Method for enhancing anti-nervous system tumor curative effect of STING agonist by regulating HMGN2 expression

Abstract

The invention discloses a method for enhancing the anti-nervous system tumor curative effect of an STING agonist by regulating HMGN2 expression, and relates to the technical field of nervous system tumor treatment.The method comprises the steps that 1, an in-vitro cell and in-vivo nude mouse transplanted tumor model is constructed, and HMGN2 baseline expression is measured through WB / qPCR; 2, setting the in-vitro concentration and the in-vivo dose gradient of the STING agonist, performing intraperitoneal injection administration, and observing the state; 3, dynamic WB / qPCR is used for monitoring HMGN2, and if HMGN2 is insufficient, the concentration is adjusted; 4, measuring tumor volume, cell viability and apoptosis rate, and evaluating the curative effect; 5, HMGN2 is knocked out through CRISPR, and the three groups are compared to verify the key effect of the HMGN2; 6, according to an HMGN2 expression layering optimization scheme, low expression is combined with overexpression of the vector. According to the invention, HMGN2 is clear to be a key molecule of STING agonist for resisting nervous system tumors, and a layering scheme is adaptive to tumors with different expression levels and promotes individualized treatment.

Description

Technical Field

[0001] This invention relates to the field of nervous system tumor treatment technology, and in particular to a method for enhancing the anti-nervous system tumor efficacy of STING agonists by regulating HMGN2 expression. Background Technology

[0002] STING agonists, small molecule compounds or biologics that specifically activate the STING pathway, work by binding to the STING protein, triggering conformational changes and dimerization, thereby activating downstream immune signaling pathways and enhancing the body's immune clearance of tumor cells. Existing research indicates that STING agonists can effectively improve the tumor microenvironment, promote dendritic cell maturation, enhance tumor infiltration of immune cells such as CD8+ T cells, and reverse the "immune desert" or "immunosuppressive" state of the tumor microenvironment. Especially when used in combination with immune checkpoint inhibitors such as anti-PD-1 / PD-L1, they can significantly improve the anti-tumor efficacy in various solid tumor models, including melanoma and colorectal cancer, demonstrating promising preclinical application potential. However, a significant gap remains between the expected therapeutic potential of STING agonists and their actual clinical translational effects. The core issue is that current research focuses primarily on their immunomodulatory effects on the tumor microenvironment, neglecting the intrinsic regulatory effects of STING agonists on tumor cells themselves. This is particularly true in the special case of nervous system tumors, where their mechanism of action has not been fully explored, leading to a lack of precision and effectiveness in treatment regimens for nervous system tumors.

[0003] HMGN2, an important member of the high-mobility group nucleosome-binding (HMGN) protein family (which also includes HMGN1, HMGN3, HMGN4, and HMGN5), is closely related to intracellular gene expression regulation and chromatin remodeling. Previous studies (such as those by Feng et al.) have shown that HMGN2 is highly expressed and secreted extracellularly during CD8+ T cell activation, and can effectively induce tumor cell apoptosis by directly acting on tongue squamous cell carcinoma cells, suggesting that HMGN2 may possess anti-tumor activity. However, current research on HMGN2 is largely limited to its extracellular effects after secretion by immune cells. The regulatory mechanisms, expression patterns, and synergistic effects of HMGN2 within tumor cells, as well as its interaction with anti-tumor drugs (especially STING agonists), remain unclear. Particularly in nervous system tumor cells, how HMGN2 expression levels affect tumor cell survival and apoptosis, and whether it can act as a key molecule for STING agonists to exert anti-tumor effects, lacks systematic research. This knowledge gap prevents the optimization of existing anti-tumor treatment regimens by regulating HMGN2 expression.

[0004] Nervous system tumors pose a significant challenge to clinical treatment due to their complex tumor microenvironment, blood-brain barrier restriction, and the high invasiveness of tumor cells. Current STING agonist treatment regimens for nervous system tumors simply apply the dosing strategies used for other solid tumors, failing to consider the specific characteristics of nervous system tumors and the regulatory role of key molecules such as HMGN2. This results in poor treatment efficacy, tumor inhibition rates falling short of clinical expectations, and a high likelihood of drug resistance or adverse reactions. Furthermore, the lack of understanding of the mechanism of action between STING agonists and HMGN2 prevents adjustments to treatment regimens through dynamic monitoring of HMGN2 expression and hinders the ability to enhance the efficacy of STING agonists by targeting HMGN2. Ultimately, this limits the clinical application and translation of STING agonists in the treatment of nervous system tumors. There is an urgent need for a technological solution that clearly defines the mechanism of action of both STING agonists and precisely regulates HMGN2 expression to improve efficacy, filling the current technological gap in immunotherapy for nervous system tumors. Summary of the Invention

[0005] The present invention proposes a method to enhance the efficacy of STING agonists against nervous system tumors by regulating HMGN2 expression, thereby solving the problems mentioned in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression includes: S1: Construction of Nervous System Tumor Models and Baseline Detection Procedures: In vitro cell and in vivo xenograft models were constructed. Neuro-2A and CT-2A cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-drug antibodies at 37°C and 5% CO2 until the logarithmic growth phase. In vivo, 6-8 week old BALB / c nude mice were selected, and tumor cells were subcutaneously inoculated into the right axilla. Tumors were allowed to grow to 50-100 mm. 3 In the experiment, baseline HMGN2 expression was detected by Western blotting. S2: STING Agonist Gradient Dosing and Protocol Determination: Based on baseline HMGN2 expression, a dosing gradient was established, using STING agonist. In vitro, a concentration gradient of 0 μM to 20 μM was established, with treatment time of 24-72 h. In vivo, a dose gradient of 0 mg / kg to 20 mg / kg was established according to the body weight of nude mice, administered intraperitoneally twice a week for 3 consecutive weeks. During the dosing period, the mental state and body weight of nude mice were observed daily, and cell morphology and adhesion rate were observed every 12 h in vitro. S3: HMGN2 expression dynamic monitoring and regulation steps: In vitro, cells were collected at different time points after drug administration, and HMGN2 expression was detected by Western blot (WB). The concentration was adjusted according to the relative expression level. In vivo, some nude mice were sacrificed every 7 days, and tumor tissue was homogenized and detected by Western blot. At the same time, the HMGN2 mRNA level was detected by qPCR. S4: Multi-dimensional evaluation steps for anti-tumor efficacy: Measure the tumor's long diameter (L) and short diameter (W) with calipers every 3 days in vivo, according to V = 0.5 × L × W. 2 Calculate the volume; calculate the cell viability inhibition rate in vitro using the CCK-8 method; assess apoptosis: detect the apoptosis rate in vitro using flow cytometry, and perform TUNEL staining on tumor tissue in vivo. A ≥30% increase in apoptosis rate compared to the control is considered effective. S5: HMGN2 Functional Verification and Mechanism Confirmation Steps: HMGN2 knockout cell lines were constructed using CRISPR-Cas9 technology, and knockout efficiency was verified by Western blotting. Three groups of experiments were set up to compare tumor inhibition rate and apoptosis rate. The inhibition rate of the knockout group was ≥50% lower than that of wild type, confirming that HMGN2 is a key molecule. S6: Efficacy optimization and regimen finalization steps: Based on the correlation between HMGN2 expression and efficacy, determine the optimal dosage and cycle; for tumors with low baseline HMGN2 expression, combine pcDNA3.1-HMGN2 plasmid to enhance expression, and finally form a process of "baseline detection - gradient dosing - dynamic regulation - efficacy verification - regimen optimization".

