cells provided by this invention... TN -GBM glioma "cure-immunity" model applied to P2ry12 High Ccl12 Low The induction and conversion of microglia subsets and the evaluation of immunotherapy are implemented as follows:

[0048] Experimental cells: G422 TN GBM cells: These cells do not expand in vitro. They are passaged alternately using subcutaneous or intracranial seeding to better maintain their biological characteristics. Cryopreservation conditions are: 80% RPMI 1640 basal medium / 10% dimethyl sulfoxide / 10% fetal bovine serum. (G422...) TN-GBM (1 million cells / 0.2 ml PBS) was inoculated subcutaneously at the right forelimb shoulder of adult male Kunming mice. When the subcutaneous tumor grew for 7 - 9 days, the subcutaneous tumor was digested into single-cell suspension with trypsin. After incubating for 24 hours, the cells were collected and counted, and 50,000 live cells were inoculated into the right side of the mouse brain or the striatum. G422 TN -The GBM cells are mouse triple-negative primary glioblastoma cells independently induced and established by the applicant of this patent application, and have been deposited with the China Center for Type Culture Collection located at Wuhan University, Wuhan, China. The name of the deposited culture is: Mouse triple-negative primary glioblastoma G422 TN -GBM; The deposit number is CCTCC No: C2020267; The deposit date is: December 17, 2020.

[0049] Experimental animals: The Kunming mice (male, 18 - 22 g) used in this invention were purchased from Beijing Speyford Biotechnology Co., Ltd., with the license number: SCXK (Jing) 2019 - 0010. All mice were housed in the Experimental Animal Management Center of Tongji Medical College in a 12-hour day-night alternating mode, with free access to food and water, and relevant experimental animal operations were carried out in accordance with the regulations issued by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.

[0050] Reagents: (1) Experimental drugs: Temozolomide (TMZ) was purchased from AbMole. Preparation method: 5 mg of sodium carboxymethyl cellulose was placed on the surface of 1 ml of physiological saline and allowed to stand for 1 day until it was fully swollen. Then, 5 mg of temozolomide powder was added and sonicated for 30 min to form a stable solid dispersion. The final concentration was 5 mg / ml. It was prepared and used immediately. TAT-Ngb was customized from Genscript Biotech. PD-1 antibody (anti-PD-1 antibody) was purchased from BioXcell (batch number: 735019J2). D-Luciferin (potassium salt) was purchased from Cayman Chemical Company. 15 mg / ml D-fluorescein potassium was prepared by dissolving 150 mg of D-fluorescein potassium in 10 ml of sterile PBS under light-protected conditions. It was prepared and used immediately. Flow cytometry antibodies: Polyclonal anti-P2Y12-PE antibody was purchased from Thermo Fisher Scientific (product #PA5-77677), and monoclonal anti-CD11b-FITC antibody was purchased from BioLegend (cloneM1 / 70). (2) Cell culture reagents: Fetal bovine serum was purchased from GEMINI, RPMI 1640 basal medium, 0.25% trypsin, and PBS (pH=7.4) were purchased from Gibco, 0.4% trypan blue staining solution and dimethyl sulfoxide were purchased from Sigma, and penicillin-streptomycin solution was purchased from HyClone.

[0051] Instruments: Stereotactic brain system (Stoelting, USA); CO2 incubator (Forma, USA); Stereomicroscope (77020, Shenzhen Ruiwode Life Technology Co., Ltd.); Animal in vivo optical imaging (Lago X); Mouse X-ray irradiator (RS2000pro); BD FACSVerse flow cytometer.

[0052] Experimental steps:

[0053] (1) Mouse in situ G422 TN -GBM model and its combined RT / TMZ / PD-1 / TAT-Ngb therapy

[0054] A. Preparation of mouse orthotopic G422 TN -GBM model. G422 TNThe GBM cells were established in our laboratory, and whole-genome sequencing confirmed that the genotype of this cell line is: IDH1 / 2. WT Chromosome 1 / 19 Intact TERT-promoter WT ATRX Mutant Trp53 Mutant It meets the subtype classification criteria for human triple-negative (TN) primary glioblastoma and is named G422. TN -GBM cells. G422 TN GBM cells can only be passaged in vivo and cannot be cultured in vitro. We established an optimized G422 cell line consisting of "subcutaneous seeding → 24-hour in vitro culture (no proliferation) → striatal seeding". TN -GBM model system, enabling all intracranial injections of 50,000 G422 TN Adult Kunming mice with GBM cells (orthotopic G422 mice) TN All mice in the GBM model died within 25 days, exhibiting short but stable survival. In the orthotopic G422 mouse model... TN - In the GBM model, we established a temozolomide concurrent chemoradiotherapy (RT / TMZ) treatment model for GBM. The combined RT / TMZ treatment was effective but not curative (survival <35 days, treatment started early on day 5). The treatment response was close to that of clinical GBM. See the literature "Clin Transl Med. 2020 Nov; 10(7):e226".

