A method and use for enhancing nk cell tumor killing

Abstract

The application relates to the field of biological medicine, and relates to a method for enhancing the tumor killing effect of NK cells and application, in particular to a method and application of fraxidin in enhancing the tumor killing effect of NK cells; through experiment verification, it is proved that the fraxidin can improve the killing ability of NK cells to tumor cells under the hypoxic tumor microenvironment, and up-regulate the expression of active molecules on the surface of NK cells and NK cell activation receptors; the fraxidin is used to regulate the NK activity for the first time, and the method has important significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and relates to a method for enhancing tumor killing effect of NK cells and application, in particular to a method and application of Fraxinellone in enhancing tumor killing effect of NK cells. BACKGROUND

[0002] Fraxinellone (FRA) is a compound isolated from the root bark of Dictamnus dasycarpus, which is a tetrahydrobenzofuran derivative and has a wide range of biological activities. Studies have shown that Fraxinellone can reduce the occurrence of liver fibrosis by reducing the protein expression of CUGBP1 and increasing the expression level of IFN-γ. Many studies have also proved that Fraxinellone has an anti-tumor effect. In osteosarcoma, Fraxinellone inhibits the proliferation and migration of osteosarcoma cells HOS and MG63 in a dose-dependent manner, induces osteosarcoma cell apoptosis and increases autophagy flow. In lung cancer, Fraxinellone inhibits the expression of PD-L1 in a dose-dependent manner and reduces the expression of HIF-1α.

[0003] Natural killer cells (NK cells) are cytotoxic lymphocytes that can recognize tumor cells in a major histocompatibility complex (MHC)-independent manner, thereby spontaneously killing various tumor cells without the need for prior sensitization and HLA or MHC matching. NK cells can also induce apoptosis of target cells through activation of exocytosis perforin and granzyme, human apoptosis-related factor ligand and TNF-related apoptosis inducing ligand (TRAIL), or through antibody-dependent cellular cytotoxicity (ADCC), thus playing a crucial role in inhibiting tumor growth and metastasis and having good clinical prospects. Currently, there are many clinical trials studying the use of NK cell immunotherapy to treat hematological and solid malignant tumors.

[0004] However, there are still considerable challenges in treating cancer with NK cells, one of the reasons is that the immunosuppressive effect of tumor microenvironment (TME) limits its therapeutic effect. Hypoxia is a common feature of the tumor microenvironment of solid tumors. Under hypoxic stress, tumor, stroma and immune cells can secrete chemotactic factors to recruit immunosuppressive cells, and mainly through inducing HIF pathway and PI3K / AKT / mTOR and NFκB pathway to promote cancer progression and chemotherapy resistance. Under hypoxic conditions, cell antitumor immune response dysfunction can further lead to NK cell dysfunction, T cell apoptosis, and facilitate tumor survival. In the hypoxic environment, the cytolytic function of NK cells is impaired due to the reduced expression of partially activated receptors.

[0005] In order to avoid the decline of NK cell growth state and activity caused by pathological factors such as tumor microenvironment and affect the quality of cell therapy, it has become a common means to enhance the killing effect of NK cells by modifying or drug treatment. At present, methods such as chimeric antigen modification and genetic engineering technology are commonly used to modify NK cells, but the process is complicated and the cost is high, and the application is limited; IL-2, IL-15 and other drugs are also used to treat NK cells to improve their killing ability, but due to the large difference in sensitivity of NK cells of different individuals to drugs, sometimes good results cannot be achieved, and high concentration of IL-2 and other substances have the risk of triggering immune factor storm during patient medication. There are also reports of using a composition including carboxymethyl β-glucan sodium, resveratrol, tert-butyl hydroquinone and anisomycin to enhance, but the composition has complex components, including carboxymethyl β-glucan sodium, resveratrol, tert-butyl hydroquinone and anisomycin, and anisomycin has a significant inhibitory effect on T cells, which may cause immune suppression of other immune cells in the body in actual use, and the influence of tumor microenvironment on drug efficacy is also not considered. SUMMARY

[0006] The purpose of the present application is to provide a method and application for enhancing the tumor killing effect of NK cells, especially to provide the application and method of jujubinone in enhancing the tumor killing ability of NK cells.

