Application of up-regulating PRDM6 gene expression level in improving NK cell anti-tumor activity

Abstract

This invention belongs to the field of biomedical technology and relates to the application of upregulating PRDM6 gene expression levels in enhancing the anti-tumor activity of NK cells. It involves inducing high expression of the endogenous PRDM6 gene in NK cells through a combination of IgG1 subtype antibody and cytokines, or by transfecting cells with an overexpressing PRDM6 gene to obtain activated NK cells. The average positive rate of PRDM6 protein in activated NK cells reaches 4.5%-6.02%, and the secretion levels of chemokines such as CCL3, CCL4, CCL5, CXCL9, and CXCL10 are increased, which can promote dendritic cell activation and TCF1. + CD8 + T-cell infiltration. These activated NK cells can be used alone or in combination with PD-1 inhibitors to prepare anti-tumor drugs, and can also serve as predictive biomarkers for the efficacy of PD-1 inhibitors, showing promising applications in immunotherapy for various tumors such as lung cancer and esophageal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of upregulating PRDM6 gene expression level in enhancing the anti-tumor activity of NK cells. Background Technology

[0002] Natural killer (NK) cells are among the most important cytotoxic effector cells in innate immunity. They can rapidly eliminate tumor cells without prior antigen sensitization by integrating activation / inhibition receptor signals to recognize stress-transformed cells. Simultaneously, they secrete cytokines such as IFN-γ and TNF, as well as various chemokines, participating in shaping the tumor immune microenvironment and influencing subsequent adaptive immune responses. Within the framework of tumor immune surveillance, NK cells not only perform the "first wave" of non-specific clearance but also drive immune cascade responses through cytokine networks. The IFN-γ axis, in particular, plays a pivotal role in anti-tumor immunity: IFN-γ promotes antigen presentation-related pathways, enhances myeloid cell immune activation, and is associated with stronger T-cell effector responses in various models. Furthermore, bidirectional communication between NK cells and dendritic cells (DCs) is considered a key bridge connecting innate and adaptive immunity: DCs from secondary lymphoid organs can drive IFN-γ secretion and proliferation / survival of NK cells, suggesting a clear functional division and temporal synergy within the DC-NK axis at the tissue level. This intercellular synergistic mechanism provides a biological basis for the therapeutic strategy of amplifying adaptive immunity with NK cells in anti-tumor immunity. Furthermore, multiple studies have shown that NK cell activation can enhance the efficacy of immune checkpoint PD-1 inhibitors. Therefore, NK cells have become immunotherapeutic cells with significant clinical application value.

[0003] At the epigenetic regulatory level, the PR / SET domain family (PRDM) comprises a group of genes whose protein products share the conserved N-terminal PR [PRDI-BF1 (positive regulatory domain I binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1)] homologous domains, structurally and functionally similar to the SET [Su(var)3-9, enhancer and trithoracic] domains catalyzed by histone methyltransferases (HMTs). PRDM family proteins participate in the epigenetic regulation of gene expression through their intrinsic HMTase activity or interactions with other chromatin-modifying enzymes. In this way, they regulate a wide range of biological processes, including proliferation and differentiation control, cell cycle progression, and the maintenance of immune cell homeostasis. PRDM family proteins are also involved in cancer development, invasion, and metastasis, and altered expression is associated with poor prognosis and clinical outcomes. Although NK cells have been widely used in tumor immunotherapy and their combined use with PD-(L)1 inhibitors has shown some potential, how to effectively activate NK cells, enhance their chemokine secretion and the immune regulation capacity of the DC-NK-T cell axis remains an unsolved technical problem in this field.

[0004] Furthermore, PRDM6 is a member of the PRDM transcriptional repressor family with histone methyltransferase activity, and its expression characteristics and functions in NK cells have not been previously reported or studied. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides an application of upregulating PRDM6 gene expression levels in enhancing the anti-tumor activity of NK cells. By upregulating PRDM6 gene expression levels in NK cells, the secretion levels of chemokines such as CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, XCL1, and LTA are increased, thereby activating TCF1 by activating dendritic cells. + CD8 + T cells exert anti-tumor effects, and the combination of activated NK cells with PD-1 inhibitors can enhance the anti-tumor efficacy.

[0006] The technical solution adopted by this invention to solve the technical problem is: The first aspect of this invention provides an application of upregulating PRDM6 gene expression levels in enhancing the anti-tumor activity of NK cells.