[0055] B. Mouse in situ G422 TN -Combined therapy of RT / TMZ / PD-1 / TAT-Ngb for GBM tumors. See the schematic diagram for the dosing pathway of RT, TMZ, PD-1 antibody, and TAT-Ngb. Figure 1 Day 0: Intracranial inoculation with G422 TN -GBM cells: Using a stereotaxic instrument (USA, STOELTING CO. 620WHEAT LANE type stereotaxic instrument), 50,000 optimized G422 cells were injected using a Hamilton microsyringe (1701RN, 33G). TNGBM cells / 1 μL (expressing luciferase) were injected into the right striatum of normal adult male Kunming mice (18-22g) (coordinates: 0.5 mm anterior to the anterior fontanelle, 2 mm lateral to the anterior fontanelle; depth 3.5 mm). Treatment period from day 7 to 18: Monotherapy or combination therapy begins on day 7 after tumor cell inoculation; on day 7, a single whole-brain irradiation is administered (RS2000pro X-ray irradiator, irradiation parameters set as: 160KV, 25mA, 1Gy / 48.4sec, total X-ray dose 10Gy; other body parts are shielded with 3mm lead plates). Four hours later, the RT / TMZ combination therapy group receives a single TMZ gavage (50mg / kg), followed by a single intraperitoneal injection of TAT-Ngb (200µg, 10mg / kg) one hour later (TAT-Ngb monotherapy or combination therapy group), and then an intraperitoneal injection of PD-1 antibody (anti-PD-1 antibody, PD-1, 200µg) one hour later (PD-1 antibody monotherapy or combination therapy group); from day 8 to 18, treatment is administered according to the dosing regimen (arrows indicate days). Figure 1 Record the generation time.

[0056] (2) Mouse in situ G422 TN - GBM model tumor re-challenge after cure and single-cell sequencing of tumor tissue from "cured-immune" mice undergoing secondary tumor re-challenge.

[0057] A. A new challenge after "curing" mouse tumors. In situ G422 TN GBM tumor-bearing mice treated with a combination of RT / TMZ / PD-1 / TAT-Ngb (days 7-18) achieved a partial cure (2 / 8 mice) (survival ≥100 days, i.e., long-term survival, LTS). Figure 2 ). A second inoculation of 50,000 doses of G422 was administered to the left striatum of the "cured" mice. TN -GBM cells (coordinates: 0.5 mm anterior to the anterior fontanelle, 2 mm lateral; depth 3.5 mm). Control mice were age-matched normal male Kunming mice, also inoculated with G422. TN GBM cells. No treatment was given, and the survival time of the animals after inoculation was recorded. One of the "cured" mice experienced spontaneous tumor regression (1 / 2), achieving long-term survival again (survival ≥100 days after secondary tumor inoculation, obtaining resistance to G422). TN -The immune capacity of GBM cells, named "Cure-Immune" mice) Figure 4 Another "cured" mouse, BLI, showed a significant tumor and survived for less than 20 days after a second tumor inoculation. Figure 4 ).

[0058] B. Secondary tumor re-challenge and single-cell sequencing of tumor tissue in "cured-immune" mice. See the experimental procedure below. Figure 5The left striatum of the above "cured-immune" mice (i.e., the LTS group) was re-inoculated with 50,000 doses of optimized G422. TN -GBM cells (coordinates: 0.5 mm anterior to the anterior fontanelle, 3 mm lateral; depth 2.5 mm). Control mice were age-matched normal male Kunming mice (n=2), also inoculated with G422. TN -GBM cells. On day 7 after tumor cell inoculation, the brain tumor tissue was perfused with pre-cooled PBS, minced, and immediately digested (collagenase IV + DNase I). Single-cell isolation was performed using Percoll in a 70% / 37% / 30% (mixed cell layer) solution (800g, 4℃, 30min). Single-cell suspensions were prepared, and cell viability and quantity were assessed to meet single-cell sequencing requirements. These suspensions were then sent to Novogene Biotech Co., Ltd. for single-cell RNA sequencing. Raw sequencing results were obtained (uploaded to https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE166525).

