Method for inhibiting amplification of MDSCs through targeted fatty acid binding protein FABP4

Abstract

The invention provides a method for inhibiting MDSCs amplification, which comprises the following steps: (1) acquiring mouse bone marrow cells, and culturing by using a culture medium; and (2) adding an FABP4 inhibitor into the culture system in the step (1), uniformly mixing, and continuously culturing. Research finds that the FABP4 inhibitor with proper concentration is added into an MDSCs differentiation induction system for treatment, so that the generation of MDSCs can be obviously inhibited, and a corresponding methodology is established. The MDSCs inhibit the immune response under many pathological conditions, the accumulation of which is closely related to the adverse clinical outcomes of various diseases, including tumors. Therefore, the method for inhibiting the generation of the MDSCs can provide a methodological basis for pathogenesis research of MDSCs related diseases and development of targeted drugs, and provides a research basis for immunotherapy taking the MDSCs as a target spot.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and particularly relates to a method for inhibiting MDSCs proliferation by targeting fatty acid binding protein FABP4. Background Art

[0002] Myeloid-derived suppressor cells (MDSCs) are pathologically activated neutrophils (PMN-MDSCs) and monocytes (M-MDSCs) with potent immunosuppressive activity. They are activated by cytokines released during long-term exposure to chronic infection, inflammation, autoimmune diseases, and cancer. These immunosuppressive cells are mostly immature myeloid cells, and their formation may be related to the blockage of neutrophil or monocyte differentiation pathways.

[0003] The phenotype of MDSCs in mice is CD11b + Gr-1 + It is mainly regulated by STAT1, STAT3, STAT6, NF-κB, endoplasmic reticulum stress signaling pathway, cAMP, cyclooxygenase (COX2), prostaglandin E synthase (PTGES), CCAAT enhancer binding protein β (CEBPβ), IRF8 and RB1 genes, and differentiates into different cell subsets to exert immunosuppressive effects.

[0004] The main characteristic of MDSCs is their ability to suppress immune responses, including those mediated by T cells, B cells, and NK cells. They modulate immune responses in many pathological conditions, and the accumulation of MDSCs is closely associated with poor clinical outcomes in cancer. Metabolic reprogramming is one of the hallmarks of cancer, where tumor cells reprogram their metabolism to maintain high energy demands to support rapid proliferation, survival, and differentiation. Competition for nutrients and oxygen in the tumor microenvironment forces immune cells to adapt their metabolism, and MDSCs can sense the environment and maintain their suppressive function against immune responses and pro-tumorigenic functions by selecting the most efficient metabolic pathways.

[0005] Therefore, inhibiting the production of MDSCs is of great value for studying the pathogenesis of MDSCs-related diseases (including tumors) and developing targeted drugs. Summary of the Invention

[0006] Based on this, the object of the present invention is to provide a method for inhibiting the proliferation of MDSCs by targeting the fatty acid binding protein FABP4, which can effectively inhibit the generation of MDSCs in an in vitro culture system.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions.

[0008] A first aspect of the present invention provides a method for inhibiting the proliferation of MDSCs, comprising the following steps:

[0009] (1) Obtaining mouse bone marrow cells and culturing them in culture medium;

[0010] (2) Add FABP4 inhibitor to the culture system of step (1), mix well, and continue culturing.

[0011] In some embodiments, the density of mouse bone marrow cells in the culture system of step (1) is 1×10 6 ~2.2×10 6 / mL.

[0012] In some embodiments, the culture medium in step (1) is RPMI 1640 culture medium comprising the following components at the following concentrations: 10 v / v% to 15 v / v% FBS, 5 ng / mL to 15 ng / mL GM-CSF.

[0013] In some embodiments, in step (2), the FABP4 inhibitor is added after the mouse bone marrow cells are cultured for 12 hours to 24 hours.

[0014] In some embodiments, the FABP4 inhibitor is BMS-309403.

[0015] In some embodiments, the final concentration of BMS-309403 in the culture system is 10 μM to 60 μM.

[0016] In some embodiments, the final concentration of BMS-309403 in the culture system is 20 μM to 40 μM.

[0017] A second aspect of the present invention provides use of a FABP4 inhibitor in preparing a kit for inhibiting MDSCs proliferation.

[0018] In some embodiments, the FABP4 inhibitor is BMS-309403.

[0019] In some embodiments, the final concentration of BMS-309403 in the MDSCs expansion system is 10 μM to 60 μM, preferably 20 μM to 40 μM.

[0020] Compared with the prior art, the present invention has the following beneficial effects.

[0021] The present invention has discovered that adding an appropriate concentration of a FABP4 inhibitor to an MDSC differentiation induction system can significantly inhibit the generation of MDSCs, and has established a corresponding methodology. MDSCs suppress immune responses in many pathological conditions, and their accumulation is closely associated with adverse clinical outcomes in a variety of diseases, including tumors. Therefore, the present method for inhibiting MDSC production can provide a methodological basis for studying the pathogenesis of MDSC-related diseases and the development of targeted drugs, providing a research foundation for immunotherapy targeting MDSCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of MDSCs ratio detection in each group are shown.

