M2 macrophage inducer and application

Abstract

The invention provides an inducer for M2 macrophages and application, the inducer for M2 macrophages is a combination of adenosine and ADORA1, and adenosine is obtained from gastric juice of a gastric cancer patient. According to the invention, adenosine obtained from gastric juice of a patient with gastric cancer is combined with ADORA1 to form an inducer of M2 macrophages, the inducer of M2 macrophages can mediate secretion of IL-6, promote M2 polarization of macrophages and remodel an immune microenvironment, and the new way is expected to improve the curative effect of immunotherapy of gastric cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an inducer of M2 macrophages and application thereof. Background Art

[0002] Gastric cancer (GC) is a common cancer. In order to improve the survival rate of gastric cancer patients, some high-incidence countries have implemented gastric cancer screening programs. However, due to limited screening efforts and the lack of clear early clinical characteristics of gastric cancer, most patients are diagnosed at an advanced stage. Chemotherapy and radiotherapy are widely used first-line treatment strategies for advanced gastric cancer, but drug resistance remains a challenge.

[0003] The tumor immune microenvironment (IME) is crucial for the occurrence and development of GC, and activation of tumor IME contributes to immunotherapy, which is a new approach to treat gastric cancer. Tumor IME contains a variety of factors that jointly affect the efficacy of immunotherapy. For example, immune cell populations with anticancer effects, such as T cells, dendritic cells (DCs), M1 macrophages, etc., improve the immune level of cancer patients in different ways to inhibit the development of cancer. Conversely, there are also anticancer immune cell populations in the microenvironment. Such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages, which work together to exert immunosuppressive effects. When cancer patients receive immunotherapy, the efficacy usually depends on the IME characteristics formed by the interaction of these cells7. In addition, the cell metabolism generated under a specific tumor IME can also reshape the IME to a certain extent, such as the inhibition of adenosine synthesis in hypoxia-induced tumor immune activity. The tumor immune microenvironment IME, which is affected by multiple factors, ultimately determines the immune level of cancer patients and the sensitivity of immunotherapy.

[0004] Tumor-associated macrophages (TAMs) are the main component of immune cells in tumor IME, accounting for 50% of the entire tumor mass. A large number of studies have found that TAMs affect cancer cell proliferation, metastasis, tumor immune activity, and resistance to chemotherapy and immune checkpoint inhibitors, and are therefore closely related to the prognosis of cancer patients. Plasticity and functional polarization are the hallmarks of TAMs, and there are two different phenotypes of TAMs in tumor IME: M1 and M213. In GC, TAMs are usually polarized into M2 macrophages, which are directly or indirectly involved in tumor malignant progression.

[0005] M0 macrophages in the tumor microenvironment polarize into M2 cells and promote cancer development under the stimulation of excessive secretion of IL-4 and IL-10. At the same time, M2 macrophages also secrete TGFB1, which in turn stimulates GC cells to activate the TAK / JUK / JUN signaling pathway and transcriptionally activate CAP2, ultimately forming a positive feedback loop that enhances M2 polarization and GC metastasis. The M2 polarization of TAMs is affected by cytokines secreted by tumor cells in the IME.

[0006] It is proposed that the expression of IL-6 in gastric cancer tissue is significantly higher than that in normal tissue and is positively correlated with the polarization level of M2 macrophages. Mechanistically, IL-6 induces the polarization of normal macrophages into M2 macrophages by activating STAT3 phosphorylation, which ultimately leads to the proliferation and migration of gastric cancer cells. Given the relationship between TAM polarization and the malignant progression of gastric cancer, it is necessary to further explore the molecular mechanisms that affect TAM polarization in order to improve the poor prognosis of gastric cancer.

[0007] Adenosine is a functionally conserved regulator of energy homeostasis and a component of some biological molecules. During cancer progression, cancer cells or tissues can produce an adenosine-rich immunosuppressive microenvironment by increasing the release of adenosine and the production of adenosine triphosphate (ATP). Specifically, a large number of CD39 and CD73 molecules are often enriched on the surface of cancer cells. ATP is transported from the cytoplasm to the extracellular space, where it first interacts with CD39, releases a phosphate group unit, and is converted into adenosine diphosphate (ADP). ADP then continues to be dephosphorylated by CD39, converted into adenosine monophosphate (AMP), and finally converted into adenosine by CD73, releasing the last phosphate group. This is why a large amount of adenosine can be detected in tumor tissues.

[0008] Adenosine can bind to A2AR to inhibit the growth and anti-cancer ability of immune cells themselves. However, whether adenosine can act through other adenosine receptors and which immune cell populations it can affect need further clarification. In addition, some key genes in the adenosine production pathway (such as CD39 and CD73) are also considered by most people to be next-generation candidate immune checkpoint inhibitors (ICI), but from another perspective, targeted inhibition of adenosine receptors may also achieve the effect of promoting immune activity. In addition, when evaluating cancer immune activity, appropriate samples for direct and accurate detection of adenosine levels have not yet been determined. Gastric juice carries a variety of gastric cancer markers, and whether it can objectively and accurately reflect adenosine levels and indicate the progression of gastric cancer is also worthy of study. Summary of the invention

[0009] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide an inducer of M2 macrophages and its application, wherein the inducer of M2 macrophages can mediate IL-6 secretion, promote M2 macrophage polarization, reshape the immune microenvironment, and improve the efficacy of gastric cancer immunotherapy.