[0059] C. A Second Challenge to G422 TN - GBM tumor single-cell RNA sequencing results, microglial cell subset characteristics and functional analysis. Common bioinformatics analysis software and packages, such as CellRanger (version 4.0.0), Seurat (version 3.0.2), and the SingleR package, were used to analyze G422 cells. TN Single-cell sequencing raw data from GBM tumor tissue were used for cell type annotation, gene expression analysis, copy number variation (inferCNV) analysis, cell communication (CellChat software) signaling pathway differentiation, and Metascape database analysis (GO signaling pathway enrichment, KEGG signaling pathway enrichment). The results identified G422. TN GBM can identify "cure-immune" mice G422. TN GBM tumor re-challenge primarily induces a P2ry12 protein. High Ccl12 Low Increased microglial subsets and T cells were positively correlated with long-term survival in tumor re-challenge of "cured-immune" mice.

[0060] (3)P2ry12 High Small colloid subset infusion therapy in mice with orthotopic G422 TN -GBM

[0061] A. Preparation of P2ry12 High Small glial subsets of live cells. Intracranial inoculation of mice with 5 × 10⁶ cells. 4 G422 TNAfter inoculation with GBM cells, patients were randomly divided into two groups: a control group (n=8) and a combined treatment group (n=20). Treatment was administered from day 5 to 16 post-inoculation. Figure 1 The treatment regimen involved a combination of temozolomide concurrent chemoradiotherapy, PD-1 antibody, and brain globulin transmembrane peptide (treatment starting on day 5 increased the cure rate to 50%, see reference 8). On day 17, tumor tissue was harvested, and 10 tumors that responded to the treatment (their volume was reduced by approximately 50% compared to the control group, consistent with the 50% cure rate) were selected. Single viable cells were isolated and co-incubated at 4°C for 30 minutes with polyclonal P2ry12-PE fluorescent antibody (10 μg / 1 million cells) and monoclonal CD11b-FITC antibody (10 μg / 1 million cells, used for microglial flow cytometry to separate surface markers). CD11b-positive cells were then sorted by flow cytometry. + High expression of P2ry12 (a marker of microglia in all microglia) High , representing P2ry12 High Ccl12 Low Small glial subset cells; Ccl12 is located inside the cell and cannot be used for live cell sorting), CD11b positive, low expression of P2ry12 (P2ry12) Low , representing P2ry12 Low Ccl12 High Small glial subpopulation cells).

[0062] B.P2ry12 High Small gels promote the survival of tumor-bearing mice. The above 200 CD11b... + P2ry12 High or CD11b + P2ry12 Low Live cells were injected into mice in situ on day 5 of G422 cells. TN -GBM organization (n=7 / group). Results show: CD11b infusion + P2ry12 High Small-particle-coated tumor-bearing mice had significantly prolonged survival time (***P<0.001, compared with CD11b). + P2ry12 Low (Group comparison).

[0063] Experimental results:

[0064] (1) The combination of brain globulin transmembrane peptide and PD-1 antibody with temozolomide and concurrent chemoradiotherapy can cure orthotopic G422 mice. TN GBM tumor

[0065] We have independently established a stable mouse orthotopic triple-negative glioblastoma (G422) variant. TN-GBM) model: In the optimized model, adult male mice from Kunming were inoculated with 50,000 G422 molecules in the striatum. TN GBM cells resulted in 100% survival of tumor-bearing mice for <25 days. In this model, concurrent chemoradiotherapy with temozolomide (RT / TMZ, X-brain irradiation, 10 Gy; TMZ: 50 mg / kg, gavage, 10 doses / 12 days) on day 7 after tumor inoculation significantly prolonged animal survival, but had no curative effect (survival <35 days) (ClinTranslMed. 2020 Nov; 10(7):e226).

