Identification kit and identification method for mouse tumor-associated macrophage subpopulation
By providing the identification kit and corresponding identification methods of mouse tumor -related macrophages, the lack of accurate screening and sorting of colorectal cancer -related macrophages in the existing technology has been solved. The accurate identification and sorting of C1Q -positive macrophages provides new targets for immunotherapy of colon cancer.
Patent Information
- Application Number
- CN202411538525.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-31
- Publication Date
- 2025-01-28
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The lack of accurate and comprehensive screening, identification and sorting methods for colorectal cancer-related macrophage subpopulations is limited in the prior art, which limits the effectiveness of tumor immunotherapy.
Provide a mouse tumor-related macrophage group appraisal kit and the corresponding identification method, and use single-cell sequencing technology and specific marking antibodies (such as F4/80 antibodies marked by algae red protein, multi-algae lutein- CD68 antibodies marked with protein-protein complexes and C1Q antibodies marked with albisotrose fluorescein signs) identification and sorting C1Q positive tumor-related macrophages.
Through this kit and method, you can accurately identify and divide the C1Q -positive macrophages in mice tumor tumors in the colorectal cancer, and determine its sources of differentiation, which significantly improves the congenital immune cell sub -in the immune microcyrosis of the intestinal tumor. The understanding of the group provides new targets for the diagnosis and treatment of colorectal cancer.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to an identification kit and an identification method for mouse tumor-associated macrophage subpopulations. Background Art
[0002] Colorectal cancer is the third most common malignant tumor in the world. It is highly occult, has a complex pathogenesis, and seriously endangers human health. At present, surgery and perioperative chemoradiotherapy are still the main treatments for patients with colorectal cancer. Although new technologies and drugs continue to enter the clinic, the survival time and quality of patients with colorectal cancer have not improved significantly. Cellular immunotherapy is currently a frontier field in the clinical precision treatment of tumors and an important step in the development strategy of tumor treatment. Immune cell infiltration into solid tumors is one of the hallmarks of colorectal cancer and plays a key role in disease progression. The tumor microenvironment may reprogram tumor-infiltrating immune cells (including monocytes-macrophages and neutrophils), causing the immune cells to accumulate in the tumor and turn to specific tumor-promoting phenotypes, such as the molecular subtype of mesenchymal CRC. However, the process of tumor-infiltrating immune cells is dynamic, and innate immune cells can also exert anti-tumor activity. Macrophages that infiltrate solid tumors are called tumor-associated macrophages (TAMs). TAM cells are a type of tumor-associated immune cells that belong to the macrophage lineage and infiltrate or are adjacent to tumor tissue areas. There are also specific M2 morphological macrophages that play an immunosuppressive and tumor-promoting role, which are defined as TAMs in a narrow sense. Tumor-associated macrophages can acquire a phenotype with anti-tumor properties, and these macrophages with special phenotypes may contain a large number of specific anti-tumor molecular targets. The main factor currently limiting tumor immunotherapy is the lack of specific immunotherapy molecular targets or immune cell activating factors. Therefore, screening colorectal cancer-sensitive macrophage subpopulations and intracellular specific molecular targets is of great significance for the immunotherapy of colorectal cancer.
[0003] Although tumor-associated macrophages play an important role in tumor immunotherapy, there are still some technical problems. An important obstacle limiting the clinical application of tumor-associated macrophages in colorectal cancer is the lack of specific colorectal cancer-associated macrophage screening, identification and sorting methods. Cell membrane surface antigens are the identity codes of cells. Cells can be classified into groups and subgroups through cell membrane surface antigen labeling. Single-cell sequencing technology can help us find cell subgroups that uniquely express a certain type of antigen in a cell group, providing conditions for studying the relationship between these subgroups and colorectal tumors. Summary of the invention
[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a mouse tumor-associated macrophage subpopulation identification kit and identification method, which solves the problem that the identification method of the C1Q tumor-associated macrophage subpopulation in the prior art is not accurate and comprehensive enough.