[0007] In order to solve the above technical problems, the present application provides the following technical solutions:

[0008] The present application provides a method for enhancing the tumor killing effect of NK cells, which comprises adding 1-500 μM of jujubinone in the cell culture solution.

[0009] Preferably, a method for enhancing the tumor killing effect of NK cells comprises the following steps:

[0010] S1, peripheral blood mononuclear cell suspension preparation;

[0011] S2, NK cell negative selection;

[0012] S3, co-culture treatment: after 7 days of self-separation culture of NK cells, continue to culture in the medium containing fraxin for 7 days.

[0013] Preferably, the concentration of fraxin in the medium is 1-500 μM.

[0014] Preferably, a method for enhancing the tumor killing effect of NK cells is briefly summarized as Figure 1 As shown, the specific method comprises the following steps:

[0015] Step 1: Preparation of peripheral blood mononuclear cell suspension

[0016] Transfer the blood to a 50 mL centrifuge tube, centrifuge at 800g for 10 minutes, discard the upper plasma, dilute the blood cells with PBS to 1.5 times the volume of the whole blood, slowly add 30 mL of blood cell diluent to a 50 mL centrifuge tube pre-packaged with 15 mL of lymphocyte separation medium (Ficoll), centrifuge at 400g for 20 minutes, discard the upper diluent and plasma with a 10 mL pipette, slowly suck the white membrane layer and transfer it to a new 50 mL centrifuge tube; add appropriate PBS to the separated PBMC to 45 mL with a 25 mL pipette, mix well, centrifuge at 400g for 5 minutes; discard the supernatant, and wash the precipitate with PBS twice to obtain the peripheral blood mononuclear cell suspension;

[0017] Step 2: NK cell negative selection

[0018] Count the peripheral blood mononuclear cell suspension, 5x10 7 cells per mL of pre-cooled PBS, add 50 μL of EasySep TM Human NK Cell Isolation Cocktail magnetic beads per mL of cell suspension; mix well, incubate at room temperature for 5 minutes in the dark, vortex for 30 seconds; add 40 μL of RapidSpheres TM to each mL of sample and mix; continue to add medium to 2.5 mL to the system, place the suspension on a magnetic stand for 5 minutes, carefully suck out the suspension, which is the NK cells obtained by negative selection, centrifuge at 300g for 5 min to discard the supernatant, and resuspend the cells in cell culture medium and culture in a 37°C cell incubator;

[0019] Step 3: Co-culture treatment

[0020] On the third day after cell sorting, transfer the cells to a new culture bottle, add NK cell culture medium again for culture, start to add fraxin on the seventh day of culture, continue to culture until the 14th day to obtain the treated NK cells.

[0021] The application also provides a use of fraxinellone in enhancing the tumor killing ability of NK cells.

[0022] Further, the use includes a use in preparing a product for enhancing the tumor killing ability of NK cells.

[0023] Further, the use includes a use in preparing a product for promoting the tumor killing ability of NK cells in vivo and / or in vitro, or promoting the tumor killing ability of NK cells under a hypoxic tumor microenvironment.

[0024] Further, the product includes a medicine, which includes an injection and an oral agent.

[0025] Further, the tumor is a tumor of the intestinal tract, endometrial cancer, hepatocellular carcinoma, melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, biliary tract cancer, colorectal cancer, gastric cancer, glioblastoma, ovarian cancer, pancreatic cancer, or triple-negative breast cancer.

[0026] The application also provides a product for enhancing the tumor killing ability of NK cells, which includes fraxinellone.

[0027] Compared with the prior art, the application has the beneficial effects and significant progress in that:

[0028] The application first discovers that fraxinellone can significantly enhance the tumor killing ability of NK cells, up-regulate the expression of active molecules on the surface of NK cells, and activate receptors, thereby providing a new method for the preparation of immune cells for tumor immunotherapy, the expansion of NK cells, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 FIG. 1 is a schematic diagram of a method for enhancing the tumor killing effect of NK cells according to the application.

[0030] Figure 2 FIG. 2 is a curve of the viability of NK cells under the action of different concentrations of fraxinellone in Example 1.

[0031] Figure 3 FIG. 3 is a curve of the proliferation of NK cells cultured under conventional conditions.

[0032] Figure 4 FIG. 4 is a curve of the proliferation of NK cells under the action of different concentrations of fraxinellone in Example 1 (fraxinellone culture).

