Use of arachidonic acid inhibitors to improve the immune microenvironment of a tumor

Abstract

The application relates to application of arachidonic acid inhibitors in preparation of drugs for improving tumor immune microenvironment. + The arachidonic acid inhibitors improve the ratio and expression amount of perforin and IFN-gamma secreted by T cells and improve the ratio and expression amount of IFN-gamma and IL-17A secreted by Treg cells.

Description

Technical Field

[0001] This invention relates to the field of tumor immunology, and more specifically to the application of arachidonic acid inhibitors in the preparation of drugs that improve the tumor immune microenvironment. Background Technology

[0002] The tumor microenvironment (TME) is a nutrient-poor, hypoxic, and acidic environment in which effector T cells can only switch from oxidative phosphorylation to aerobic glycolysis. However, due to nutrient deficiency and insufficient glucose, the survival and function of effector T cells are hindered. Regulatory T cells (Tregs), on the other hand, rely on oxidative phosphorylation and can utilize lactate and fatty acid metabolism, and are considered to have proliferative and functional advantages within the TME. Because of the suppressive effect of Treg cells on immune cells, Treg cells in the tumor immune microenvironment are now considered an obstacle to effective anti-tumor immunity.

[0003] As the main regulatory cells of the immune system, Treg cells can trigger harmful autoimmune responses due to systemic depletion. Therefore, inhibiting Treg cell function while avoiding Foxp3 expression dysregulation is crucial in the anti-tumor process. The phenomenon of upregulated IFN-γ secretion in Treg cells without a significant decrease in Foxp3 expression is known as Treg cell fragility, a state between stability and functional imbalance. Fragile Treg cells cannot inhibit CD8. + T cells secrete IFNg, restoring CD8 + The tumor-killing function of T cells. Existing literature indicates that effective tumor therapies against PD1 and GITR exhibit a fragile Treg cell phenotype; therefore, inducing Treg cells to acquire fragility and increasing CD8+ are crucial. + The effector function of T cells may be an important means to improve the tumor immune microenvironment and enhance the efficacy of tumor immunotherapy.

[0004] In recent years, mounting evidence has shown that inflammation and angiogenesis in the tumor microenvironment are associated with tumorigenesis. Endogenous lipid compounds and locally acting small molecule mediators, collectively known as arachidonic acid (AA), play a central role in inflammation and tissue homeostasis, and are related to cancer development. The most well-known AAs are metabolites of arachidonic acid (AA), including prostaglandins, leukotrienes, and cytochrome P450 (CYP)-derived metabolites. These AAs may be involved in tumor survival and growth, as well as in mechanisms that promote tumor progression.

[0005] Arachidonic acid (ARA) is an essential fatty acid stored in membrane phospholipids and is an ω-6 polyunsaturated fatty acid. ARA metabolites are ligands in the PPAR pathway, capable of regulating the PPAR pathway and thus altering T cell differentiation fate. Prostaglandins and leukotrienes, as ARA metabolites, are known as PPAR agonists and have the function of regulating PPAR pathway activity. PPAR is the peroxisome proliferator-activated receptor, a subfamily of the transcription factor nuclear hormone receptor superfamily, including three subtypes: PPARα, PPARγ, and PPARδ (also known as PPARβ). As a key metabolic regulator, PPAR guides the differentiation, proliferation, and fate of various immune cell types. Therefore, ARA may affect the fate of immune cells in a tumor environment by influencing PPAR pathway activity-related proteins through metabolism. Summary of the Invention

[0006] This invention was made in view of the above-mentioned prior art, and its purpose is to provide the application of arachidonic acid inhibitors in the preparation of drugs that improve the tumor immune microenvironment.

[0007] To address the aforementioned problems, the inventors conducted in-depth research. Their results represent the first discovery that the use of arachidonic acid inhibitors can restore CD8 levels by inducing fragility in Treg cells within the tumor immune microenvironment. + The tumor-killing function of T cells can improve the tumor immune microenvironment. Therefore, this invention provides the following aspects:

[0008] 1. Application of arachidonic acid inhibitors in the preparation of drugs that improve the tumor immune microenvironment.

[0009] 2. The application according to item 1, wherein the arachidonic acid inhibitor enhances the effect of CD8. + The ratio and expression levels of perforin and IFN-γ secreted by T cells.

[0010] 3. The application according to item 1, wherein the arachidonic acid inhibitor increases the ratio and expression level of IFN-γ and IL-17A secreted by Treg cells.