[0007] Preferably, the method of upregulating PRDM6 gene expression level includes any of the following: (1) Inducing endogenous PRDM6 expression using IgG1 subtype antibody combined with cytokines; or (2) Overexpression of PRDM6 via gene transfection.

[0008] A second aspect of the present invention provides activated NK cells (PRDM6 cells) that highly express the PRDM6 gene. + NK cells (NK cells) showed upregulated PRDM6 gene expression levels compared to unactivated or low-expressing PRDM6 cells, with an average PRDM6 protein positivity rate of 4.5%-6.02%, and possessed at least the following functional characteristics: (1) Increased secretion levels of one or more of the chemokines CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, XCL1, LTA, and LTB; (2) Promotes dendritic cell activation and TCF1 + CD8 + T-cell infiltration.

[0009] Preferably, the activated NK cells are activated by any of the following methods: (a) Induction of endogenous PRDM6 gene expression using IgG1 subtype antibody in combination with cytokines; or (b) Overexpression of the PRDM6 gene via gene transfection.

[0010] A third aspect of the present invention provides the application of the activated NK cells described above in the preparation of antitumor drugs.

[0011] Preferably, the tumor is a lung cancer tumor or an esophageal cancer tumor.

[0012] The fourth aspect of this invention provides the application of the aforementioned activated NK cells combined with a PD-1 inhibitor in the preparation of an antitumor drug.

[0013] The fifth aspect of the present invention provides the use of the activated NK cells described above as a detection marker in the preparation of a kit for predicting the efficacy of PD-1 inhibitor therapy.

[0014] Preferably, the PD-1 inhibitor is an anti-PD-1 antibody (anti-PD1).

[0015] The advantages and positive effects of this invention are: (1) This invention is the first to discover and utilize a novel target on NK cells—the PRDM6 gene. By upregulating the expression level of the PRDM6 gene in NK cells, activated NK cells (PRDM6) are obtained. + Increased secretion levels of one or more of the chemokines CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, XCL1, LTA, and LTB in NK cells can enhance their anti-tumor effects. (2) PRDM6 upregulates PRDM6 gene expression level+ NK cells can promote local TCF1 in tumors + CD8 + T-cell infiltration, and its combination with PD-1 inhibitors, can enhance anti-tumor efficacy; (3) PRDM6 + NK cells can improve the clinical efficacy of cancer patients receiving PD-1 antibody therapy and have a synergistic effect on PD-1 antibody therapy. PRDM6 in neoadjuvant cancer patients... + High NK cell expression is associated with improved treatment efficacy, suggesting that PRDM6... + NK cells can serve as a prognostic factor for the effectiveness of PD-1 inhibitor therapy. Attached Figure Description

[0016] Figure 1 The graph shows the results of PRDM6 protein expression testing in activated NK cells obtained by different activation methods; where A is a flow cytometry gating plot and B is a statistical bar chart.

[0017] Figure 2 For the detection of IgG1-NK cells and IgG1-NK cells by flow cytometry PRDM6oe Cells, PRDM6 + A comparison of the secretion of cytokines and the chemokine CCL3 in the NK-induced group; where A is a flow cytometry gating plot and B is a bar chart.

[0018] Figure 3 For the detection of IgG1-NK cells and IgG1-NK cells by flow cytometry PRDM6oe Cells, PRDM6 + A comparison of the secretion of cytokines and the chemokine CCL4 in the NK-induced group; where A is a flow cytometry gating plot and B is a bar chart.

[0019] Figure 4 For the detection of IgG1-NK cells and IgG1-NK cells by flow cytometry PRDM6oe Cells, PRDM6 + A comparison of cytokine and chemokine CCL5 secretion in the NK-induced group; where A is a flow cytometry gating plot and B is a bar chart.

[0020] Figure 5 For the detection of IgG1-NK cells and IgG1-NK cells by flow cytometry PRDM6oe Cells, PRDM6 + A comparison of cytokine and chemokine CXCL9 secretion in the NK-induced group; where A is a flow cytometry gating plot and B is a bar chart.

[0021] Figure 6For the detection of IgG1-NK cells and IgG1-NK cells by flow cytometry PRDM6oe Cells, PRDM6 + Comparison of cytokine and chemokine CXCL10 secretion in the NK-induced group; where A is a flow cytometry gating plot and B is a bar chart.

[0022] Figure 7 For the detection of IgG1-NK cells and IgG1-NK cells by flow cytometry PRDM6oe Cells, PRDM6 + A comparison of the secretion of cytokines and the chemokine LTA in the NK-induced group; where A is a flow cytometry gating plot and B is a bar chart.

