Application of chemokine CCL23 in the preparation of breast cancer immunotherapy drugs

Abstract

The present application provides the use of chemokine CCL23 in the preparation of drugs for breast cancer immunotherapy, which relates to the fields of molecular biology and immunology. Regarding the use of chemokine CCL23 in the preparation of drugs for breast cancer immunotherapy, the chemokine CCL23 of the present application can induce the M1 polarization of TAMs in breast cancer, secrete cytokines such as IL-12, CXCL9, and CXCL10, promote the chemotaxis and function of Th1 cells and CD8+ T cells, enhance the formation of the anti-tumor immune microenvironment, and thus may serve as a new strategy for breast cancer immunotherapy.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies. Specifically, it relates to the use of chemokine CCL23 in the preparation of breast cancer immunotherapy drugs. Background Art

[0002] Currently, the conventional treatment methods for tumors mainly include surgical resection, radiotherapy, and chemotherapy. Although the conventional therapies have been widely applied for many years, their limitations are also very obvious. For some organs, such as tumors in the pancreas, the surgical difficulty is extremely high, and the surgical process is likely to cause damage to the patient's organs. In addition, surgery cannot effectively remove residual tumor cells and prevent tumor metastasis; radiotherapy and chemotherapy often have relatively large side effects, resulting in a decline in the patient's overall immune function and causing symptoms such as pain, nausea, and fatigue. Tumor immunotherapy aims to activate the patient's own anti-tumor immune response to achieve the purpose of clearing tumor cells. Compared with conventional therapies such as surgery, chemotherapy, and radiotherapy, it has irreplaceable advantages such as high specificity in recognizing tumors, small toxic and side effects, and little damage to the patient's organs. With the development of immunological theories and technologies, a series of breakthroughs have been made in tumor immunotherapy in clinical practice. However, while tumor immunotherapy shows great application prospects, the obstacles it faces are becoming increasingly prominent. Especially in the process of treating solid tumors, problems such as the ineffective infiltration of effector T cells into the local tumor and the immunosuppressive microenvironment inside the tumor have become the main factors restricting the efficacy of tumor immunotherapy.

[0003] Once normal cells transform into tumor cells, they become "non-self" components and should be recognized and cleared by the host's immune system. However, tumors can evade and inhibit the anti-tumor immune response through various mechanisms. For example, while tumor cells are rapidly proliferating, they will promote the formation of a microenvironment that supports their growth and invasion, called the tumor microenvironment (Tumor Microenvironment, TME). The shaping of the tumor microenvironment is similar to the process of tissue repair and regeneration, which is characterized by angiogenesis, matrix remodeling, and immunosuppression. The immunosuppression of the TME will lead to the infiltration and impaired function of anti-tumor effector T cells and natural killer cells, greatly limiting the anti-tumor response mediated by the immune system. Therefore, finding ways to overcome the local immunosuppressive microenvironment of tumors is of great significance for the efficacy of tumor immunotherapy.

[0004] During the occurrence and development of solid tumors, various immune cells in the tumor stroma are usually "tricked" by tumor cells to promote their growth, invasion and metastasis, including macrophages, myeloid-derived suppressor cells (MDSCs), immature dendritic cells (iDCs) and regulatory T cells (Tregs). Among them, tumor-associated macrophages (TAMs) are usually the most abundant immune cells in solid tumors and can promote tumor growth or metastasis through multiple mechanisms. TAMs are considered to be M2-like macrophages, and their most significant characteristics and modes of action are that they can inhibit the activation of Th1 cells and induce the differentiation of regulatory T cells (Tregs) by secreting cytokines such as IL-10, TGF-β, arginase-1, etc., thereby preventing CD8 + The lysis of T cells and natural killer cells (NK) against tumors inhibits the anti-tumor immune response. M2-like TAMs can also produce pro-angiogenic factors such as VEGF and IL-1β, as well as matrix metalloproteinases (MMPs), directly or indirectly promoting angiogenesis in tumor tissues. Macrophages in the M1 polarization state can promote the differentiation or infiltration of Th1 cells and CD8 + T cells, playing an anti-tumor immune-enhancing effect. In the actual physiological environment, TAMs have strong heterogeneity, presenting a mixed and complex gene expression profile, expressing both M2-related genes and M1-related genes. It's just that cytokine signals or environmental factors such as hypoxia in the tumor microenvironment (TME) can more strongly activate M2-related transcription factors, such as STAT3, STAT6, PPARγ, etc., so that TAMs show characteristics biased towards M2 polarization. Inducing the deflection of TAMs from the M2 phenotype to the M1 phenotype can enhance the anti-tumor effector function of TAMs and at the same time reduce their immunosuppressive ability, achieving the purpose of tumor immunotherapy. Therefore, finding the key molecules that affect the polarization and function of TAMs in the tumor microenvironment and clarifying their mechanism of action are of great significance for the development of new anti-tumor drug targets. Summary of the Invention