[0011] 4. The application according to any one of items 1 to 3, wherein the arachidonic acid inhibitor is methyl arachidonic fluorophosphonate.

[0012] 5. The application according to any one of items 1 to 3, wherein the concentration of arachidonic acid inhibition is 1 to 5 mg / kg.

[0013] 6. The application according to any one of items 1 to 3, wherein the tumor is selected from melanoma, liver cancer, and subcutaneous tumor.

[0014] According to the present invention, arachidonic acid inhibitors can restore CD8 by inducing fragility in Treg cells in the tumor immune microenvironment. + T cells' tumor-killing function can improve the tumor immune microenvironment, thus having a positive impact on improving tumor immunity and laying a theoretical foundation for improving the clinical efficacy of tumor treatment.

[0015] Brief description of the attached diagram

[0016] Figure 1 This image shows the tumor phenotype results in a liver cancer model using different concentrations of arachidonic acid inhibitors in Example 1 of this invention. (A is a photograph of the tumor size; B is the ratio of tumor weight to mouse weight)

[0017] Figure 2 In Example 2 of this invention, CD4 in the tumor immune microenvironment after using different concentrations of arachidonic acid inhibitors in a liver cancer model were analyzed. + T cells and CD8 + The status of T cells. (A represents CD4) + T cell proportion and number; B is CD8 + (T cell ratio and number)

[0018] Figure 3 In Example 2 of this invention, CD4 in the tumor immune microenvironment after using different concentrations of arachidonic acid inhibitors in a liver cancer model were analyzed. + The differentiation of T cells. (A represents CD4) + The proportion and number of T cells differentiating into Th1 cells; B is CD4. + (The proportion and number of T cells differentiating into Th17 cells)

[0019] Figure 4 In Example 2 of this invention, CD8 in the tumor immune microenvironment after using different concentrations of arachidonic acid inhibitors in a liver cancer model were analyzed. + The molecular composition of T cells as effector cells. (A represents effector CD8) + The proportion and expression of Gzmb in T cells; B represents effector CD8. + The proportion and expression of perforin in T cells; C represents effector CD8. + (Proportion and expression of IFN-γ in T cells)

[0020] Figure 5 This diagram illustrates the Treg cell-related conditions in the tumor immune microenvironment after using different concentrations of arachidonic acid inhibitors in a liver cancer model according to Example 2 of this invention. (A represents the proportion and number of Treg cells; B represents the proportion and expression level of IFN-γ secreted by Treg cells; C represents the proportion and expression level of IL17-A secreted by Treg cells.)

[0021] Figure 6 The images show the tumor phenotype results in the subcutaneous tumor model of Example 3 of this invention, after injection of 1×PBS solvent in the control group and after treatment with arachidonic acid inhibitor. (A is a photograph of tumor size; B is a growth curve of subcutaneous tumor volume; C is tumor weight)

[0022] Figure 7 In Example 3 of this invention, the control group injected with 1×PBS solvent and the group treated with arachidonic acid inhibitors were compared to the CD4+ group in the tumor immune microenvironment of the subcutaneous tumor model. + T cells and CD8 + The status of T cells. (A represents CD4) + T cell proportion and number; B is CD8 + (T cell ratio and number)

[0023] Figure 8 In Example 3 of this invention, the control group injected with 1×PBS solvent and the group treated with arachidonic acid inhibitors were compared to the CD4+ group in the tumor immune microenvironment of the subcutaneous tumor model. + The differentiation of T cells. (A represents CD4) + The proportion and number of T cells differentiating into Th1 cells; B is CD4. + (The proportion and number of T cells differentiating into Th17 cells)

[0024] Figure 9 In Example 3 of this invention, the control group injected with 1×PBS solvent and the group treated with arachidonic acid inhibitors were compared to CD8+ in the tumor immune microenvironment. + The molecular composition of T cells as effector cells. (A represents effector CD8) + The proportion and expression of IFN-γ in T cells; B represents effector CD8. + (Perforin proportion and expression in T cells)

[0025] Figure 10 This section describes the Treg cell-related conditions in the tumor immune microenvironment of the subcutaneous tumor model in Example 3 of this invention, after injection of 1×PBS solvent in the control group and after use of arachidonic acid inhibitor. (A represents the proportion and number of Treg cells; B represents the proportion and expression level of IFN-γ secreted by Treg cells; C represents the proportion and expression level of IL17-A secreted by Treg cells).