[0066] We previously demonstrated that endogenous neuroglobin (Ngb) plays an anti-cancer role in liver cancer cells (Mol Pharmacol. 2013 May; 83(5):1109-19); and that purified exogenous neuroglobin transmembrane peptide (TAT-Ngb, i.e., a fusion protein of TAT polypeptide containing YGRKKRRQRRR and human neuroglobin) can enter cells and promote neuronal neurite regeneration (Cell Death Dis. 2018 Feb 7; 9(2):163). The construction of the pET28a-TAT-Ngb prokaryotic expression vector can be found in Dr. Xiong Xinxin's dissertation "The Role and Mechanism of Neuroglobin in Promoting Axonal Growth After Neuronal Ischemia Injury" (Huazhong University of Science and Technology, DOI: 10.7666 / d.D01313107). We used G422... TN GBM cells also demonstrated that overexpression of Ngb prolonged the survival time of tumor-bearing mice, while knockdown of G422... TN Endogenous Ngb in GBM cells shortened the survival time of tumor-bearing mice, but the survival time was less than 25 days (results not shown).

[0067] Based on the above results, we speculate that the combination of RT / TMZ with TAT-Ngb and PD-1 antibodies may have the potential to cure G422. TN -GBM effect. Therefore, we tested it in stable G422 mice. TN -GBM model was given RT / TMZ / PD-1 / TAT-Ngb combination therapy, with various single-agent or combination therapy controls ( Figure 1 The survival curve shows () Figure 2(n=8 / group): The survival time of tumor-bearing mice treated with TAT-Ngb and PD-1 antibody monotherapy was not different from that of the control group (no treatment); RT / TMZ treatment significantly prolonged the survival of tumor-bearing mice (compared to the control group); TAT-Ngb / PD-1 combination therapy significantly prolonged the survival time of animals compared with TAT-Ngb and PD-1 antibody monotherapy; RT / TMZ combined with TAT-Ngb or PD-1 antibody monotherapy did not prolong the survival of animals (compared to the RT / TMZ group); RT / TMZ combined with TAT-Ngb and PD-1 antibody significantly prolonged the survival of animals (compared to the RT / TMZ, RT / TMZ / TAT-Ngb, and RT / TMZ / PD-1 groups), and 25% of tumor-bearing mice (2 / 8) achieved "cure" (survival for more than 100 days after tumor implantation, meeting the criteria for glioma cure in mice).

[0068] During the treatment process, on day 20 (post-inoculation), luciferase in vivo imaging was performed on animals in the RT / TMZ / PD-1 / TAT-Ngb and RT / TMZ / PD-1 treatment groups to visualize tumor size. Figure 3 The results showed that the intracranial tumors in the RT / TMZ / PD-1 / TAT-Ngb treatment group were relatively small. Statistical analysis showed that the mean optical density of luciferase in the intracranial tumors of mice in the RT / TMZ / PD-1 / TAT-Ngb treatment group was significantly lower than that in the RT / TMZ / PD-1 treatment group. For specific methods, please refer to the literature "Clin Transl Med. 2020 Nov; 10(7):e226".

[0069] Two "cured" mice from the RT / TMZ / PD-1 / TAT-Ngb treatment group were subjected to a tumor rechallenge experiment. The left striatum of the "cured" mice was re-injected with 50,000 units of G422. TN GBM cells were not treated. Age-matched normal mice were inoculated with an equal amount of tumor cells as a control (Control, n=4). On day 10 post-inoculation, luciferase in vivo imaging was performed, and the results were... Figure 4 The results showed that the tumor in one of the "cured" mice was significantly smaller than that in the control group, and the survival curve showed that the tumor in the brain of this "cured" mouse regressed spontaneously (achieving a reduction in G422). TN The mice with improved immunity to GBM cells survived for more than 100 days and were named "cured-immune" mice; while another "cured" mouse had a significant tumor (and did not gain immunity to G422). TN -GBM cell immunity), animal survival <20 days; control mouse survival <20 days.

[0070] (2) "Cure-immune" mice were re-challenged by tumor tissue, which induced microglial cell subset transformation and T cell increase.

[0071] In our mouse in situ G422 TN In the GBM model, all untreated animals survived less than 25 days. While simulated clinical standard chemoradiotherapy (RT / TMZ) effectively prolonged survival, all animals still survived less than 35 days. This indicates that the tumor in this model is highly malignant and resistant to RT / TMZ treatment, making it the most stable preclinical treatment model currently available. Therefore, the "cured" mice obtained through combined RT / TMZ / PD-1 / TAT-Ngb treatment accurately reflect the therapeutic effect. In tumor rechallenge experiments with these "cured" mice, only half of the animals achieved long-term survival again without treatment (LTS, i.e., "cured-immune" mice). This indicates that the brain microenvironment of these LTS mice has changed, becoming an immune microenvironment capable of treating tumors. Within this therapeutic immune microenvironment, the increased non-tumor cell components can be identified as having immunotherapeutic effects.

