Detection kit for detecting CXCR4+CD8 + T cells for predicting tumor immunotherapy

Abstract

The invention discloses a detection kit used for detecting CXCR4 < + > CD8 < + > T cells and used for predicting tumor immunotherapy, a prediction biomarker is expression chemokine receptor 4 (CXCR4) on the CD8 < + > T cells, and the prediction biomarker is expression chemokine receptor 4 (CXCR4) on the CD8 < + > T cells. Researches find that the expression level of CXCR4 < + > CD8 < + > T cells in tumors is related to the clinical immunotherapy reaction effect. The application comprises the steps that the expression change level of CXCR4 + CD8 + T cells in tumor tissue of a patient before tumor immunotherapy is detected and analyzed through the kit, the patient is divided into a high expression group and a low expression group according to the relative expression quantity of CXCR4 protein and CD8 protein, the low expression group is poor in immunotherapy effect, and the patient is low in immunotherapy reaction; the high expression group is an immunotherapy sensitive patient; the total survival time and the progression-free survival time of a high-expression group after immunotherapy are longer than those of a low-expression group, and the method has important clinical value in the aspects of promoting precise treatment of tumor patients and improving prognosis.

Description

Technical Field

[0001] The present invention relates to the fields of medical technology and biological detection, and in particular to a detection kit for detecting CXCR4+CD8+T cells for predicting tumor immunotherapy. Background Art

[0002] Tumor immunotherapy is a treatment method that mobilizes the body's immune system to enhance anti-tumor immunity in order to control and kill tumor cells. In recent years, it has become one of the important treatments for lung cancer, breast cancer, gastric cancer and other tumors. More and more cancer patients benefit from immunotherapy. Currently, the most commonly used immunotherapy drugs are mainly PD-1 and PD-L1 antibody-type immune checkpoint inhibitors. + T cells are the key executors of anti-tumor immune responses, and the effectiveness of tumor immunotherapy also depends on CD8 + The persistent killing effect of T cells. Currently, the common biomarkers for predicting the efficacy of immunotherapy in clinical practice include PD-L1, microsatellite instability (MSI) and tumor mutation burden (TMB), which have shown certain predictive efficacy. However, there are still many limitations, including high detection costs and lack of unified measurement standards.

[0003] PD-L1 quantified by immunohistochemistry is currently the most widely validated, used, and accepted biomarker to guide patient selection for anti-PD-1 or anti-PD-L1 therapy. However, many challenges remain in the clinical application of these assays, including the necessity of using different companion diagnostic assays for specific drugs, high levels of inter-assay variability in performance and cut-off points, and the lack of prospective comparisons between PD-L1+ disease diagnosed by each assay and clinical outcomes.

[0004] TMB refers to the number of somatic mutations in the tumor genome after removing germline mutations. In general, the higher the TMB in most tumor cells, the more neoantigens may be produced and the higher the tumor immunogenicity, suggesting that the benefit from PD-1 / PD-L1 immune checkpoint inhibitors (ICIs) treatment is more significant. TMB can be affected by many factors, including different tumor types, intrinsic genetic / molecular factors, changes in the tumor microenvironment, external carcinogens, different detection methods and different thresholds may cause TMB to lack heterogeneity, making it unable to serve as an accurate response predictor.

[0005] Human mismatch repair genes (MMR genes) can express corresponding mismatch repair proteins after transcription and translation (that is, the process of gene expression into protein). If any MMR protein expression is problematic, it can cause defects in the cell's repair function, resulting in the loss of repair function for base mismatches during DNA replication and accumulation, leading to the occurrence of microsatellite instability (MSI). It is common in colorectal cancer, endometrial cancer, and gastric cancer. MSI testing is of great significance for patients with a variety of solid tumors, including Lynch syndrome screening, guiding the selection of 5-FU chemotherapy drugs, prognostic stratification, and screening for people who benefit from immune checkpoint inhibitors. MSI can be divided into three categories according to the degree: microsatellite high instability (MSI-high, MSI-H), microsatellite low instability (MSI-low, MSI-L), and microsatellite stability (microsatellite stability, MSS). Although MSI-H status is associated with the response rate of immunotherapy, not all MSI-H tumor patients will respond well to immunotherapy. In addition, some patients with microsatellite stability (MSS) or low instability (MSI-L) may also respond to immunotherapy, which limits the application of MSI-H as a single predictive marker.

