CCL25 and application thereof in evaluating liver cancer immune microenvironment and predicting liver cancer immunotherapy curative effect in combination with CD8 + T cell and M1 macrophage

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to CCL25 and application of the CCL25 combined with CD8 + T cells and M1 macrophages to evaluation of a liver cancer immune microenvironment and prediction of a liver cancer immunotherapy curative effect. Based on TCGA liver cancer tissue transcriptome data, liver cancer tissue slice multiple immunohistochemistry, liver cancer tissue MRS score calculation and ROC curve multiple verification, it is proved that the chemotactic factor CCL25 expression level can serve as a biomarker for evaluating the liver cancer immune microenvironment. Furthermore, by analyzing data of liver cancer patients receiving anti-PD-1 treatment, a combined prediction model containing the CCL25 expression level, the CD8 + T cell infiltration level and the M1 macrophage infiltration level is established, and the prediction effect of the combined prediction model is better than that of independent use of immune checkpoint indexes. The technical scheme of the invention provides a potential new method for predicting the curative effect of immunotherapy of clinical liver cancer patients, and has a certain clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to CCL25 and its combination with CD8. + Application of T cells and M1 macrophages in assessing the immune microenvironment of liver cancer and predicting the efficacy of immunotherapy for liver cancer. Background Technology

[0002] Functional assessment of the immune microenvironment in hepatocellular carcinoma (HCC) is crucial for predicting immunotherapy response and patient prognosis. However, existing clinical prediction methods suffer from significant technical limitations and application constraints. Currently, the main clinically relied-upon immune checkpoint marker, PD-L1, suffers from insufficient reliability in predicting efficacy due to the high heterogeneity of HCC tissues and standardization differences between different detection platforms, making it difficult to serve as a stable predictor of treatment efficacy. Simultaneously, genomic-based prediction methods such as tumor mutational burden (TMB) and microsatellite instability (MSI) have extremely low positive rates in HCC (only 1-2% and 0.2%, respectively), limiting their clinical value. More importantly, these traditional indicators only reflect local tumor characteristics, while the efficacy of immunotherapy essentially depends on the overall activation state of multiple immune cells within the tumor microenvironment. Therefore, a comprehensive assessment of the activation level of the immune microenvironment is essential for accurately predicting immunotherapy response. Summary of the Invention

[0003] The purpose of this invention is to provide CCL25 and its combination with CD8. + The application of T cells and M1 macrophages in assessing the immune microenvironment of liver cancer and predicting the efficacy of liver cancer immunotherapy is highly specific and sensitive.

[0004] This invention provides the use of reagents for detecting biomarkers in the preparation of products for assessing the immune microenvironment of liver cancer and / or predicting the efficacy of liver cancer immunotherapy, wherein the biomarkers include the chemokine CCL25.

[0005] Preferably, the reagents for detecting the chemokine CCL25 include reagents for detecting the expression level of the chemokine CCL25 mRNA.

[0006] Preferably, the biomarker also includes CD8. + T cells and / or M1 macrophages.

[0007] Preferably, the CD8 is detected. + The reagents for T cells include the detection of CD8. + Reagents for assessing T cell infiltration levels; The reagents for detecting the M1 macrophages include reagents for detecting the infiltration level of the M1 macrophages.

[0008] Preferably, the hepatocellular carcinoma immune microenvironment includes an immune-activated hepatocellular carcinoma microenvironment.

[0009] Preferably, the efficacy of the liver cancer immunotherapy includes the efficacy of anti-PD-1 therapy for liver cancer.

[0010] Preferably, the product includes a model.

[0011] This invention provides a biomarker for assessing the immune microenvironment of liver cancer and / or predicting the efficacy of immunotherapy for liver cancer, the biomarker comprising a first biomarker and a second biomarker; The first biomarker includes the chemokine CCL25; The second marker includes CD8 + T cells and / or M1 macrophages.