[0026] Detailed Implementation of the Invention

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0028] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0029] The mice used in this experiment were Wild-type (WT)C57BL / 6, with a genotype of Foxp3. -YFP-cre They are raised under normal conditions, with a 12-hour light and dark cycle, and free access to food and water.

[0030] In this experiment, melanoma was used as the tumor, specifically B16-F10 melanoma cells (purchased from ATCC). These B16-F10 melanoma cells were cultured in RPMI-1640 (C3010-0500, VivaCell) medium containing 10% fetal bovine serum (C04001-020, BI) and 1% triple antibody (S120JV, BasalMedia) at 37°C in a 5% CO2 incubator. After approximately 80% confluence, the cells were passaged to obtain the B16-F10 cell line. All subsequent experiments used cells exhibiting exponential growth.

[0031] The arachidonic acid inhibitor used in this experiment was methyl arachidonicylfluorophosphonate (MAFP, 188404-10-6, Cayman). Due to the low concentration used, 1×PBS buffer (BL302A, Biosharp) was used as the solvent to dissolve the drug.

[0032] Example 1: Phenotypic observation of the therapeutic effects of different concentrations of arachidonic acid inhibitors on tumors

[0033] To determine whether arachidonic acid inhibitors are effective in treating tumors, wild-type mice bearing tumors were first treated with different concentrations of arachidonic acid inhibitors, and the phenotypic changes of the tumors in the mice after treatment with arachidonic acid inhibitors were observed.

[0034] 1. Experimental materials and treatment

[0035] MAFP working solution: The initial concentration of MAFP is 10 mg / kg. According to the reagent instructions, use 1×PBS to dilute the concentration to obtain working solutions of different concentrations. Prepare fresh solution before use.

[0036] 2. Experimental Methods

[0037] 2.1 Establishing a tumor model

[0038] Mice were anesthetized, and a 1cm incision was made in the epidermis and peritoneum below the left rib using surgical scissors. The spleen was gently pulled out of the abdominal cavity, and 5 × 10⁶ B16-F10 cells were injected into the spleen using a 1mL insulin syringe. 5 Each mouse was injected with a volume of 100 μL. Five minutes were allowed after injection to allow sufficient time for the cells to circulate to the liver via the bloodstream. Five minutes later, the blood vessels at both ends of the spleen were ligated with absorbable surgical sutures, and the spleen was immediately removed. The endothelium was then sutured with absorbable surgical sutures, and finally the outer skin was secured with a skin suture clamp. The mice were kept under anesthesia throughout the entire procedure, and aseptic techniques were followed.

[0039] 2.2 Use of MAFP for tumor treatment

[0040] Starting from the fifth day after tumor establishment, mice were treated with MAFP via intraperitoneal injection at a frequency of once every two days for a total of five times. The injection concentrations were set at 0 mg / kg, 1 mg / kg, 5 mg / kg, and 10 mg / kg, with an injection volume of 200 μL.

[0041] 2.3 Observation of anti-tumor effects

[0042] After treatment, on day 14 of tumor establishment, the mice were sacrificed. Before sacrifice, the mice's body weight was measured, the liver organs were photographed, and the weight of the liver tumor was measured to observe the phenotype of the liver tumor.

[0043] 3. Results Analysis

[0044] The results are as follows Figure 1 Mice injected with MAFP at a concentration of 10 mg / kg could not tolerate the drug stimulation and all died. In contrast, mice treated with 1 mg / kg and 5 mg / kg MAFP showed lower levels of liver cancer development and exhibited a better anti-tumor phenotype; the results are as follows. Figure 1 B, Compared with the blank control group of 0 mg / kg, mice treated with 5 mg / kg MAFP had a lower ratio of liver tumor to body weight, milder tumor symptoms, and a better anti-tumor phenotype.

[0045] Example 2: Analysis of the effects of different concentrations of arachidonic acid inhibitors on the tumor immune microenvironment

[0046] Experiment 1 has shown that arachidonic acid inhibitors have good anti-tumor effects. In order to analyze their impact on the tumor immune microenvironment, this experiment carried out flow cytometry analysis of lymphocytes in the tumor environment.

[0047] 1. Experimental materials and treatment

[0048] Related flow cytometry antibodies:

[0049] PE:Gzmb(MHGB04,eB)IL17A(12-7177-81,eB)

[0050] Percp-cy5.5:CD44(560570, BD)IFNg(45-7311-80, eB)

[0051] PE-cy7:CD44(25-0441-82, eB)

[0052] APC:CD45.2(109814,Biolegend)Perforin(154304,Biolegend)

[0053] APC-eFluor 780:CD45.2 (47-0454-82, eB)

[0054] PB450:NK1.1 (562921, BD)

[0055] KO525: CD4 (563106, BD) CD8 (563068, BD)

[0056] RP10 medium: Add 10% fetal bovine serum to 1640 medium to obtain RP10 medium.