[0072] Based on the above analysis, we again intracranially injected G422 into the above "cured-immune" mice (LTS, n=1). TN -GBM cells (Second tumor re-challenge, see experimental procedure) Figure 5 Considering the rapid regression of intracranial tumors in "cured-immune" mice on day 10 after the first tumor re-challenge, we hypothesized that the anti-cancer components of the tumor microenvironment might have been fully induced within a week. Therefore, on day 7 of the second tumor re-challenge, we harvested tumor tissue for single-cell RNA sequencing (scRNA-seq). Simultaneously, we used brain tumor tissue from an untreated, age-matched control mouse as a control group.

[0073] Single-cell RNA sequencing results showed that ( Figure 6 The total number of effective cells sequenced was 7547 in control mice and 4465 in LTS mice, consistent with the smaller tumor tissue observed in the "cured-immune" mice (LTS group). Tumor tissue cells could be divided into 17 subpopulations (Cluster 0-16), with the cell number decreasing sequentially. Compared to the control, only Cluster 8 (microglia) and Cluster 7 (T cells) were increased in the tumor tissue of the "cured-immune" mice. Clearly, the increase in these two cell groups is the cause of the G422 tumor in the "cured-immune" mice. TN- Cellular components involved in GBM tumor healing. Therefore, the Cluster8 glial subset is a group of glial cells with therapeutic effects on GBM. In previous glial models, the therapeutic effects were not stable, and the cure results were mostly not verified by tumor re-challenge experiments. Therefore, the correlation between the tumor microenvironment and the cure results could not be determined. Furthermore, glial subgroups themselves have high heterogeneity, making it difficult to identify or determine glial cells with therapeutic effects in previous animal models.

[0074] (3) Chromosomal copy number variation analysis determined that the Cluster 0, 1, 3, 4, and 6 cell subsets in single-cell sequencing were G422. TN -GBM cells

[0075] Currently, there are no identified marker genes for tumor cell subsets in mouse gliomas; therefore, the original analysis results above did not show tumor cells. Figure 6 CNV (chromosomal copy number variation) analysis is a method for identifying tumor cells using scRNA-seq. Compared with normal cells (References), Clusters 0, 1, 3, 4, and 6 showed significant deletions and increases in chromosome copy number. Figure 7 The brain tissue lacked Fibroblasts, and Erythrocytes showed little or no transcripts, with the majority being removed. Therefore, Clusters 0, 1, 3, 4, and 6 can be identified as G422. TN -GBM cells. Cluster 2 and 5 normal cells showed no significant changes in chromosome copy number. Figure 7 A) is consistent with the cell type shown in the original analysis results. Figure 7 B).

[0076] (4) Differential gene expression analysis of each subgroup showed that the top 5 highly expressed characteristic genes in Cluster 8 microglia were P2ry12, Cx3cr1, Btg2, Hexb, and Cstd.

[0077] In the cells analyzed by the above scRNA-seq, G422 TN GBM tumor cells were the most numerous (Cluster 0, 1, 3, 4, 6), followed by microglia (Cluster 2, 8, 9). The top 1-5 tag genes for each of the microglia subsets in Cluster 2, 8, and 9 are as follows: Figure 8The following groups were identified: C1qc, C1qa, C1qb, Ccl12, Cstd (Cluster2), P2ry12, Cx3cr1, Btg2, Hexb, Cstd (Cluster8), Aif1, C1qc, C1qb, Tmsb4x, and Ccl12 (Cluster9). Among these, P2ry12 showed high expression (P2ry...). High ) and low expression of Ccl12 (Ccl12) Low The effect was more pronounced in the Cluster 8 gel. Therefore, we named the Cluster 8 gel P2ry12. High Clc12 Low Microglial subpopulations. Using the markers P2ry12 and Clc12, combined with other universal markers for microglia and monocytes such as CD11b and CD45, the Cluster 8 microglial subpopulation can be classified and purified for further research or therapeutic applications. (Note: The tag genes for the first 6-10 positions of each of the Cluster 2, 8, and 9 microglial subpopulations are Egr1, etc.) High Fcer1g High Tryobp High Grn High Cstz High Mafb High Tmem119 High Hexb High Zfp36 High Jun High Trem2 High Hexb High Lgmn High Ifi30 High Ly6a High 。 )

[0078] (5) The main characteristic that distinguishes Cluster8 from Cluster2 in “cured-immune” mice is high expression of P2ry12 and low expression of Clc12.