[0023] Figure 8 For the detection of IgG1-NK cells and IgG1-NK cells by flow cytometry PRDM6oe Cells, PRDM6 + Figure 1 shows the results of PRDM6 protein expression assay in the NK-induced group; where A is a flow cytometry gating plot and B is a bar chart.

[0024] Figure 9 For PRDM6 + Flowcharts of mouse treatment models using NK cells alone and in combination with anti-PD1 therapy; Figure 10 For PRDM6 + In vivo imaging images of mice at different time points after NK cell therapy alone and in combination with anti-PD1; Figure 11 This is a fluorescence bar chart of the four groups of cells on day 21 after adoptive treatment in Example 3; P<0.05 P<0.01 P<0.001, P<0.0001.

[0025] Figure 12 For PBS, IgG1-NK, PRDM6 + NK, PRDM6 + CD8 of NK+anti-PD1 group + T cells at CD45 + The flow cytometry results show the percentage of immune cells; where A is a flow cytometry gating chart and B is a bar chart.

[0026] Figure 13For PBS, IgG1-NK, PRDM6 + NK, PRDM6 + IFNγ in NK+anti-PD1 group + CD8 + T cells at CD45 + The flow cytometry results show the percentage of immune cells; where A is a flow cytometry gating chart and B is a bar chart.

[0027] Figure 14 For PBS, IgG1-NK, PRDM6 + NK, PRDM6 + TCF1 of NK+anti-PD1 group + CD8 + T cells at CD45 + The flow cytometry results show the percentage of immune cells; where A is a flow cytometry gating chart and B is a bar chart.

[0028] Figure 15 For PBS, IgG1-NK, PRDM6 + NK, PRDM6 + NK+anti-PD1 group PD1 + CD8 + T cells at CD45 + The flow cytometry results show the percentage of immune cells; where A is a flow cytometry gating chart and B is a bar chart.

[0029] Figure 16 For PBS, IgG1-NK, PRDM6 + NK, PRDM6 + CD11c of NK+anti-PD1 group + XCR1 + DC cells in CD45 + The flow cytometry results show the percentage of immune cells; where A is a flow cytometry gating chart and B is a bar chart.

[0030] Figure 17 For PBS, IgG1-NK, PRDM6 + NK, PRDM6 + CD80 of NK+anti-PD1 group + CD86 + The flow cytometry results show the proportion of cells in cCD1 immune cells; where A is a flow cytometry cell gating plot and B is a bar chart.

[0031] Figure 18 PRDM6 for esophageal cancer neoadjuvant therapy patients +The correlation analysis results of NK cell infiltration and treatment response are shown in the figure, where A represents the PRDM6 concentration in tumor tissues of neoadjuvant therapy patients in the MPR and non-MPR groups. + Schematic diagram of NK cell expression; B represents PRDM6 in tumor tissues of neoadjuvant therapy patients in the MPR and non-MPR groups. + Comparison of NK cell expression. Detailed Implementation

[0032] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0033] Example 1 PRDM6 + In vitro induction, isolation and identification of NK cells A method for preparing PRDM6 by Herceptin + cytokine induction + The NK cell method involves the following steps: 1. Isolation of human peripheral blood mononuclear cells (PBMCs): (1) Take peripheral blood from healthy volunteers and add it to a 15mL centrifuge tube. Dilute it 1:1 with PBS buffer (product number 03.15018C, pH=7.4, EallBio) to obtain diluted blood. (2) Add human peripheral blood lymphocyte separation solution (product number P8610, Solarbio) to another centrifuge tube. Add the diluted blood slowly along the centrifuge tube wall to the upper layer of the lymphocyte separation solution at a ratio of lymphocyte separation solution to diluted blood of 1:1~2. (3) Centrifuge at 1800 rpm at room temperature for 20 minutes, setting the acceleration to 9 and the deceleration to 0. After centrifugation, aspirate the white membrane layer (PBMC layer) with a sterile pipette and transfer it to a new centrifuge tube. Add PBS buffer to wash the cells, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Aspirate the cell pellet and resuspend the cell pellet with PBS buffer to obtain the PBMC cell suspension. Adjust the cell concentration to 2 × 10⁻⁶ cells / mL. 6 cell / mL.