[0005] To solve the above technical problems, the purpose of this application is to provide an application of chemokine CCL23 in the preparation of breast cancer immunotherapy drugs. The chemokine CCL23 of this application has the effect of enhancing anti-tumor immunity, and its mechanism lies in promoting the M1 phenotype polarization of macrophages.

[0006] This application solves its technical problems by adopting the following technical solutions.

[0007] By analyzing the The Cancer Genome Atlas (TCGA) database, all currently known members of the chemokine family were screened, and it was identified that CCL23 / m9 was significantly positively correlated with the survival of breast cancer patients (i.e., high expression indicated a good prognosis), while its expression was significantly decreased in breast cancer patients, suggesting that the decreased expression of CCL23 / m9 might promote the progression of breast cancer. Since in mice, the homologous protein of CCL23 is CCL9, thus in this application, it is all written as CCL23 / m9. Through further analysis and experiments, it was found that: (1) The expression of CCL23 / m9 in breast cancer was significantly positively correlated with the infiltration of the main anti-tumor effector cells - CD4 + Th1 cells and CD8 + T cells, that is, tumors with high expression of CCL23 / m9 showed characteristics of immune activation; (2) Macrophages were the main target cells and main secreting cells of CCL23 / m9 in breast cancer; (3) Intravenous injection of CCL23 / m9 alleviated the growth of breast cancer in mice and up-regulated the expression of characteristic genes of M1 macrophages in tumor tissues (M1 macrophages can promote the infiltration and function of CD4 + Th1 cells and CD8 + T cells); (4) Treatment with CCL23 / m9 in vitro could enhance the expression of M1 characteristic genes in macrophages. The above data indicated that CCL23 / m9 could promote the chemotaxis and function of Th1 cells and CD8 + T cells, and enhance the formation of the anti-tumor immune microenvironment by inducing the M1 polarization of TAMs in breast cancer, secreting cytokines such as IL-12, CXCL9, and CXCL10, thus potentially serving as a new strategy for breast cancer immunotherapy. Based on this, this application further utilized a mouse tumor model, combined with in vitro cell and molecular mechanism experiments, to clarify the functional effect of CCL23 / m9 in promoting the formation of the anti-tumor immune microenvironment by remodeling the function of TAMs. And through the method of combining clinical patient samples with bioinformatics analysis, it provided a theoretical basis, experimental evidence, and clinical feasibility evidence for the immunotherapy strategy based on CCL23 / m9.

[0008] Compared with the prior art, the embodiments of this application at least have the following advantages or beneficial effects:

[0009] 1. The application of the chemokine CCL23 provided in this application in the preparation of breast cancer immunotherapy drugs, for the first time, proposed that: (1) The expression of CCL23 / m9 in breast cancer was significantly positively correlated with the infiltration of the main anti-tumor effector cells - CD4 + Th1 cells and CD8 +The infiltration of T cells was significantly positively correlated, that is, tumors with high expression of CCL23 / m9 exhibited immune activation characteristics; (2) Macrophages were the main target cells and main secreting cells of CCL23 / m9 in breast cancer; (3) Intravenous injection of CCL23 / m9 alleviated the growth of murine breast cancer and upregulated the expression of characteristic genes of M1 macrophages in tumor tissues (M1 macrophages can promote the infiltration and function of CD4 + Th1 cells and CD8 + T cells); (4) Treatment with CCL23 / m9 in vitro could enhance the expression of M1 characteristic genes in macrophages.