[0012] This invention provides a model for evaluating the immune microenvironment of liver cancer and / or predicting the efficacy of liver cancer immunotherapy, including a chemokine CCL25 detection module and CD8... + T-cell detection module and M1 macrophage detection module; The CCL25 chemokine detection module is used to detect the expression level of the CCL25 chemokine mRNA. The CD8 + The T-cell detection module is used to detect CD8. + T cell infiltration level; The M1 macrophage detection module is used to detect the infiltration level of the M1 macrophages.

[0013] Preferably, the model further includes a scoring module; the scoring module is based on the chemokine CCL25 detection module and CD8... + The comprehensive evaluation value is obtained by calculating the correlation coefficient between the evaluation indicators of the T cell detection module and the M1 macrophage detection module. The comprehensive evaluation value = 0.892 × CD8 + The T cell infiltration level was +2.787 × M1 macrophage infiltration level +0.618 × CCL25 expression level -1.448; when the comprehensive evaluation value was ≥-0.867, it was determined to be an immune-activated liver cancer microenvironment, and the liver cancer immunotherapy efficacy was good.

[0014] Beneficial effects: This invention discovers that the chemokine CCL25 expressed by liver cancer cells interacts with CD8 in the liver cancer immune microenvironment. +The infiltration levels of T cells and M1 macrophages (Mφ) are significantly positively correlated. Based on TCGA liver cancer tissue transcriptome data, multiplex immunohistochemistry of liver cancer tissue sections, calculation of liver cancer tissue MRS scores, and multiple validations of ROC curves, it was confirmed that CCL25 expression level can serve as a biomarker for assessing the immune microenvironment of liver cancer. The technical solution of this invention provides a potential new method for predicting the efficacy of immunotherapy in clinical liver cancer patients, and has certain clinical application prospects.

[0015] Furthermore, by analyzing data from liver cancer patients receiving anti-PD-1 therapy, this invention establishes a combined predictive model that includes CCL25 expression level, CD8+ T cell infiltration level, and M1 macrophage infiltration level, which has better predictive performance than using immune checkpoint indicators alone. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 This is the result of the prognostic analysis of CCL25 expression in liver cancer in Example 1; Figure 2 The results of in situ immunohistochemical staining of non-tumor liver tissue and hepatocellular carcinoma tissue in Example 1 are shown. Figure 3 Example 2: CCL25 mRNA expression level and CD8 expression in liver cancer tissue. + A bubble plot showing the correlation between T cell and M1 macrophage infiltration levels; where the bubble color changes from purple to yellow to represent a gradual change in the correlation r-value from negative to positive, and the bubble size can indicate the correlation. P The magnitude of the value; *, P <0.05;** P <0.01; ***, P <0.001; ****, P <0.0001; Figure 4 The results of multiplex immunohistochemistry (mIHC) in Example 2 are shown; where A represents staining results; B-E represent total T cells, CD8+, and CD8+, respectively. + T cells, total macrophages, CD169 + Statistical results of macrophage infiltration density;* P <0.05;** P <0.01; ***, P <0.001; Figure 5 A pie chart showing the in situ myeloid reaction characteristics of liver cancer tissues with low and high CCL25 expression in Example 2. Figure 6 The correlation between CCL25 expression level and myeloid response score (MRS) in liver cancer tissue is shown in Example 2. Figure 7 ROC curves for predicting the efficacy of immunotherapy for liver cancer using different indicators. Detailed Implementation

[0018] This invention provides the use of reagents for detecting biomarkers in the preparation of products for assessing the immune microenvironment of liver cancer and / or predicting the efficacy of liver cancer immunotherapy, wherein the biomarkers include the chemokine CCL25.

[0019] The amino acid sequence of the chemokine CCL25 described in this invention is registered in NCBI under accession number NP_001188288.1. The mRNA sequence of the chemokine CCL25 described in this invention is registered in NCBI under accession number NM_001201359.2.