[0023] Figure 2 These are the results of cell apoptosis detection in each group. DETAILED DESCRIPTION

[0024] The experimental methods in the following examples of the present invention, where specific conditions are not specified, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0025] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0026] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0027] The following describes the method in conjunction with specific embodiments.

[0028] Example 1

[0029] This embodiment provides a method for inhibiting the proliferation of MDSCs in vitro, comprising the following steps:

[0030] 1. Preparation of Mouse Bone Marrow Single Cell Suspension

[0031] Six- to eight-week-old C57 mice were sacrificed by dislocation and soaked in 75% alcohol for approximately 30 seconds. The hind limbs were dissected, the muscles removed, and the femur and tibia separated. The femur and tibia were then soaked in 75% alcohol for 1-2 minutes. The surface of the 75% alcohol was then cleaned with D-Hank's solution. The ends of each femur and tibia were cut to expose the bone marrow cavity. A 5ml syringe was used to draw an appropriate amount of D-Hank's solution to flush out all cells in the bone marrow cavity. The cells were then transferred to a 15ml centrifuge tube. The cells were centrifuged at 600g and 4°C for 5 minutes. The supernatant was discarded, and the cells were resuspended in an appropriate amount of red blood cell lysis buffer and allowed to stand for 5 minutes. The cells were then lysed by adding an appropriate amount of D-Hank's solution. The cells were centrifuged at 600g and 4°C for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1ml of D-Hank's solution. The bone marrow cell suspension was filtered through a 200-mesh sieve and counted using an abalone counting chamber.

[0032] 2. Induction of MDSCs

[0033] In a 48-well plate, 1 × 10 6 Bone marrow cells were diluted to 1 ml with RPMI 1640 medium containing 10 v / v% FBS, and GM-CSF (Cat: 315-03-20UG, peproTech) at a final concentration of 10 ng / ml was added to the culture system for stimulation. The 48-well plate was placed in a 37°C, 5% CO2 incubator to culture MDSCs.

[0034] After MDSCs were induced and cultured in vitro for 12 h (Day 1), DMSO, 20 μM (final concentration in the culture system), and 40 μM (final concentration in the culture system) FABP4 inhibitor BMS-309403 (Cat: HY-101903, MCE) were added, respectively; on the third day (Day 3), the medium was changed and the same concentration of BMS-309403 was supplemented; on the fifth day (Day 5), cells were collected for flow cytometry detection.

[0035] 3. Flow Cytometry

[0036] On the fifth day of induction culture, cells from each group were collected and flow cytometry was used to detect MDSCs (CD11b + Gr-1 + ; CD11b, Cat: 101216, Biolegend; Gr-1, Cat: 108408, Biolegend) ratios are as follows:

[0037] (1) Collect cells from each group, place them in 5 ml flow cytometry tubes, centrifuge at 3500 rpm / min for 5 min, and discard the supernatant;

[0038] (2) Add approximately 5 ml of pre-chilled PBS and resuspend the cells by pipetting to remove residual serum and other impurities. Centrifuge at 3500 rpm / min for 5 min and discard the supernatant.

[0039] (3) Dilute the flow cytometry antibodies at a ratio of 1:400, i.e., add 100 μl PBS and 0.25 μl flow cytometry antibodies (CD11b and Gr1) to each sample; store the antibodies in the dark and centrifuge briefly before use; pipette the cells to mix thoroughly and incubate them in the dark at 4°C for 30 min;

[0040] (4) Add approximately 4 ml of pre-chilled PBS to terminate staining, centrifuge at 3500 rpm / min for 5 min, and discard the supernatant;

[0041] (5) Resuspend the cells in 500 μl of PBS containing 0.5 v / v% FBS per tube, filter through a 100-mesh cell sieve, and then detect using a flow cytometer.

[0042] The results of flow cytometry detection were as follows Figure 1 As shown, the left figure is a representative flow cytometry staining image, and the right figure is a bar graph. It can be seen that compared with the DMSO-treated group (solvent control group), the FABP4 inhibitor BMS-309403-treated group significantly inhibited the generation of MDSCs in the induction system and reduced the proportion of MDSCs in a concentration-dependent manner. The proportion of MDSCs in the induction system decreased with increasing BMS-309403 treatment concentration (DMSO: 50.7±1.212%; 20μM BMS-309403: 43.03±2.359%; 40μM BMS-309403: 22.37±1.692%).

[0043] The above results show that adding FABP4 inhibitor BMS-309403 to the MDSCs induction system can significantly inhibit the generation of MDSCs and reduce the proportion of MDSCs.