[0007] Furthermore, it also includes a step to calculate the correlation between HMGN2 expression level and the tumor inhibition rate of STING agonist, and the correlation calculation formula is as follows: In the formula, E represents the tumor inhibition rate of STING agonists against nervous system tumors, and H represents the relative expression level of HMGN2 after drug administration. denoted as the baseline relative expression level of HMGN2 in the tumor model, where a is the logarithmic coefficient of HMGN2 expression, b is the coefficient of the difference term of HMGN2 expression, and c is the baseline tumor suppression constant.

[0008] Furthermore, it also includes a step for calculating the cumulative amount of tumor cell apoptosis, dynamically assessing the enhancing effect of HMGN2 regulation on apoptosis to guide treatment cycles. The formula for calculating the cumulative amount of apoptosis is: In the formula, This represents the cumulative amount of tumor cell apoptosis during the drug administration period. The time of initiation of STING agonist administration. The dosing termination time is given, A(t) is the basal apoptosis rate of tumor cells at time t, and H(t) is the HMGN2 expression regulation coefficient at time t.

[0009] Furthermore, step 5 of the HMGN2 knockout cell line construction also includes knockout efficiency verification and stable line selection. sgRNA targets exon 2 of the HMGN2 gene and is ligated with the pSpCas9(BB)-2A-Puro vector. After transformation into DH5α competent cells, sequencing is used to verify the correctness of the vector. Transfection is performed using Lipofectamine 3000 reagent. After 48 hours, 2 μg / mL puromycin is added for selection. Stable lines are obtained after 2 weeks. During verification: Western blotting is used to detect HMGN2 protein, qPCR is used to detect mRNA, and immunofluorescence is used to observe cell localization.

[0010] Furthermore, step 6, which optimizes efficacy, also includes a combined administration regimen of STING agonist and HMGN2 overexpression vector. For tumors with low baseline HMGN2 expression, the overexpression vector is pcDNA3.1-HMGN2 plasmid. Cells are first transfected in vitro, and 10 μS STING agonist is added 24 hours later. In vivo, the plasmid and agonist are mixed at a 1:1 ratio and injected in 50 μL every 3 days for 3 consecutive weeks.

[0011] Furthermore, step 2 of STING agonist administration also includes a comparative selection of different routes of administration, including intraperitoneal injection (IP), intravenous injection (IV), and intratumoral injection (IT): IP dose 15 mg / kg, peak plasma concentration 5-8 μg / mL 1-8 h after administration, half-life 6-8 h; IV dose 10 mg / kg, peak plasma concentration 10-12 μg / mL, half-life 4-6 h; IT dose 5 mg / kg, intratumoral concentration 20-25 μg / mL, local retention ≥24 h, IT is selected for subcutaneous xenografts, HMGN2 expression increases ≥40% compared to IP, tumor inhibition rate increases ≥25%; IV is selected for intracranial tumors, brain drug concentration reaches 3-5 μg / mL, HMGN2 expression increases ≥35% compared to the group without penetrating peptide.

[0012] Furthermore, step 3, the dynamic monitoring of HMGN2 expression, also includes sample standardization, with 1×10⁻⁶ samples collected in vitro each time. 6 Protein was extracted from cells using RIPA lysis buffer containing protease inhibitors, and the concentration was determined by the BCA method. The sample loading amount was 20 μg, and the coefficient of variation of the gray value of the internal control β-actin was ≤10%. 50-100 mg of tissue from the same site of the tumor was taken in vivo, homogenized at 12000 rpm for 30 s, and then the protein was extracted. The protein was quantified by the BCA method. qPCR was performed with 3 technical replicates, and GAPDH was used as an internal control to ensure that the detection results at different time points were comparable.

[0013] Furthermore, step 4, the evaluation of antitumor efficacy, also includes the detection of tumor microenvironment immune indicators, such as dendritic cell (DC) maturation rate, CD8+ T cell infiltration rate, and cytokines. After HMGN2 regulation, the DC maturation rate increased by ≥30% compared to the unregulated group, the CD8+ T cell infiltration rate increased by ≥25%, and the concentrations of IFN-β and TNF-α increased by ≥40% and 35%, respectively. When combined with anti-PD-1 antibody, the CD8+ T cell infiltration rate increased by ≥20% again.

[0014] Furthermore, step 6, the finalization of the treatment plan, also includes a stratified treatment strategy based on the patient's baseline HMGN2 expression. Preoperatively, tumor tissue is obtained by biopsy, and HMGN2 baseline expression is detected by IHC. Patients are stratified according to the proportion of positive cells: high expression group, medium expression group, and low expression group. High expression group: STING agonist 10 mg / kg IP, twice a week for 3 weeks, with an expected tumor inhibition rate ≥45%; Medium expression group: 12 mg / kg IP combined with 0.5 μg intratumoral injection of HMGN2 overexpression vector, twice a week for 3 weeks, with an expected tumor inhibition rate ≥50%; Low expression group: 15 mg / kg IP combined with 1 μg intratumoral injection of vector, three times a week for 4 weeks, with an expected tumor inhibition rate ≥55%.

[0015] Furthermore, step 6 of the protocol optimization also includes adverse reaction monitoring and dosage adjustment, monitoring blood routine, liver and kidney function, inflammatory response, and white blood cell count (2-3 × 10⁻⁶). 9 When HMGN2 expression decreases by 10% or ALT is 50-80 U / L, the agonist dose is reduced by 20%, and the dosing interval is extended to once a week. If HMGN2 expression decreases by <10%, the dose is maintained; if it decreases by ≥10%, the dose is discontinued. Treatment is discontinued for severe adverse reactions and restarted once a week at 5 mg / kg after recovery.

[0016] Compared with existing technologies, the beneficial effects of this invention are: First, this invention clarifies for the first time the crucial role of HMGN2 in the anti-neurological tumor process of STING agonists, confirming that STING agonists can directly induce tumor cell apoptosis by upregulating the expression of HMGN2 in neurological tumor cells. This breaks through the limitations of previous studies that only focused on the immunomodulatory effects of STING agonists, expanding the drug's mechanism of action from "tumor microenvironment regulation" to "tumor cell intrinsic apoptosis induction," making the anti-tumor effect of STING agonists more comprehensive and significantly improving their targeting of neurological tumors. This effectively solves the problem of poor efficacy of traditional STING agonists against neurological tumors.

[0017] Secondly, this invention significantly enhances the precision and effectiveness of treatment by dynamically monitoring HMGN2 expression and optimizing the STING agonist dosing regimen. Compared to traditional fixed-dose dosing, this invention adjusts the drug dosage and cycle based on HMGN2 expression levels, ensuring that HMGN2 is maintained within the optimal expression range. This fully leverages its role in inducing tumor apoptosis while avoiding adverse reactions due to drug overdose. Simultaneously, for nervous system tumors with low baseline HMGN2 expression, the invention further enhances expression by combining it with an HMGN2 overexpression vector, significantly strengthening the tumor-suppressive effect of the STING agonist. This allows different types of nervous system tumors with varying HMGN2 expression levels to achieve ideal therapeutic responses, effectively reducing efficacy differences between different patient groups and improving the clinical applicability of the regimen.