[0034] 2. Herceptin combined with cytokines induces PRDM6 + Experimental steps for NK cells: Preparation of antibody incubation solutions: 10 μL of human IgG1 antibody (1 μg / mL, Sigma-Aldrich) was diluted to 10 mL with PBS buffer and thoroughly mixed. This antibody incubation solution was designated IgG1-NK. 0.5 mL of Herceptin antibody (1 μg / mL, Roche Shanghai) was diluted to 10 mL with PBS buffer and thoroughly mixed. This antibody incubation solution was designated PRDM6.+ NK-induced group; (1) Add the above two antibody incubation solutions into T25 cell culture flasks, spread them evenly on the bottom of the flasks, and freeze them overnight at 4°C for antibody coating; (2) Take out the antibody-coated bottle from step (2), aspirate the antibody incubation solution, wash the bottom of the bottle with PBS buffer, discard the PBS wash solution, and then add 10 mL of PBMC suspension, for a total of 2 × 10⁻⁶. 7 Add serum-free culture medium (GT-T551 H3, Takara) containing cytokines IL-2 (500-1000 IU / mL, Peprotech) and IL-15 (50-100 ng / mL, Peprotech) to each flask.

[0035] (3) After culturing at 37℃ for 7-10 days, perform cell counting, supplement with the corresponding cytokines and culture medium, and adjust the cell concentration to 1.0×10⁻⁶. 6 cell / mL, continue culturing; (4) Adjust the cell concentration every 2-3 days and supplement with fresh culture medium containing the same concentration of cytokines until 14 days of culture, then harvest the cells for phenotypic and functional testing.

[0036] Setting up the negative control PBMC-NK group: Take 10 mL of the isolated PBMC suspension, totaling 2 × 10⁻⁶. 7 Cells were added to serum-free medium (GT-T551 H3, Takara) containing cytokines IL-2 (500-1000 IU / mL, Peprotech) and IL-15 (50-100 ng / mL, Peprotech) to adjust the cell concentration to 1×10⁻⁶ cells / mL. 6 Cells were cultured at 37°C for 7-10 days, with fresh culture medium containing the same concentration of cytokines added every 2-3 days to adjust the cell concentration to 1.0 × 10⁻⁶ cells / mL. 6 cell / mL. This group was not treated with antibody coating and served as a negative control.

[0037] 3. PRDM6 + Flow cytometry identification of NK cells PRDM6 + The flow cytometry identification steps for NK cells are as follows: Take the three groups of single-cell suspensions prepared in step 2, and put 1×10⁻⁶ cells into each flow cytometry tube. 6Cells were dissolved in 100 mL of PBS buffer. First, cell surface staining was performed by adding surface staining antibodies (live / dead, anti-CD3, anti-CD56) sequentially. After standing for 20 min, an appropriate amount of PBS was added, and the cells were centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was discarded after centrifugation. Next, nucleation was performed by adding 1 mL of nucleation buffer (catalog number 00-5523-00, Invitrogen) to each flow cytometry tube. After standing for 20 min, 3 mL of nucleation washing buffer was added, and the cells were centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was discarded after centrifugation. Anti-PRDM6 antibody was added sequentially, and staining was performed in the dark for 30 min. Finally, an appropriate amount of PBS buffer was added, and the cells were centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was discarded after centrifugation. Cells were resuspended in 200 μL of staining buffer, protected from light with aluminum foil, and stored at 4 °C for analysis.

[0038] Test results as follows Figure 1 As shown, the PBMC-NK group hardly expresses PRDM6. + The PRDM6 positive expression rate in the NK induction group was significantly higher than that in the IgG1-NK control group, indicating that NK cells with high PRDM6 expression were successfully obtained. + (NK cells). The differences between the groups were statistically significant.

[0039] Example 2: Construction of IgG1-NK cells by overexpressing PRDM6 in lentivirus PRDM6oe Cells and their functional verification NK cells were activated and expanded by using antibody incubation combined with viral transfection to increase PRDM6 expression levels, as detailed below: (1) Preparation of IgG1 antibody incubation solution: human IgG1 antibody (1ug / mL, Sigma-Aldrich) was added to PBS and diluted to 10mL. The solution was mixed thoroughly and set aside. This group was designated as IgG1-NK group. (2) Increase the expression level of PRDM6 in NK cells through viral transfection to prepare IgG1-NK cells. PRDM6oe cell; 1) Take the PBMC cell suspension from step 1 of Example 1, and aliquot 2 mL of cell suspension into 15 mL sterile centrifuge tubes, for a total of 4 portions; 2) Infect the target cells with lentivirus carrying the PRDM6 gene at a dose of 10 MOI, and add 8 μL / mL of the infection enhancer HitransGA (REVG004, Jikai Gene); 3) Place the cell suspension containing the lentivirus carrying the PRDM6 gene back into the incubator and incubate for 20 minutes; 4) After incubation, centrifuge at 800g, 32℃ for 30 minutes; 5) Remove the supernatant virus solution and resuspend the infected cells in 2 mL of fresh culture medium. Transfer each cell suspension to each well of a 6-well plate and incubate for 18 hours (this can be extended to 72 hours or more depending on the cell condition).