[0010] To achieve the above object and other related objects, the present invention provides an inducer of M2 macrophages, wherein the inducer of M2 macrophages is a combination of adenosine and ADORA1, and the adenosine is obtained from the gastric juice of gastric cancer patients.

[0011] Adenosine is highly expressed in the gastric juice and tissues of gastric cancer patients, and no significant difference was detected in the blood. Adenosine has a strong binding relationship with ADORA1, and the expression level of ADORA1 is positively correlated with the adenosine concentration. In addition, the expression of ADORA1 in gastric cancer cells is significantly positively correlated with M2 macrophage polarization and IL-6 secretion. Adenosine is upregulated in the gastric juice of gastric cancer patients, and it forms an inducer of M2 macrophages by binding to ADORA1. This M2 macrophage inducer can mediate IL-6 secretion, promote macrophage M2 polarization, and reshape the immune microenvironment. This new pathway is expected to improve the efficacy of immunotherapy for gastric cancer.

[0012] Preferably, the combination of adenosine and ADORA1 is obtained from gastric cancer AGS cells.

[0013] Preferably, the combination of adenosine and ADORA1 is a culture medium or a kit for preparing a complex of adenosine and ADORA1.

[0014] The present invention also provides a use of the above-mentioned M2 macrophage inducer in the preparation of gastric cancer immunotherapy drugs.

[0015] Preferably, the inducer of M2 macrophages is used to mediate IL-6 secretion and promote the polarization of M2 macrophages.

[0016] The present invention also provides a biomarker for gastric cancer immunotherapy, which is adenosine obtained from the gastric juice of gastric cancer patients. Adenosine in the gastric juice of gastric cancer patients is suitable as a biomarker for gastric cancer, and one of its potential immunosuppressive effects is to activate M2 macrophage polarization.

[0017] The present invention also provides a use of the above-mentioned gastric cancer immunotherapy biomarker in the preparation of gastric cancer immunotherapy drugs.

[0018] ADOR1 is an adenosine receptor, and the activation of ADOR1 is closely related to gastric cancer tumor immunity. Therefore, this application analyzes ADORA1 as a downstream target of adenosine.

[0019] As described above, the present invention has the following beneficial effects: by combining adenosine obtained from the gastric juice of gastric cancer patients with ADORA1 to form an inducer of M2 macrophages, the inducer of M2 macrophages can mediate IL-6 secretion, promote macrophage M2 polarization, and reshape the immune microenvironment. This new approach is expected to improve the efficacy of gastric cancer immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shown is the detection of gastric fluid metabolites in gastric cancer patients and healthy subjects by untargeted metabolomics sequencing.

[0021] Figure 2 Shown are the expressions of adenosine in gastric juice (A), tissue (B), and blood (C) of gastric cancer patients and healthy subjects; * indicates P < 0.05.

[0022] Figure 3 Shown is the molecular docking simulation of the binding relationship between adenosine and ADORA1.

[0023] Figure 4 Shown are the CETSA-WB experimental results of the ADORA1 group and the adenosine and ADORA1 co-treated group.

[0024] Figure 5 Shown is the mRNA expression level of ADORA1 in cells treated with different concentrations of adenosine detected by qPCR; * represents P < 0.05.

[0025] Figure 6 Shown is the protein expression level of ADORA1 in cells treated with different concentrations of adenosine detected by WB; * represents P < 0.05.

[0026] Figure 7 Shown are the results of bioinformatics analysis of the correlation between ADORA1 and M2 macrophage infiltration.

[0027] Figure 8 Shown are the mRNA expression levels of M1 macrophage markers (CD86) and M2 macrophage markers (CD206) in each treatment group detected by qPCR, * represents P < 0.05.

[0028] Fig. 9 Shown are the protein expression levels of M1 macrophage markers (CD86) and M2 macrophage markers (CD206) in each treatment group detected by WB, * represents P < 0.05.

[0029] Fig.10 Shown are flow cytometric plots of the expression levels of M1 macrophage surface markers (CD68-positive and CD86-positive) and M2 macrophage surface markers (CD68-positive and CD206-positive) in each treatment group.

[0030] Fig.11 Shown are quantitative graphs of the expression levels of M1 macrophage surface markers (CD68 positive and CD86 positive) and M2 macrophage surface markers (CD68 positive and CD206 positive) in each treatment group.

[0031] Fig.12 Shown are the ELISA test results of M1 macrophage cytokines (IL-23) and M2 macrophage cytokines (TGF-β) in each treatment group, * represents P < 0.05.

[0032] Fig.13 Shown are the results of bioinformatics analysis of the correlation between ADORA1 and IL-6.

[0033] Fig.14 Shown are the expressions of IL-6 in the control group (sh NC) and the ADORA1 knockout group (sh-ADORA1).