[0006] In summary, the main disadvantages of existing technologies (TMB detection, PD-L1 detection and MSI detection) are: 1. The predictive power of efficacy is limited; 2. High cost, resulting in a high medical burden; 3. There are certain differences in the gene sets selected by the gene panels of different institutions, making it difficult to make unified measurements and comparisons; 4. The threshold setting standards are not unified; 5. It is difficult to promote clinically.

[0007] Therefore, there is still a need in the art to more efficiently and accurately identify methods and tools suitable for predicting the efficacy of cancer immunotherapy.

[0008] CXCR4 is a G protein-coupled receptor (GPCR) with 7 transmembrane structures, consisting of 352 amino acids, and its encoding gene is located on chromosome 2q21. As a member of the GPCR family, the GPCR superfamily group has been proven to be the pharmacological target of many drugs (click to view the research progress of GPCR antibody drugs). CXCR4 is expressed on most tissues and organs in the body, including the surface of cells derived from bone marrow, umbilical cord blood and mobilized peripheral blood, as well as the surface expression of a variety of non-hematopoietic stem cells. In recent years, studies have found that tumor cell metastasis to specific organs is the result of different organs attracting specific types of tumor cells to home through chemotaxis. Among the many chemokine receptors, CXCR4 has enhanced expression in many tumors. Current studies have shown that there are more than 23 types of cancers associated with the chemokine receptor CXCR4, which can also promote angiogenesis, cell metastasis, growth or survival. In addition, it is also involved in HIV infection. These biological functions determine that CXCR4 targeted drugs can be used to treat diseases such as tumors, hematopoietic stem cell mobilization, and HIV.

[0009] CXCR4, as the most commonly expressed chemokine receptor in tumor cells, is associated with the development and metastasis of a variety of tumors, including breast cancer, ovarian cancer, lung cancer, colorectal cancer, primary brain tumors (such as gliomas), pancreatic cancer, prostate cancer, multiple myeloma (MM) ADDIN EN.CITE ADDIN EN.CITE.DATA , acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), etc.

[0010] In terms of hematological tumors, CXCR4 has an indisputable clinical relevance to a variety of hematological tumor diseases. The first CXCR4 antagonist, Plerixafor, was approved in Europe and the United States in 2008 and introduced into China in 2018. Currently, Plerixafor can be used in combination with G-CSF for hematopoietic stem cell mobilization in NHL and MM patients undergoing autologous hematopoietic stem cell transplantation (ASCT). A number of CXCR4 targeted inhibitors are also being studied. A phase Ib / II trial (NCT01359657) of an anti-CXCR4 antibody ulocuplumab (BMS-936564) confirmed that blocking CXCR4-CXCL12 in combination with lenalidomide and dexamethasone for the treatment of relapsed or refractory myeloma patients has a high response rate. In addition, the humanized CXCR4 antibody PF-06747143 showed strong anti-tumor effects in a variety of hematological tumor models including NHL, AML and MM.

[0011] In terms of solid tumors, CXCR4 inhibitors have also been shown to have significant anti-cancer potential. Several clinical trials are evaluating the clinical benefits of CXCR4 inhibitors for patients with gliomas. In addition, a Phase I / II trial (NCT01977677) showed that Plerixafor inhibited CXCL4-mediated angiogenesis and enhanced the effect of radiotherapy; a CXCR4 peptide antagonist (LY2510924) showed anti-tumor activity in various solid tumors and preclinical models of metastatic breast cancer.

[0012] CXCR4 not only plays a key role in tumors, but is also closely related to some immune diseases (such as systemic lupus erythematosus, rheumatic diseases, etc.), the genetic disease WHIM syndrome, and the viral disease HIV. Other reports indicate that CXCR4 mediates axon regeneration of damaged optic nerves during embryonic development, which may provide a new strategy for visual therapy. Therefore, targeting CXCR4 will bring new approaches to drug development for a variety of diseases, especially in the immunotherapy of hematological tumors and solid tumors. Summary of the invention

[0013] The present invention discloses a detection kit for predicting tumor immunotherapy biomarkers to solve the problems existing in the above-mentioned prior art. The biomarkers can effectively predict the efficacy of tumor immunotherapy, which has important clinical value and significance for early evaluation of the efficacy of immunotherapy in clinical tumor patients and formulation of individualized treatment plans, and ultimately improving the survival rate of tumor patients.