[0040] 6) On the second day after viral infection, collect the infected cells by centrifugation and resuspend them in 2 mL of fresh culture medium. Transfer the cells to a new culture dish and incubate overnight.

[0041] 7) After 72 hours of infection, collect cells and, if necessary, add serum-free medium containing cytokines IL-2 (500-1000 IU / mL, Peprotech) and IL-15 (50-100 ng / mL, Peprotech) for amplification culture, adjusting the cell concentration to 1.0 × 10⁻⁶. 6 cell / mL. This group was counted as IgG1-NK. PRDM6oe cell.

[0042] (3) PRDM6 + NK-induced group (same as Example 1) Flow cytometry was used to detect IgG1-NK group and IgG1-NK PRDM6oe Cell group, PRDM6 + The secretion of cytokines and chemokines in the NK-induced group was determined as follows: Take the three groups of prepared single-cell suspensions mentioned above, and add 1×10 to each flow cytometry tube. 6One cell was dissolved in 1 mL of 1640 buffer. 1 μL / mL of GolgiStop™ Protein Transport inhibitor (catalog number 554724, BDBioscience) was added to each tube. The tubes were then placed in a 37°C incubator for 4-6 hours in the dark with aluminum foil. After centrifugation, the cells were centrifuged at 1500 rpm for 5 min at 4°C. The supernatant was discarded after centrifugation. Cell surface staining was then performed by adding surface staining antibodies (live / dead, anti-CD3, anti-CD56) in sequence. After standing for 20 min, an appropriate amount of PBS buffer was added, and the cells were centrifuged at 1500 rpm for 5 min at 4°C. The supernatant was discarded after centrifugation. Then, the cells were perforated. 250 μL of intracellular fixation and perforation buffer (catalog number 00-8222-49, eBioscience) was added to each flow cytometry tube. After standing for 20 min, 750 μL of perforation washing buffer (catalog number 00-8333-56, eBioscience) was added, and the tubes were centrifuged at 1500 rpm for 5 min at 4°C. After centrifugation, the supernatant was discarded, and cytokine antibodies (anti-CCL3, anti-CCL4, anti-CCL5, anti-CXCL9, anti-CXCL10, anti-LTA) were added sequentially for staining in the dark for 30 min. Finally, an appropriate amount of perforation washing buffer was added, and the tubes were centrifuged at 1500 rpm for 5 min at 4°C. After centrifugation, the supernatant was discarded. The cells were resuspended in 200 μL of staining buffer, protected from light with aluminum foil, and stored at 4°C for analysis.

[0043] Figure 2-8 For the detection of IgG1-NK cells and IgG1-NK cells by flow cytometry PRDM6oe Cells, PRDM6 + A comparison of cytokine and chemokine secretion in the NK-induced group. Results show that IgG1-NK... PRDM6oe Cells significantly increased the secretion levels of chemokines CCL3, CCL4, CCL5, CXCL9, CXCL10, and LTA, indicating that upregulating PRDM6 gene expression can significantly enhance the chemotactic capacity of NK cells.

[0044] Example 3 PRDM6 + Mouse models of NK cell therapy alone and in combination with anti-PD1 therapy like Figure 9 The flowchart of the mouse treatment model shown in Figure A illustrates the timeline and operational procedures of the animal experiment, as detailed below: 1. Establishment of experimental animal and tumor models: A549 lung cancer cells cultured to the logarithmic growth phase were collected, digested with trypsin, and resuspended in sterile PBS buffer to adjust the cell suspension concentration to 5 × 10⁻⁶ cells / mL. 6 1 / 100μL PBS.

[0045] Using female severely immunodeficient (NOD / scid-IL-2Rγc) nul NSG mice (4 weeks old, Beijing Spefol Biotechnology Co., Ltd.) were injected with 100 μL (5 × 10⁻⁶) of A549 lung cancer cell suspension on day 0. 6 (One A549 lung cancer cell), during the procedure, avoid leakage and intramuscular injection; inject until a small subcutaneous bulge appears to establish a humanized tumor model. Continuously observe the subcutaneous tumor formation in mice; when the tumor grows to 50-100 mm... 3 The mice were randomly divided into groups.