[0044] Example 2

[0045] This example detects cell apoptosis after treatment with the FABP4 inhibitor BMS-309403.

[0046] 1. Preparation of Mouse Bone Marrow Single Cell Suspension

[0047] Six- to eight-week-old C57 mice were sacrificed by dislocation and soaked in 75% alcohol for approximately 30 seconds. The hind limbs were dissected, the muscles removed, and the femur and tibia separated. The femur and tibia were then soaked in 75% alcohol for 1-2 minutes. The ends of each femur and tibia were cut to expose the bone marrow cavity. A 5ml syringe was used to draw an appropriate amount of D-Hank's solution to flush out all cells in the bone marrow cavity. The cells were then transferred to a 15ml centrifuge tube. The cells were centrifuged at 600g and 4°C for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1ml of D-Hank's solution. The cells were then centrifuged at 600g and 4°C for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1ml of D-Hank's solution. The bone marrow cell suspension was filtered through a 200-mesh sieve and counted using an abalone counting chamber.

[0048] 2. Induction of MDSCs

[0049] In a 48-well plate, 1 × 10 6 Bone marrow cells were diluted to 1 ml with RPMI 1640 medium containing 10 v / v% FBS, and GM-CSF (Cat: 315-03-20UG, peproTech) was added to stimulate the culture system at a final concentration of 10 ng / ml. The 48-well plate was placed in a 37°C, 5% CO2 incubator to induce MDSCs.

[0050] On the first day (Day 1) of MDSCs in vitro induction, DMSO and 40 μM (final concentration in the culture system) FABP4 inhibitor BMS-309403 (Cat: HY-101903, MCE) were added for treatment; on the third day (Day 3), the medium was replaced and the same concentration of BMS-309403 was supplemented.

[0051] 3. Apoptosis Detection

[0052] After five days of culture, cells were harvested and assayed for early, late, and total apoptosis using the Annexin V-FITC Apoptosis Detection Kit (Cat: AP101, Lianke Biotech). The assay steps were as follows: cells and culture supernatant were collected into flow cytometry tubes, centrifuged at 600g for 5 minutes at 4°C, and the supernatant discarded. For flow cytometry, cell surface staining was performed according to the instructions for the apoptosis detection kit. Cells were resuspended in Binding Buffer, and Annexin V and PI dyes were added. After mixing, the cells were incubated at room temperature in the dark for 10 minutes and analyzed immediately by flow cytometry.

[0053] Apoptosis detection results Figure 2As shown, the left figure is a representative flow cytometry staining image, and the right figure is a bar graph. It can be seen that compared with the DMSO-treated group, after BMS-309403 treatment, the proportion of total apoptosis, early apoptosis, and late apoptosis in the induced system were significantly increased (total apoptosis: DMSO: 52.33±3.041%; BMS-309403: 83.52±4.496. Early apoptosis: DMSO: 47.80±3.251%; BMS-309403: 73.90±4.530. Late apoptosis: DMSO: 4.527±0.2136%; BMS-309403: 9.620±1.431)

[0054] The above results indicate that BMS-309403 treatment can promote cell apoptosis in the induced system.

[0055] In summary, the method of inhibiting the production of MDSCs of the present invention can provide a methodological basis for the study of the pathogenesis of MDSCs-related diseases and the development of targeted drugs, and provide a research basis for immunotherapy targeting MDSCs.

[0056] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0057] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for inhibiting the proliferation of MDSCs, characterized in that: The following steps are involved: (1) Obtaining mouse bone marrow cells and culturing them in culture medium; (2) Add FABP4 inhibitor to the culture system of step (1), mix well, and continue culturing.

2. The method according to claim 1, wherein The density of mouse bone marrow cells in the culture system in step (1) is 1×10 6 ~2.2×10 6 / mL.

3. The method according to claim 1, wherein The culture medium in step (1) is RPMI 1640 culture medium containing the following components at the following concentrations: 10 v / v% to 15 v / v% FBS, 5 ng / mL to 15 ng / mL GM-CSF.

4. The method according to claim 1, wherein In step (2), the FABP4 inhibitor is added after the mouse bone marrow cells are cultured for 12 hours to 24 hours.

5. The method according to any one of claims 1 to 4, characterized in that The FABP4 inhibitor is BMS-309403.

6. The method according to claim 5, wherein The final concentration of BMS-309403 in the culture system is 10 μM to 60 μM.

7. The method according to claim 6, wherein The final concentration of BMS-309403 in the culture system is 20 μM to 40 μM.

8. Application of FABP4 inhibitors in the preparation of a kit for inhibiting MDSCs proliferation.

9. The use according to claim 8, characterized in that The FABP4 inhibitor is BMS-309403.

10. The use according to claim 9, characterized in that The final concentration of BMS-309403 in the MDSCs expansion system is 10 μM to 60 μM, preferably 20 μM to 40 μM.

Images