[0014] The prediction kit comprises: CXCR4 primary antibody, CD8 primary antibody, horseradish peroxidase labeled secondary antibody, 2-4% H2O2 solution, 0.5-1.5% BSA blocking solution, DAB colorimetric reagent, hematoxylin, xylene and ethanol.

[0015] Specifically, the CXCR4 primary antibody and the CD8 primary antibody are monoclonal antibodies or polyclonal antibodies, and the horseradish peroxidase-labeled secondary antibody is a goat anti-rabbit IgG labeled with horseradish peroxidase. In the embodiment of the present invention, the CXCR4 primary antibody and the CD8 primary antibody are CXCR4 primary antibody and CD8 primary anti-rabbit polyclonal antibodies purchased from abcam; the horseradish peroxidase-labeled secondary antibody is a goat anti-rabbit IgG labeled with horseradish peroxidase purchased from Sewell.

[0016] Wherein, the immunotherapy is anti-PD-1 and anti-CTLA-4 immunotherapy. The specific drugs for the anti-PD-1 immunotherapy include carrelizumab, pembrolizumab, and sintilimab, and the specific drug application methods include single drug and combined chemotherapy. The specific drugs for the anti-CTLA-4 immunotherapy include ipilimumab, tesimumab, cardunilizumab, belatacept, and abatacept, and the specific drug application methods include single drug and combined chemotherapy.

[0017] The present invention also provides a specific method for using CXCR4 protein and CD8 protein as molecular markers, detecting the relative expression of CXCR4 protein and CD8 protein in tumor tissue by using the prediction kit, and judging the sensitivity of a patient to immunotherapy according to the relative expression of CXCR4 protein and CD8 protein in tumor tissue, comprising the following steps: (a) performing immunohistochemical staining on the sliced ​​tumor tissue using the prediction kit; (b) photographing the tumor tissue after immunohistochemical staining in step (a) using a microscope and an imaging device; (c) Use biological image processing software to analyze the staining intensity of tumor tissue and the proportion of positive cells and give a score; (d) The relative expression levels of CXCR4 protein and CD8 protein in tumor tissues were calculated according to the scoring criteria.

[0018] Among them, in step (d), the scoring criteria are divided into a staining intensity scoring standard and a positive cell area percentage scoring standard (Note: when predicting the staining intensity and the percentage of positive cells, only the tumor tissue in the field of view of the high-power microscope (200×) is included).

[0019] Specifically, the staining intensity scoring standard is: blue is defined as negative staining as 0 points, light yellow is defined as weak positive staining as 1 point, brown is defined as moderate positive staining as 2 points, and dark brown is defined as strong positive staining as 3 points; when scoring the percentage of positive cells in the area of ​​the tissue, a positive cell area ratio of <25% is defined as 1 point, 25%≤positive cell area ratio<50 is defined as 2 points, 50%≤positive area ratio<75% is defined as 3 points, and a positive area ratio of ≥75% is defined as 4 points; the product of the staining intensity score and the score of the percentage of positive cells in the area of ​​the tissue is used as the immunohistochemical score, and a score greater than or equal to 8 points is high expression, and a score less than 8 points is low expression.

[0020] Wherein, in step (d), the relative expression of CXCR4 protein and CD8 protein in the tumor tissue is recorded as the product of the staining intensity score and the percentage score of the area of ​​positive cells in the tissue, that is, the immunoreactivity score (IRS), the IRS scores of CXCR4 protein and CD8 protein are added, and the score range is 0 to 24 points, and the standard score is 8 points; when the immunoreactivity score ≤ the standard score: CXCR4 protein and CD8 protein are lowly expressed, and the patient is not sensitive to immunotherapy; when the immunoreactivity score > the standard score: CXCR4 protein and CD8 protein are highly expressed, and the patient is sensitive to immunotherapy.