[0046] 2. Immune cell infusion On day 7, 0.1 mL of PBMC suspension from Example 1 was injected to establish a humanized mouse model. On day 9, NK cell infusion therapy was initiated, and the mice were divided into the following groups: 1) PBS group: 100 μL of PBS was infused into mice via the tail vein. 2) IgG1-NK control group: NK cells (5 × 10⁻⁶) activated with human IgG1 antibody and cultured with cytokines were infused via the tail vein of mice. 6 (cells / 100μL PBS) (IgG1-NK cells from Example 1); 3) PRDM6 + NK cell group: PRDM6 activated by Herceptin combined with cytokines via tail vein infusion in mice. + NK cells (5×10) 6 cells / 100μL PBS (PRDM6 in Example 1) + NK cells); 4) PRDM6 + NK+anti-PD1 treatment group: PRDM6 activated by Herceptin combined with cytokines was infused via tail vein in mice. + NK cells (5×10) 6 cells / 100μL PBS), and anti-PD1 antibody (150ug / animal, once every 3 days, for a total of 4 times) was injected via tail vein.

[0047] The four groups of cells were monitored regularly, with mice dynamically monitored twice a week using an in vivo imaging system to record tumor fluorescence intensity. In vivo imaging results at different time points (day 7, day 14, and day 21) are shown below. Figure 10As shown, in vivo imaging results clearly validated our combination therapy hypothesis. Data showed that PRDM6 + The NK cell group showed a significant tumor-suppressive effect compared to the PBS group and the IgG1-NK control group. Meanwhile, PRDM6... + The NK+anti-PD1 group exhibited more potent and durable anti-tumor activity: from the mid-treatment stage, the tumor fluorescence signal intensity in this group was significantly lower than that in the other three groups; by the end of the treatment, the tumor burden in the combination therapy group was suppressed to the lowest level. Figure 11 This is a statistical bar chart of fluorescence on day 21 after adoptive cell therapy in four groups. P<0.05 P<0.01 P<0.001, P<0.0001, indicating PRDM6. + The NK cell group showed statistically significant differences compared to other control groups.

[0048] 3. When the tumor volume in mice exceeds 1000 mm 3 If the tumor length and diameter reach 1.5 cm, the mouse is considered to have reached the end of its life and should be euthanized. If this criterion is not met, the mice are euthanized at a uniform time point, and the tumor tissue is collected for single-cell suspension preparation for flow cytometry analysis.

[0049] Preparation of single-cell suspension from mouse tumor tissue: Mice were euthanized by CO2 inhalation, and their limbs were fixed to a foam box. The abdominal skin of the mice was cut open, and the tumor tissue was removed and placed into pre-prepared 24-well plates containing 1 mL of 1640 basal culture medium (catalog number 03.4007C, EallBio). The cells were then arranged neatly according to groups and photographed. The weighed tumor tissue was then transferred back into 6-well plates and cut into 1 mm pieces using scissors. 3To digest and break down tumor tissue, collagenase 1 (1 mg / mL, Sigma-Aldrich) and DNA hydrolase 1 (500 μg / mL, Sigma-Aldrich) were added to 6-well plates. The plates were placed in a 37°C incubator, and tumor tissue was aspirated three times every 10 minutes using a dropper to ensure no adhesion between the tumor tissues. After digestion, complete culture medium was added to the 6-well plates to stop the digestion, resulting in a single-cell suspension of tumor cells. The single-cell suspension was filtered through a 70 μm sieve. Any incompletely digested tumor tissue was gently ground with the syringe handle, and washed with 1640 basal medium or DMEM medium. The final single-cell suspension was counted under a microscope, and the cells were resuspended in PBS to adjust the A549 lung cancer cell concentration to 1 × 10⁻⁶. 7 The cell density is 1 cell / mL, and the resulting cell suspension can be used for flow cytometry staining. Each tube contains 1 × 10 cells / mL. 6 cell.