[0010] 2. By constructing a murine tumor model and combining in vitro cell and molecular mechanism experiments, this application elucidated the functional effect of CCL23 / m9 in promoting the formation of an anti-tumor immune microenvironment by remodeling the function of TAM. And through the combination of clinical patient samples and bioinformatics analysis, this application provided a theoretical basis, experimental evidence, and clinical feasibility evidence for the immunotherapy strategy based on CCL23 / m9. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] To more clearly illustrate the technical solutions of the embodiments of this application, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative efforts.

[0012] Figure 1 It is a result diagram of analyzing the chemokine family in the TCGA database in Example 1 of this application;

[0013] Among them, Figure 1 A shows the expression changes (abscissa) of the chemokine family in breast cancer patients and the correlation with the OS of patients (ordinate); Figure 1 B shows the pan-cancer expression changes of CCL23 / m9;

[0014] Figure 2 It is the analysis result of the TCGA database and the ROC analysis result in Example 1 of this application; among them, Figure 2 A shows the correlation between the CCL23 / m9 level in the TCGA database and the OS, DFS, and PFI of breast cancer patients; Figure 2 B shows the analysis result of the GEO database; Figure 2 C shows the area under the curve of the ROC analysis of the CCL23 / m9 expression in breast cancer patients

[0015] Figure 3 It is for CCL23 / m9 in Example 1 of this application hi group and CCL23 / m9low Differences in gene expression profiles of tumor tissues of a group of breast cancer patients and results of KEGG / GO enrichment analysis; among them Figure 3 A is for CCL23 / m9 hi group and CCL23 / m9 low Differences in gene expression profiles of tumor tissues of breast cancer patients in group; Figure 3B is the result of KEGG / GO enrichment analysis;

[0016] Figure 4 Correlation analysis of the expression level of CCL23 / m9 with immune infiltration of T cells, NK cells, macrophages and DCs in Example 1 of this application; among them, Figure 4 A is the result of correlation analysis of the expression abundance of CCL23 / m9 in breast cancer with immune infiltration of Th1 cells and Th2 cells; Figure 4 B is the result of correlation analysis of the expression abundance of CCL23 / m9 in breast cancer with immune infiltration of Th17 cells and Tregs; Figure 4 C is the result of correlation analysis of the expression abundance of CCL23 / m9 in breast cancer with immune infiltration of CD8 + T cells and NK cells; Figure 4 D is the result of correlation analysis of the expression abundance of CCL23 / m9 in breast cancer with immune infiltration of dendritic cells (DCs) and macrophages (Macrophages);

[0017] Figure 5 Analysis of the correlation between CCR1, the receptor of CCL23 / m9, and the signatures of various immune cell subsets in the TCGA database in Example 1 of this application;

[0018] Figure 6 Analysis of the expression distribution of CCR1 in breast cancer patients (6A) and mice (6B) in the single-cell sequencing dataset in Example 1 of this application;

[0019] Figure 7 Tumor growth in mice in Example 2 of this application; among them, Figure 7 A is the tumor growth curve of mice; Figure 7 B is the change in tumor weight of mice;

[0020] Figure 8 Results of QPCR detection of the expression of M1 macrophage marker genes in Example 2 of this application;

[0021] Figure 9 In Example 2 of this application, BMDM was stimulated with CCL23 / m9 and IFN-γ-induced M1 BMDM, and the expression of corresponding genes by QPCR. Detailed implementation manners

[0022] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0023] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. This application will be described in detail below with reference to specific embodiments.

[0024] An embodiment of this application provides an application of chemokine CCL23 in the preparation of a breast cancer immunotherapy drug.