[0021] Furthermore, the specific steps of the above method are: (1) Prepare and cut 4 mm paraffin-embedded tumor tissue sections; (2) dewaxing the slices obtained in step (1), specifically, the following steps: xylene I for 10 min → xylene II for 10 min → xylene III for 10 min → 100% ethanol for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min → ddH2O for 5 min; (3) Inactivating the enzyme: adding 2-4% volume fraction of H2O2 solution to the slices treated in step (2) and incubating at room temperature for 20 min; (4) ddH2O washing for 5 min × 3; (5) Place the sections washed with ddH2O in step (4) in 1×EDTA antigen retrieval solution and boil for 30 min; (6) Cool naturally to room temperature and wash with ddH2O for 5 min × 3; (7) After blocking with 1% BSA at 37°C for 30 min, the blocking solution was discarded, and the primary antibody was added directly without washing (CXCR4 and CD8 primary antibodies were rabbit polyclonal antibodies, abcam, dilution ratio 1:500) and placed in a humidified box at 4°C overnight. (8) Take out from 4°C refrigerator, warm to room temperature for 15 min, and wash with 0.01 mol / L PBS buffer for 5 min × 4; (9) Add secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG antibody, Sewell Biotechnology, dilution ratio 1:200) and incubate at 37°C for 45 min; (10) After washing with 0.01 mol / L PBS buffer for 5 min × 4, the cells were developed with DAB colorimetric reagent for 2-10 min and observed under a microscope; (11) Rinse with ddH2O to stop color development, and then counterstain with hematoxylin for 10 seconds; (12) After differentiation, tap water turns blue and is soaked in distilled water; (13) Dehydration, transparency, and sealing: After soaking in distilled water, the sections were placed in 80% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 10 min, xylene I for 10 min, and xylene II for 10 min. After taking out, drip sealing agent into the sections and place them in a fume hood to dry. (14) The sealed sections were placed under a microscope to observe positive staining, and three fields of view were randomly selected from the tumor tissue and photographed; (15) Semi-quantitative determination of staining results: Semi-quantitative determination was performed by comprehensively evaluating the staining intensity and the percentage of positive cells in the tissue area under a high-power microscope (200×).

[0022] Compared with the prior art, the present invention has the following beneficial effects: The immunohistochemical method is used to detect the expression levels of CXCR4 protein and CD8 protein in the tissues of tumor patients applying immunotherapy, so as to judge the sensitivity of the patients to immunotherapy. Based on the correlation between the expression levels of CXCR4 protein and CD8 protein and immunotherapy of tumor patients, the present invention uses CXCR4 protein and CD8 protein as molecular markers to detect their expression levels, which can be used as a predictive indicator for the efficacy of immunotherapy of tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of the expression levels of CXCR4 protein and CD8 protein detected by immunohistochemical staining of the recipient's tumor specimens; Figure 2 This is the Kaplan-Meier survival analysis diagram of CXCR4 gene expression on prognosis. Among them, AC is the effect of CXCR4 gene expression on the overall survival of tumor patients who received PD-1 immunotherapy, and DE is the effect of CXCR4 protein expression on the overall survival of tumor patients who received CTLA-4 immunotherapy; Figure 3 The Kaplan-Meier survival analysis diagram of CXCR4 and CD8 gene expression on prognosis. Among them, AC is the effect of CXCR4 and CD8 gene expression on the overall survival of tumor patients who received PD-1 immunotherapy, and DF is the effect of CXCR4 and CD8 gene expression on the overall survival of tumor patients who received CTLA-4 immunotherapy; Figure 4 This is the Kaplan-Meier survival analysis diagram of CXCR4 protein expression on prognosis. Among them, AC is the effect of CXCR4 protein expression on the progression-free survival of tumor patients who received PD-1 immunotherapy, and DE is the effect of CXCR4 protein expression on the progression-free survival of tumor patients who received CTLA-4 immunotherapy; Figure 5The Kaplan-Meier survival analysis diagram of CXCR4 and CD8 protein expression on prognosis. Among them, AC is the effect of CXCR4 and CD8 protein expression on progression-free survival of tumor patients who received PD-1 immunotherapy, and DF is the effect of CXCR4 and CD8 protein expression on progression-free survival of tumor patients who received CTLA-4 immunotherapy. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The present invention provides a kit for predicting tumor immunotherapy, wherein the detection reagent is used to detect CXCR4 protein and CD8 protein; The predictive immunotherapy mentioned herein refers to predicting the sensitivity of tumor patients to immunotherapy before they receive immunotherapy.