[0034] Fig.15 Shown are the mRNA expression levels of M1 macrophage markers (CD86) and M2 macrophage markers (CD206) in each co-culture system detected by qPCR.

[0035] Fig.16 Shown are the protein expression levels of M1 macrophage markers (CD86) and M2 macrophage markers (CD206) in each co-culture system detected by WB.

[0036] Fig.17 Shown are flow cytometric plots of the expression of M1 macrophage surface markers (CD68-positive and CD86-positive) and M2 macrophage surface markers (CD68-positive and CD206-positive) in each co-culture system.

[0037] Fig.18 Shown are quantitative plots of the expression of M1 macrophage surface markers (CD68 positive and CD86 positive) and M2 macrophage surface markers (CD68 positive and CD206 positive) in each co-culture system.

[0038] Fig.19 Shown are the expressions of M1 macrophage cytokines (IL-23) and M2 macrophage cytokines (TGF-β) in each co-culture system.

[0039] Fig. 20 Shown are the chemotaxis and migration levels of M2 macrophages in each co-culture system, * represents P < 0.05.

[0040] Fig.21 Shown are images of gastric wall nodules in each treatment group.

[0041] Fig. 22 Shown is the mRNA expression level of ADORA1 in each treatment group detected by qPCR.

[0042] Fig.23 Shown is the protein expression of ADORA1 in each treatment group obtained by WB analysis.

[0043] Fig.24 Shown is the protein expression of ADORA1 in each treatment group obtained by IHC analysis.

[0044] Fig.25 Shown are flow cytometry plots of the expression of M1 macrophage surface markers (CD11b positive and CD86 positive) and M2 macrophage surface markers (CD11a positive and CD206 positive) in each treatment group.

[0045] Fig.26 Shown are quantitative graphs of the expression of M1 macrophage surface markers (CD11b positive and CD86 positive) and M2 macrophage surface markers (CD11a positive and CD206 positive) in each treatment group.

[0046] Fig. 27 Shown are the expressions of IL-6, M1 macrophage cytokine (IL-23), and M2 macrophage cytokine (TGF-β) in each treatment group; *P<0.05. DETAILED DESCRIPTION

[0047] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0048] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0049] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0050] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0051] 1.1 Clinical sample collection The clinical sample collection of the embodiment of the present application selected 19 patients, including 10 gastric cancer patients and 9 healthy subjects. Gastric cancer patients had no other underlying diseases and had not received chemotherapy or radiotherapy. Gastric cancer patients provided cancer tissue (4 samples), adjacent tissue (4 samples), blood (4 samples) and gastric juice (6 samples). Healthy subjects provided blood (3 samples) and gastric juice (9 samples), all of which were stored at -80 degrees for future use. Sample testing was carried out at LUOXiMedical (China). The project has been certified by the Ethics Committee. All experiments based on patient samples have been approved by the committee. In addition, written informed consent has been obtained from all patients.

[0052] 2. Cell culture The human gastric cancer cell line AGS and the mouse gastric cancer cell line MFC used in the examples of the present application were cultured in RPMI-1640 medium. The mononuclear cell line THP-1 and the M0, M1 and M2 macrophages induced or polarized later were stored in RPMI-1640 complete medium. 1% P / S double antibody was added to all culture media, and the cells were placed in a constant temperature incubator at 37°C, about 90% humidity and 5% carbon dioxide concentration. When the relative cell count reaches 80-90%, the culture medium needs to be aspirated and rinsed twice with phosphate buffered saline (PBS) buffer. Then, 1 mL of 0.25% trypsin (Gibco, USA) was introduced for 3 minutes for subculture. Subculture was performed every 2 to 3 days. Adenosine A1 receptor (ADORA1) was enriched from gastric cancer cell AGS or MFC lysate by co-immunoprecipitation technology (CO-IP), and adenosine was obtained from patient gastric juice or gastric cancer cell culture fluid by high performance liquid chromatography, and the two were combined to prepare M2 macrophage inducer. Human gastric cancer cell line AGS (Catalog No. CL-0022), mouse gastric cancer cell line MFC (Catalog No. CL-0156), and monocytic cell line THP-1 (Catalog No. CL-0233) were purchased from Procell Life Science Technology Co., Ltd., China.

[0053] 3. Cell transfection The cell transfection in the examples of this application includes silent expression and overexpression. Ruibo Biotech Co., Ltd. synthesized and constructed small interfering RNA (siRNA-ADORA1) targeting ADORA1 and pcDNA3.1 vector overexpressing ADORA1 gene. AGS cells were seeded in 6-well plates (1×10 6 The cells were cultured overnight. When the cell density reached 70%-80%, the original culture medium was removed. The above plasmid products were transfected into AGS cells using UltraFection 3.0 high-efficiency transfection reagent (4ABIOTECH, China) for subsequent experiments.