[0025] The application of chemokine CCL23 provided by this application in the preparation of a breast cancer immunotherapy drug first proposed: (1) The expression of CCL23 / m9 in breast cancer is significantly positively correlated with the infiltration of the main anti-tumor effector cells - CD4 + Th1 cells and CD8 + T cells, that is, tumors with high expression of CCL23 / m9 exhibit immune activation characteristics; (2) Macrophages are the main target cells and main secreting cells of CCL23 / m9 in breast cancer; (3) Intravenous injection of CCL23 / m9 alleviates the growth of breast cancer in mice and upregulates the expression of characteristic genes of M1 macrophages in tumor tissues (M1 macrophages can promote CD4 + Th1 cells and CD8 + T cell infiltration and function); (4) In vitro treatment with CCL23 / m9 can enhance the expression of M1 characteristic genes in macrophages.

[0026] This application constructs a mouse tumor model and combines in vitro cell and molecular mechanism experiments to clarify the functional effect of CCL23 / m9 in promoting the formation of an anti-tumor immune microenvironment by remodeling the function of TAM. And through the method of combining clinical patient samples with bioinformatics analysis, it provides a theoretical basis, experimental evidence, and clinical feasibility evidence for the immunotherapy strategy based on CCL23 / m9.

[0027] In some embodiments of this application, the target cells of the above chemokine CCL23 in the breast cancer microenvironment include macrophages.

[0028] In some embodiments of this application, the source cells of the above chemokine CCL23 in the breast cancer microenvironment are macrophages.

[0029] The features and properties of this application will be further described in detail below with reference to the embodiments.

[0030] Example 1

[0031] Expression changes and prognostic value of chemokine family members in breast cancer

[0032] Screening of chemokines: Using the TCGA database, the expression changes (compared with adjacent cancer tissues) of all members of four chemokine families (CCL family, CXCL family, CX3CL family, and XCL family) and their correlations with patient prognosis were analyzed, and the analysis results are as Figure 1 shown.

[0033] Figure 1 Among them, the CCL family (orange bubbles), the CXCL family (blue bubbles), the CX3CL family (green bubbles), and the XCL family (yellow bubbles). From Figure 1 It can be seen from A that the expressions of a total of 9 chemokines (above the horizontal line) were significantly correlated with the overall survival (OS) of patients (p < 0.05, that is, -log10(p) > 1.30). Figure 1 In A, taking the Figure 1 horizontal line in A as the benchmark, those above the horizontal line had significant differences (p < 0.05), and those below the horizontal line had no significant differences (p > 0.05). In addition, the middle part enclosed by the two vertical lines had certain differences but no 2-fold difference changes, and the chemokines in the remaining parts all had 2-fold or more difference changes. From Figure 1 A, it can be seen that there were 4 chemokines in the left part (downregulated chemokines) above the horizontal line and 2 chemokines in the right part (upregulated chemokines) above the horizontal line, that is, a total of 6 chemokines had expression change multiples (cancer tissue vs normal tissue) exceeding 2-fold. And CCL23 / m9 was the only chemokine with a decrease in expression exceeding 2-fold and was positively correlated with the OS of breast cancer patients. From Figure 1 It can be seen from B that except for breast cancer (BRCA), the expression of CCL23 / m9 decreased in various types of cancer tissues (cancer tissue vs normal tissue), indicating that the downregulation of CCL23 / m9 in tumors was a relatively common phenomenon.