[0057] 100% Percoll: Mix 100% Percoll by mixing Percoll stock solution (17089109, Cytiva) with 10×PBS buffer (BL316A, Biosharp) at a ratio of 9:1.

[0058] 42% Percoll: Mix 42 mL of 100% Percoll with 58 mL of 1×PBS to prepare a 42% Percoll solution. Prepare and use immediately.

[0059] 70% Percoll: Mix 70 mL of 100% Percoll with 30 mL of 1×PBS to prepare a 70% Percoll solution. Prepare and use immediately.

[0060] FACS buffer: Add 5% fetal bovine serum to 1×PBS buffer to obtain FACS buffer.

[0061] 2. Experimental Methods

[0062] 2.1 Isolation of liver tumor lymphocytes

[0063] Wild-type mice bearing tumors were treated with arachidonic acid inhibitors at concentrations corresponding to the group names, with the specific procedures being the same as in Example 1. On day 14 of tumor establishment, the mice were sacrificed, and liver tumor tissue was extracted.

[0064] Place a 200-mesh stainless steel sieve on a 10cm cell culture dish, then place the liver on the sieve and add a small amount of RP10 medium to grind it. Transfer the grinding solution to 50mL centrifuge tubes and add an appropriate amount of RP10 medium to make each tube contain 40mL of liquid.

[0065] Invert the centrifuge tube to mix the liquid, then immediately centrifuge at 4°C, 50g for 1 minute. Transfer the supernatant to a new 50mL centrifuge tube. Make up the volume to 40mL, centrifuge at 4°C, 500g for 5 minutes, discard the supernatant, and keep the precipitate in the centrifuge tube.

[0066] First, add 3 mL of 70% Percoll to a new 15 mL centrifuge tube, then resuspend the precipitate obtained in the previous step with 3 mL of 42% Percoll. After mixing thoroughly, gently add it to the 70% Percoll.

[0067] Perform density gradient centrifugation. Centrifuge settings: room temperature 23℃, 750g, ramp rate 6, drop rate 2, centrifuge for 30 min.

[0068] After centrifugation, gently remove the centrifuge tube from the centrifuge and use a 3mL Pasteur dropper to aspirate the lymphocytes in the middle white membrane layer. Transfer the cells to a new 15mL centrifuge tube, add an appropriate amount of RP10 culture medium to bring the liquid to 10mL, invert the tube to mix, and centrifuge at 4℃, 500g for 5min. Discard the supernatant and keep the liver lymphocytes precipitated at the bottom of the centrifuge tube.

[0069] Add 1 mL of red blood cell lysis buffer, resuspend and mix the cells, let stand for 3 min, add RP10 medium to a volume of 10 mL, mix by inverting, centrifuge at 4°C, 500 g for 5 min, and discard the supernatant.

[0070] Resuspend the precipitated cells in 1 mL of FACS buffer and filter to transfer to a new 1.5 mL EP tube for subsequent cell counting and flow cytometry staining.

[0071] 2.2 Flow cytometry detection of cell surface molecules

[0072] Add 100 μL of the cell suspension obtained in step 2.1 to each well of a 96-well plate.

[0073] Centrifuge at 4℃, 1500 rpm for 5 min, discard the supernatant, and collect the cell pellet at the bottom of a 96-well plate.

[0074] The external standard antibody was mixed with FACS buffer at a certain ratio and added to each well of a 96-well plate, 100 μL per well. The cells were resuspended and incubated at 4°C in the dark for 40 min, with the mixture being resuspended by pipetting every 20 minutes.

[0075] After incubation, add 100 μL of FACS buffer to each well of a 96-well plate, centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant, and the cell pellet will settle at the bottom of the 96-well plate.

[0076] Resuspend the precipitated cells in FACS buffer, add 200 μL to each well, mix well, filter through a 0.45 μm filter, and then transfer to a flow cytometer.

[0077] Adjust the parameters according to the flow cytometer cell detection instructions and perform the detection.

[0078] 2.3 Flow cytometry detection of intracellular molecules

[0079] Add 100 μL of the cell suspension obtained in step 2.1 to each well of a 96-well plate.