[0054] 4. Macrophage polarization First, purchased monocytic THP-1 cells (Catalog No. CL-0233, Wuhan Pronocell Biotechnology Co., Ltd., China) were differentiated into M0 macrophages. The specific steps were as follows: THP-1 cells were centrifuged in the logarithmic growth phase and resuspended in RPMI-1640 medium. The density of THP-1 cells was adjusted to 5 × 10 5cells / mL. Then, THP-1 cells were seeded in six-well plates with 2 mL per well. PMA was added to the six-well plates to a final concentration of 100 ng / mL. Under normal conditions, THP-1 cells were cultured for 48 hours, and the results of macrophage induction were observed under a microscope. Next, the polarization of M0 macrophages to M1 or M2 macrophages was analyzed by an indirect co-culture system of cancer cells and macrophages. Specifically, AGS cells of gastric cancer were inoculated into the upper chamber (5 × 10 4 cells / well) and added M0 macrophages to the lower chamber. After incubation for 24 h at 37°C and 5% CO2, the polarization of M0 macrophages in the lower chamber was determined by flow cytometry.

[0055] 5. Reverse transcription-polymerase chain reaction Total RNA was extracted from cells using the TRIzol kit (Kangwei Century, China). Subsequently, total RNA was synthesized into cDNA using the Hifair® II 1st Strandc DNA Synthesis Kit (YESEN, China). Finally, cDNA was subjected to qPCR using the SYBR Green (Biorad, USA) kit. Primer sequences were purchased from Qingke Biotechnology Co., Ltd. (China).

[0056] The primer sequences for ADORA1 were 5'-ATTGCTGGACCGCTAC-3' and 5'-CTCAGATTGTTCCAGCAAAC-3'. The reference primer sequences for GAPDH were 5'-GAGGTCGGAGTCAACGGAT-3' and 5'-CCTGAGAGAGGTGATGGGAT-3'. The RNA expression levels of the genes were based on the RNA expression of the internal reference gene GAPDH, and the expression levels were quantified using 2 -ΔΔCt Relative gene expression was calculated.

[0057] 6. Western Blot Transfected cells were collected and lysed with RIPA buffer (Thermo Scientific, USA) to extract total protein. Protein concentration was measured using a BCA assay kit (Beyotime, China). Equal amounts of protein samples were separated on SDS-PAGE and transferred to PVDF membranes (Thermo Fisher Scientific, USA). After blocking with 5% skim milk for 1 h, the PVDF membrane was covered with primary antibodies and incubated overnight at 4 °C. After rinsing with TBST, the proteins were incubated with secondary antibodies at room temperature for 4 h. After washing again with TBST, the proteins were developed using an ECL kit (P0018M, Beyotime, China), and finally the protein bands were analyzed using a chemiluminescence imaging system. The primary antibodies included ADORA1 antibody (A5219, ABclonal, China), CD86 antibody (A1199, ABclone, China), and CD206 (A8301, ABclonaly, China). The secondary antibody was goat anti-rabbit IgG (ab302644, Abcam, UK).

[0058] 7. Immunohistochemistry (IHC) We cut serial sections from each tissue block and dewaxed them with xylene and a series of graded ethanol solutions (100%, 95%, and 70%) to water, respectively. Next, the sections were immersed in an antigen retrieval buffer containing EDTA and boiled in a microwave for 20 min for antigen retrieval. After blocking with peroxidase blocking solution for 10 min, the sections were blocked with 10% skim milk for 30 min and then incubated with the primary antibody ADORA1 (A5219, ABclonal, USA) at 4°C overnight. After rinsing three times with PBS, we incubated with the secondary antibody goat anti-rabbit IgG (A0208, Beyotime, China) at 37°C for 30 min. Subsequently, the sections were developed, restained with hematoxylin, and mounted for analysis.

[0059] 8. Non-targeted metabolomics sequencing for adenosine detection The collected clinical samples were subjected to non-targeted metabolomics sequencing by using a high-resolution mass spectrometer (LUOXI Medical Technology, China). Metabolites were first extracted from each gastric juice sample using an organic reagent protein precipitation method. Next, the extracted samples were randomly sorted and tested, and pre-prepared quality control samples were inserted before, during, and after the samples as experimental technical repeat evaluations. Finally, the samples were scanned and detected using a mass spectrometer in positive and negative ion modes.

[0060] (IX) Adenosine detection kit Adenosine levels in each sample were assessed using an adenosine assay kit (Cell Biolabs, USA). Working reaction solutions were prepared according to the recommended ratios, including two types (with and without adenosine deaminase). Then, samples for detection were prepared. Serum and gastric fluid samples were centrifuged at 10,000 rpm for 5 min, and the supernatant was diluted with PBS. After GC tissue samples were lysed, centrifuged and diluted. After all samples were prepared, the two working reagents were added to the black microtiter plate required for the fluorescence plate reader in a 1:1 ratio, and each group was repeated three times. With thorough mixing, the samples were incubated at room temperature in the dark for 15 min. After the reaction was completed, we read the plate with a fluorescence plate reader. Fluorescence was excited in the range of 530-570 nm, and the emission light signal was received in the range of 590-600 nm. We subtracted the signal value of the sample well without adenosine deaminase from the signal value of the sample well with adenosine deaminase to obtain the difference, which represents the adenosine level.