[0079] Figure 8 The microglia subpopulations in the tumor tissues included cells from "cure-immune" (i.e., LTS group) and control mice. Due to the differences in tumor microenvironments between "cure-immune" and control mice, and the high transcriptional heterogeneity of microglia, gene expression of the same microglia subpopulations in tumor tissues from "cure-immune" and control mice may also differ. Therefore, we further compared the expression of Cluster8 and Cluster2 (…) in tumor tissues from "cure-immune" mice. Figure 9The main differentially expressed genes in the small gel subsets were determined. Results showed that in "cured-immune" mice, compared to Cluster 2 gels, Cluster 8 gels exhibited significantly higher expression of P2ry12 and lower expression of Ccl12. Figure 9 In "cured-immune" mice, Cluster2 cells still constitute the largest number. Therefore, using both high expression of P2ry12 and low expression of Clc12 simultaneously will help distinguish Cluster8 glial cells from the other two glial subpopulations.

[0080] (6) The main characteristic gene that distinguishes Cluster 8 from Cluster 9 in “cured-immune” mice is the high expression of P2ry12.

[0081] Comparison of Cluster 8 and Cluster 9 in tumor tissues of "cured-immune" mice Figure 10 The main differentially expressed genes in the small gel subsets. Results showed that in "cured-immune" mice, compared to Cluster9 gels, Cluster8 gels showed significantly higher expression of P2ry12. Figure 10 ). Combination Figure 9 As a result, it is clear that P2ry12 is highly expressed (P2ry12 High P2ry12 is the most important biomarker for Cluster8 glial cells in "cured-immune" mice. It is known that P2ry12 is the main receptor molecule for phagocytosis and release of reactive oxygen species (ROS) in normal brain tissue glial cells. Clearly, Cluster8 glial cells with high P2ry12 expression can also phagocytose and release ROS to kill tumor cells through the P2ry12 molecule.

[0082] (7) The functions of Cluster 8 capsules are significantly different from those of Cluster 2 and Cluster 9 capsules.

[0083] We further compared the effects of differentially expressed genes from the Cluster2, Cluster8, and Cluster9 microglia subpopulations on cell communication, and the results showed that ( Figure 11The cell-mediated signaling pathways in the Cluster 8 glial subset are mostly significantly weakened compared to Cluster 2, with the GAS signaling network being absent in Cluster 8; while the IL-2 signaling network shows a trend of enhancement. Normal brain tissue is known to be an immune organ; however, tumors grow rapidly in control mice; clearly, the most numerous and predominant Cluster 2 glial subsets represent the immunosuppressive power of glial cells. Therefore, the weakened signaling pathways in Cluster 8 glial subsets (such as the GAS signaling network) mainly reflect the relief of their immunosuppressive function. IL-2 is known to be a potent cytotoxic T cell inducing factor, promoting tumor immunity and possessing tumor therapeutic effects. The enhanced IL-2 signaling pathways in Cluster 8 glial subsets are consistent with the increased Cluster 8 glial subsets and T cell counts in "cured-immune" mice, reflecting the immunotherapeutic effect of Cluster 8 glial subsets.

[0084] (8) Cluster8 gels mainly play an immunotherapeutic role in the tumor microenvironment of "heal-immune" mice.

[0085] Cluster glial cells are known to be highly heterogeneous, and their phenotype and function are closely related to their microenvironment and regulated by numerous pathological factors. Cluster 8 glial cells are significantly increased in "cure-immune" mice, and their function may also be influenced by the microenvironment. We compared the gene expression of the Cluster 8 glial subset in "cure-immune" and control mice, and the results showed differences in the expression of many genes in the Cluster 8 glial subset between the two groups. Figure 12 In this study, Ccl12 expression in Cluster 8 microglia of "cured-immune" mice was lower than that in the control group. This result also supports the use of low Ccl12 expression as a biomarker for Cluster 8 in "cured-immune" mice.