[0026] The prediction kit comprises: CXCR4 and CD8 polyclonal antibodies, 2-4% H2O2 solution, 1% BSA blocking solution, DAB colorimetric reagent, hematoxylin, horseradish peroxidase-labeled goat anti-rabbit IgG, xylene, and ethanol.

[0027] In the following examples, the preparation of the immunohistochemical test reagents used is as follows: 1. Preparation of gradient ethanol solutions: 75%, 80%, 85%, and 95% ethanol solutions are prepared by diluting anhydrous ethanol and distilled water.

[0028] 2. Preparation of 1×EDTA antigen retrieval solution (pH 9.0): Take 121.1g Tris, add water to 1L, then take 186.1g EDTA, add water to 1L. Take 1mL 1mol / L Tris and 2mL 500mmol / L EDTA, adjust the pH to 9.0, and add water to 1000mL.

[0029] In this example, immunohistochemical staining of tumor tissue before immunotherapy was performed, and the steps were as follows: (1) Prepare and cut 4 mm paraffin-embedded tumor tissue sections; (2) dewaxing the slices obtained in step (1), specifically, the following steps: xylene I for 10 min → xylene II for 10 min → xylene III for 10 min → 100% ethanol for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min → ddH2O for 5 min; (3) Inactivating the enzyme: adding 2-4% volume fraction of H2O2 solution to the slices treated in step (2) and incubating at room temperature for 20 min; (4) ddH2O washing for 5 min × 3; (5) Place the sections washed with ddH2O in step (4) in 1×EDTA antigen retrieval solution and boil for 30 min; (6) Cool naturally to room temperature and wash with ddH2O for 5 min × 3; (7) After blocking with 1% BSA at 37°C for 30 min, shake off the blocking solution, do not wash, and directly add primary antibody for incubation (CXCR4 and CD8 primary antibodies are rabbit polyclonal antibodies, abcam, dilution ratio 1:500). Place in a humidified box at 4°C overnight for 16 hours; (8) Take out from 4°C refrigerator, warm to room temperature for 15 min, and wash with 0.01 mol / L PBS buffer for 5 min × 4; (9) Add secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG antibody, Sewell Biotechnology, dilution ratio 1:200) and incubate at 37°C for 45 min; (10) After washing with 0.01 mol / L PBS buffer for 5 min × 4, the cells were developed with DAB colorimetric reagent for 2-10 min and observed under a microscope; (11) Rinse with ddH2O to stop color development, and then counterstain with hematoxylin for 10 seconds; (12) After differentiation, tap water turns blue and is soaked in distilled water; (13) Dehydration, transparency, and sealing: After soaking in distilled water, the sections were placed in 80% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 10 min, xylene I for 10 min, and xylene II for 10 min. After taking out, drip sealing agent into the sections and place them in a fume hood to dry. (14) The sealed sections were placed under a microscope to observe positive staining, and three fields of view were randomly selected from the non-small cell lung cancer tissues and photographed; (15) Semi-quantitative determination of staining results: Semi-quantitative determination was performed by comprehensively evaluating the staining intensity and the proportion of positive cells under a high-power microscope (200×).