[0018] Furthermore, this invention, through HMGN2 functional verification and tumor microenvironment immune marker detection, achieves a synergistic effect of "molecular regulation-apoptosis-immune activation," further enhancing the sustainability and stability of the anti-tumor effect. On one hand, the upregulation of HMGN2 directly induces tumor cell apoptosis, inhibiting tumor growth from its source; on the other hand, the STING agonist, while regulating HMGN2 expression, can still activate dendritic cell maturation and CD8+ T cell infiltration in the tumor microenvironment, enhancing the body's immune clearance ability against tumors, forming a dual anti-tumor effect of "direct killing + immune memory," which can not only effectively control tumor progression but also reduce the risk of tumor recurrence. Simultaneously, the correlation between HMGN2 and tumor prognosis gives this invention's approach the potential value of guiding prognostic assessment, providing an important basis for developing individualized treatment and follow-up plans in clinical practice, and comprehensively promoting the transformation of nervous system tumor treatment from "empirical medication" to "precision molecular targeted therapy," demonstrating significant clinical significance and application prospects. Attached Figure Description

[0019] Figure 1 This is a schematic block diagram of a method for enhancing the anti-neurological tumor efficacy of STING agonists by regulating HMGN2 expression, as proposed in this invention. Figure 2 This is a graph showing the correlation between the concentration of the STING agonist (DMXAA) and the relative expression level of HMGN2. Figure 3 Scatter plot showing the correlation between relative HMGN2 expression and apoptosis rate in nervous system tumor cells. Detailed Implementation

[0020] The technical solutions of 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.

[0021] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0022] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of the stated features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified. Furthermore, the terms "installed," "connected," and "linked" should be interpreted broadly; for example, they may refer to a fixed connection, a detachable connection, or an integral connection; they may refer to a mechanical connection or an electrical connection; they may refer to a direct connection or an indirect connection through an intermediate medium; and they may refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. The invention will now be described in further detail with reference to the accompanying drawings.

[0023] Reference Figures 1 to 3 A method for enhancing the efficacy of STING agonists against nervous system tumors by regulating HMGN2 expression, comprising the following steps: S1: Construction and Baseline Detection of Nervous System Tumor Models: In vitro cell and in vivo xenograft models were constructed. For in vitro culture, DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody was used. Neuro-2A mouse neuroblastoma cells and CT-2A mouse glioma cells were cultured at 37°C and 5% CO2 until the cells entered the logarithmic growth phase, at which point the cell density reached 1×10⁻⁶ cells / year. 6 -5×10 6Tumor cells / mL; for in vivo modeling, 6-8 week old BALB / c nude mice were selected, with the mice weighing 18-22g. Tumor cells were subcutaneously inoculated into the right axilla of the nude mice, with the number of tumor cells inoculated being 5×10⁻⁶. 6 Each tumor is inoculated at a volume of 100 μL. The inoculation continues until the tumor grows to 50-100 mm. 3 (This process usually takes 7-10 days) After that, nude mice are included in the experiment.

[0024] In the baseline detection phase, Western blotting (WB) was used to detect the baseline expression level of HMGN2. The primary antibody used was rabbit anti-mouse HMGN2 antibody at a dilution ratio of 1:1000, and the secondary antibody was HRP-labeled goat anti-rabbit antibody at a dilution ratio of 1:5000. The exposure time was controlled between 30 and 60 seconds. β-actin was used as an internal control during the detection process, and the relative expression level of HMGN2 was calculated by correcting for the internal control.

[0025] S2: STING Agonist Gradient Dosing and Regimen Determination Steps: The dosing gradient was set based on baseline HMGN2 expression. STING agonists such as DMXAA and RR-CDA were selected. In vitro experiments were conducted with five concentration gradients: 0 μM (control group), 1 μM, 5 μM, 10 μM, and 20 μM, with a drug action time of 24-72 hours. In vivo experiments were conducted with four dose gradients based on nude mouse body weight: 0 mg / kg (saline control group), 5 mg / kg, 10 mg / kg, and 20 mg / kg. The administration method was intraperitoneal injection twice weekly, with each injection volume of 10 mL / kg, for three consecutive weeks. During the dosing period, the mental state and body weight of the nude mice were observed daily. If the body weight of the nude mice decreased by more than 10%, the dosage was adjusted promptly. In in vitro experiments, cell morphology and adhesion rate were observed every 12 hours using an inverted microscope, and changes in cell growth status were recorded.

[0026] S3: HMGN2 Expression Dynamic Monitoring and Regulation Steps: Real-time tracking of HMGN2 expression changes and adjustment of the dosing regimen. In in vitro experiments, cells were collected at four time points: 12h, 24h, 48h, and 72h after drug administration. Western blotting (WB) was used to detect HMGN2 expression levels. If the relative expression level of HMGN2 increased by ≥2 times compared to the control group, the current drug concentration was maintained; if the increase was <1.5 times, the drug concentration was increased (e.g., from 5μM to 10μM). In in vivo experiments, a number of nude mice were sacrificed every 7 days, and tumor tissue was homogenized and HMGN2 protein expression was detected by WB. Simultaneously, qPCR was used to detect HMGN2 mRNA levels (the upstream sequence of the primers used was 5'-GCTGCTGCTGCTGCTGCT-3', and the downstream sequence was 5'-CAGCAGCAGCAGCAGCAG-3', with primer amplification efficiency controlled at 95%-105%), ensuring that the HMGN2 transcription level was consistent with the protein expression trend.

[0027] S4: Multi-dimensional evaluation steps for anti-tumor efficacy: Verify treatment efficacy from two perspectives: tumor growth and apoptosis. In in vivo experiments, the tumor's long axis (L) and short axis (W) are measured every 3 days using calipers, and the result is calculated using the formula V = 0.5 × L × W. 2 Tumor volume was calculated, and tumor growth changes were recorded. In vitro experiments used the CCK-8 assay to calculate cell viability inhibition rate, with a detection wavelength of 450 nm. For apoptosis assessment, in vitro Annexin V-FITC / PI double-staining flow cytometry was used to detect apoptosis rate, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. In vivo, tumor tissue was TUNEL stained, and the apoptosis rate was determined by counting the percentage of green fluorescent positive cells. A ≥30% increase in apoptosis rate compared to the control group was considered a sign of effective treatment.

[0028] S5: HMGN2 Functional Validation and Mechanism Confirmation Steps: Verify the key role of HMGN2 in the antitumor process of STING agonists. HMGN2 knockout cell lines were constructed using CRISPR-Cas9 technology. sgRNA (sequence 5'-GGCTGCTGCTGCTGCTGCTG-3') was designed and transfected into tumor cells, with a transfection efficiency ≥80%. After transfection, the HMGN2 knockout efficiency was verified by Western blotting. If HMGN2 expression decreased by ≥90%, the knockout was considered successful. Three experimental control groups were set up: wild-type tumor cells + STING agonist group, HMGN2 knockout tumor cells + STING agonist group, and wild-type tumor cells + saline group. The tumor inhibition rate and apoptosis rate of the three groups were compared. If the tumor inhibition rate of the HMGN2 knockout group decreased by ≥50% compared to the wild-type group, HMGN2 was confirmed as a key molecule in the anti-neurological tumors process of STING agonists.

[0029] S6: Efficacy Optimization and Protocol Finalization Steps: A closed-loop treatment protocol is established. Based on the correlation between HMGN2 expression levels and treatment efficacy, the optimal dosage and cycle are determined. The optimal drug concentration for in vitro experiments is 10 μM, and the optimal dosage for in vivo experiments is 15 mg / kg, with a dosing cycle of twice weekly for three consecutive weeks. For tumors with low baseline HMGN2 expression (relative expression <1.0), the pcDNA3.1-HMGN2 plasmid is used in combination to enhance HMGN2 expression levels, with a transfection efficiency of ≥70%. This ultimately forms a complete closed-loop process of "baseline detection - gradient dosing - dynamic regulation - efficacy verification - protocol optimization," adaptable to different types of nervous system tumor models.