[0050] The list of antibodies for flow cytometry detection is shown in Table 1: Table 1 List of antibodies for flow cytometry detection

[0051] Flow cytometry was used to quantitatively analyze the proportion of immune cells in the tumor microenvironment of each group, including PBS, IgG1-NK, and PRDM6. + NK, PRDM6 + CD8 of the four groups of NK+anti-PD1 + T, IFNγ + CD8 + T, TCF1 + CD8 + T cells, PD1 + CD8 + T cells, CD11c + XCR1 + DC cells at CD45 + The flow cytometry results of the proportion of immune cells are as follows: Figure 12-16 As shown, the four groups of CD80 + CD86 + The flow cytometry results of the proportion of cells in cCD1 immune cells are as follows: Figure 17 As shown. It can be seen that PRDM6 + CD8 infiltrated in NK+anti-PD1 + T cells not only highly expressed effector molecules such as IFN-γ, but also significantly upregulated the expression of stemness / memory potential-related transcription factors such as TCF-1. This result indicates that the combination therapy successfully induced a group of CD8 cells possessing both stemness and cytotoxic capabilities. +T cell subsets lay the cellular foundation for long-term anti-tumor immunity.

[0052] Example 4: Multicolor immunohistochemical staining experiment on neoadjuvant therapy specimens To determine PRDM6 + To investigate NK cell expression in human tumor tissues, we performed multiplex immunofluorescence staining (mIHC) on 30 tumor tissue sections from patients with esophageal squamous cell carcinoma (ESCC), including 17 patients in the major pathological response (MPR) group and 13 patients who did not achieve a major pathological response (non-MPR) after neoadjuvant anti-PD1 therapy. The mIHC assay was designed as follows: primary antibodies were rabbit anti-human NCAM1 (CD56) antibody (catalog number 99746, CST Biotechnology) and rabbit anti-human PRDM6 antibody (catalog number 503105, Boster Biotechnology); the Opal multiplex immunofluorescence IHC kit (catalog number NEL811001KT, Akoya) was used.

[0053] The multicolor immunohistochemical staining experiment and slide scanning procedures are as follows: (1) Baking the slices The two sets of tissue sections were placed in a stainless steel staining rack and baked in an electric thermostatic drying oven set to 70°C for more than 2 hours to ensure that the tissues adhered fully to the glass slides and to help soften the paraffin, which would facilitate the subsequent dewaxing process.

[0054] (2) Dewaxing and rehydration Immediately after baking, the sections were dewaxed by immersing them sequentially in xylene I, xylene II, and xylene III for 10 minutes each to completely remove paraffin. Following this, a gradient ethanol rehydration process was performed, placing the sections in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, and 75% ethanol for 5 minutes each, allowing the tissue to gradually regain its hydration. After rehydration, the sections were transferred to a plastic staining jar and rinsed three times with sterile water for 3 minutes each time to remove residual organic solvents.

[0055] (3) Organizational Refixation To further stabilize the tissue structure, the sections were fixed in 10% neutral formalin for 15 minutes in the dark. After fixation, the sections were rinsed three times with sterile water for three minutes each time to remove excess fixative.

[0056] (4) Antigen retrieval Dilute 50× immunohistochemistry antigen retrieval buffer (pH 9.0) with sterile water to a final volume of 1×, for a total volume of 200 mL. Pour the retrieval buffer into the antigen retrieval chamber, seal it, and microwave on high for 3 minutes. Once the retrieval buffer boils, add the slides and continue heating on low for 15 minutes to perform antigen retrieval. After retrieval, remove the slides and allow them to cool naturally to room temperature.

[0057] (5) Sealing treatment After the sections have cooled, rinse once with sterile water (3 minutes), then rinse once with 1×TBST buffer (3 minutes). Gently shake off any excess liquid from the surface of the sections and draw circles along the tissue edges with a hydrophobic immunohistochemistry pen. Then, add the blocking solution from the Opal 7-Color IHC kit to the tissue area, completely covering the tissue, and incubate at room temperature for 10 minutes to block non-specific binding sites.

[0058] (6) After the primary antibody incubation and blocking are completed, dilute the primary antibody according to the appropriate ratio as needed for the experiment. Discard the blocking solution and add sufficient primary antibody to the tissue area to completely cover the tissue. Place the slide in a humidified chamber and incubate overnight at 4°C in the dark to enhance the specificity and stability of antibody binding.

[0059] (7) Secondary antibody incubation The following day, the sections were removed from the 4°C environment and allowed to warm to room temperature for 30 minutes. They were then rinsed three times with 1×TBST for 3 minutes each time. After shaking off the surface liquid, the sections were completely covered with the polyhortradiction peroxidase (HRP)-labeled secondary antibody (rabbit and mouse universal type) provided in the Opal 7-Color IHC kit and incubated in a humidified chamber at room temperature for 10 minutes.