[0034] Furthermore, the correlations between the CCL23 / m9 level and the OS, DFS, and PFI of breast cancer patients were analyzed through the TCGA database, and the results are as Figure 2 shown in A. Figure 2 CCL23 in A low represents breast cancer patients with low expression of CCL23 / m9, and CCL23 hi represents breast cancer patients with high expression of CCL23 / m9. From Figure 2As can be seen from A, except for OS, CCL23 / m9 was also significantly positively correlated with the disease-free survival (DFS) and progression-free interval (PFI) of breast cancer patients (p < 0.05). The correlation between CCL23 / m9 levels and OS in breast cancer patients was analyzed through the GEO database, and the results are shown in Figure 2B (p < 0.01). Figure 2 In B, low represents breast cancer patients with low CCL23 / m9 expression, and high represents breast cancer patients with high CCL23 / m9 expression. It was also found that the OS of breast cancer patients with high CCL23 / m9 expression was significantly better than that of patients with low CCL23 / m9 expression. The above results indicate that CCL23 / m9 can be used as an effective indicator for evaluating the prognosis of breast cancer. In addition, the diagnostic efficacy of CCL23 / m9 was evaluated by the area under the ROC curve (AUC). Among them, the AUC value was used to represent the accuracy of the prediction. When AUC > 0.5, the closer the AUC is to 1, the better the diagnostic effect. When AUC is between 0.7 and 0.9, there is a certain accuracy, and when AUC is above 0.9, there is a high accuracy. The results are shown in Figure 2C. As can be seen from Figure 2 C, its AUC value was 0.858, indicating that CCL23 / m9 expression can also be used as an excellent indicator for the diagnosis of breast cancer.

[0035] Furthermore, by studying the differences in gene expression profiles between the CCL23 / m9 high-expression group (CCL23 / m9 hi ) and the CCL23 / m9 low-expression group (CCL23 / m9 low ), performing KEGG / GO enrichment analysis, and analyzing the differences in the tumor immune microenvironment between CCL23 / m9 hi and CCL23 / m9 low patients, the effect of CCL23 / m9 expression level on the gene expression profile of tumor tissues in breast cancer patients was further analyzed. The analysis results are as shown in Figure 3 . As can be seen from Figure 3 A, compared with the tumors in the CCL23 / m9 low-expression group (CCL23 / m9 hi ), there were 1478 significantly upregulated genes and 2659 significantly downregulated genes in the tumors of the CCL23 / m9 high-expression group (CCL23 / m9 low ) (p < 0.05, |log2(FC)| ≥ 1). GO / KEGG visualization network analysis was performed on the upregulated genes in the CCL23 / m9 hi group, and the results are as shown in Figure 3 B. It can be found that multiple entries related to tumor immunity were enriched, mainly involving T cell functions.

[0036] Due to the differentiation balance of T cell subsets (mainly referring to CD4+ T cells) have a key impact on the outcome of tumor immunity. Therefore, the correlation between CCL23 / m9 and the characteristic transcription factors of four major CD4 + T cell subsets (Th1, Th2, Th17, Treg) and CD8 + T cells was further analyzed by immune infiltration, and the analysis results are as follows Figure 4 shown. The Spearman rank correlation coefficient Spearman (r) was used as the evaluation criterion. r = 0 indicates no correlation between the two. The closer |r| is to 1, the stronger the correlation. r < 0 indicates negative correlation, and r > 0 indicates positive correlation. Generally, r > 0.5 indicates strong correlation, and r < -0.5 indicates weak correlation. As Figure 4 can be seen, the expression abundance of CCL23 / m9 in breast cancer has a strong correlation with Th1 cells and CD8 + T cell Signature (p < 0.001), while the correlation with Th17 cells and Treg Signature is relatively weak (p = 0.003, p < 0.001), and there is no significant correlation with Th2 cell Signature (p = 0.411); in addition to T cell subsets, CCL23 / m9 also has a strong correlation with Macrophages and Dendritic cell (DC) Signature (p < 0.001), while the correlation with NK cell Signature is weak (p < 0.001). The above results indicate that CCL23 / m9 can enhance anti-tumor immunity by promoting the chemotaxis and function of Th1 cells and CD8 + T cells and enhancing the formation of the anti-tumor immune microenvironment.