[0020] As one implementation method, the reagent for detecting the chemokine CCL25 includes a reagent for detecting the expression level of the chemokine CCL25 mRNA. This invention elucidates for the first time the regulatory role of chemokine CCL25 in the immune microenvironment of hepatocellular carcinoma. Chemokine CCL25 expression is significantly negatively correlated with myeloid response score (MRS). Multiplex immunohistochemical analysis further confirms that hepatocellular carcinoma tissues with high CCL25 expression have CD8... + T cells and CD169 + Macrophage infiltration levels were significantly increased. Chemokine CCL25 can serve as a novel immunomodulatory marker independent of MRS, filling a research gap in this field.

[0021] As one implementation method, the biomarker of the present invention also includes CD8. + T cells and / or M1 macrophages. As one embodiment, the present invention detects the CD8+. + The reagents for T cells include the detection of CD8. + A reagent for detecting T cell infiltration levels. As one embodiment, the reagent for detecting the M1 macrophages of the present invention includes a reagent for detecting the infiltration level of the M1 macrophages.

[0022] This invention integrates CCL25 and CD8 + Three key immune markers, including T cells and M1 macrophages, were analyzed based on CCL25 expression levels and CD8+. + T cell infiltration and M1 macrophage infiltration have better predictive power than CCL25 alone.

[0023] In one embodiment, the liver cancer immune microenvironment of the present invention includes an immune-activated liver cancer microenvironment. In one embodiment, the liver cancer immunotherapy efficacy of the present invention includes the liver cancer treatment efficacy of anti-PD-1 therapy. In one embodiment, the product of the present invention includes a model.

[0024] This invention provides a biomarker for assessing the immune microenvironment of liver cancer and / or predicting the efficacy of immunotherapy for liver cancer, the biomarker comprising a first biomarker and a second biomarker; The first biomarker includes the chemokine CCL25; The second marker includes CD8 + T cells and / or M1 macrophages.

[0025] This invention provides a model for evaluating the immune microenvironment of liver cancer and / or predicting the efficacy of liver cancer immunotherapy, including a chemokine CCL25 detection module and CD8... + T-cell detection module and M1 macrophage detection module; The CCL25 chemokine detection module is used to detect the expression level of the CCL25 chemokine mRNA. The CD8 + The T-cell detection module is used to detect CD8. + T cell infiltration level; The M1 macrophage detection module is used to detect the infiltration level of the M1 macrophages.

[0026] As one implementation, the model of the present invention further includes a scoring module; the scoring module is based on the chemokine CCL25 detection module and CD8... + The comprehensive evaluation value is obtained by calculating the correlation coefficient between the evaluation indicators of the T cell detection module and the M1 macrophage detection module. The comprehensive evaluation value = 0.892 × CD8 + The T cell infiltration level was +2.787×M1 macrophage infiltration level +0.618×CCL25 expression level -1.448; when the comprehensive evaluation value was ≥-0.867, it was determined to be an immune-activated liver cancer microenvironment, and the liver cancer immunotherapy efficacy was good.

[0027] This invention is based on the expression level of chemokine CCL25 mRNA and CD8. + A predictive model for T cell infiltration and M1 macrophage infiltration levels was established, overcoming the limitations of existing predictive methods. This provides a new combination of molecular markers for assessing the immune microenvironment of liver cancer and predicting the efficacy of liver cancer immunotherapy, and establishes a more accurate predictive system, which is of great value for improving the existing clinical predictive system.

[0028] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, explains the CCL25 and its combination with CD8 provided by the present invention. + The application of T cells and M1 macrophages in assessing the immune microenvironment of liver cancer and predicting the efficacy of liver cancer immunotherapy is described in detail, but should not be construed as limiting the scope of protection of this invention.