[0061] 10. Cellular thermal shift assay Cellular Thermal Shift Assay (CETSA) was performed based on the change in target protein stability induced by ligand binding to verify the binding relationship between ligand and receptor. In this project, Cellular Thermal Shift Assay-Western Blotting (CETSA-WB) was mainly used to determine whether adenosine interacts with ADORA1. Briefly, GC AGS cells were cultured in T175 culture flasks. The experimental group was treated with medium containing adenosine (1 mM) for 12 h, while the control group was treated with solvent (DMSO) for 12 h. After treatment, the cells were detached with trypsin, centrifuged, and resuspended in PBS. The cell suspension was divided into 5 PCR tubes and heated at a gradient temperature from 50°C to 95°C for 3 min. Subsequently, we lysed the cells with liquid nitrogen and performed two cycles of freeze-thaw. After centrifugation at 17000g for 20 min, the precipitated protein was separated from the soluble fraction, and ADORA1 protein expression was analyzed using WB. GAPDH protein was used as an internal reference without heat treatment.

[0062] 11. Flow cytometer Flow cytometry was applied to determine the protein expression of surface markers of M1 and M2 macrophages. First, macrophages were collected from the indirect co-culture system and centrifuged at 300 × g for 5 min. The supernatant was discarded. Then, the cells were rinsed once with PBS and gently resuspended. The cells were counted. Next, we took 1 × 10 5The cells were resuspended and centrifuged again. After washing the cells, we seeded them in 6-well plates, incubated with antibodies for 20-30 minutes at room temperature in the dark, and then immediately analyzed the cells using NovoCyte Penteon. The detection antibodies were PE-CD68 (79594), FITC-CD86 (E-AB-F1269C), APC-CD206 (E-ABF1161E), APC-CD11b (41249), FITC-CD 86 (99879), and PE-CD206 (E-ABF1135D), purchased from Cell Signaling Technology (USA) and Elabscience (China).

[0063] 12. Enzyme-linked immunosorbent assay Follow the instructions of the ELISA kit. Human and mouse TGF-β kits were provided by Jianglaibio (China), and human and mouse IL-6 and IL-23 kits were obtained from Elabscience (China). Specifically, cells were collected by centrifugation at 1000 × g for 20 min at 4 °C. The supernatant was taken as a sample. Then, we supplemented the sample and reaction solution in sequence, sealed the reaction wells with sealing film, and incubated at 37 °C for 60 min. Subsequently, we washed the plate 4-5 times with a plate washer and then added the reaction solution again according to the instructions. The plate was incubated at 37 °C for 30 min and then washed. Finally, the reaction solution and the stop solution were added to each well, and after 10-15 min in the dark, the optical density (OD) value of each well was measured at a wavelength of 450 nm.

[0064] 13. Transwell experiment Transwell assays were performed on 24-well plates to verify the chemotaxis and migration levels of M2 macrophages in the co-culture system (Corning, USA). Specifically, serum-free macrophage suspensions were seeded into Transwell plates (Corning, USA) with a pore size of 5 μm in the upper chamber (5 × 10 4 The cancer cells were seeded in the lower chamber and incubated at 37°C and 5% CO2 for 24 hours. During the test, the remaining cells in the upper chamber were removed with a cotton swab, and the macrophages transferred to the lower side of the membrane were fixed with 1% paraformaldehyde for 10 minutes and stained with crystal violet for 5 minutes. The cells were then counted under a microscope.

[0065] 14. Bioinformatics analysis The SeeSAR software package was used to perform docking simulations of adenosine and ADORA1 to analyze the binding relationship between adenosine and the receptor ADORA1. The TIMER algorithm was used to analyze the correlation between the expression level of ADORA1 in GC cells and markers related to immune cell infiltration to evaluate the correlation between ADORA1 and the level of macrophage immune infiltration. In addition, based on the public GC expression data, the Pearson correlation analysis algorithm was used to evaluate the levels of ADORA1 and IL-6.

[0066] 15. Animal Experimentation Female C57BL / 6 mice aged 6 to 8 weeks were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. 6 The suspension of MFC GC cells was transplanted into the subserosa of the mouse stomach to construct a GC mouse model. Then, all mice were randomly divided into four groups, with six mice in each group. The four groups of mice were regularly administered and cultured for 30 days. Drug treatment included: oral drinking water, oral adenosine (30 mg / kg), intravenous injection of DPCPX, and intravenous injection of oral adenosine-based DPCPX (DPCPX: an ADORA1 inhibitor). During the culture period, the growth of mouse tumors was closely observed. After 30 days of culture, the mice were euthanized to extract tumor tissues, and the tumor tissues were fixed with paraformaldehyde for subsequent experiments.

[0067] 16. Data processing and analysis Data were statistically analyzed using GraphPadPrism (GraphPad Software, USA) and expressed as mean ± SD. Student's t-test and one-way analysis of variance (ANOVA) were used to determine the significance of differences between two or more groups. Statistical analysis was performed using data collected from at least three independent experiments. Differences were considered significant when P value < 0.05.

[0068] In order to further study the abnormalities of gastric cancer patients compared with healthy subjects and explore biomarkers related to gastric cancer, gastric fluid samples from 6 gastric cancer patients and 6 healthy subjects were collected in this application example. The contents of various metabolites in the two gastric fluid samples were analyzed by non-targeted metabolomics sequencing. The results of mass spectrometry analysis are shown in Figure 2. Figure 1 As shown, there were significant differences in gastric fluid metabolites between gastric cancer patients and healthy subjects, and huge changes in adenosine content were detected.