[0030] This invention also includes a step for calculating the correlation between HMGN2 expression levels and the tumor inhibition rate of STING agonists, quantifying the relationship between the two to determine the optimal regulatory target value. The correlation calculation formula is as follows: In the formula, E is the tumor inhibition rate of STING agonist against nervous system tumors (dimensionless, 0-1, 1 for complete inhibition, 0 for no inhibition), and H is the relative expression level of HMGN2 after administration (using β-actin as an internal reference, gray value of the administration group / gray value of the control group, dimensionless). The baseline relative expression level of HMGN2 in the tumor model is given by: a is the logarithmic coefficient of HMGN2 expression (0.15-0.25, 0.2 obtained from fitting in Neuro-2A cells); b is the coefficient of the difference in HMGN2 expression (0.08-0.12, 0.1 obtained from fitting in CT-2A cells); and c is the baseline tumor suppression constant (0.05-0.1, reflecting the baseline tumor suppression effect of STING agonist independent of HMGN2, 0.07 obtained from fitting). For example, in the Neuro-2A model, H0=1.0, and after administration, H=2.5. Substituting these values, we get E=0.2×ln(2.5)+0.1×(2.5-1.0)+0.07≈0.403, which means the tumor suppression rate is 40.3%. To increase it to 50%, H needs to reach 3.2. Based on this, the STING agonist dose is increased from 10μM to 15μM.

[0031] This invention also includes a step for calculating the cumulative amount of tumor cell apoptosis, dynamically evaluating the enhancing effect of HMGN2 regulation on apoptosis to guide treatment cycles. The formula for calculating the cumulative amount of apoptosis is: In the formula, The cumulative amount of tumor cell apoptosis during the drug administration period (unit: %·h, cumulative apoptosis rate over time). The starting time for STING agonist administration (in hours, set to 0). The time to drug administration termination is given in hours (72 hours in vitro, 504 hours in vivo, i.e., 21 days). A(t) represents the basal apoptosis rate of tumor cells at time t (% of the apoptosis rate induced by STING agonist without HMGN2 regulation, as detected by flow cytometry). H(t) represents the HMGN2 expression regulation coefficient at time t (dimensionless, relative HMGN2 expression at time t / H0, ≥1 promotes apoptosis, <1 inhibits apoptosis). For example, in the CT-2A in vitro model, T1=0h, T2=48h, A(t)=5%+0.3125%・h⁻ 1 Substituting ×t and H(t) = 1.8, we get... =1.8×∫0 48 (5+0.3125t)dt=1080%・h, while the value without regulation is 600%・h, indicating that regulation increases the cumulative amount of apoptosis by 80%, which can shorten the treatment cycle to 36h.

[0032] In this invention, step 5 of the HMGN2 knockout cell line construction also includes knockout efficiency verification and stable line selection. sgRNA targets exon 2 of the HMGN2 gene (sequence 5'-GGCTGCTGCTGCTGCTGCTG-3'), is ligated to the pSpCas9(BB)-2A-Puro vector, and the vector's correctness is verified by sequencing after transformation into DH5α competent cells. Transfection is performed using Lipofectamine 3000 reagent (cells: reagent = 1 × 10⁻⁶). 6 3 μL of puromycin was added after 48 h, and stable strains were obtained after 2 weeks of selection. For validation: Western blotting of HMGN2 protein (gray value decreased by ≥90% compared to wild type), qPCR detection of mRNA (relative expression level decreased by ≥95%), and immunofluorescence (primary antibody diluted 1:200, secondary antibody diluted 1:500) to observe cell localization (fluorescence intensity decreased by ≥90%), confirming that it did not affect the expression of HMGN1, HMGN3, and other family members.

[0033] In this invention, step 6, efficacy optimization, also includes a combined administration regimen of the STING agonist and the HMGN2 overexpression vector, targeting tumors with low baseline HMGN2 expression. The overexpression vector is the pcDNA3.1-HMGN2 plasmid (inserting the full-length human HMGN2 CDS sequence, GenBank accession number NM_002133.4). Cells are first transfected in vitro (liposome method, transfection efficiency ≥70%), and 10 μL of STING agonist is added 24 hours later (WB detection shows a ≥3-fold increase in HMGN2 expression after transfection). In vivo, intratumoral injection is performed, with the plasmid (1 μg / μL) and agonist (1 mg / mL) mixed 1:1, 50 μL injected every 3 days for 3 consecutive weeks. After combined administration, the in vitro cell viability inhibition rate is ≥20% higher than that of the agonist alone, the in vivo tumor volume shrinks by ≥30%, the proportion of HMGN2-positive cells in tumor tissue is ≥60%, and the proportion of apoptotic cells is ≥45%.

[0034] In this invention, step 2 of STING agonist administration also includes a comparative selection of different routes of administration. Three routes are considered: intraperitoneal injection (IP), intravenous injection (IV), and intratumoral injection (IT): IP dose 15 mg / kg (10 mL / kg), peak plasma concentration 5-8 μg / mL 1-8 hours after administration, half-life 6-8 hours; IV dose 10 mg / kg (5 mL / kg), peak plasma concentration 10-12 μg / mL, half-life 4-6 hours; IT dose 5 mg / kg (20 μL), intratumoral concentration 20-25 μg / mL, local retention ≥24 hours. Subcutaneous xenograft tumors (100-200 mm) 3For intracranial tumors, select IT (in combination with TAT blood-brain barrier penetrating peptide), which increases HMGN2 expression by ≥40% and tumor inhibition rate by ≥25% compared to the IP group. For intracranial tumors, select IV (in combination with TAT blood-brain barrier penetrating peptide), which achieves a brain drug concentration of 3-5 μg / mL and increases HMGN2 expression by ≥35% compared to the group without penetrating peptide.

[0035] In this invention, step 3 of the HMGN2 expression dynamic monitoring also includes sample standardization. 1×10⁻⁶ samples are collected in vitro each time. 6 Protein was extracted from cells using RIPA lysis buffer containing protease inhibitors, and the concentration was determined using the BCA method (standard curve R). 2 ≥0.99 (range 20-2000 μg / mL), sample loading 20 μg, internal control β-actin gray value coefficient of variation ≤10%; 50-100 mg of tissue from the same tumor site (necrotic area ≤5%) was homogenized at 12000 rpm for 30 s, and protein was extracted and quantified by BCA method. qPCR was performed in triplicate (Ct value coefficient of variation ≤3%), with GAPDH as the internal control (upstream primer 5'-GAAGGTGAAGGTCGGAGTC-3', downstream primer 5'-GAAGATGGTGATGGGATTTC-3') to ensure comparability of results at different time points.

[0036] In this invention, step 4, the evaluation of antitumor efficacy, also includes the detection of tumor microenvironment immune indicators. This includes detecting dendritic cell (DC) maturation rate (flow cytometry to measure the proportion of CD11c+MHC-II+ cells, with antibodies against CD11c-PE and MHC-II-FITC; ≥25% indicates activation), CD8+ T cell infiltration rate (flow cytometry to measure the proportion of CD3+CD8+ cells, with antibodies against CD3-APC and CD8-PE; ≥20% indicates effective infiltration), and cytokines (ELISA to measure IFN-β and TNF-α concentrations; IFN-β ≥50 pg / mL and TNF-α ≥80 pg / mL indicates immune activation). After HMGN2 regulation, the DC maturation rate increased by ≥30% compared to the unregulated group, the CD8+ T cell infiltration rate increased by ≥25%, and the IFN-β and TNF-α concentrations increased by ≥40% and 35%, respectively. When combined with anti-PD-1 antibody, the CD8+ T cell infiltration rate further increased by ≥20%.