[0060] (8) Color development by fluorescent dyes After secondary antibody incubation, wash three times with 1×TBST for 3 minutes each time. Then add fluorescent dye diluted 1:100, ensuring coverage of the entire tissue area, and incubate at room temperature in the dark for 10 minutes. From this step onwards, all subsequent operations must be performed in the dark to prevent fluorescence quenching.

[0061] (9) Second round of antigen retrieval After fluorescent labeling, wash three times with 1×TBST. Then repeat the antigen retrieval step: heat the prepared retrieval solution to boiling, place the slides in the solution, heat on low for 15 minutes, and then allow to cool naturally to room temperature. This step removes the bound primary and secondary antibodies, preparing for the next round of staining.

[0062] (10) Multiple rounds of cyclic staining Repeat the above-described blocking, primary antibody incubation, secondary antibody incubation, fluorescent dye incubation, and antigen retrieval process (steps 5-9) to complete two rounds of staining for different targets in order to achieve multiplex immunofluorescence detection.

[0063] (11) Nuclear staining After completing the final round of antigen retrieval and cooling, rinse once with sterile water (3 minutes), then rinse once with 1×TBST (3 minutes). Subsequently, add DAPI dye prepared at a ratio of 1:10 (diluted with 1×TBST) to completely cover the tissue, and incubate in a humidified chamber at room temperature in the dark for 10 minutes to label the cell nuclei.

[0064] (12) Sealing and preservation After DAPI incubation, rinse once with 1×TBST (3 minutes), then rinse once with sterile water (3 minutes). Gently shake off excess liquid, add anti-fluorescence attenuation mounting medium, and mount the slide, avoiding air bubbles when covering with a coverslip. After mounting, seal along the edge of the coverslip with clear nail polish and allow to air dry in the dark. Finally, place the slides in a slide box and store at -20°C for long-term preservation and subsequent observation.

[0065] (13) Multispectral Imaging and Analysis The stained sections were scanned according to the manufacturer's instructions. Im3 image cubes were generated using the Mantra system (PerkinElmer) for subsequent processing. Spectral data of all included fluorescent dyes were analyzed using the inForm image analysis tool (v2.4.4, PerkinElmer). For each section, images were acquired from 10 fields of view enriched with immune cell populations; the results are as follows. Figure 18 As shown, A represents PRDM6 in MPR and non-MPR patients. + CD56 + NK cell expression status, B represents PRDM6 in MPR and non-MPR patients. + CD56 + Comparative analysis of NK cell expression. Compared with non-MPR patients, MPR patients showed higher levels of PRDM6. + The proportion of NK cells was significantly higher in the non-MPR group than in the other group (P<0.05). White fluorescent markers represent PRDM6 expression, red fluorescent markers represent CD56 expression, and blue fluorescent DAPI represents nuclear expression. This suggests that PRDM6... + NK cell infiltration can serve as a clinical predictor of the effectiveness of PD-1 inhibitor therapy.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the inventive concept, and these all fall within the protection scope of the present invention.

Claims

1. Application of upregulating PRDM6 gene expression level in enhancing the anti-tumor activity of NK cells.

2. Use according to claim 1, characterized in that, The methods for upregulating PRDM6 gene expression levels include any of the following: (1) Inducing endogenous PRDM6 gene expression using IgG1 subtype antibody combined with cytokines; or (2) Overexpression of PRDM6 gene through gene transfection.

3. An activated NK cell highly expressing a PRDM6 gene, characterized in that, Compared with inactive or low-expressing PRDM6 NK cells, the activated NK cells showed upregulated PRDM6 gene expression, with an average PRDM6 protein positivity rate of 4.5%-6.02%, and possessed at least the following functional characteristics: (1) Increased secretion levels of one or more of the chemokines CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, XCL1, LTA, and LTB; (2) promote dendritic cell activation and TCF1 + CD8 + T cell infiltration.

4. The activated NK cell of claim 3, wherein, The activated NK cells are activated through any of the following methods: (a) Induction of endogenous PRDM6 gene expression using IgG1 subtype antibody in combination with cytokines; or (b) Overexpression of the PRDM6 gene via gene transfection.

5. The use of activated NK cells as described in claim 3 or 4 in the preparation of antitumor drugs.

6. Use according to claim 5, characterized in that, The tumor is either a lung cancer tumor or an esophageal cancer tumor.

7. The use of activated NK cells combined with a PD-1 inhibitor as described in claim 3 or 4 in the preparation of an antitumor drug.

8. The use of activated NK cells as a detection biomarker as described in claim 3 or 4 in the preparation of a kit for predicting the efficacy of PD-1 inhibitor therapy.

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