[0069] To further elucidate the levels of adenosine in different gastric cancer samples, additional samples were added and adenosine expression in tumor tissue and blood was further tested. Figure 2 As shown. Figure 2 As can be seen from (A) in the figure, the adenosine content in the gastric juice of patients with gastric cancer is increased. Figure 2As can be seen from (B) in the figure, the adenosine concentration in gastric cancer tissue is significantly higher than that in adjacent non-cancerous tissue. Figure 2 As can be seen in (C), there is no significant difference in the serum adenosine between gastric cancer patients and healthy subjects. Overall, adenosine showed high expression in tumor tissues and gastric fluid of gastric cancer patients.

[0070] Adenosine exerts its effects by binding to corresponding receptors, and ADORA1 is one of the adenosine binding receptors. In order to clarify the relationship between adenosine and its receptor ADORA1 in GC, the present application example firstly performed a molecular docking experiment to analyze the binding relationship between adenosine and ADORA1. Figure 3 The results showed that adenosine has a strong binding affinity to ADORA1. In order to verify its accuracy and reliability, CETSA was used to further confirm the binding relationship between adenosine and ADORA1. Figure 4 The CETSA-WB test results revealed that with the increase of temperature (55-95℃), the ADORA1 protein expression level in the ADORA1 and adenosine co-treatment group did not change much, while the ADORA1 protein expression in the adenosine-free group showed a downward trend. In the ADORA1 and adenosine co-treatment group, ADORA1 had a strong protein thermal stability, indicating that there was a binding relationship between adenosine and ADORA1. In addition, in order to explore the expression relationship between adenosine and ADORA1, gastric cancer AGS cells were treated with gradient concentrations of adenosine (0, 0.125, 0.25, 0.5, 1, 2 mM) for 24 hours, and qPCR and WB experiments were used to analyze the expression relationship between adenosine and ADORA1 in each group. Figure 5 The qPCR test results and Figure 6 The WB results showed that with the increase of adenosine concentration, the mRNA expression and protein expression of ADORA1 gradually increased in a dose-dependent manner. In general, adenosine and ADORA1 have a binding relationship in GC, and their expression levels are positively correlated.

[0071] In order to clarify the effect of adenosine receptor ADORA1 on GC, bioinformatics analysis was performed in the present embodiment. The results are as follows Figure 7 As shown. Figure 7 It can be seen that ADORA1 is positively correlated with genes related to macrophage M2 polarization in GC. In order to verify its accuracy, relevant cell experiments were performed on the relationship between ADORA1 and macrophages.

[0072] First, three cell groups were established based on the GC cell line AGS, including a control group (oe-NC+DMSO), an ADORA1 overexpression group (oe-ADORA1+DMSO), and an ADORA1 overexpression and its antagonist (DPCPX) co-treatment group (oe-APORA1+DPCPX). The three groups of cells were indirectly co-cultured with induced M0 macrophages. The DPCPX antagonist can inhibit the activation of ADORA1 by adenosine by binding to the ADORA1 receptor, thereby inhibiting the function of the inducer (a combination of adenosine and ADORA1). This application uses the ADORA1 antagonist to prove that the receptor that interacts with adenosine is ADORA1 rather than other substances. qPCR and WB were used to analyze the expression levels of M1 and M2 macrophage surface markers in the co-culture system to evaluate the polarization of macrophages. Combined with Figure 8 The qPCR results shown and Fig. 9 The WB results shown found that there was no significant difference in the mRNA and protein expression levels of the M1 macrophage marker CD86 among the three co-culture systems, while the mRNA and protein expression levels of the M2 macrophage marker CD206 changed significantly. Compared with the control co-culture system, the mRNA and protein levels of CD206 in the ADORA1 overexpression co-culture system were significantly increased, while the CD206 expression level in the co-treatment co-culture system roughly returned to the original trend. This preliminary result suggests that ADORA1 can promote the polarization of M0 macrophages to M2 macrophages, but has no effect on the polarization of M0 macrophages toward M1 macrophages. In addition, flow cytometry experiments and ELISA also confirmed this conclusion. Fig.10 and Fig.11 The flow cytometry results shown in the figure show that compared with the control co-culture system, there was no significant difference in the proportion of surface markers (CD68 positive and CD86 positive) of M1 macrophages in the ADORA1 overexpression co-culture system, while the proportion of surface markers (CD68 positive and CD206 positive) of M2 macrophages in this system increased significantly. The proportion of surface markers (CD68 positive and CD86 positive) of M1 macrophages in the co-treatment co-culture system was still not significantly different from the first two groups, and the proportion of surface markers (CD68+ and CD206+) of M2 macrophages in this system was reduced compared with the ADORA1 overexpression co-culture system. Fig.12 The ELISA results shown indicate that the changes in the levels of M1 macrophage-associated cytokines (IL-23) and M2 macrophage-associated cytokines (TGF-β) in the three co-culture systems were consistent with the trends observed in the flow cytometry results.