[0086] Further functional analysis was performed by comparing differentially expressed genes in the "cure-immunity" and control mouse Cluster8 microglia subsets. GO signaling pathway enrichment was also conducted. Figure 13 The results showed that the Cluster8 microparticle subset in "cured-immune" mice was mainly concentrated in immune response or immune regulation functions, such as "positive regulation of immune response," "acquired immune response," and "defense response against other microorganisms." KEGG signaling pathway enrichment was also observed. Figure 14 The results showed that the Cluster 8 subpopulation in "cure-immune" mice was mainly concentrated in immune responses or immunomodulatory functions, such as "natural killer cell-mediated cytotoxicity," "cytokine-cytokine receptor interactions," and "antigen processing and presentation." Similarly, the Cluster 9 subpopulation in "cure-immune" and control mice was compared. Figure 15 Cluster2 Figure 16 Differentially expressed genes in the small colloid subpopulation were analyzed, and the results showed that the KEGG enrichment signaling pathway was concentrated and not related to the immune pathway.

[0087] Through single-cell RNA sequencing, we confirmed the presence of P2ry12 in glioma tissues of "cured-immune" mice. High Ccl12 Low The (i.e., Cluster8) small glial subset is positively correlated with its spontaneous regression. Clearly, P2ry12 was induced during the first combined treatment in the "cure-immune" mice. High Ccl12 Low Small colloids, and the subsequent tumor re-challenge is to re-stimulate P2ry12. High Ccl12 Low The process of glioma formation. Therefore, we extracted CD11b+P2ry12 from glioma tissue that had been effectively treated with combined therapy (temozolomide concurrent chemoradiotherapy + PD-1 antibody + brain globulin transmembrane peptide). High Small glue (CD11b) + To distinguish microglia from other cells, P2ry12 High Distinguishing P2ry12 High Ccl12 Low (small colloid subgroup), infused into mouse orthotopic G422 TN -GBM organization to confirm P2ry12 High Ccl12 Low The function of small colloid subgroups was demonstrated by the infusion of CD11b. + P2ry12 High Small gel significantly prolonged the survival time of tumor-bearing mice (***P<0.001, compared with CD11b). + P2ry12 Low (Group comparison). The above results prove that P2ry12 High Ccl12 Low Small glial cells are a therapeutic subgroup of gliomas that can be used in clinical trials.

Claims

1. A therapeutic microglial cell subset for glioma, characterized in that, It is a microglia subset induced by the methods described in steps (a) to (c) with high expression of the purinergic receptor P2ry12 and low expression of the chemokine ligand Ccl12: (a) Inoculation with tumor cells: Lethal doses of G422 were injected into the brains of mice. TN -GBM cells, namely G422 TN GBM cells have been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No.: C2020267; the culture name is mouse triple-negative primary glioblastoma G422. TN -GBM; Accession number: CCTCCNo:C2020267; Accession date: December 17, 2020; (b) Combined radiotherapy and chemotherapy with immunotherapy: Treatment on day 7 after tumor cell inoculation: Mice inoculated with tumor cells were given a single whole-brain X-ray irradiation; temozolomide was administered by gavage 4 hours after whole-brain X-ray irradiation; brain globulin transmembrane peptide was injected intraperitoneally 1 hour later; and PD-1 antibody was injected intraperitoneally 1 hour after that. Treatment on the 8th day after tumor cell inoculation: Temozolomide was administered by gavage once, followed by an intraperitoneal injection of brain globulin transmembrane peptide one hour later; Treatment on the 9th day after tumor cell inoculation: Temozolomide was administered by gavage once, followed by intraperitoneal injection of brain globulin transmembrane peptide 1 hour later, and then intraperitoneal injection of PD-1 antibody 1 hour later. Treatment on day 10 after tumor cell inoculation: Administer the medication as described on day 8 after tumor cell inoculation; Treatment on day 11 after tumor cell inoculation: Administer the medication as described on day 9 after tumor cell inoculation; Days 12-13 after tumor cell inoculation: No treatment administered; Treatment on day 14 after tumor cell inoculation: Administer the medication as described on day 9 after tumor cell inoculation; Treatment on day 15 after tumor cell inoculation: Administer the medication as described on day 8 after tumor cell inoculation; Treatment on day 16 after tumor cell inoculation: Administer the medication as described on day 9 after tumor cell inoculation; Treatment on day 17 after tumor cell inoculation: Administer the medication as described on day 8 after tumor cell inoculation; Treatment on day 18 after tumor cell inoculation: Administer the medication as described on day 9 after tumor cell inoculation; Record the survival status of tumor-bearing mice; mice that achieve long-term survival are considered cured. (c) Re-injecting a lethal dose of G422 into the brain of long-term surviving mice that achieved a cure in step (b). TN Mice were injected with GBM cells without treatment, and then those that survived for ≥100 days underwent a second tumor rechallenge trial, which involved a third inoculation of the mice's brains with a lethal dose of G422. TN Mice that, after inoculating GBM cells without treatment, achieve long-term survival after receiving tumor cell inoculation are considered to have acquired immunity to G422. TN -GBM cell immunity, acquired G422 TN Immunocure of GBM tumors: Long-term surviving mice identified as P2ry12 High Ccl12 Low Microglia subset induction was successful.