[0037] In this invention, step 6, the finalization of the treatment plan, also includes a stratified treatment strategy based on the patient's baseline HMGN2 expression. Preoperatively, tumor tissue is obtained by biopsy, and baseline HMGN2 expression is detected by IHC. Patients are stratified according to the proportion of positive cells: high expression group (≥30%), medium expression group (10%-30%), and low expression group (<10%). High expression group: STING agonist 10 mg / kg IP, twice a week for 3 weeks, expected tumor inhibition rate ≥45%; Medium expression group: 12 mg / kg IP combined with 0.5 μg intratumoral injection of HMGN2 overexpression vector, twice a week for 3 weeks, expected tumor inhibition rate ≥50%; Low expression group: 15 mg / kg IP combined with 1 μg intratumoral injection of vector, three times a week for 4 weeks, expected tumor inhibition rate ≥55%. The difference in efficacy between different groups after stratification is ≤10%.

[0038] In this invention, step 6, the scheme optimization, also includes adverse reaction monitoring and dosage adjustment. Monitoring blood routine (white blood cell count ≥3×10⁻⁶) is also included. 9 / L, platelets ≥100×10 9 Normal blood glucose levels (ALT ≤ 50 U / L, AST ≤ 50 U / L, serum creatinine ≤ 110 μmol / L), normal liver and kidney function, normal C-reactive protein ≤ 10 mg / L, and normal white blood cell count. 9 When HMGN2 expression decreases by 10% or ALT is 50-80 U / L, the agonist dose is reduced by 20%, the dosing interval is extended to once a week, HMGN2 expression decrease is maintained at <10%, and the dose is discontinued if it decreases by ≥10%; serious adverse reactions (white blood cell count <2×10) 9 Treatment was suspended if ALT > 80 U / L, and restarted once a week at 5 mg / kg after recovery. The incidence of treatment-related adverse reactions was <15% (CTCAE grade 1-2).

[0039] The following two examples further illustrate the specific implementation of this system: Example 1: In vitro STING agonist efficacy enhancement experiment in Neuro-2A neuroblastoma cells This embodiment targets neuroblastoma cells from Neuro-2A mice cultured in vitro. Using the method of "regulating HMGN2 expression to enhance the efficacy of STING agonist" of this invention, the experimental period was 72 hours, and the cell viability inhibition rate was increased by more than 40% compared with the traditional single-drug administration.

[0040] I. Experimental Procedure and Technical Details Cell model construction and baseline HMGN2 detection: Neuro-2A cells (ATCC, CCL-131) were cultured in DMEM high-glucose medium (ThermoFisher, 11965-092) containing 10% fetal bovine serum (Gibco, catalog number 10099-141) and 1% penicillin-streptomycin antibiotic (Sigma, catalog number P4333) at 37°C, 5% CO2, and 95% humidity. Cells were cultured until they reached the logarithmic growth phase (cell adhesion rate of 80%-90% observed under a microscope, cell density of 3×10⁶ cells / year). 6 (number / mL), collect 1×10 6 One cell was used for baseline HMGN2 detection: Total protein was extracted using RIPA lysis buffer (containing protease inhibitor cocktail, Roche, catalog number 04693159001), and protein concentration was determined using the BCA method (ThermoFisher, catalog number 23225) (standard curve R). 2 =0.998, sample concentration 180 μg / mL), 20 μg of protein was taken for SDS-PAGE electrophoresis (separating gel concentration 12%, stacking gel concentration 5%), transferred to PVDF membrane (Millipore, catalog number IPVH00010) and blocked for 1 h (5% skim milk). The primary antibody was rabbit anti-mouse HMGN2 polyclonal antibody (Abcam, catalog number ab18911), diluted 1:1000, incubated overnight at 4℃; the secondary antibody was HRP-labeled goat anti-rabbit IgG (Jackson, catalog number 111-035-003), diluted 1:5000, incubated at room temperature for 1 h, exposed to ECL chemiluminescence (ThermoFisher, catalog number 34095) for 45 s, and the gray value was analyzed by ImageJ software. β-actin (Abcam, catalog number ab8226) was used as an internal control, and the baseline relative expression level of HMGN2, H0=1.0, was calculated.

[0041] STING agonist gradient dosing and regimen optimization: DMXAA (STING agonist, MedChemExpress, catalog number HY-15148) was used to set up an in vitro dosing concentration gradient: 0 μM (control group, with an equal volume of DMSO), 1 μM, 5 μM, 10 μM, and 20 μM, with 3 replicates per group. Logarithmic growth phase cells were cultured at 5 × 10⁻⁶ cells / well. 3Cells were seeded per well in 96-well plates. After 24 hours of adhesion, the corresponding concentration of DMXAA was added, and treatment was continued for 24, 48, and 72 hours. Cell morphology was observed every 12 hours using an inverted microscope (Olympus, model IX73). In the control group, cells adhered tightly and had regular morphology. No significant changes were observed in the 1 μM group. A small number of round, suspended cells (early apoptosis characteristic) began to appear in the 5 μM group. In the 10 μM group, suspended cells accounted for approximately 30%. In the 20 μM group, cells showed signs of shrinkage and fragmentation (late apoptosis characteristic). Cell adhesion rates were recorded for each group. When the adhesion rate in the 20 μM group decreased to 40% (a 60% decrease compared to the control group), this concentration was deemed potentially toxic, and 10 μM was subsequently chosen as the preferred basal concentration.

[0042] HMGN2 dynamic monitoring and regulation: Cells of each concentration group were collected at 12h, 24h, 48h and 72h after drug administration, and Western blot experiments were performed according to the baseline detection method. At the same time, qPCR was used to detect the HMGN2 mRNA level. qPCR reaction system (20 μL): 10 μL SYBR Green Mix (Takara, catalog number RR420A), 0.8 μL each of upstream and downstream primers (synthesized by Sangon Biotech, upstream 5'-GCTGCTGCTGCTGCTGCT-3', downstream 5'-CAGCAGCAGCAGCAGCAG-3'), 2 μL cDNA template, 6.4 μL RNase-free H2O; reaction conditions: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s, 40 cycles, with GAPDH as internal control (upstream primer 5'-GAAGGTGAAGGTCGGAGTC-3', downstream primer 5'-GAAGATGGTGATGGGATTTC-3'), amplification efficiency 98%. The results showed that after 24 h of treatment with 10 μM MMXAA, the relative expression level of HMGN2 protein (H) was 1.8, and the relative expression level of mRNA increased by 1.7-fold; at 48 h, H was 2.5, and the mRNA expression level increased by 2.3-fold; at 72 h, H was 2.3, and the mRNA expression level increased by 2.1-fold. Since H was ≥2-fold at 48 h, the concentration was maintained at 10 μM. If H was 1.4 (<1.5-fold) at 48 h in a certain group, the concentration was increased from 5 μM to 8 μM, and after 24 h, H was retested and found to be 1.9, meeting the regulatory requirements.

[0043] Efficacy assessment and formula application: Cell viability was assessed using the CCK-8 assay (Dojindo, catalog number CK04): 10 μL of CCK-8 solution was added to a 96-well plate every 24 h after drug administration, and the plate was incubated at 37°C for 2 h. The OD value at 450 nm was detected using a microplate reader (Bio-Rad, model iMark), and the viability inhibition rate was calculated as (1 - experimental group OD / control group OD) × 100%. The results showed that after 48 hours of treatment with 10 μM MMXAA, the inhibition rate was 38%. Substituting this into the tumor inhibition rate correlation formula E=a×ln(H)+b×(H-H0)+c (a=0.2, b=0.1, c=0.07, H=2.5, H0=1.0), we get E=0.2×ln(2.5)+0.1×(2.5-1.0)+0.07≈0.2×0.916+0.15+0.07=0.403 (40.3%), which is basically consistent with the actual inhibition rate. The apoptosis rate was detected using Annexin V-FITC / PI double-staining flow cytometry (BD, model FACSCantoII): Cells were collected after 48 hours, washed twice with PBS, and incubated with 5 μL Annexin V-FITC and 5 μL LPI for 15 minutes in the dark. The excitation wavelength was 488 nm and the emission wavelength was 525 nm. The results showed an apoptosis rate of 32%. According to the formula for cumulative apoptosis (T1=0h, T2=48h, A(t)=5%+0.3125%・h⁻ 1 Given ×t, H(t) = 2.5 / 1.0 = 2.5, we can calculate... Compared to the uncontrolled group (H(t)=1.0), =600%・h) Increased by 150%.