[0073] In summary, in gastric cancer, adenosine receptor ADORA1 promotes the polarization of M0 macrophages to M2 macrophages, but this process does not affect the polarization of M0 macrophages to M1 macrophages.

[0074] In order to further clarify the mechanism between adenosine receptor ADORA1 and gastric cancer macrophage M2 polarization, the present application example Fig.13 The bioinformatics analysis results shown in the figure show that there is a positive correlation between ADORA1 and IL-6. To verify the bioinformatics results, a cell group was established based on the GC AGS cell line, including a control group (sh NC) and an ADORA1 knockout group (sh-ADORA1). The expression of IL-6 in the two groups was detected by ELISA. The results are shown in Fig.14 As shown. Fig.14 It can be seen that the IL-6 expression level in the ADORA1 knockout group was significantly reduced compared with the control group. Next, the present application studied the relationship between ADORA1 / IL-6 and macrophage polarization.

[0075] Based on the GC AGS cell line, we constructed cell groups, including a control group (sh-NC+DMSO), an ADORA1 knockout group (sh-ADORA1+DMSO), and a silencing ADORA1 and IL-6 treatment group (sh-APORA1+IL-6). The cells in each treatment group were co-cultured with induced differentiated M0 macrophages, and qPCR was used to analyze the mRNA expression of M1 macrophage markers (CD86) and M2 macrophage markers (CD206) in each co-culture system. There was no significant difference in the mRNA expression level of CD86 in the three co-culture systems.

[0076] However, if Fig.15 As shown, compared with the control co-culture system, the mRNA expression level of CD206 in the ADORA1 silencing co-culture system was significantly reduced, but it was restored in the co-treated co-culture system. Subsequently, the present application used WB to determine the M1 macrophage marker (CD86) and M2 macrophage marker (CD206) in the three co-culture systems. Fig.16 The results shown indicate that changes in protein levels of M1 and M2 macrophages in the three groups of co-culture lines showed the same trend as changes in mRNA levels.

[0077] This indicates that ADORA1 activation promotes the polarization of M0 macrophages to M2 macrophages by activating downstream IL-6 expression, but does not affect the polarization of M0 macrophages to M1 macrophages. Flow cytometry and ELISA experiments were used to measure the relevant indicators of M1 and M2 macrophages in the three co-culture systems, which also verified this conclusion. Fig.17 and Fig.18The flow cytometry results shown showed that there was no difference in the proportion of surface markers of M1 macrophages (CD68 positive and CD86 positive) among the three co-culture systems. However, compared with the control co-culture system, the proportion of surface markers of M2 macrophages (CD68 positive and CD206 positive) in the ADORA1 silencing co-culture system was significantly reduced, and this reduction was alleviated after the addition of IL-6.

[0078] Fig.19 The ELISA results shown show that the changes in cytokines (IL-23 for M1 macrophages and TGF-β for M2 macrophages) in the three co-culture systems follow the same trend as the flow cytometry results. In addition, the Transwell assay was used to assess the chemotactic migration level of M2 macrophages in each co-culture system. Fig. 20 The results showed that there was no significant difference in the chemotactic migration level of M2 macrophages in the three co-culture systems. In conclusion, ADORA1 in GC cells upregulated the expression of IL-6, thereby enhancing the polarization of M0 macrophages to M2 macrophages. This process did not affect the polarization of M0 macrophages to M1 macrophages, nor did it enhance the chemotactic ability of M2 macrophages.

[0079] The above experiments show that adenosine combined with ADORA1 can enhance the secretion of IL-6 at the cellular level and mediate the polarization of M0 macrophages into M2 macrophages. The specific role of this mechanism in animals remains to be confirmed. In view of this, the present application embodiment designs a series of animal experiments to further confirm this.

[0080] First, mice with gastric cancer in the orthotopic mouse model were divided into four groups and treated with different treatments, including the control group (NC), adenosine group (adenosine), ADORA1 receptor antagonist group (DPCPX), and adenosine and receptor antagonist combined treatment group (adenosine + DCPX). After 30 days of culture, the mice were euthanized, and the physiological changes of the gastric wall in each treatment group were observed and photographed. Fig.21 As shown. Fig.21 It can be seen that compared with the control group, the number of gastric wall nodules in the adenosine group increased, while that in the antagonist group decreased. Compared with the adenosine group, the malignancy of the tumor in the combined treatment group was restored to a certain extent.

[0081] Next, qPCR was performed to evaluate the mRNA expression levels of ADORA1 in the four treatment groups. Fig. 22 The results showed that compared with the antagonist group, the ADORA1 mRNA level in the adenosine group was significantly increased, the antagonist group was significantly downregulated, and the combined treatment group was partially restored. The present application example also used WB and IHC to detect and evaluate the protein expression level of ADORA1 in the four treatment groups. The results were as follows: Fig.23 and Fig.24 As shown. Compared with the control group, the ADORA1 protein level was significantly increased in the adenosine group, significantly decreased in the antagonist group, and partially restored in the combined treatment group compared with the antagonist group. The results showed that adenosine binds to ADORA1 and upregulates its expression level in the animal gastric cancer model. Adenosine / ADOR1 signaling enhances gastric cancer progression.