[0037] Furthermore, by studying the cell-specific expression pattern of CCL23 / m9 and its receptor CCR1 in breast cancer, the main producing cells and target cells of action were determined. The correlation between CCR1 and the signatures of each immune cell subset was analyzed through the TCGA database, and the results are as follows Figure 5 shown. The analysis results of the TCGA database show that the level of CCR1 in breast cancer patients has the highest correlation with the Macrophages signature among all covered immune cells. In addition, by analyzing single-cell sequencing data, it was found that the positions of CCR1-positive cells and Mono / Macro in breast cancer patients highly overlap ( Figure 6 A). Further, single-cell sequencing was performed on breast cancer mice, and the results are as follows Figure 6As shown in Figure B, it can be found that CCR1 is mainly expressed in monocytes / macrophages, while its expression levels in T cells, B cells, tumor cells, endothelial cells, and fibroblasts are extremely low. The above results indicate that CCL23 / m9 mainly acts on macrophages, thereby affecting the formation of the anti-tumor immune microenvironment in breast cancer, and this mode of action is relatively conserved between humans and mice.

[0038] Example 2

[0039] The bioinformatics analysis results of Example 1 indicated that CCL23 / m9 might have the effect of enhancing anti-tumor immunity. To verify this conjecture, an E0771 breast cancer mouse model was constructed and treated with intraperitoneal injection of CCL23 / m9.

[0040] The specific operation steps are as follows:

[0041] 1. Construction of a mouse tumor model

[0042] The E0771 mouse breast cancer cells were passaged one day before tumor inoculation to achieve a cell confluence of approximately 80% on the day of inoculation. The cells were digested with trypsin, and after terminating the digestion, the cells were collected into a 15 ml centrifuge tube and centrifuged at 500 rcf for 5 min. The supernatant was discarded. The cells were resuspended with 1 ml of PBS and counted using a hemocytometer. The cell density was diluted and adjusted to 3×10 6 cells / ml with PBS to mix the cell suspension evenly. 100 μl of the cell suspension was aspirated with an insulin syringe and injected subcutaneously into 6-8-week-old C57BL / 6 mice, that is, 3×10 5 cells were injected into each mouse. The day of subcutaneous injection was defined as day 0. Thereafter, the tumor growth was observed every 2 days, and the tumor size of the tumor-bearing mice was measured using a vernier caliper. The long diameter (L) and short diameter (W) of the tumor were recorded. The formula for calculating the tumor volume (V) is as follows: Vtumor = (long diameter L × short diameter W / 2). After 3 - 4 weeks, the lungs and livers of the mice were taken to observe the tumor metastasis situation and count the number of metastatic foci.

[0043] 2. CCL23 / m9 treatment

[0044] The E0771 breast cancer cells were subcutaneously inoculated at 3×10 5 cells into 6-8-week-old C57BL / 6 mice. When the tumor was palpable (about 7 days), mouse CCL9 was diluted with 200 μL of PBS and intraperitoneally injected into the tumor-bearing mice at a dose of 2 μg / mouse, once every 3 days. The control group mice were given intraperitoneal injection of 200 μL of PBS.

[0045] Monitor the tumor growth curve of the mice, and the results are as Figure 7As shown, *p < 0.05; **p < 0.01; ***p < 0.001; the expression of M1 macrophage marker genes in their tumor tissues was detected by QPCR, and the results are as Figure 8 shown. From Figure 7 A and Figure 7 B, it can be seen that compared with the PBS group, the tumor growth of mice receiving CCL23 / m9 injection was significantly inhibited. Moreover, through QPCR analysis, it was found that the expressions of M1 macrophage characteristic cytokines TNF-α, IL-12, CXCL9, and CXCL10 in the tumor tissues of CCL23 / m9 group mice were significantly up-regulated compared with those of PBS group mice, as Figure 8 shown, **p < 0.01; ***p < 0.001.