[0029] Example 1 1. Using transcriptomic data from 371 hepatocellular carcinoma (HCC) tissues in the TCGA database and corresponding clinicopathological data, a prognostic analysis of CCL25 expression was performed. Results showed that CCL25 expression levels in patient tumors were positively correlated with prognosis. Figure 1 ).

[0030] 2. To clarify the expression and localization of the chemokine CCL25 in hepatocellular carcinoma (HCC) tissues, the expression level of CCL25 mRNA in paired non-tumor liver tissues and HCC tissues was detected by RT-PCR, and immunohistochemical staining was performed on paraffin sections of HCC tissues. The results showed that tumor cells in some HCC tissues could express CCL25 (… Figure 2 ).

[0031] Example 2 1. To preliminarily explore the regulatory significance of CCL25 on the immune microenvironment of hepatocellular carcinoma (HCC), we first analyzed the immune cell subset infiltration of 371 HCC tissues from the TCGA database using the CIBERSORT algorithm. The correlation between CCL25 expression and immune infiltration was revealed at the transcriptomic level. The results showed that CCL25 expression level was correlated with CD8+ expression in the tumor microenvironment. + The infiltration of T cells and M1 macrophages was positively correlated. Figure 3 ) 2. Due to CD169 + Mφ is a subset of macrophages identified in liver cancer with anti-tumor effects. It possesses a strong ability to phagocytose and apoptotic tumor cells and exhibits classic antigen-presenting cell-related functions, inducing T cell proliferation. Furthermore, its infiltration level in tumor tissue is positively correlated with patient prognosis, primarily through CD169... + Using Mφ as a validation indicator, multiple immunohistochemical staining (mIHC) was performed on paraffin sections of the tissue. The results showed that, compared with CCL25-negative (CCL25-) hepatocellular carcinoma tissue, CCL25-high expression (CCL25+) hepatocellular carcinoma tissue had CD8... + T cells and CD169 + Mφ infiltration level increased significantly ( Figure 4 CD8 + The median number of T cells was 60 / mm². 2 CD169 + The median number of Mφ is 150 / mm. 2These results suggest that CCL25 expression in liver cancer may be related to the antitumor properties of the tissue microenvironment.

[0032] 3. CCL25 expression and CD8 expression in liver cancer + T cells and CD169 + The infiltration levels of immune-activated cell subsets such as Mφ were significantly correlated, suggesting that CCL25 expression may be related to the dynamic balance of immunosuppression / activation in the cancer microenvironment. To more comprehensively assess the impact of CCL25 on the hepatocellular carcinoma immune microenvironment, the relationship between CCL25 expression and myeloid response score (MRS) was further analyzed. MRS is based on CD11b in cancer nests. + Cells and CD169 + The risk score constructed from cell count was significantly correlated with the risk of postoperative recurrence and death in patients, serving as an independent prognostic indicator for liver cancer and outperforming several currently used clinical indicators. Analysis of CCL25 mRNA expression results from transcriptome sequencing data of 20 liver cancer tissues, along with in situ myeloid reaction characteristics data from paired cases, showed a significant negative correlation between CCL25 expression levels and MRS (metastatic reactivity score). Figure 5 and Figure 6 This result suggests that high expression of CCL25 in liver cancer may regulate the immune balance of the tissue microenvironment towards the anti-tumor side and indicate a good prognosis for patients.