[0082] In addition, flow cytometry was performed in the present embodiment to evaluate the expression of surface markers of M1 macrophages and M2 macrophages in the four treatment groups. The results are as follows: Fig.25 and Fig.26 As shown. Among the four treatment groups, the proportion of M1 macrophage surface markers (CD11b positive and CD86 positive) did not change significantly, while the percentage of M2 macrophage surface markers (CD11b positive and CD206 positive) showed a significant trend. Compared with the control group, the proportion of M2 macrophage surface markers in the adenosine group increased significantly, while that in the antagonist group decreased significantly. Compared with the antagonist group, the trend of M2 polarization attenuation in the combined treatment group was partially alleviated.

[0083] In addition, the present invention also uses ELISA to determine the secretion of IL-6, M1 macrophage factor (IL-23) and M2 macrophage factor (TGF-β) in each treatment group. Fig. 27 The results showed that compared with the control group, the IL-6 level in the adenosine group was significantly increased, while the IL-6 level in the antagonist group was significantly decreased. Compared with the antagonist group, the combined treatment group showed a recovery trend in the secretion of the corresponding cytokines. In addition, there was not much change in the polarization-related cytokines (IL-23) of M1 macrophages in each group. In conclusion, in the GC animal model, adenosine can enhance IL-6 secretion and promote M2 polarization of GC macrophages by binding to the ADORA1 receptor, ultimately promoting the malignant progression of GC.

[0084] This study found that adenosine expression was upregulated in gastric juice and tissues of gastric cancer patients, but there was no significant difference in the blood, which means that the quality of gastric juice as a gastric cancer biomarker sample is better than blood, which indicates the potential of gastric juice as a GC detection sample. According to the reported adenosine mechanism and receptors, cell experiments confirmed that adenosine binds to ADORA1 and upregulates its expression in GC. Bioinformatics analysis found that ADORA1 is associated with M2 polarization of GC-associated macrophages. In addition, in vivo and in vitro experiments confirmed that adenosine binding to ADORA1 in GC can enhance IL-6 secretion and promote M2 polarization of macrophages, and has no effect on the chemotaxis and polarization of M1 macrophages, thereby reshaping the IME of GC. In summary, this application links adenosine and its receptor ADORA1 to IL-6 secretion by GC cells and M2 polarization of macrophages.

[0085] This application verified the mechanism by which adenosine binds to ADORA1 to promote macrophage M2 polarization through in vivo and in vitro experiments, confirming that adenosine and ADORA1 have a strong binding affinity in GC, ADORA1 plays a key role in promoting the malignant progression of GC, and ADORA1 is positively correlated with genes related to macrophage M2 polarization in GC. In vitro cell experiments showed that high expression of ADORA1 induced polarization of M0 macrophages into M2 macrophages, but did not affect the polarization of M0 macrophages into M1 macrophages. Adding the ADORA1 antagonist DPCPX to the high ADORA1 group can reduce the effect on macrophage M2 polarization.

[0086] This application study found that the combination of adenosine and ADORA1 enhances the M2 polarization of macrophages in GC, which is one of the mechanisms by which adenosine-ADORA1 reshapes GC IME. This application is the first to verify the effect of adenosine and its receptor ADORA1 on the M2 polarization of macrophages in GC, which is one of the innovations of this application.

[0087] This application study found that adenosine is closely related to GC IME remodeling. At the molecular level, adenosine binds to ADORA1 to promote GC cell secretion of IL-6 and M2 macrophage polarization. The results of this application emphasize the potential role of adenosine and ADORA1 as targets for GC immunotherapy. In addition, this application shows that gastric fluid may be a potential high-quality sample for collecting GC biomarkers. In many cases, blood samples may not provide the necessary tumor markers for GC patients, and the collection of tumor tissue samples requires strict requirements (failure to accurately collect tumor tissue may result in false negative results). The method used in this study to detect adenosine in gastric fluid may be a new way to solve this dilemma.

[0088] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. An inducer of M2 macrophages, characterized in that The inducer of M2 macrophages is a combination of adenosine and ADORA1, and the adenosine is obtained from the gastric juice of gastric cancer patients.

2. The inducer of M2 macrophages according to claim 1, characterized in that: The combination of adenosine and ADORA1 is obtained from gastric cancer AGS cells.

3. The inducer of M2 macrophages according to claim 1, characterized in that: The combination of adenosine and ADORA1 is a culture medium or a kit for the complex of adenosine combined with ADORA1.

4. Use of the M2 macrophage inducer according to any one of claims 1 to 3 in the preparation of gastric cancer immunotherapy drugs.

5. The use according to claim 4, characterized in that: The M2 macrophage inducer is used to mediate IL-6 secretion and promote the polarization of M2 macrophages.

6. A biomarker for gastric cancer immunotherapy, characterized in that: The gastric cancer immunotherapy biomarker is adenosine obtained from the gastric juice of gastric cancer patients.

7. Use of the gastric cancer immunotherapy biomarker according to claim 6 in the preparation of gastric cancer immunotherapy drugs.

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