[0033] Figure 5 FIG. 5 is a verification result of the killing rate of K562 cells by NK cells in Example 2.

[0034] Figure 6 FIG. 6 is a verification result of the killing rate of MGC-803 cells by NK cells under normoxia in Example 2.

[0035] Figure 7 To verify the results of the killing rate of NK cells on MGC-803 cells in the hypoxic tumor microenvironment in Example 2. DETAILED DESCRIPTION

[0036] To make the purposes, technical solutions, beneficial effects and significant progress of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application.

[0037] Obviously, all the described embodiments are only part of the embodiments of the present application, not all the embodiments; all other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative labor belong to the scope of protection of the present application.

[0038] It should be understood that:

[0039] For those of ordinary skill in the art, the specific meanings of the above-mentioned terms in the present application can be understood according to specific circumstances.

[0040] It should also be noted that the following specific embodiments can be combined with each other, and the same or similar concepts or processes in some embodiments may not be described again.

[0041] The technical solutions of the present application will be described in detail below with specific embodiments.

[0042] Example 1: Effect of fraxinellone on the proliferation activity of NK cells

[0043] (1) Detection of cell viability and proliferation efficiency of NK cells

[0044] NK cells isolated from the peripheral blood of healthy volunteers were cultured, and after 7 days of self-isolation and culture, the NK cells were further cultured in medium containing or not containing 100 μM fraxinellone (medium preparation method: ALyS505 medium + 5% FBS + 1000 U IL-2, and the medium involved in the following examples is this medium) for 7 days. On days 0, 3, 5, 7, 9, 11 and 14 of culture, samples were taken for counting to obtain cell proliferation curve and cell viability data.

[0045] (2) Identification of NK cells

[0046] On the 14th day of cell culture, cell immunophenotype determination analysis was performed.

[0047] About 1.0 × 10 6The cells were centrifuged at 300 g for 5 min, resuspended in PBS after removing the supernatant, incubated with fluorescently labeled CD3 and CD56 antibodies (purchased from Thermo) and PBS resuspended cells, then washed twice with PBS, discarded the supernatant, resuspended in 0.5 mL PBS, and the treated cells were detected and analyzed by flow cytometry.

[0048] The experimental results prove that fraxinellone does not produce cytotoxicity to NK cells at a higher concentration (100 μM) and does not affect the proliferation of NK cells, as shown in Figures 2-4 ; the purity (after sorting of NK cells, a small amount of T cells and other impurity cells are still contained, if the culture conditions are not good, the impurity cells may be expanded and the purity of NK cells may decrease, so the purity here refers to the proportion of NK cells in total cells) after culture in the medium with or without fraxinellone is 88.56% and 91.36% respectively, and there is no obvious change, indicating that fraxinellone treatment also does not affect the purity of NK cells.

[0049] Example 2: Effect of fraxinellone on the killing activity of NK cells in normoxic and hypoxic tumor microenvironment

[0050] (1) Effect of fraxinellone on the killing of K562 tumor cells by NK cells

[0051] Experimental method: K562 (purchased from Fenghui Biotechnology) was used as target cells, the cell concentration was adjusted to 5 x 10 4 cells / mL, and was added to a 96-well plate at 100 μL per well; NK cells cultured in medium containing 10 μM, 30 μM and 100 μM fraxinellone (the medium was ALyS505 medium added with 5% FBS and 1000 U interleukin-2) for 7 days were used as effector cells; the effector: target ratio was 10:1, 5:1, 1:1, and 100 μL was added to each well; the control group had two groups, which were NK cell group cultured without drug (fraxinellone) and medium group containing 30 μM fraxinellone but without NK cells, each effector: target ratio had three replicate wells, and effector cell and target cell control wells were set in each group. After 6 hours of culture at 37°C and 5% CO2, centrifugation at 400 g for 5 min, the supernatant was aspirated, 100 μL of medium and 10 μL of CCK8 (ZETA) were added, and the culture was continued for 1.5 hours, the OD value at 450 nm was measured, the NK cell killing rate was calculated according to the following formula, and the different condition experimental groups and corresponding killing rates were plotted:

[0052] Killing rate (%) = {1-[(effector cell + target cell OD value) - effector cell OD value] / target cell OD value} x 100%

[0053] Experimental results: the results are as shown in Figure 5As shown, after NK cells were cultured with different concentrations of fraxinellone for 7 days, fraxinellone could enhance the killing effect of NK cells on K562 cells. Compared with the control group, when the concentration of fraxinellone was ≥30 μM, the killing activity of NK cells on K562 cells could be significantly improved under different effector-target ratios (p<0.05); the killing activity of fraxinellone combined with NK cells on K562 cells was much higher than that of fraxinellone alone.