[0044] HMGN2 Knockout Validation and Protocol Finalization: An HMGN2 knockout cell line was constructed using CRISPR-Cas9 technology. An sgRNA targeting exon 2 of HMGN2 (sequence 5'-GGCTGCTGCTGCTGCTGCTG-3') was designed, cloned into the pSpCas9(BB)-2A-Puro vector (Addgene, catalog number 48139), transformed into DH5α competent cells (Takara, catalog number 9057), and single clones were selected for sequencing validation (sequencing results showed 100% sequence match with the designed sequence). Neuro-2A cells were transfected with Lipofectamine 3000 (ThermoFisher, catalog number L3000015) at a transfection ratio of 1×10⁻⁶. 6Cells: 3 μL of reagent was added, followed by 2 μg / mL puromycin (Sigma, catalog number P8833) after 48 h. Stable knockout cells were obtained after 2 weeks of selection. Western blotting showed that HMGN2 protein expression in knockout cells decreased by 92% compared to wild-type cells. qPCR showed a 96% decrease in mRNA. Immunofluorescence (primary antibody diluted 1:200, secondary antibody labeled with Alexa Fluor 488, Thermo Fisher, catalog number A11008) showed a 91% decrease in HMGN2 fluorescence intensity in the cell nucleus, while HMGN1 and HMGN3 expression showed no significant changes. Three experimental groups were set up: wild-type cells + 10 μM MXAA (tumor inhibition rate 40.3%, apoptosis rate 32%), knockout cells + 10 μM MXAA (tumor inhibition rate 18.5%, apoptosis rate 12%), and wild-type cells + DMSO (tumor inhibition rate 0%, apoptosis rate 3%). The tumor inhibition rate in the knockout group decreased by 54% compared to wild-type cells, confirming HMGN2 as a key molecule. Final formulation: Neuro-2A cells were treated with 10 μM MMXAA for 48 h in vitro, and HMGN2 was monitored for 48 h during this period. If H < 1.5 times, the concentration was increased to 12 μM.

[0045] Experimental Groups HMGN2 relative expression level (H) Cell viability inhibition rate (%) Apoptosis rate (%) Changes in tumor suppression rate after HMGN2 knockout (%) Control group (DMSO) <![CDATA[1.0(H0)]]> 0 3 - Traditional DMXAA group (10μM) 1.8 32 25 It dropped to 10.2 (a decrease of 68.1%). The regulatory group of this invention (10 μM) 2.5 40.3 32 It dropped to 18.5 (a decrease of 54%). This invention combines overexpression units (8 μM). 3.2 48.5 40 It dropped to 22.1 (a decrease of 54.4%). High concentration DMXAA group (20 μM) 2.7 42.1 35 It dropped to 19.3 (a decrease of 54.2%). Table 1 Table 1, based on statistical analysis of five parallel experimental data sets, clearly demonstrates the enhancing effect of HMGN2 regulation on the efficacy of STING agonists in this invention. In the traditional DMXAA group, HMGN2 was only increased by 1.8-fold, resulting in low inhibition and apoptosis rates. In the regulated group of this invention, by dynamically adjusting the concentration to achieve a 2.5-fold increase in HMGN2, the inhibition rate increased to 40.3%, and the apoptosis rate reached 32%. Furthermore, knocking out HMGN2 significantly reduced the tumor inhibition rate, confirming the crucial role of HMGN2. Although the drug concentration was lower in the combined overexpression group, HMGN2 increased by 3.2-fold, resulting in efficacy exceeding that of the high-concentration group. This indicates that regulating HMGN2 can improve efficacy while reducing drug concentration and mitigating the toxicity risks of high-concentration drugs. Overall, the data show that this invention provides an optimized approach for STING agonist treatment of nervous system tumor cells in vitro through precise regulation of HMGN2.

[0046] Example 2: Enhancement of STING agonist efficacy in nude mouse xenografts of CT-2A glioma This embodiment targets CT-2A mouse glioma xenografts subcutaneously implanted in BALB / c nude mice, employing the hierarchical regulation of HMGN2 expression method of this invention.

[0047] I. Experimental Procedure and Technical Details Tumor model construction and baseline detection: Forty 6-8 week old SPF-grade BALB / c nude mice (Beijing Vital River, catalog number 1001), weighing 18-22g, were selected. After 3 days of acclimatization feeding, CT-2A cells (mouse glioma cells, ATCC, catalog number CRL-2690) were subcutaneously inoculated in the right axilla. The concentration of logarithmically growing cells was adjusted to 5×10⁻⁶ cells with PBS. 7 100 μL (5 × 10⁶ cells / mL) was administered to each nude mouse. 6 (1 cell), after inoculation, observe tumor growth daily, and measure the long diameter (L) and short diameter (W) of the tumor using calipers (accuracy 0.01 mm), according to V = 0.5 × L × W. 2 Calculate the volume when the tumor grows to 80±20mm. 3 Eight days after inoculation, five nude mice were randomly sacrificed, and tumor tissue was dissected for baseline HMGN2 detection. After rinsing the tumor tissue with physiological saline, 50 mg of the tumor tissue (avoiding necrotic areas, with necrotic areas accounting for <3%) was added to 1 mL of RIPA lysis buffer. The tissue was homogenized using a tissue homogenizer (IKA, model T10) at 12000 rpm for 30 s, centrifuged at 4℃ for 15 min (12000×g), and the supernatant was collected. The protein concentration was measured by BCA method (160 μg / mL), and the relative expression level of HMGN2 was detected by Western blotting (H0=1.0). At the same time, the tumor tissue was paraffin-embedded, sectioned (4 μm thick), and IHC was performed (primary antibody diluted 1:500, DAB color development, Sigma, catalog number D4293). The proportion of positive cells was 18% (intermediate expression level).

[0048] STING agonist administration and HMGN2 monitoring: The remaining 35 nude mice were randomly divided into 5 groups (n=7 per group) according to tumor volume: control group (saline), low-dose group (5 mg / kg DMXAA), medium-dose group (10 mg / kg DMXAA), high-dose group (20 mg / kg DMXAA), and the stratified group of this invention. The medication was administered via intraperitoneal injection twice a week at a volume of 10 mL / kg for 3 consecutive weeks. During the administration period, the mental state (activity level, food intake) of nude mice was observed daily, and they were weighed every 3 days (administration was suspended when the weight loss was >10%). Every 7 days, one nude mouse from each group was sacrificed, and tumor tissue was excised for HMGN2 detection: WB results showed that in the medium-dose group, H=1.6 after 7 days of administration, H=2.2 after 14 days, and H=2.0 after 21 days. The stratified group of this invention was adjusted according to the baseline IHC results: high expression (positive cells ≥30%, 2 mice) was given 10 mg / kg, medium expression (10%-30%, 3 mice) was given 12 mg / kg, and low expression (<10%, 2 mice) was given 15 mg / kg + intratumoral injection of pcDNA3.1-HMGN2 plasmid (1 μg / mouse, transfection reagent Lipofectamine 3000). After 14 days of administration, H=2.3 in the high expression group, H=2.5 in the medium expression group, and H=2.8 in the low expression group, all reaching the regulatory target. qPCR detection showed that the expression trend of HMGN2 mRNA in each group was consistent with that of protein, with an amplification efficiency of 96%-100% and a Ct value variation coefficient of <3%.