[0033] Example 3 Sensitivity and specificity detection Based on the conclusions of Examples 1 and 2, this study utilized the overall mRNA expression profiles of tumor tissues from hepatocellular carcinoma (HCC) patients receiving anti-PD-1 therapy in the open-source databases GSE202069 and PRJEB34724 to investigate the impact of CCL25 expression levels and the infiltration levels of immune activation-related immune cell subsets on the patients' immunotherapy response. The CIBERSORT algorithm was used to analyze the immune cell subset infiltration of 52 HCC tissues from the databases, obtaining CD8+... + The infiltration of T cells and M1 macrophages was then analyzed. This was followed by analysis of CCL25 expression levels, PDCD1 (the gene for the PD-1 protein) expression levels, and cell subsets (CD8+). + The infiltration levels of T cells (CD8T) and M1 macrophages (M1) were analyzed using multivariate ROC curve analysis to explore their predictive role in treatment efficacy. Predictive score = 0.892 × CD8T. + T cell infiltration level +2.787×M1 macrophage infiltration level +0.618×CCL25 expression level -1.448 (≥-0.867 was classified as an immune-activated microenvironment). Results are shown in Table 1 and Figure 7 As shown.

[0034] Table 1. Predictive effect of different detection indicators on the efficacy of immunotherapy

[0035] According to Table 1 and Figure 7 It can be seen that CCL25 expression level has an important predictive role in the predictive effect of anti-PD-1 therapy in HCC patients. Using CCL25 expression level and CD8+... + The combined approach of T cell infiltration level and M1 macrophage infiltration level is more effective in predicting the efficacy of anti-PD-1 therapy in HCC patients.

[0036] Based on the above, it can be seen that CCL25, or CCL25 combined with CD8, is involved in liver cancer. + T cells and M1 macrophages are indicative of an immune-activated tumor microenvironment and play an important predictive role in the efficacy of immunotherapy in patients.

[0037] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting a biomarker in the manufacture of a product for evaluating the immune microenvironment of a liver cancer and / or predicting the efficacy of immunotherapy for a liver cancer, characterized in that, The biomarker includes a chemokine CCL25.

2. Use according to claim 1, characterized in that, The reagent for detecting the chemokine CCL25 includes a reagent for detecting the mRNA expression level of the chemokine CCL25.

3. Use according to claim 1, characterized in that, The biomarkers also include CD8 + T cells and / or M1 -type macrophages.

4. Use according to claim 3, characterized in that, reagents for detecting the CD8 + reagents for detecting the CD8 + T cell infiltration levels; The reagent for detecting the M1 type macrophage includes a reagent for detecting the infiltration level of the M1 type macrophage.

5. The use according to claim 1, characterized in that, The liver cancer immune microenvironment includes an immune activation type liver cancer microenvironment.

6. Use according to claim 1, characterized in that, The liver cancer immunotherapy efficacy includes the treatment efficacy of liver cancer treated by anti-PD-1.

7. The use according to any one of claims 1 to 6, characterized in that, The product includes a model. 8.A biomarker for evaluating an immune microenvironment of a liver cancer and / or predicting an efficacy of an immunotherapy for a liver cancer, characterized in that, The biomarker includes a first marker and a second marker; The first marker includes a chemokine CCL25; The second marker comprises CD8 + T cells and / or M1 -type macrophages. 9.A model for evaluating an immune microenvironment of a liver cancer and / or predicting an efficacy of an immunotherapy for the liver cancer, characterized in that, comprising a chemokine CCL25 detection module, a CD8 + T cell detection module and a M1 type macrophage detection module; The chemokine CCL25 detection module is used for detecting the mRNA expression level of the chemokine CCL25. the CD8 + The T cell detection module is configured to detect the CD8 + T cell infiltration level; The M1 type macrophage detection module is used for detecting the infiltration level of the M1 type macrophage.

10. The model of claim 9, wherein, The model further comprises a scoring module; the scoring module calculates a comprehensive evaluation value according to the correlation coefficients between the evaluation indexes of the chemokine CCL25 detection module, the CD8 + T cell detection module and the M1 type macrophage detection module. The comprehensive evaluation value = 0.892 x CD8 + T cell infiltration level + 2.787 x M1 macrophage infiltration level + 0.618 x CCL25 expression level - 1.448; when the comprehensive evaluation value is greater than or equal to -0.867, it is determined that the liver cancer microenvironment is immune activated, and the liver cancer immunotherapy effect is good.

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