[0030] The specific scores are as follows: 1. Specifically, the staining intensity scoring standard is: blue is defined as negative staining as 0 points, light yellow is defined as weak positive staining as 1 point, brown is defined as moderate positive staining as 2 points, and dark brown is defined as strong positive staining as 3 points; when scoring the percentage of positive cells in the area of ​​the tissue, the positive cell area ratio <25% is defined as 1 point, 25%≤positive cell area ratio <50 is defined as 2 points, 50%≤positive area ratio <75% is defined as 3 points, and positive area ratio ≥75% is defined as 4 points; the product of the staining intensity score and the score of the percentage of positive cells in the area of ​​the tissue is used as the immunohistochemical score, and a score greater than or equal to 8 points is high expression, and a score less than 8 points is low expression; 2. The relative expression of CXCR4 protein and CD8 protein in tumor tissue is recorded as the product of the staining intensity score and the percentage score of positive cells in the tissue area, that is, the immunoreactivity score (IRS). The IRS scores of CXCR4 protein and CD8 protein are added together, with a score range of 0 to 24 points and a standard score of 8 points. When the immunoreactivity score is ≤ the standard score, CXCR4 protein and CD8 protein are lowly expressed, and the patient is insensitive to immunotherapy. When the immunoreactivity score is greater than the standard score, CXCR4 protein and CD8 protein are highly expressed, and the patient is sensitive to immunotherapy.

[0031] The experimental results are as follows Figure 1 Figure A shows high expression of CXCR4 protein, Figure B shows low expression of CXCR4 protein, Figure C shows high expression of CD8 protein, and Figure D shows low expression of CD8 protein. Figure 2-3 To investigate the effects of CXCR4 and CD8 gene expression on the overall survival of cancer patients treated with anti-PD-1 and anti-CTLA-4 immunotherapy. Figure 4-5 To investigate the effects of CXCR4 and CD8 protein expression on progression-free survival in tumor patients who received anti-PD-1 and anti-CTLA-4 immunotherapy.

[0032] In summary, the expression levels of CXCR4 and CD8 proteins are closely related to the efficacy of immunotherapy, and the expression levels of CXCR4 and CD8 proteins can be used as markers to predict the efficacy of immunotherapy.

[0033] The present invention provides a kit for predicting tumor immunotherapy, wherein the detection reagent is used to detect CXCR4 protein and CD8 protein. There are many application methods and approaches for realizing the prediction of tumor immunotherapy. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

[0034] The above is only a specific implementation of the present invention, but the protection scope of the embodiments of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the embodiments of the present invention, which should be included in the protection scope of the embodiments of the present invention. Therefore, the protection scope of the embodiments of the present invention shall be based on the protection scope of the claims.

Claims

1. A detection kit for detecting CXCR4+CD8+T cells for predicting tumor immunotherapy, characterized in that: The kit comprises a solid phase carrier and a detection reagent coated on the solid phase carrier, and the detection reagent is used for detecting CXCR4 protein and CD8 protein.

2. The kit according to claim 1, characterized in that The tumor tissue is a tumor puncture specimen before immunotherapy or a tumor tissue after surgery.

3. The kit according to claim 1, characterized in that The kit comprises reagents for immunohistochemical quantitative detection of CXCR4 protein and CD8 protein and corresponding labels or instructions.

4. The use according to claim 1, characterized in that: The prediction kit comprises: CXCR4 primary antibody, CD8 primary antibody, horseradish peroxidase labeled secondary antibody, 2-4% H2O2 solution, 0.5-1.5% BSA blocking solution, DAB color developing reagent, hematoxylin, xylene and ethanol.

5. The use according to claim 1, characterized in that: The immunotherapy is anti-PD-1 and anti-CTLA-4 immunotherapy.

6. The use according to claim 1, characterized in that: CXCR4 protein and CD8 protein are used as molecular markers to detect the relative expression levels of CXCR4 protein and CD8 protein in tumor tissues, and the patient's sensitivity to immunotherapy is predicted based on the relative expression levels of CXCR4 protein and CD8 protein in tumor tissues.

7. The use according to claim 5, characterized in that: The steps include: (a) performing immunohistochemical staining on the sliced ​​tumor tissue using the prediction kit; (b) photographing the tumor tissue after immunohistochemical staining in step (a) using a microscope and an imaging device; (c) Use biological image processing software to analyze the staining intensity of tumor tissue and the proportion of positive cells and give a score; (d) The relative expression levels of CXCR4 protein and CD8 protein in tumor tissues were calculated according to the scoring criteria.

8. The use according to claim 1, characterized in that: The malignant tumors are breast cancer, ovarian cancer, lung cancer, colorectal cancer, primary brain tumors (such as gliomas), pancreatic cancer, prostate cancer, multiple myeloma (MM), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), etc.

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