[0046] Furthermore, mouse bone marrow-derived macrophages (BMDM) were stimulated with CCL23 / m9 in vitro, and the specific operation steps are as follows:

[0047] Mice were sacrificed by cervical dislocation, and four leg bones of the mice were taken, and bone marrow cells were blown out with a syringe. After centrifugation and lysis of red blood cells, they were plated in a 6-well plate, and DMEM medium containing 10 ng / ml macrophage colony-stimulating factor (M-CSF) was added for culture. On the third day, 2 mL of fresh medium was supplemented, and the medium was completely changed on the fifth day. Macrophages matured on the seventh day. After digestion with trypsin (containing EDTA), the cells were counted and replated. After overnight incubation, different concentrations (10 ng / ml, 20 ng / ml, 50 ng / ml) of CCL23 / m9 were given for stimulation, the expression of M1-type genes of macrophages was detected, and the expression of CCL23 / m9 was detected. The results are shown in Figure 9, *p < 0.05; **p < 0.01; ***p < 0.001.

[0048] It can be Figure 9 seen that by stimulating mouse BMDM with CCL23 / m9 in vitro, it can be found that CCL23 / m9 treatment can up-regulate the expression levels of TNF-α, IL-12, CXCL9, and CXCL10 in BMDM; after stimulating IFN-γ-induced M1 macrophages with CCL23 / m9, the effect of CCL23 / m9 in promoting the expression of the above molecules was further enhanced; however, CCL23 / m9 treatment inhibited the expression of the immunosuppressive cytokine IL-10. The above experimental results indicate that CCL23 / m9 can promote anti-tumor immune responses and delay tumor progression by enhancing the M1 polarization of macrophages.

[0049] In summary, for the application of chemokine CCL23 in the preparation of breast cancer immunotherapy drugs according to the embodiments of the present application, by analyzing the The Cancer Genome Atlas (TCGA) database, all known members of the chemokine family were screened, and it was identified that CCL23 / m9 was significantly positively correlated with the survival of breast cancer patients (i.e., high expression indicated a good prognosis), while its expression was significantly decreased in breast cancer patients, suggesting that the decreased expression of CCL23 / m9 might promote the progression of breast cancer. Through further analysis and experiments, it was found that: (1) The expression of CCL23 / m9 in breast cancer was significantly positively correlated with the infiltration of the main anti-tumor effector cells - CD4 + Th1 cells and CD8 + T cells, that is, tumors with high expression of CCL23 / m9 showed characteristics of immune activation; (2) Macrophages were the main target cells and main secreting cells of CCL23 / m9 in breast cancer; (3) Intravenous injection of CCL23 / m9 alleviated the growth of breast cancer in mice and up-regulated the expression of characteristic genes of M1 macrophages in tumor tissues (M1 macrophages could promote the infiltration and function of CD4 + Th1 cells and CD8 + T cells); (4) Treatment with CCL23 / m9 in vitro could enhance the expression of M1 characteristic genes in macrophages. The above data indicated that CCL23 / m9 could promote the chemotaxis and function of Th1 cells and CD8 + T cells and enhance the formation of an anti-tumor immune microenvironment by inducing M1 polarization of TAMs in breast cancer and secreting cytokines such as IL-12, CXCL9, and CXCL10, and thus might be used as a new strategy for breast cancer immunotherapy. Lactate in the tumor microenvironment promoted tumor immune escape by inhibiting the expression of CCL23 / m9 in macrophages. Based on this, the present application further used a mouse tumor model and combined in vitro cell and molecular mechanism experiments to clarify the functional effect of CCL23 / m9 in promoting the formation of an anti-tumor immune microenvironment by remodeling the function of TAMs. And through the method of combining clinical patient samples with bioinformatics analysis, a theoretical basis, experimental evidence, and clinical feasibility evidence for the immunotherapy strategy based on CCL23 / m9 were provided.

[0050] The embodiments described above are some, but not all, of the embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application claimed, but merely represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without making creative efforts fall within the scope of protection of the present application.

Claims

1. Use of chemokine CCL23 in the preparation of breast cancer immunotherapy drugs, characterized in that, By increasing the expression level of CCL23, tumor-associated macrophages are induced to polarize towards the M1 phenotype in the macrophage immune microenvironment, thereby promoting the anti-tumor immune response.

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