2. The therapeutic microglial subpopulation for glioma according to claim 1, characterized in that, The step (a) involves inoculating the mouse brain with a lethal dose of G422. TN GBM cells are a lethal number of G422 cells. TN GBM cells were injected into the striatum of the mouse brain.

3. The therapeutic microglial subpopulation for glioma according to claim 2, characterized in that, The striatum mentioned is the right striatum.

4. The therapeutic microglial subpopulation for glioma according to claim 1, characterized in that, In the treatment on the 7th day after tumor cell inoculation in step (b): the parameters of the single whole brain X-ray irradiation were set as 160KV, 25mA, 1Gy / 48.4sec, and the total X-ray irradiation dose was 10Gy; the dosage of temozolomide administered by gavage was 50mg / kg; the dosage of brain globulin transmembrane peptide was 10mg / kg; and the dosage of PD-1 antibody was 200 micrograms.

5. The therapeutic microglial subpopulation for glioma according to claim 1, characterized in that, In the treatment on day 8 after tumor cell inoculation in step (b): the dosage of temozolomide was 50 mg / kg; the dosage of brain globulin transmembrane peptide was 10 mg / kg.

6. The therapeutic microglial subpopulation for glioma according to claim 1, characterized in that, In the treatment on day 9 after tumor cell inoculation in step (b): the dosage of temozolomide was 50 mg / kg; the dosage of brain globulin transmembrane peptide was 10 mg / kg; and the dosage of PD-1 antibody was 200 micrograms.

7. The therapeutic microglial subpopulation for glioma according to claim 1, characterized in that, In step (c), the long-term surviving mice that achieved a cure in step (b) were re-inoculated intracerebrally with a lethal dose of G422. TN -GBM cells, specifically, a lethal number of G422 cells were injected into the striatum of the brains of long-term surviving mice that had achieved a cure. TN -GBM cells.

8. The therapeutic microglial subset for glioma according to claim 7, characterized in that, The striatum mentioned is the left striatum.

9. The therapeutic microglial subset for glioma according to claim 1, 2, or 7, characterized in that, The lethal dose of G422 TN -GBM cells consist of 50,000 G422 cells TN -GBM cells.

10. The therapeutic microglial subpopulation for glioma according to claim 1 or 7, characterized in that, Long-term survival is defined as survival for more than 100 days.

11. The therapeutic microglial subpopulation for glioma according to any one of claims 1 to 8, characterized in that, The top 5 marker genes for microglia that are highly expressed are P2ry12, Cx3cr1, Btg2, Hexb, and Cstd.

12. The therapeutic microglial subpopulation for glioma according to claim 9, characterized in that, The top 5 marker genes for microglia that are highly expressed are P2ry12, Cx3cr1, Btg2, Hexb, and Cstd.

13. The therapeutic microglial subpopulation for glioma according to claim 10, characterized in that, The top 5 marker genes for microglia that are highly expressed are P2ry12, Cx3cr1, Btg2, Hexb, and Cstd.

14. An inducible P2ry12 High Ccl12 Low The method for constructing an in situ glioma "cure-immunity" animal model based on microglial cell subsets is characterized by, Includes steps (a) to (c) of the induction method described in claim 1.

15. The construction method according to claim 14, characterized in that, It also includes the definition of step (a), step (b) or step (c) as described in any one of claims 2 to 10.

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