[0005] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a kit for identifying mouse tumor-associated macrophage subpopulations, the identification kit comprising a packaging box and a PBS phosphate buffer reagent bottle, a cell digestion solution reagent bottle, a red blood cell lysis solution reagent bottle, an antigen blocking solution reagent bottle, a phycoerythrin (PE) labeled F4 / 80 antibody A reagent bottle, a peridinin-chlorophyll-protein complex (PerCP) labeled CD68 antibody B reagent bottle, and a fluorescein isothiocyanate (FITC) labeled C1Q antibody C reagent bottle.
[0006] Furthermore, the above-mentioned mouse tumor-associated macrophage subpopulation identification kit contains 50 ml of 10-fold concentrated PBS phosphate buffer in the PBS phosphate buffer reagent bottle, which is stored at 4°C; the specification of the cell digestion solution reagent bottle is 40 ml, which is stored at 4°C; the specification of the red blood cell lysis solution reagent bottle is 50 ml, which is stored at 4°C; the specification of the antigen blocking solution reagent bottle is 10 ml, which is prepared with bovine serum albumin and PBS to a liquid with a mass concentration of 1%, and filtered with a 0.22 micron filter membrane Then packaged into 2 ml each tube and stored at -20℃; the volume of the phycoerythrin (PE) labeled F4 / 80 antibody A reagent bottle is 30μL, and it is sealed and stored in the dark at -20℃; the volume of the CD68 antibody B reagent bottle labeled with peridinin-chlorophyll-protein complex (PerCP) is 30μL, and it is sealed and stored in the dark at -20℃; the volume of the C1Q antibody C reagent bottle labeled with fluorescein isothiocyanate (FITC) is 30μL, and it is sealed and stored in the dark at -20℃.
[0007] Furthermore, a method for identifying a mouse tumor-associated macrophage subpopulation is provided, using the above-mentioned identification kit for identification, comprising the following steps:
[0008] S1: retrieve data, retrieve colorectal cancer single-cell transcriptomics data through the NCBI-GEO database, and analyze the innate immune cell subpopulations specifically expressed in the colorectal tumor immune microenvironment;
[0009] S2: Cell clustering, labeling and annotating each cell, and classifying cells into various cell groups, including macrophages, according to existing marker genes;
[0010] S3: Perform differential analysis on genes in each cell population in the sample, define genes with differential fold FC>2 times or FC<0.5 times and pvalue<0.05 as differential genes, and find new marker genes specifically in each cell population through differential genes, as well as the cell subpopulation represented by the marker gene;
[0011] S4: Targeting tumor-associated macrophages, we discovered the tumor-associated macrophage marker gene C1Q and the cell population that expressed this marker gene positively, namely the C1Q-positive macrophage subpopulation;
[0012] S5: Use the identification kit to identify or sort the C1Q-positive macrophage subpopulation in colorectal tumor tissue in colorectal tumor-bearing mice, and identify and sort whether the subpopulation is derived from bone marrow differentiation or tissue-resident macrophage differentiation.
[0013] Furthermore, the identification method of the above mouse tumor-associated macrophage subpopulation, S5 is specifically as follows:
[0014] S51: Establish an orthotopic colorectal cancer mouse model, dissect the mouse, obtain colorectal tumor tissue, and rinse the tissue surface with PBS;
[0015] S52: Cut the colorectal tumor tissue into pieces with dissecting scissors, immerse the colorectal tumor tissue in 1 mL of cell digestion solution, prepare a 100-mesh cell sieve, grind the tissue with a 2 mL syringe stopper for 3-5 minutes, and place the ground tissue in a 15 mL centrifuge tube to form a cell suspension;
[0016] S53: Add 2 mL of cell digestion solution to the cell suspension and digest for 45 min at 37°C and 40 rpm on a shaker.
[0017] S54: Centrifuge the cell suspension at 1000-2000g for 10 minutes at room temperature, collect the cell pellet, and wash once with 2 mL of phosphate buffered saline (PBS);
[0018] S55: discard the supernatant, add 2 mL of red blood cell lysis buffer, lyse the red blood cells at 4°C for 15 minutes, collect the cells by centrifugation, and resuspend in PBS;
[0019] S56: Take some cells and place them in a 1.5 mL EP tube, block with 150 μL 1% BSA at room temperature for 30 minutes, and mix by inverting every 10 minutes;
[0020] S57: Use 1% BSA to prepare the antibody. Each sample requires 150 μL of antibody working solution. Add the prepared working solution into the EP tube containing the cell suspension and stain at 4°C for 1 hour in the dark.