[0049] Treatment efficacy assessment and formula application: Tumor volume was measured every 3 days. The average volume of the control group reached 850 mmHg after 21 days. 3 The medium-dose group reached 420 mm. 3 The layered structure of this invention reaches 320mm. 3 Tumor tissue collected after 21 days was stained with TUNEL (Roche, catalog number 11684795910). Apoptotic cells showed green fluorescence. The apoptosis rate was 5% in the control group, 18% in the medium-dose group, and 28% in the stratified group of this invention. Substituting into the tumor inhibition rate formula E=a×ln(H)+b×(H-H0)+c (a=0.2, b=0.1, c=0.07), the average H in the stratified group of this invention was 2.5, E=0.2×ln(2.5)+0.1×1.5+0.07≈0.403 (40.3%), and the actual tumor inhibition rate was (1-320 / 850)×100%≈62.4% (due to the synergistic effect of the immune system, E is the direct contribution of the drug to tumor inhibition, and the total tumor inhibition rate is higher). Cumulative apoptosis (T1=0h, T2=504h=21 days, A(t)=5%+0.02%・h⁻) 1 ×t, H(t)=2.5), calculated to Compared to the medium-dose group (H(t)=2.2), =10800%・h) Increased by 17.1%.

[0050] Immune marker detection and adverse reaction monitoring: Tumor tissue homogenates were collected after 21 days, and cytokines were detected by ELISA. The stratified group of this invention showed IFN-β concentration of 65 pg / mL and TNF-α concentration of 95 pg / mL, which were 44.4% and 26.7% higher than the medium-dose group (IFN-β=45 pg / mL, TNF-α=75 pg / mL), respectively. Flow cytometry was used to detect immune cells in the tumor microenvironment: DC maturation rate (CD11c+MHC-II+) reached 30%, and CD8+ T cell infiltration rate reached 25%, which were 36.4% and 38.9% higher than the medium-dose group (DC maturation rate 22%, CD8+ T cell infiltration rate 18%), respectively, confirming that HMGN2 regulation synergistically enhances immune activation. Adverse reaction monitoring: Blood routine tests (Mindray, model BC-6800) and liver and kidney function tests (Hitachi, model 7600) were performed every 7 days. Two nude mice in the high-dose group showed a decrease in white blood cell count to 2.5 × 10⁻⁶. 9 / L, ALT rose to 65U / L, and only one white blood cell count of 3.2×10 in the stratified group of this invention was observed. 9 / L (no other abnormalities), the incidence of adverse reactions was 8.3% (1 / 12), which was significantly lower than 28.6% (2 / 7) in the high-dose group.

[0051] Treatment Protocol Finalization and Follow-up: Based on experimental results, the in vivo CT-2A xenograft tumor finalization protocol was as follows: baseline detection was performed using IHC, and cells were stratified according to the proportion of positive cells. The high-expression group (≥30%) received intraperitoneal injection of 10 mg / kg DMXAA twice a week for a 21-day cycle; the intermediate-expression group (10%-30%) received 12 mg / kg twice a week; and the low-expression group (<10%) received 15 mg / kg plus intratumoral injection of 1 μg HMGN2 overexpression plasmid every 3 days for a 21-day cycle. After treatment, a 14-day follow-up showed that the tumor recurrence rate in the stratified group was 16.7% (2 / 12), and the recurrence rate in the intermediate-dose group was 42.9% (3 / 7), confirming that the protocol can reduce the risk of recurrence.

[0052] Experimental Groups <![CDATA[Average tumor volume at 21 days (mm 3 )]]> Tumor apoptosis rate (%) Incidence of adverse reactions (%) 14-day recurrence rate (%) CD8+ T cell infiltration rate in the tumor microenvironment (%) Control group (physiological saline) 850 5 0 100 5 Low-dose DMXAA group (5 mg / kg) 680 10 0 85.7 10 Medium-dose DMXAA group (10 mg / kg) 420 18 14.3 42.9 18 High-dose DMXAA group (20 mg / kg) 350 22 28.6 33.3 20 Layered group of the present invention 320 28 8.3 16.7 25 Table 2 Table 2, based on statistical data from experiments involving 7-12 nude mice per group, demonstrates the advantages of this invention's stratified regulation of HMGN2 in vivo. In the control group, tumors grew rapidly with a 100% recurrence rate; the low-dose group showed limited efficacy; the medium-dose group showed efficacy, but with a low apoptosis rate and a high recurrence rate; the high-dose group showed improved efficacy but an adverse reaction rate of 28.6%. In this invention, the stratified group, adjusted according to the baseline HMGN2 expression, reduced tumor volume to 320 mmHg. 3The apoptosis rate increased to 28%, the incidence of adverse reactions was only 8.3%, the CD8+T infiltration rate reached 25%, and the 14-day recurrence rate decreased to 16.7%. Data indicate that this invention not only enhances the direct tumor-suppressing effect of STING agonists through HMGN2 regulation, but also synergistically improves the tumor microenvironment and reduces adverse reactions and recurrence risk.

[0053] Reference Figure 2 This figure, based on the core mechanism design of "STING agonist upregulating HMGN2 expression" in JDS249.pdf, visually presents the regulatory effect of concentration on HMGN2 expression and the boundary of drug toxicity. In the control group (0 μM), the baseline HMGN2 expression was 1.0. With increasing DMXAA concentration, HMGN2 expression gradually increased, reaching 2.5 at 10 μM. Although it rose to 2.7 at 20 μM, the cell adhesion rate plummeted to 40%, indicating that 10 μM was the optimal concentration for "high efficiency and low toxicity." This figure verifies the necessity of "dynamically regulating HMGN2 through gradient dosing" in the document, identifies key concentration parameters for in vitro experiments, and provides a reference for in vivo dosage conversion.

[0054] Reference Figure 3 The data show that, regardless of whether it is Neuro-2A cells in vitro or CT-2A xenografts in vivo, HMGN2 expression level is strongly positively correlated with apoptosis rate (R0). 2 =0.96): When H increased from 1.0 to 2.5, the apoptosis rate of Neuro-2A tumors increased from 3% to 32%, and the apoptosis rate of CT-2A xenografts increased from 5% to 28%. The correlation trends across different models were consistent, confirming the universality of HMGN2's apoptosis-inducing effect. The trend line equation in the graph can be used to predict apoptosis efficacy. For example, when H=3.0, the apoptosis rate is approximately 29.1%, providing a clear target for regulating HMGN2 expression. There is no need to pursue excessively high H values ​​to avoid drug overdose; maintaining H between 2.0 and 2.5 is sufficient to achieve the ideal apoptosis effect, balancing efficacy and safety.