[0080] Centrifuge at 4℃, 1500 rpm for 5 min, discard the supernatant, and collect the cell pellet at the bottom of a 96-well plate.

[0081] The external standard antibody was mixed with FACS buffer at a certain ratio and added to each well of a 96-well plate, 100 μL per well. The cells were resuspended and incubated at 4°C in the dark for 40 min, with the mixture being resuspended by pipetting every 20 minutes.

[0082] Using the Transcription Factor Staining Buffer Set (2511819, ThermoFisher Scientific), prepare the membrane permeation solution fresh according to the instructions, add 100 μL to each well of a 96-well plate, resuspend the precipitated cells, and incubate at 4°C in the dark for 1 hour, mixing once every 30 minutes.

[0083] After cell disruption, add 100 μL of diluted Wash Buffer (from the kit) to each well of a 96-well plate. Centrifuge at 2500 rpm for 5 min at 4°C, discard the supernatant, and allow the cell pellet to settle at the bottom of the 96-well plate.

[0084] Mix the internal standard antibody with FACS buffer at a certain ratio and add 100 μL to each well of a 96-well plate. Resuspend the cell pellet and incubate at 4°C in the dark for 40 min, mixing once every 20 minutes.

[0085] After incubation, add 100 μL of FACS buffer to each well of a 96-well plate, centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant, and the cell pellet will settle at the bottom of the 96-well plate.

[0086] Resuspend the precipitated cells in FACS buffer, add 200 μL to each well, mix well, filter through a 0.45 μm filter, and then transfer to a flow cytometer.

[0087] Adjust the parameters according to the flow cytometer cell detection instructions and perform the detection.

[0088] 2.4 Flow cytometry detection of transcription factors

[0089] Add 250 μL of cell suspension to a flow cytometer, followed by 250 μL of RP10 medium to make a 500 μL system. Then add PMA (final concentration 30 ng / mL), iomycin (final concentration 1 μg / mL), monensin (final concentration 2.5 μg / mL), and BFA (final concentration 0.6 μL / mL), mix well, and incubate at 37°C in a 5% CO2 incubator for 5-6 hours.

[0090] After stimulation, centrifuge at 4°C and 1500 rpm for 5 min, discard the supernatant, and the cell pellet settles at the bottom of the flow cytometer.

[0091] Resuspend the cell pellet in FACS buffer, adding 200 μL to each tube, mix well, and transfer to a 96-well plate. Centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant, and the cell pellet will settle at the bottom of the 96-well plate.

[0092] The external standard antibody was mixed with FACS buffer at a certain ratio and added to each well of a 96-well plate, 100 μL per well. The cells were resuspended and incubated at 4°C in the dark for 40 min, with the mixture being resuspended by pipetting every 20 minutes.

[0093] Using the Transcription Factor Staining Buffer Set kit, prepare the membrane permeation solution fresh according to the instructions, add 100 μL to each well of a 96-well plate, resuspend the precipitated cells, and incubate at 4°C in the dark for 1 hour, mixing once every 30 minutes.

[0094] After cell disruption, add 100 μL of diluted Wash Buffer (from the kit) to each well of a 96-well plate. Centrifuge at 2500 rpm for 5 min at 4°C, discard the supernatant, and allow the cell pellet to settle at the bottom of the 96-well plate.

[0095] Mix the internal standard antibody with FACS buffer at a certain ratio and add 100 μL to each well of a 96-well plate. Resuspend the cell pellet and incubate at 4°C in the dark for 40 min, mixing once every 20 minutes.

[0096] After incubation, add 100 μL of FACS buffer to each well of a 96-well plate, centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant, and the cell pellet will settle at the bottom of the 96-well plate.

[0097] Resuspend the precipitated cells in FACS buffer, add 200 μL to each well, mix well, filter through a 0.45 μm filter, and then transfer to a flow cytometer.

[0098] Adjust the parameters according to the flow cytometer cell detection instructions and perform the detection.