[0021] S58: Centrifuge the EP tube in S57 at 2000 rpm for 10 minutes using a conditioned shaker, discard the supernatant, wash once with PBS, and finally resuspend with 1 mL of PBS and analyze by flow cytometry. Use phycoerythrin (PE)-labeled F4 / 80 antibody A as a bone marrow-derived macrophage marker, use peridinocyanin-chlorophyll-protein complex (PerCP)-labeled CD68 antibody B as a tissue-resident macrophage marker, and use fluorescein isothiocyanate (FITC)-labeled FITC antibody C as a C1Q-positive macrophage marker.
[0022] Furthermore, in the above-mentioned method for identifying mouse tumor-associated macrophage subpopulations, in S57, the antibody working solution includes a bone marrow-derived macrophage marker flow cytometry antibody, a tissue-resident macrophage marker flow cytometry antibody and a C1Q macrophage subpopulation marker flow cytometry antibody, the combination of antibody A and antibody C can identify and sort bone marrow-derived C1Q-positive macrophage subpopulations, and the combination of antibody B and antibody C can identify and sort tissue-resident C1Q-positive macrophage subpopulations.
[0023] The beneficial effects of the present invention are as follows: the present invention discovers a special tumor-associated macrophage population in colorectal tumor tissue through colorectal cancer single-cell transcriptome data mining, which is significantly related to tumor immune microenvironment regulation and tumor immunotherapy. Since this subpopulation cell membrane surface uniquely expresses C1Q, we name it a C1Q-positive tumor-associated macrophage subpopulation. The F4 / 80 antibody labeled with phycoerythrin (PE) is used as a bone marrow-derived macrophage marker, the CD68 antibody labeled with polydinoflagellates-chlorophyll-protein complex (PerCP) is used as a tissue-resident macrophage marker, and the FITC antibody labeled with fluorescein isothiocyanate (FITC) is used as a C1Q-positive macrophage marker. The present invention can construct a kit for identifying and sorting mouse tumor-associated macrophage subpopulations. It can identify and sort C1Q-positive tumor-associated macrophages in mouse colorectal cancer tumors, and identify whether its differentiation source belongs to bone marrow-derived macrophage differentiation or tissue-resident macrophage differentiation. The present invention provides a model and conditions for further studying colorectal cancer tumor immunity, tumor immunotherapy, and the regulation and application of tumor-associated macrophages on tumor immunity. There is currently no report on the kit for this effect. The reagent and antibody combination provided by the present invention can quickly and conveniently identify or sort C1Q-positive tumor-associated macrophages in relatively simplified steps. The invention can do the following: 1. Identify whether C1Q-positive tumor-associated macrophages appear in colorectal cancer tumor tissue; 2. Identify whether C1Q-positive tumor-associated macrophages are differentiated from bone marrow-derived macrophages; 3. Identify whether C1Q-positive tumor-associated macrophages are differentiated from tissue-resident macrophages; The present invention can significantly improve the understanding of innate immune cell subsets in the colorectal tumor immune microenvironment, provide new targets for the diagnosis and treatment of colorectal cancer, and is of great significance to research in related fields. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Schematic diagram of the flow cytometric sorting and identification method for mouse tumor-associated macrophage subsets;
[0025] Figure 2 Schematic diagram of flow cytometry identification of bone marrow-derived C1Q-positive tumor-associated macrophage subsets in intestinal tissues of mice with colorectal inflammation and colorectal cancer;
[0026] Figure 3 Schematic diagram of flow cytometry identification of tissue-resident C1Q-positive tumor-associated macrophage subsets in intestinal tissues of mice with colorectal inflammation and colorectal cancer. DETAILED DESCRIPTION
[0027] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.