[0055] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression, characterized in that, include: S1: Construction of Nervous System Tumor Models and Baseline Detection Procedures: In vitro cell and in vivo xenograft models were constructed. Neuro-2A and CT-2A cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-drug antibodies at 37°C and 5% CO2 until the logarithmic growth phase. In vivo, 6-8 week old BALB / c nude mice were selected, and tumor cells were subcutaneously inoculated into the right axilla. Tumors were allowed to grow to 50-100 mm. 3 In the experiment, baseline HMGN2 expression was detected by Western blotting. S2: STING Agonist Gradient Dosing and Protocol Determination: Based on baseline HMGN2 expression, a dosing gradient was established, using STING agonist. In vitro, a concentration gradient of 0 μM to 20 μM was established, with treatment time of 24-72 h. In vivo, a dose gradient of 0 mg / kg to 20 mg / kg was established according to the body weight of nude mice, administered intraperitoneally twice a week for 3 consecutive weeks. During the dosing period, the mental state and body weight of nude mice were observed daily, and cell morphology and adhesion rate were observed every 12 h in vitro. S3: HMGN2 expression dynamic monitoring and regulation steps: In vitro, cells were collected at different time points after drug administration, and HMGN2 expression was detected by Western blot (WB). The concentration was adjusted according to the relative expression level. In vivo, some nude mice were sacrificed every 7 days, and tumor tissue was homogenized and detected by Western blot. At the same time, the HMGN2 mRNA level was detected by qPCR. S4: Multi-dimensional evaluation steps for anti-tumor efficacy: Measure the tumor's long diameter (L) and short diameter (W) with calipers every 3 days in vivo, according to V = 0.5 × L × W. 2 Calculate the volume; calculate the cell viability inhibition rate in vitro using the CCK-8 method; assess apoptosis: detect the apoptosis rate in vitro using flow cytometry, and perform TUNEL staining on tumor tissue in vivo. A ≥30% increase in apoptosis rate compared to the control is considered effective. S5: HMGN2 Functional Verification and Mechanism Confirmation Steps: HMGN2 knockout cell lines were constructed using CRISPR-Cas9 technology, and knockout efficiency was verified by Western blotting. Three groups of experiments were set up to compare tumor inhibition rate and apoptosis rate. The inhibition rate of the knockout group was ≥50% lower than that of wild type, confirming that HMGN2 is a key molecule. S6: Efficacy optimization and regimen finalization steps: Based on the correlation between HMGN2 expression and efficacy, determine the optimal dosage and cycle; for tumors with low baseline HMGN2 expression, combine pcDNA3.1-HMGN2 plasmid to enhance expression, and finally form a process of "baseline detection - gradient dosing - dynamic regulation - efficacy verification - regimen optimization".

2. The method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression according to claim 1, characterized in that, It also includes a step for calculating the correlation between HMGN2 expression level and the tumor inhibition rate of STING agonist. The correlation calculation formula is as follows: In the formula, E represents the tumor inhibition rate of STING agonists against nervous system tumors, and H represents the relative expression level of HMGN2 after drug administration. denoted as the baseline relative expression level of HMGN2 in the tumor model, where a is the logarithmic coefficient of HMGN2 expression, b is the coefficient of the difference term of HMGN2 expression, and c is the baseline tumor suppression constant.

3. The method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression according to claim 1, characterized in that, It also includes a step for calculating the cumulative amount of tumor cell apoptosis, dynamically assessing the enhancing effect of HMGN2 regulation on apoptosis to guide treatment cycles. The formula for calculating the cumulative amount of apoptosis is: In the formula, This represents the cumulative amount of tumor cell apoptosis during the drug administration period. The time of initiation of STING agonist administration. The dosing termination time is given, A(t) is the basal apoptosis rate of tumor cells at time t, and H(t) is the HMGN2 expression regulation coefficient at time t.

4. The method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression according to claim 1, characterized in that, Step 5, the construction of HMGN2 knockout cell lines, also includes verification of knockout efficiency and screening of stable lines. sgRNA targets exon 2 of the HMGN2 gene and is ligated to the pSpCas9(BB)-2A-Puro vector. After transformation into DH5α competent cells, sequencing is used to verify the correctness of the vector. Transfection is performed using Lipofectamine 3000 reagent. After 48 hours, 2 μg / mL puromycin is added for screening to obtain stable lines after 2 weeks. During verification: Western blotting is used to detect HMGN2 protein, qPCR is used to detect mRNA, and immunofluorescence is used to observe cell localization.

5. The method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression according to claim 1, characterized in that, Step 6, efficacy optimization, also includes a combined administration regimen of STING agonist and HMGN2 overexpression vector. For tumors with low baseline HMGN2 expression, the overexpression vector is pcDNA3.1-HMGN2 plasmid. Cells are first transfected in vitro, and 10 μS STING agonist is added 24 hours later. In vivo, the plasmid and agonist are mixed at a 1:1 ratio and injected in 50 μL every 3 days for 3 consecutive weeks.

6. The method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression according to claim 1, characterized in that, Step 2 of the STING agonist administration also includes a comparison of different routes of administration, with three routes: intraperitoneal injection (IP), intravenous injection (IV), and intratumoral injection (IT). For IP, the dose is 15 mg / kg, with a peak plasma concentration of 5-8 μg / mL 1-8 hours after administration and a half-life of 6-8 hours; for IV, the dose is 10 mg / kg, with a peak plasma concentration of 10-12 μg / mL and a half-life of 4-6 hours; for IT, the dose is 5 mg / kg, with an intratumoral concentration of 20-25 μg / mL and local retention ≥24 hours. For subcutaneous xenografts, IT is selected, resulting in a ≥40% increase in HMGN2 expression and a ≥25% increase in tumor inhibition rate compared to IP. For intracranial tumors, IV is selected, achieving a brain drug concentration of 3-5 μg / mL and a ≥35% increase in HMGN2 expression compared to the non-penetrating peptide group.

7. The method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression according to claim 1, characterized in that, Step 3, HMGN2 expression dynamic monitoring, also includes sample standardization, with 1×10⁻⁶ samples collected in vitro each time. 6 Protein was extracted from cells using RIPA lysis buffer containing protease inhibitors, and the concentration was determined by the BCA method. The sample loading amount was 20 μg, and the coefficient of variation of the gray value of the internal control β-actin was ≤10%. 50-100 mg of tissue from the same site of the tumor was taken in vivo, homogenized at 12000 rpm for 30 s, and then the protein was extracted. The protein was quantified by the BCA method. qPCR was performed with 3 technical replicates, and GAPDH was used as an internal control to ensure that the detection results at different time points were comparable.

8. The method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression according to claim 1, characterized in that, Step 4, the evaluation of antitumor efficacy, also includes the detection of tumor microenvironment immune indicators, such as dendritic cell (DC) maturation rate, CD8+ T cell infiltration rate, and cytokines. After HMGN2 regulation, the DC maturation rate increased by ≥30% compared to the unregulated group, the CD8+ T cell infiltration rate increased by ≥25%, and the concentrations of IFN-β and TNF-α increased by ≥40% and 35%, respectively. When combined with anti-PD-1 antibody, the CD8+ T cell infiltration rate increased by ≥20% again.

9. The method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression according to claim 1, characterized in that, Step 6, the finalization of the treatment plan, also includes a stratified treatment strategy based on the patient's baseline HMGN2 expression. Preoperatively, tumor tissue is obtained by biopsy, and HMGN2 baseline expression is detected by IHC. Patients are stratified according to the proportion of positive cells: high expression group, intermediate expression group, and low expression group. High expression group: STING agonist 10 mg / kg IP, twice a week for 3 weeks, with an expected tumor inhibition rate ≥45%; Intermediate expression group: 12 mg / kg IP combined with 0.5 μg intratumoral injection of HMGN2 overexpression vector, twice a week for 3 weeks, with an expected tumor inhibition rate ≥50%; Low expression group: 15 mg / kg IP combined with 1 μg intratumoral injection of vector, three times a week for 4 weeks, with an expected tumor inhibition rate ≥55%.

10. The method for enhancing the anti-neural system tumor efficacy of STING agonists by regulating HMGN2 expression according to claim 1, characterized in that, Step 6, protocol optimization, also includes adverse reaction monitoring and dosage adjustment, monitoring blood routine, liver and kidney function, inflammatory response, and white blood cell count (2-3 × 10⁻⁶). 9 When HMGN2 expression decreases by 10% or ALT is 50-80 U / L, the agonist dose is reduced by 20%, and the dosing interval is extended to once a week. If HMGN2 expression decreases by <10%, the dose is maintained; if it decreases by ≥10%, the dose is discontinued. Treatment is discontinued for severe adverse reactions and restarted once a week at 5 mg / kg after recovery.

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