Application of the OLFM4-CCL2 signaling axis in regulating the tumor immune microenvironment

Abstract

This invention discloses the application of the OLFM4-CCL2 signaling axis in regulating the tumor immune microenvironment, belonging to the field of biomedical technology. This invention reveals for the first time the complete mechanism by which OLFM4 upregulates CCL2 expression by activating the MAPK-AP1 signaling pathway, thereby recruiting immature neutrophils and inducing NETosis, thus reshaping the immunosuppressive microenvironment. Experiments demonstrate that OLFM4 knockout significantly inhibits tumor growth, reduces immature neutrophil infiltration, lowers NETosis levels, and restores CD8+. + T cell function. Based on this mechanism, this invention provides the application of CCL2 as a target in the preparation of drugs for treating biliary tract tumors, as well as pharmaceutical compositions using CCL2 inhibitors in combination with PD-1 inhibitors. In vivo experiments have demonstrated that the combination therapy of α-CCL2 and α-PD-1 has a synergistic anti-tumor effect and significantly increases CD8⁺ T cell infiltration. This invention provides new targets and strategies for the immunotherapy of biliary tract tumors and has important clinical translational value.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to a method for regulating the tumor immune microenvironment through the OLFM4-MAPK-AP1-CCL2 signaling axis, and its application in drug screening, preparation of drug compositions, and treatment of biliary tract tumors based on this signaling axis. Background Technology

[0002] Biliary tract carcinoma (BTC) is a highly heterogeneous group of malignant tumors originating from the epithelium of the biliary system, including intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and gallbladder cancer. Due to the insidious nature of early symptoms, most patients are diagnosed at a locally advanced stage or with distant metastases, thus losing the opportunity for radical surgery. Although chemotherapy regimens, such as gemcitabine combined with cisplatin, have been established as the first-line standard of care, the objective response rate remains below 30%, and drug resistance is widespread. In recent years, immunotherapy, represented by immune checkpoint inhibitors, has achieved breakthroughs in various solid tumors, but the overall response rate in BTC is only 10%-20%, suggesting a unique immunosuppressive characteristic in its tumor microenvironment. Therefore, in-depth analysis of the key molecular events of immune escape in BTC and the discovery of core regulatory factors driving tumor microenvironment remodeling are of significant clinical importance for developing novel combination therapy strategies.

[0003] Tumor-associated neutrophils (TAN) are among the most abundant myeloid immune cells in the tumor microenvironment, and their role in cancer progression has recently gained renewed attention. Contrary to the traditional understanding of neutrophils as short-lived, terminally differentiated cells and "bystanders," recent research reveals significant functional heterogeneity and differentiation plasticity in neutrophils. Immature neutrophils have been shown to possess potent pro-cancer activity, promoting tumor cell proliferation, angiogenesis, metastasis, and immune evasion through mechanisms such as secretion of cytokines and growth factors, and the formation of extracellular neutrophil traps. NETosis, a programmed cell death mechanism unique to neutrophils, has been shown to promote tumor progression through various mechanisms, including directly killing tumor cells, promoting tumor cell invasion and metastasis, and encapsulating tumor cells to evade recognition and attack by cytotoxic immune cells. However, in biliary tract tumors, the specific molecular signals that dominate the intratumoral recruitment of immature neutrophils and their NETosis induction remain poorly understood.

[0004] CC motif chemokine ligand 2 is a prototype member of the CC family of chemokines, whose classic function is to recruit monocytes / macrophages to inflammatory sites by binding to CC motif chemokine receptor 2. In recent years, the functional spectrum of CCL2 has been significantly expanded. In the field of tumor immunology, CCL2 has been shown to participate in tumor-associated macrophage recruitment, myeloid-derived suppressor cell mobilization, angiogenesis, and the upregulation of immune checkpoint molecules. However, whether CCL2 participates in neutrophil recruitment and its upstream regulatory mechanisms remains a blank in biliary tract tumors.

[0005] The mitogen-activated protein kinase (MAPK) signaling pathway is an evolutionarily conserved serine / threonine protein kinase cascade system, widely recognized as one of the most important oncogenic driving pathways in tumor biology. The MAPK signaling pathway can be activated by various stress signals, amplifying these signals through cascading phosphorylation to ultimately regulate cell proliferation, differentiation, survival, and inflammatory responses. Activator protein-1 (AP1) is a key downstream transcription factor complex of the MAPK pathway, regulating multiple cellular processes, including differentiation, apoptosis, and proliferation. However, whether the MAPK-AP1 signaling axis participates in regulating CCL2 expression and its role in the remodeling of the immune microenvironment in biliary tract tumors remains unreported.

[0006] Oligosin 4 (OLFM4) is a member of the olfactory protein family. In our previous research on gallbladder cancer, we found that OLFM4 mediates tumor cell immune escape by activating the MAPK-AP1 signaling axis and upregulating PD-L1 expression. However, whether OLFM4 affects neutrophil recruitment by regulating chemokines, especially its molecular association with CCL2 and its function in immune microenvironment remodeling, remains a blank.