[0028] Example 1
[0029] The present embodiment provides a kit for identifying mouse tumor-associated macrophage subpopulations, comprising 10 ml of antigen blocking solution, 30 μL of phycoerythrin (PE)-labeled F4 / 80 antibody, 30 μL of peridinin-chlorophyll-protein complex (PerCP)-labeled CD68 antibody, 30 μL of fluorescein isothiocyanate (FITC)-labeled C1Q antibody, 50 ml of PBS phosphate buffer (10×), 40 ml of cell digestion solution and 50 ml of red blood cell lysis solution, all of which are separately packed into sterile sealed bottles, and the following purposes are achieved by combining the reagents: 1. Identifying whether C1Q-positive tumor-associated macrophages appear in colorectal cancer tumor tissue; 2. Identifying whether C1Q-positive tumor-associated macrophages are differentiated from bone marrow-derived macrophages; 3. Identifying whether C1Q-positive tumor-associated macrophages are differentiated from tissue-resident macrophages.
[0030] Example 2
[0031] like Figure 1 As shown, this example uses an identification kit to identify and sort C1Q-positive tumor-associated macrophage subpopulations in the intestinal tissue of colorectal cancer mice. The components of the identification kit are the same as those in Example 1. The identification method includes the following steps:
[0032] S1. The in situ colorectal chronic inflammation mouse model was established by submucosal injection of azoxymethane / dextran sulfate sodium (AOM / DSS), and the in situ colorectal cancer mouse model was established by submucosal injection of CT26.WT mouse colon cancer cells. The mice were dissected to obtain colorectal tumor tissues, and the tissue surface was rinsed with PBS.
[0033] S2. Cut the tissue into pieces with dissecting scissors, immerse in 1 mL of cell digestion solution, prepare a 100-mesh cell sieve, grind the tissue with a 2 mL syringe stopper for 3-5 minutes, and place the ground tissue in a 15 mL centrifuge tube to form a cell suspension;
[0034] S3. Add 2 mL of cell digestion solution to the cell suspension and digest at 37°C and 40 rpm for 45 minutes.
[0035] S4, centrifuge the cell suspension at 1000-2000g for 10 minutes at room temperature, collect the cell pellet, and wash once with 2 mL of phosphate buffered saline (PBS);
[0036] S5. Discard the supernatant, add 2 mL of red blood cell lysis buffer, lyse the red blood cells at 4°C for 15 minutes, collect the cells by centrifugation, and resuspend them in PBS;
[0037] S6. Take some cells (about 2×107 cells) and place them in a 1.5 mL EP tube. Block them with 150 μL 1% BSA at room temperature for 30 minutes, and mix them by inverting every 10 minutes.
[0038] S7. Prepare the antibody with 1% BSA according to the recommended ratio. Each sample needs 150 μL of antibody working solution. Add the prepared working solution into the EP tube containing the cell suspension and stain at 4°C for 1 hour in the dark.
[0039] S8, centrifuge at 2000 rpm for 10 min, discard the supernatant, wash once with PBS, and finally resuspend in 1 mL PBS and analyze by flow cytometry. Use phycoerythrin (PE)-labeled F4 / 80 antibody as a bone marrow-derived macrophage marker, and use fluorescein isothiocyanate (FITC)-labeled C1Q antibody as a C1Q-positive macrophage marker.
[0040] S9. Using healthy mice as negative controls, cell sorting and collection can be set up while identifying cells. Using F4 / 80 antibody and C1Q antibody positivity as markers, use flow cytometry collection tubes to collect F4 / 80 antibody and C1Q antibody positive cells, and you can collect the C1Q-positive tumor-associated macrophage subpopulation derived from bone marrow differentiation.
[0041] like Figure 2 As shown, the number 1 is the ordinate, which indicates the identification of the F4 / 80 antibody labeled with phycoerythrin (PE), and the number of positive cells increases from bottom to top; the numbers 2, 3 and 4 are the abscissas, which respectively indicate the labeling of the C1Q antibody labeled with fluorescein isothiocyanate (FITC) in healthy mice (2), mice with chronic colorectal inflammation (3), and mice with in situ colorectal cancer (4), and the number of positive cells increases from left to right. Therefore, the black box in the upper right corner of the figure is the F4 / 80 antibody-positive and C1Q antibody-positive cells, that is, the C1Q-positive tumor-associated macrophage subpopulation identified as bone marrow-differentiated. It can be seen that the cells are less in the intestinal tissue of healthy mice (2), increase under mouse intestinal inflammation, and significantly increase in the intestinal tissue of mouse in situ colorectal cancer (3,4). The F4 / 80 antibody-positive and C1Q antibody-positive cells are collected by flow cytometry collection tubes to obtain the C1Q-positive tumor-associated macrophage subpopulation derived from bone marrow differentiation. In this group of samples, approximately 1.5×10 7cells, and 20.6% of them were C1Q-positive tumor-associated macrophages derived from bone marrow differentiation, that is, a maximum of 3.1×10 6 A subset of C1Q-positive tumor-associated macrophages derived from bone marrow differentiation.