[0099] 3. Results Analysis

[0100] like Figure 2 As shown, MAFP does not affect CD4 in the tumor immune microenvironment. + T cells and CD8 + The proportion and number of T cells. Simultaneously... Figure 3 Display CD4 + There was no significant difference in the proportion and number of Th1 and Th17 cells differentiated from T cells. However, Figure 4 The results showed that the detection effect CD8 + Functional molecules of T cells showed that, compared with the control group of 0 mg / kg, treatment with 5 mg / kg MAFP resulted in increased effector CD8+. + The proportions of perforin and IFN-γ secreted by T cells were significantly increased, and their expression levels showed an upward trend. This indicates that injection of 5 mg / kg MAFP for tumor treatment can significantly increase CD8 levels. + The anti-tumor function of T cells. Subsequently, this experiment analyzed Treg cells, which play an important regulatory role in the tumor immune microenvironment, and the results are as follows: Figure 5 As shown, although there was no significant difference in the proportion of Treg cells among the groups, compared with the control group, treatment with 5 mg / kg MAFP resulted in Treg cells secreting a significantly higher proportion of IFN-γ and IL-17A. This indicates that injection of 5 mg / kg MAFP for tumor treatment can significantly increase the fragility of Treg cells in the tumor environment and reduce the immunosuppressive function of Treg cells.

[0101] Example 3: Detecting the effect of arachidonic acid inhibitors on improving the tumor immune microenvironment in different tumor models

[0102] Examples 1 and 2 have demonstrated the therapeutic effect of the arachidonic acid inhibitor MAFP on improving the tumor immune microenvironment in liver cancer. To verify the generalizability of MAFP's therapeutic effect, wild-type mice were subcutaneously tumor-bearing using B16-F10 cells to observe the tumor therapeutic effect of MAFP and its effect on improving the tumor immune microenvironment under different tumor models. In Example 3, the control group (Vehicle) received 200 μL of 1×PBS, while the experimental group received MAFP at a concentration of 5 mg / kg.

[0103] 1. Experimental Methods

[0104] 1.1 Establishing a tumor model

[0105] The fur on the abdomen and back of the mice was shaved off with a shaver. B16-F10 cells were then injected subcutaneously using a 1mL insulin syringe, at a concentration of 1×10⁶ cells per mouse. 5 Each injection volume is 100 μL.

[0106] 1.2 Tumor volume tracking measurement

[0107] Starting from the fifth day after tumor formation, the tumor was measured every two days using vernier calipers. The tumor volume formula is V = (length × width × width) / 2.

[0108] 1.3 Phenotypic observation of the therapeutic effect of MAFP on tumors

[0109] On day 14 of tumor establishment, mice were sacrificed, and subcutaneous tumors were photographed to observe tumor size.

[0110] 1.3 Flow Cytometry Analysis of the Tumor Immune Microenvironment

[0111] Following the method in Example 2, flow cytometry was used to detect the proportions and expression of surface molecules, internal molecules, and transcription factors of lymphocytes in a tumor environment.

[0112] 2. Results Analysis

[0113] The results are as follows Figure 6 As shown, compared with the control group injected with 1×PBS solvent, mice treated with MAFP showed a trend towards smaller tumor volume and weight. Specific analysis of the tumor immune microenvironment revealed results consistent with those of the liver cancer model. Figure 7 As shown, CD4 + T CD8 + There was no significant difference in the proportion and quantity of T. CD4 + T cell differentiation status, such as Figure 8 As shown, there was no significant difference in the proportion and number of Th1 and Th17 cells. The effector CD8 was detected. + Functional molecules of T cells, such as... Figure 9As shown, compared with the control group, the MAFP treatment group had effector CD8 in the tumor immune microenvironment. + The proportion of IFN-γ secreted by T cells was significantly increased, and the proportion of perforin showed an increasing trend. Analysis of Treg cells yielded the following results: Figure 10 As shown, although there was no significant difference in the proportion of Treg cells between the two groups, the proportion and expression of IFN-γ and IL-17A in Treg cells were significantly higher in the MAFP treatment group than in the control group. This indicates that MAFP can enhance CD8 expression in the subcutaneous tumor model. + The effector function of T cells makes Treg cells in the tumor immune microenvironment fragile and improves the tumor immune microenvironment. This result is consistent with the liver cancer model and has universality.

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of arachidonic acid inhibitors in the preparation of drugs that improve the tumor immune microenvironment, wherein the arachidonic acid inhibitor is methyl arachidonic fluorophosphonate, and wherein the tumor is melanoma.

2. The application according to claim 1, wherein the arachidonic acid inhibitor enhances the effect of CD8. + The ratio and expression levels of perforin and IFN-γ secreted by T cells.

3. The application according to claim 1, wherein the arachidonic acid inhibitor increases the ratio and expression level of IFN-γ and IL-17A secreted by Treg cells.

4. The application according to any one of claims 1 to 3, wherein the concentration of arachidonic acid inhibition is 1 to 5 mg / kg.

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