[0007] Therefore, elucidating the molecular mechanisms by which OLFM4 regulates the tumor immune microenvironment and identifying key modifiable molecular nodes is of significant scientific and clinical value for developing therapeutic strategies targeting the immune microenvironment. This invention, based on a systematic study of the OLFM4-MAPK-AP1-CCL2 signaling axis, reveals for the first time the complete mechanism by which OLFM4 upregulates CCL2 expression through activation of the MAPK-AP1 pathway, thereby recruiting immature neutrophils and inducing NETosis, thus reshaping the immunosuppressive microenvironment. This provides a novel intervention target and strategy for the treatment of biliary tract tumors. Summary of the Invention

[0008] The purpose of this invention is to provide an application of the OLFM4-CCL2 signaling axis in regulating the tumor immune microenvironment, specifically involving the molecular mechanism by which OLFM4 regulates the expression of the ligand CCL2 through the MAPK-AP1 signaling axis, thereby affecting the recruitment and function of immune cells.

[0009] Another object of the present invention is to provide a drug screening method and its application based on this signal axis.

[0010] Another object of the present invention is to provide the use of CCL2 inhibitors in the preparation of drugs for treating biliary tract tumors, particularly a synergistic treatment strategy in combination with immune checkpoint inhibitors.

[0011] 1. Elucidation of the molecular mechanism This invention reveals for the first time the existence of the OLFM4-MAPK-AP1-CCL2 signaling axis and its core role in the remodeling of the tumor immune microenvironment. Through a series of molecular biology and cell biology experiments, this invention confirms that: (1) OLFM4 regulates CCL2 expression. Through cross-species and cross-cell line transcriptome sequencing analysis, after knocking down OLFM4 in RBE and KPC cells, the intersection analysis of differentially expressed genes showed 318 co-downregulated genes. GO enrichment analysis showed that these genes were significantly enriched in tumor microenvironment remodeling pathways such as epithelial-mesenchymal transition and angiogenesis, among which the chemokine CCL2 was significantly downregulated. Western blot validation showed that OLFM4 and CCL2 expression were positively correlated.

[0012] (2) OLFM4 regulates CCL2 through the MAPK-AP1 signaling axis. Western blot experiments confirmed that OLFM4 deficiency can inhibit the phosphorylation levels of key MAPK pathway molecules ERK and JNK, as well as the activity of downstream transcription factor AP1. Combined with previous studies by our research group, it was confirmed that OLFM4 upregulates CCL2 expression by activating the MAPK-AP1 signaling axis.

[0013] (3) OLFM4 recruits immature neutrophils via CCL2. Flow cytometry analysis showed that immature neutrophils (CD11b) in tumor tissues of Olfm4 wild-type mice... + Ly6G + The proportion of Ly6Chi was significantly higher in the OLFM4 knockdown group than in the Olfm4 knockout group; pseudo-time series analysis showed that wild-type neutrophils were enriched in the early stages of the differentiation trajectory. Transwell chemotaxis assays confirmed that the migration ability of neutrophils in the OLFM4 knockdown group was significantly reduced, and the effect was comparable to that in the CCL2 chemotactic signal loss group, indicating that OLFM4 mediates neutrophil chemotaxis through CCL2.

[0014] (4) OLFM4 induces NETosis through CCL2. Single-cell transcriptome GSEA analysis showed that the set of NETosis-related genes was significantly enriched in wild-type neutrophils; in situ immunofluorescence of tissues showed that there were large areas of MPO / cit-H3 double-positive NETosis in wild-type tumor tissues; ELISA showed that the level of MPO-DNA complex in the peripheral blood of wild-type mice was significantly increased; Western blot confirmed that the level of MPO protein in wild-type tumor tissues was significantly increased.

[0015] (5) OLFM4 inhibits T cell function by remodeling the immune microenvironment. Tumor-immune cell co-culture experiments showed that cells derived from the wild-type tumor microenvironment significantly inhibited CD8. + T cell IFNγ secretion and proliferation activity.

[0016] (6) Validation of the in vivo therapeutic effect of targeting CCL2. In a subcutaneous tumor-bearing mouse model, α-CCL2 monotherapy significantly inhibited tumor growth. The combination therapy of α-CCL2 and α-PD-1 had a synergistic anti-tumor effect, manifested by minimizing tumor burden and increasing CD8 concentration in tumor tissue. + T-cell infiltration was the most significant.

[0017] Based on the above findings, this invention proposes a working model of the “OLFM4-MAPK-AP1-CCL2” signaling axis: OLFM4, which is highly expressed in tumor cells, activates the MAPK-AP1 signaling axis and upregulates the expression of its ligand CCL2. As a key effector molecule, CCL2 recruits immature neutrophils to the tumor microenvironment on the one hand, and induces them to undergo NETosis on the other hand. At the same time, it synergistically upregulates PD-L1, thereby shaping a deeply immunosuppressive tumor microenvironment through both innate and adaptive immune pathways, driving the malignant progression of biliary tract tumors.