[0054] (2) Effect of fraxinellone on the killing effect of NK cells on MGC-803 gastric cancer cells

[0055] Experimental method: The MGC-803 target cells (Fenghui Biological) were adjusted to 8×10 4 cells / mL, 100 μL / well was added to the 96-well plate, and after incubation for 48 hours under normoxic and hypoxic conditions, the NK cells cultured in the medium containing 10 μM, 30 μM and 100 μM fraxinellone for 7 days were added as effector cells according to the effector-target ratio of 10:1, 5:1 and 1:1, 100 μL per well; the control group was 2, which was the NK cell group cultured without drug (fraxinellone) and the medium group containing 30 μM fraxinellone without NK cells, 3 replicate wells were set for each effector-target ratio, and effector and target control wells were set for each group. According to the original normoxic and hypoxic grouping, it was placed in 37°C, 5% CO2 for 6 hours, 100 μL supernatant was aspirated, 10 μL CCK8 was added, and then it was incubated for 1.5 h, the OD value at 450 nm was measured, the killing rate of NK cells was calculated, and the different condition experimental groups and corresponding killing rates were plotted.

[0056] Experimental results: After NK cells were cultured with fraxinellone for 7 days, fraxinellone could enhance the killing effect of NK cells on MGC-803 cells. Compared with the control group, when the concentration of fraxinellone was ≥30 μM, the killing activity of NK cells on MGC-803 cells could be significantly improved under different effector-target ratios (p<0.05), as shown in Figure 6 and Figure 7 .

[0057] The experimental results prove that fraxinellone can significantly improve the killing activity of NK cells on tumor cells K562 and MGC-803.

[0058] Example 3: Effect of fraxinellone on the phenotype and activation molecule expression of NK cells

[0059] Experimental method: flow cytometry detection of NK cell surface molecules. NK cells were cultured in medium containing different concentrations of fraxinellone (final concentration 25, 50, 100 μM) for 7 days, and a control group was set up at the same time, and the medium was changed every 2 days. The cells were collected, washed twice with PBS, and then 0.25% BSA was added for blocking for 30 minutes, followed by washing twice with PBS, and then monoclonal antibodies CD3, CD56, CD69, CD107a, CD314 (Thermo) were added for incubation for 30 minutes, and then the cells were washed twice with PBA, and then flow cytometry analysis was performed.

[0060] Experimental results: the flow cytometry detection results of cell phenotype are as follows:

[0061] Table 1. NK cell phenotype and activation molecule antibody expression percentage

[0062]

[0063] The experimental results prove that fraxinellone can improve the ability of NK cells to secrete perforin and granzyme by up-regulating the expression of CD69, CD107a and granzyme B, and the ability of NK cells to recognize tumor cells, and ultimately achieve the effect of improving the ability of NK cells to kill tumor cells.

[0064] Finally, it should be noted that:

[0065] The above embodiments are only used to illustrate the technical solutions of the present application, and are not a limitation thereof;

[0066] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or some or all of the technical features can be replaced by equivalents, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application. Non-essential improvements and adjustments or replacements made by those skilled in the art based on the content of the present application are within the scope of the present application.

Claims

1. A method for enhancing the tumor-killing effect of NK cells, characterized in that, The method includes adding morrhuone during NK cell culture.

2. The method according to claim 1, characterized in that, The concentration of morrhuone is 1-500 μM.

3. The method according to claim 1, characterized in that, The enhanced tumor-killing effect of NK cells includes: promoting the tumor-killing ability of NK cells in vitro, or promoting the tumor-killing ability of NK cells in a hypoxic tumor microenvironment.

4. The method according to claim 1, characterized in that, The tumors include intestinal tumors, endometrial cancer, hepatocellular carcinoma, melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, urothelial carcinoma, biliary tract cancer, gastric cancer, glioblastoma, ovarian cancer, pancreatic cancer, or triple-negative breast cancer.

5. The method according to claim 4, characterized in that, The intestinal tumors include colorectal cancer.

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