[0018] 2. Drug screening methods Based on the above molecular mechanism, this invention provides a method for screening candidate substances that regulate the tumor immune microenvironment, comprising the following steps: a) Contact the test substance with tumor cells expressing OLFM4; b) Detect the expression level of CCL2 or the activity of the MAPK-AP1 signaling pathway in the cells; and c) If, compared with the control, the test substance can downregulate the expression level of CCL2 or inhibit the activity of the MAPK-AP1 signaling pathway, then the test substance is identified as a candidate substance for regulating the tumor immune microenvironment.

[0019] The detection methods described in step b) include, but are not limited to, qRT-PCR, Western Blot, ELISA, immunofluorescence, or reporter gene assays.

[0020] 3. Application This invention provides the application of CCL2 as a target in the preparation of drugs for the treatment of biliary tract tumors, wherein the drugs target CCL2 and block the formation of an immunosuppressive microenvironment mediated by the OLFM4-CCL2 signaling axis.

[0021] This invention provides the use of a CCL2 inhibitor in the preparation of a medicament for treating biliary tract tumors, wherein the CCL2 inhibitor inhibits the recruitment of immature neutrophils and / or the formation of NETosis by blocking the chemotactic function of CCL2.

[0022] Preferably, the CCL2 inhibitor is used in combination with an immune checkpoint inhibitor; the immune checkpoint inhibitor is preferably a PD-1 inhibitor or a PD-L1 inhibitor.

[0023] The present invention provides a pharmaceutical composition for treating biliary tract tumors, comprising an effective amount of a CCL2 inhibitor and an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier; wherein the CCL2 inhibitor and the immune checkpoint inhibitor work synergistically to enhance the anti-tumor immune response.

[0024] The CCL2 inhibitor may be selected from one or more of the following groups: neutralizing antibodies targeting CCL2, antagonists of the CCL2 receptor CCR2, siRNA, shRNA, antisense oligonucleotides, CRISPR gene editing systems, and small molecule compounds targeting CCL2; the immune checkpoint inhibitor may be selected from one or more of the following groups: anti-PD-1 antibody, anti-PD-L1 antibody.

[0025] This invention also provides the application of OLFM4 or CCL2 as biomarkers in the preparation of kits for predicting the prognosis of biliary tract tumors. The prognosis of patients can be assessed by detecting the expression level of OLFM4 or CCL2 in tumor tissue or by detecting the activity of the OLFM4-CCL2 signaling axis.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to discover and demonstrate that OLFM4 upregulates CCL2 expression through the MAPK-AP1 signaling axis, thereby recruiting immature neutrophils and inducing NETosis, reshaping the immunosuppressive microenvironment. This discovery extends the function of OLFM4 from its intrinsic effects on tumor cells to the level of tumor immune microenvironment regulation, providing a new paradigm for understanding immune escape in biliary tract tumors.

[0027] 2. This invention reveals that CCL2, as a key downstream effector molecule of OLFM4, is a core node regulating neutrophil recruitment and NETosis. Targeting CCL2 can effectively block the formation of an immunosuppressive microenvironment, providing a theoretical basis for the development of novel immunotherapeutic drugs.

[0028] 3. This invention demonstrates through in vivo drug intervention experiments that the combined treatment of α-CCL2 and α-PD-1 has a synergistic anti-tumor effect, significantly superior to monotherapy. This discovery provides a new strategy for immunotherapy of biliary tract tumors and has significant clinical translational value.

[0029] 4. Through clinical sample validation and survival analysis, this invention confirms that the activity of the OLFM4-CCL2 signal axis is closely related to the poor prognosis of patients with biliary tract tumors, suggesting that this signal axis can serve as a biomarker for judging patient prognosis and provide a basis for precision clinical treatment. Attached Figure Description

[0030] Figure 1 The effect of Olfm4 gene knockout on the growth of subcutaneous xenografts of cholangiocarcinoma in mice; (A) gross photograph of tumor tissue; (B) tumor weight distribution; (C) tumor volume growth curve; Figure 2 Flow cytometry analysis of tumor-infiltrating neutrophil subsets; including (A) flow cytometry analysis of the maturity of tumor-infiltrating neutrophils; and (B) statistical analysis of the proportion of neutrophil subsets. Figure 3 Pseudo-temporal analysis was used to analyze the differentiation trajectory of neutrophils; (A) pseudo-temporal analysis was used to analyze the differentiation trajectory; (B) pseudo-time-related gene expression heatmaps were used. Figure 4 Single-cell transcriptome GSEA analysis showed the enrichment of NETosis-related gene sets; (A) neutrophil module score; (B) enrichment analysis of NETosis-related gene sets; Figure 5 O lfm4 WT and O lfm4 - / - Differential analysis of transcriptome and regulator enrichment; including (A) differential gene expression analysis; (B) specificity analysis of transcription factor regulator enrichment; Figure 6 O lfm4 WT and O lfm4 - / - Remodeling analysis of malignant cell → neutrophil communication; Figure 7 OLFM4 vs CD8 + The regulatory role of T cell effector function and proliferation activity; among which (A) CFSE staining detection of CD8 +T cell proliferation activity; (B) CD8 + Ifng + Cell proportion statistics; (C)CD8 + Validation of the association between T cell proliferation and effector function; Figure 8 OLFM4 promotes protein and tissue-level validation of tumor-associated neutrophil NETosis; including (A) in situ MPO / cit-H3 double staining immunofluorescence detection in tumor tissue; (B) ELISA detection of peripheral blood MPO-DNA complex quantification; (C) MPO protein expression in tumor tissue. Figure 9 Transwell chemotaxis assay to verify the effect of OLFM4 on neutrophil migration ability; including (A) neutrophil migration assay; (B) migration cell count statistics; Figure 10 Downstream conserved genes regulated by OLFM4 and their functional enrichment analysis; including (A) cross-cell line differential gene intersection analysis; (B) GO functional enrichment analysis of 318 commonly downregulated genes; (C) mRNA level validation of key candidate genes. Figure 11 Construction of OLFM4 knockdown cell lines and verification of knockdown efficiency; (A) qPCR verification of OLFM4 mRNA level; (B) Western Blot verification of OLFM4 protein level; Figure 12 Effects of OLFM4 knockdown on the proliferation, migration and invasion of cholangiocarcinoma cells; (A) cell growth curve analysis; (B) Transwell assay of RBE cell line migration ability; (C) Matrigel assay of RBE and KPC cell lines invasion ability. Figure 13 The mechanism by which OLFM4 regulates neutrophil recruitment through CCL2 was verified; (A) in vitro neutrophil chemotaxis experiment; (B) Western Blot analysis of the effect of OLFM4 on CCL2 protein expression. Figure 14 In vivo validation of CCL2-targeted combined anti-PD-1 immunotherapy in biliary tract tumors; (A) gross tumor photograph; (B) tumor growth curve; (C) tumor weight distribution; (D) CD45 of tumor tissues in different groups. + CD8 + Cell statistics. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0032] Example 1: Effect of OLFM4 gene knockout on the growth of biliary tract tumors 1. Experimental Objective This embodiment aims to verify the promoting effect of OLFM4 on the in vivo growth of biliary tract tumors, and to provide in vivo phenotypic evidence for subsequent mechanism analysis.

[0033] 2. Experimental Materials (1) Cells: Mouse cholangiocarcinoma cell line KPC was purchased from QuiCell Cell Bank of Shanghai Kuisai Biotechnology Co., Ltd.

[0034] (2) Animals: Five 6-8 week old male Olfm4 wild-type mice with C57BL / 6J background and five Olfm4 knockout mice were purchased from Nanjing Jicui Pharmaceutical Co., Ltd. and housed in an SPF-grade animal facility.

[0035] (3) Main reagents: DMEM medium, fetal bovine serum, penicillin-streptomycin, PBS, 4% paraformaldehyde.

[0036] (4) Main instruments: CO2 constant temperature incubator, ultra-clean workbench, electronic vernier caliper.

[0037] 3. Experimental Methods KPC cells in the logarithmic growth phase were collected, washed twice with PBS, and resuspended in sterile PBS to adjust the cell concentration to 5 × 10⁻⁶. 6 Cells / ml. Using a 1ml insulin syringe, 100μl of cell suspension (i.e., 5×10⁶ cells / ml) was subcutaneously injected into the right back of mice. 5 (cells / animal). Starting on day 7 post-inoculation, the long and short diameters of the tumor were measured every 3 days using electronic calipers, and calculated using the formula V = 0.5 × long diameter × short diameter. 2 Calculate tumor volume. Measure continuously until day 28 post-inoculation and plot tumor growth curves. Sacrifice mice on day 28 post-inoculation, completely dissect the tumor tissue, remove surrounding connective tissue, weigh, and photograph the tumor.

[0038] 4. Experimental Results The experimental results showed that the tumor growth rate in the Olfm4 knockout group was significantly slower than that in the wild-type group. From day 14 post-inoculation, the tumor volume in the knockout group was significantly smaller than that in the wild-type group, and this difference persisted at subsequent time points. Tumor weight analysis showed that the tumor weight in the knockout group was significantly lower than that in the wild-type group. Figure 1 This result directly confirms that OLFM4 has the ability to promote the in vivo growth of biliary tumor cells.

[0039] Example 2: Regulation of Immature Neutrophil Recruitment by OLFM4 1. Experimental Objective This embodiment aims to investigate the effects of OLFM4 on neutrophil subsets and their differentiation status in the tumor microenvironment.

[0040] 2. Experimental Materials (1) Animal-derived tumor tissue: Subcutaneous tumor tissue from wild-type and Olfm4 knockout mice in Example 1.

[0041] (2) Main reagents: collagenase IV, hyaluronidase, DNA enzyme I, red blood cell lysis buffer, Fc receptor blocker, flow cytometry antibodies (FITC-anti-CD45, APC-anti-CD11b, PE-anti-Ly6G, PerCP-Cy5.5-anti-Ly6C).

[0042] (3) Main instruments: flow cytometer, constant temperature shaker, cell filter.

[0043] 3. Experimental Methods 3.1 Preparation of single-cell suspension from tumor tissue Take approximately 100 mg of fresh tumor tissue, place it in 1640 culture medium containing 2% FBS, and cut it into 1 mm pieces. 3 Fragmented tissue was added to a tissue digestion solution containing collagenase IV (1 mg / ml), hyaluronidase (0.1 mg / ml), and DNase I (0.1 mg / ml). The tissue was digested at 37°C in a shaker for 45 minutes, with pipetting every 15 minutes. After digestion, the tissue was passed through 70 μm and 40 μm cell filters sequentially. The filtrate was centrifuged at 400 g for 5 minutes at 4°C, and the supernatant was discarded. Red blood cell lysis buffer was added and the tissue was treated at room temperature for 3 minutes. Lysis was terminated by adding PBS containing 2% FBS. The tissue was then centrifuged again, washed, and resuspended in flow cytometry staining buffer.

[0044] 3.2 Flow cytometry detection Add 100 μl of single-cell suspension to a flow cytometry tube, add Fc receptor blocker, and incubate at 4°C for 10 minutes. Then add a surface antibody mixture: FITC-anti-CD45, APC-anti-CD11b, PE-anti-Ly6G, and PerCP-Cy5.5-anti-Ly6C, and incubate at 4°C in the dark for 30 minutes. After washing, analyze the sample. Immature neutrophils are defined as CD45 cells. + CD11b + Ly6G + Ly6Chi cell subset.

[0045] 3.3 Quasi-Time Series Analysis The CD11b⁺Ly6G⁺ cell population sorted by flow cytometry was imported into R software, and pseudo-time series analysis was performed using the Monocle2 package. Using the expression levels of Ly6G and CD11b as reference indicators of differentiation status, differentiation trajectory diagrams were plotted, and the differences in the distribution of neutrophils in the two groups along the differentiation trajectory were compared.

[0046] 4. Experimental Results Flow cytometry results showed that in the tumor microenvironment of the wild-type group, the proportion of immature neutrophils with low CD101 expression was significantly higher than that in the knockout group. Figure 2 Pseudo-time series analysis showed that wild-type neutrophils were more distributed at earlier nodes of the differentiation trajectory, while the knockout group showed a relatively mature differentiation bias. Figure 3 The above results indicate that OLFM4 promotes the accumulation of immature subsets in the tumor by regulating the differentiation and maturation process of neutrophils.

[0047] Example 3: The regulatory effect of OLFM4 on NETosis 1. Experimental Objective This embodiment aims to investigate the effect of OLFM4 on the ability of tumor-associated neutrophils to form NETosis.

[0048] 2. Experimental Materials (1) Animal-derived tumor tissue and serum: Subcutaneous tumor tissue and peripheral blood were obtained from wild-type and Olfm4 knockout mice in Example 1.

[0049] (2) Major antibodies: rabbit anti-mouse MPO primary antibody, mouse anti-human citrullinated histone H3 primary antibody, goat anti-rabbit IgG labeled with Alexa Fluor 488, and goat anti-mouse IgG labeled with Alexa Fluor 594.

[0050] (3) Main reagents: MPO-DNA ELISA kit, RIPA lysis buffer, BCA protein quantification kit.

[0051] (4) Main instruments: Leica TCS SP8 confocal microscope, microplate reader, Western Blot electrophoresis system.

[0052] 3. Experimental Methods 3.1 Immunofluorescence double-labeling staining Paraffin sections of subcutaneous tumor tissue from mice bearing tumors, 4 μm thick, were dewaxed, hydrated, and antigen-retrieved. Rabbit anti-mouse MPO primary antibody and mouse anti-human cit-H3 primary antibody were added simultaneously, and the sections were incubated overnight at 4°C. The following day, after washing, Alexa Fluor 488-labeled goat anti-rabbit IgG and Alexa Fluor 594-labeled goat anti-mouse IgG were added, and the sections were incubated at 37°C in the dark for 1 hour. Cell nuclei were counterstained with DAPI, and the sections were mounted and observed under a confocal microscope.

[0053] 3.2 ELISA detection Peripheral blood was collected from mice and allowed to coagulate at room temperature for 30 minutes. Serum was then separated by centrifugation at 3000 rpm for 15 minutes. The level of NETosis markers in the serum was detected using the MPO-DNA ELISA kit. The absorbance value was read at 450 nm according to the instructions, and the sample concentration was calculated based on the standard curve.

[0054] 3.3 Western Blot Detection Total protein was extracted from tumor tissue by lysis and quantified using the BCA method. 30 μg of protein was then subjected to SDS-PAGE electrophoresis, transferred to a membrane, and incubated with MPO primary antibody and HRP-labeled secondary antibody. ECL luminescence was then performed, and GAPDH was used as an internal control for semi-quantitative analysis.

[0055] 4. Experimental Results Immunofluorescence assays showed that the wild-type group tumor tissue contained large, patchy, reticular MPO / cit-H3 double-positive structures, mainly distributed in the tumor stroma; while the knockout group only showed scattered, punctate double-positive staining, with significantly reduced fluorescence intensity. Figure 8 A). ELISA analysis showed that the level of MPO-DNA complex in the peripheral blood of wild-type mice was significantly higher than that in the knockout group ( Figure 8 B). Western blot analysis showed that the MPO protein level in the wild-type group tumor tissue was significantly higher than that in the knockout group (B). Figure 8 C). The above results confirm that OLFM4 expression can promote NETosis in tumor-associated neutrophils.

[0056] Example 4: Regulation of CD8⁺ T cell function by OLFM4 1. Experimental Objective This embodiment aims to explore the regulatory role of OLFM4 in adaptive immune response, particularly the effector function of CD8⁺ T cells.

[0057] 2. Experimental Materials (1) Cell source: Subcutaneous tumor tissue from wild-type and Olfm4 knockout mice in Example 1; spleen from mice of the same strain.

[0058] (2) Main reagents: magnetic bead sorting kit, IL-2, anti-CD3 / CD28 Dynabeads, CFSE working solution, Live / Dead dye, flow cytometry antibodies (PE-anti-CD8a, APC-anti-CD4, PE-Cy7-anti-IFNγ).

[0059] (3) Main instruments: flow cytometer, magnetic rack, CO2 constant temperature incubator.

[0060] 3. Experimental Methods 3.1 Primary cell isolation Subcutaneous tumor tissue from mice bearing tumors was collected, and a single-cell suspension was prepared according to the method in Example 2 to serve as the source cells for the tumor microenvironment in the co-culture system. Simultaneously, spleens from mice of the same strain were collected, ground, and lysed with erythrocytes to obtain spleen cell suspensions. CD8+ cells were isolated from the spleen cells using a magnetic bead sorting kit. + T cells were activated for 48 hours in a complete culture medium containing IL-2 and anti-CD3 / CD28 Dynabeads for later use.

[0061] 3.2 CFSE-labeled co-culture system Activated CD8 + T cells were washed with PBS, and CFSE working solution (final concentration 5 μM) was added and incubated at 37°C in the dark for 15 minutes. The staining was then terminated by adding an equal volume of culture medium containing 10% FBS. CFSE-labeled T cells and primary cells derived from tumor tissue were mixed at a ratio of 10:1 and seeded in 96-well round-bottom culture plates and cultured for 72 hours.

[0062] 3.3 T cell function detection After co-culture, cells were collected for flow cytometry analysis. Live / dead cells were distinguished from dead cells, and after surface staining, cells were fixed, perforated, and IFNγ stained intracellularly. CD8+ was assessed by the dilution of CFSE fluorescence intensity. + T cell proliferation activity, with CD8 + The proportion of IFNγ-positive cells in T cells reflects the effector function of T cells.

[0063] 4. Experimental Results The experimental results showed that, compared with the knockout group, the expression level of IFNγ in wild-type tumor microenvironment-derived cells was significantly reduced after co-culturing with T cells, indicating that CD8+ expression was significantly lower. + The effector function of T cells was significantly suppressed. Meanwhile, CD8+ in the wild-type co-culture system... + T cells exhibited significant fluorescence intensity retention, and the dilution of CFSE was significantly reduced, indicating that their proliferative activity was significantly inhibited. Figure 7 The above results indicate that a strong immunosuppressive signal exists in the OLFM4 wild-type tumor microenvironment, which can effectively inhibit CD8. + Clonal expansion of T cells and the exertion of their effector functions.

[0064] Example 5: Molecular mechanism of OLFM4 regulating CCL2 expression 1. Experimental Objective This embodiment aims to explore the molecular mechanism by which OLFM4 regulates CCL2 expression, especially the role of the MAPK-AP1 signaling axis.

[0065] 2. Experimental Materials (1) Cells: human cholangiocarcinoma cell line RBE, human gallbladder cancer cell line GBC-SD, and mouse cholangiocarcinoma cell line KPC.

[0066] (2) Main reagents: TRIzol reagent, reverse transcription kit, SYBR Green qPCR Master Mix, RIPA lysis buffer, protease inhibitor, phosphatase inhibitor, BCA protein quantification kit, ECL chemiluminescence kit.

[0067] (3) Main antibodies: anti-OLFM4, anti-CCL2, anti-p-ERK, anti-ERK, anti-p-JNK, anti-JNK, anti-AP1, anti-GAPDH.

[0068] (4) Main instruments: qPCR instrument, Western Blot electrophoresis system, gel imaging system.

[0069] 3. Experimental Methods 3.1 Construction of stable knockdown cell lines In RBE, GBC-SD, and KPC cells, stable OLFM4 knockdown cell lines and corresponding control cells were constructed by transfecting shRNA with lentivirus. The knockdown efficiency was verified at the transcriptional and protein levels using qRT-PCR and Western blotting, respectively.

[0070] 3.2 Transcriptome Sequencing Analysis Total RNA was extracted from RBE and KPC control cells and OLFM4 knockdown cells for transcriptome sequencing. Differential expression analysis was performed using the DESeq2 package, with selection criteria of |log2FC|≥1 and corrected P<0.05. Genes downregulated in both cell lines were analyzed using Venn diagram intersection analysis, and GO functional enrichment analysis was performed on the intersecting genes using the clusterProfiler package.

[0071] 3.3 Western Blot Validation Cells were collected after treatment, and total protein was extracted for Western blotting. The assay indicators included OLFM4, CCL2, p-ERK, ERK, p-JNK, JNK, and AP1. GAPDH was used as an internal control to analyze changes in the expression of each protein.

[0072] 4. Experimental Results Transcriptome sequencing analysis showed that OLFM4 knockdown in KPC cells resulted in the downregulation of 2358 genes, while in RBE cells 808 genes were downregulated, with a total of 318 genes downregulated in both cell lines. Figure 10 A). GO enrichment analysis showed that these genes were significantly enriched in tumor microenvironment remodeling pathways such as EMT and angiogenesis. Figure 10 B), in which the chemokine CCL2 was significantly downregulated.

[0073] Western blot analysis showed that the activities of p-ERK, p-JNK, and AP1 were significantly decreased in the OLFM4 knockdown group, while the level of CCL2 protein was also significantly downregulated. Figure 13 B). OLFM4 expression was positively correlated with CCL2 expression. These results indicate that OLFM4 upregulates CCL2 expression by activating the MAPK-AP1 signaling axis.

[0074] Example 6: OLFM4 regulates neutrophil chemotaxis via CCL2 1. Experimental Objective This embodiment aims to verify whether OLFM4 regulates the chemotactic migration ability of neutrophils through CCL2.

[0075] 2. Experimental Materials (1) Cells: mouse bone marrow neutrophils, RBE cells and OLFM4 knockdown cells.

[0076] (2) Main reagents: magnetic bead sorting kit, Transwell plate (5 μm pore size), chemokines CXCL1 / CXCL2.

[0077] (3) Main instruments: CO2 constant temperature incubator, flow cytometer.

[0078] 3. Experimental Methods 3.1 Isolation and purification of bone marrow neutrophils Six- to eight-week-old C57BL / 6J mice were used. The femurs and tibias were aseptically dissected bilaterally, and the bone marrow cavity was flushed with RPMI 1640 medium containing 2% FBS. Bone marrow cell suspensions were collected. CD11b was purified using a magnetic bead sorting kit. + Ly6G + Neutrophils.

[0079] 3.2 Transwell chemotaxis experiment Chemotaxis assays were performed using 24-well Transwell plates with 5 μm pore size. 600 μl of culture supernatant from different treatment groups (control cells or OLFM4 knockdown cells) or culture medium containing chemokines was added to the lower chamber. 100 μl of purified neutrophil suspension (containing 1 × 10⁻⁶ cells) was added to the upper chamber. 5 (1 cell), incubated at 37°C for 2 hours. After incubation, cells from the lower chamber were collected for counting.

[0080] 4. Experimental Results Transwell assay results showed that, compared with the control group, the number of migrating neutrophils in the OLFM4 knockdown group was significantly reduced; at the same time, the experimental group without CCL2 chemokine also showed the same degree of decrease in migration ability. Figure 13 A). Western blot analysis showed that CCL2 protein levels were correspondingly elevated in the control group with higher OLFM4 expression, while CCL2 expression was significantly downregulated in the OLFM4 knockdown group. Figure 13 B). The above results indicate that OLFM4 affects the chemotactic recruitment capacity of neutrophils by regulating CCL2 expression.

[0081] Example 7: In vivo antitumor effect of CCL2 targeting combined with PD-1 blockade 1. Experimental Objective This embodiment aims to verify the therapeutic potential of targeting CCL2 in combination with immune checkpoint blockade in biliary tract tumors.

[0082] 2. Experimental Materials (1) Animals: 6-8 week old male C57BL / 6J mice.

[0083] (2) Cells: mouse cholangiocarcinoma cell line KPC.

[0084] (3) Main reagents: α-PD-1 antibody, α-CCL2 antibody, isotype control IgG.

[0085] (4) Main instruments: electronic vernier caliper, flow cytometer.

[0086] 3. Experimental Methods 3.1 Animal Model and Grouping A subcutaneous KPC cell tumor model was established. Mice were randomly divided into four groups of three mice each: isotype control IgG group, α-PD-1 monotherapy group (200 μg / mouse), α-CCL2 monotherapy group (100 μg / mouse), and α-CCL2 and α-PD-1 combination therapy group (same dosage as the monotherapy group). Starting from day 3 post-inoculation, the corresponding drugs were administered intraperitoneally every three days until the end of the experiment.

[0087] 3.2 Tumor growth monitoring Tumor volume was measured every 2 days after inoculation, and growth curves were plotted. Mice were sacrificed on day 21 after inoculation, and tumor tissue was removed and weighed.

[0088] 3.3 Analysis of Immune Cell Infiltration Fresh tumor tissue was used to prepare single-cell suspensions, which were then analyzed by flow cytometry to detect CD45 levels in each group of tumor tissues. + White blood cells and CD8 + The proportion and absolute number of T cells.

[0089] 4. Experimental Results Tumor growth curves and final tumor weight results showed that, compared with the IgG control group, the tumor volume and weight of mice in the α-PD-1 monotherapy group and the α-CCL2 monotherapy group were significantly reduced; the tumor burden of mice in the α-CCL2 and α-PD-1 combination therapy group was significantly lower than that of the other three groups, showing the smallest tumor volume and the lightest tumor weight. Figure 14 AC). Tumor tissue immune cell infiltration analysis showed that the combination therapy group exhibited the most significant CD8⁺ T cell infiltration enrichment (AC). Figure 14 D). The above results indicate that targeting CCL2 in combination with PD-1 blockade has a synergistic anti-tumor effect, enhancing adaptive anti-tumor immune responses by reshaping the tumor immune microenvironment.

[0090] Example 8: Working Model of OLFM4-CCL2 Signal Axis Based on the results of the above embodiments, the present invention proposes a working model for the "OLFM4-MAPK-AP1-CCL2" signal axis: Tumor cells highly express OLFM4, which activates the MAPK-AP1 signaling axis. On one hand, it upregulates PD-L1 expression, directly inhibiting CD8⁺ T cell function; on the other hand, it upregulates CCL2 expression, promoting the recruitment of immature neutrophils and NETosis. Through both innate and adaptive immune pathways, it shapes a deeply immunosuppressive tumor microenvironment, thereby driving the malignant progression of biliary tract tumors. Targeting CCL2 in combination with PD-1 blockade can simultaneously target both innate and adaptive immune inhibitory pathways, achieving a comprehensive remodeling of the immune microenvironment and producing a synergistic anti-tumor effect.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of the OLFM4-CCL2 signaling axis in regulating the tumor immune microenvironment, characterized in that, The signal axis regulates CCL2 expression through OLFM4, thereby affecting the function and composition of immune cells in the tumor microenvironment.

2. The application according to claim 1, characterized in that, The OLFM4 regulates CCL2 expression by activating the MAPK-AP1 signaling pathway; The MAPK-AP1 signaling pathway includes phosphorylation activation of ERK and / or JNK, and upregulation of transcriptional activity of the downstream transcription factor AP1.

3. The application according to claim 1 or 2, characterized in that, The functions and composition of immune cells in the tumor microenvironment include: (1) Recruiting immature neutrophils; and / or (2) Inducing NETosis in neutrophils; and / or (3) Inhibit the proliferation and effector function of CD8⁺T cells.

4. A method for regulating the tumor immune microenvironment based on the OLFM4-CCL2 signaling axis, characterized in that, By regulating the expression or activity of OLFM4 or CCL2, the tumor immune microenvironment can be remodeled. The regulation includes upregulating or downregulating the expression or activity of OLFM4 or CCL2.

5. The application of CCL2 as a target in the preparation of drugs for treating biliary tract tumors, characterized in that, The drug targets CCL2, blocking the formation of an immunosuppressive microenvironment mediated by the OLFM4-CCL2 signaling axis.

6. The use of CCL2 inhibitors in the preparation of drugs for treating biliary tract tumors, characterized in that, The CCL2 inhibitors inhibit the recruitment of immature neutrophils and / or the formation of NETosis by blocking the chemotactic function of CCL2.

7. The application according to claim 6, characterized in that, The CCL2 inhibitor is used in combination with an immune checkpoint inhibitor; The immune checkpoint inhibitor is preferably a PD-1 inhibitor or a PD-L1 inhibitor.

8. A pharmaceutical composition for treating biliary tract tumors, characterized in that, It contains effective amounts of CCL2 inhibitors and immune checkpoint inhibitors, as well as pharmaceutically acceptable carriers; The CCL2 inhibitor works synergistically with immune checkpoint inhibitors to enhance the anti-tumor immune response.

9. The pharmaceutical composition according to claim 8, characterized in that, The CCL2 inhibitor is selected from one or more of the following groups: neutralizing antibodies targeting CCL2, antagonists of the CCL2 receptor CCR2, siRNA, shRNA, antisense oligonucleotides targeting CCL2, CRISPR gene editing systems, and small molecule compounds; The immune checkpoint inhibitor is selected from one or more of the following groups: anti-PD-1 antibody, anti-PD-L1 antibody.

10. A method for screening candidate substances that regulate the tumor immune microenvironment, characterized in that, The method includes the following steps: a) Contact the test substance with tumor cells expressing OLFM4; b) Detect the expression level of CCL2 or the activity of the MAPK-AP1 signaling pathway in the cells; and c) If, compared with the control, the test substance can downregulate the expression level of CCL2 or inhibit the activity of the MAPK-AP1 signaling pathway, then the test substance is identified as a candidate substance for regulating the tumor immune microenvironment.

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