[0042] Example 3
[0043] This example uses an identification kit to identify and sort C1Q-positive tumor-associated macrophage subpopulations in the intestinal tissue of colorectal cancer mice. The components of the identification kit are the same as those in Example 1. The identification method includes the following steps:
[0044] S1. The in situ colorectal chronic inflammation mouse model was established by submucosal injection of azoxymethane / dextran sulfate sodium (AOM / DSS), and the in situ colorectal cancer mouse model was established by submucosal injection of CT26.WT mouse colon cancer cells. The mice were dissected to obtain colorectal tumor tissues, and the tissue surface was rinsed with PBS.
[0045] S2. Cut the tissue into pieces with dissecting scissors, immerse in 1 mL of cell digestion solution, prepare a 100-mesh cell sieve, grind the tissue with a 2 mL syringe stopper for 3-5 minutes, and place the ground tissue in a 15 mL centrifuge tube to form a cell suspension;
[0046] S3. Add 2 mL of cell digestion solution to the cell suspension and digest at 37°C and 40 rpm for 45 minutes.
[0047] S4, centrifuge the cell suspension at 1000-2000g for 10 minutes at room temperature, collect the cell pellet, and wash once with 2 mL of phosphate buffered saline (PBS);
[0048] S5. Discard the supernatant, add 2 mL of red blood cell lysis buffer, lyse the red blood cells at 4°C for 15 minutes, collect the cells by centrifugation, and resuspend them in PBS;
[0049] S6. Take some cells (about 2×107 cells) and place them in a 1.5 mL EP tube. Block them with 150 μL 1% BSA at room temperature for 30 minutes, and mix them by inverting every 10 minutes.
[0050] S7. Prepare the antibody with 1% BSA according to the recommended ratio. Each sample needs 150 μL of antibody working solution. Add the prepared working solution into the EP tube containing the cell suspension and stain at 4°C for 1 hour in the dark.
[0051] S8, centrifuge at 2000 rpm for 10 min, discard the supernatant, wash once with PBS, and finally resuspend in 1 mL PBS and analyze by flow cytometry. CD68 antibody labeled with peridinin-chlorophyll-protein complex (PerCP) was used as a tissue-resident macrophage marker, and C1Q antibody labeled with fluorescein isothiocyanate (FITC) was used as a C1Q-positive macrophage marker.
[0052] S9. Using healthy mice as negative controls, cell sorting and collection can be set up while identifying cells. Using CD68 antibody and C1Q antibody positivity as markers, use flow cytometry collection tubes to collect CD68 antibody and C1Q antibody positive cells, and you can collect C1Q-positive tumor-associated macrophage subpopulations of tissue-resident differentiation origin.
[0053] like Figure 3 As shown, the number 1 is the ordinate, which indicates the CD68 antibody labeled with dinoflavin-chlorophyll-protein complex (PerCP), and the number of positive cells increases from bottom to top; the numbers 2, 3 and 4 are the abscissas, which respectively indicate the labeling of the C1Q antibody labeled with fluorescein isothiocyanate (FITC) in healthy mice (2), mice with chronic colorectal inflammation (3), and mice with in situ colorectal cancer (4), and the number of positive cells increases from left to right. Therefore, the black box in the upper right corner of the figure is the F4 / 80 antibody-positive and C1Q antibody-positive cells, that is, the C1Q-positive tumor-associated macrophage subpopulation identified as tissue-resident differentiation. It can be seen that the cells are less in the intestinal tissue of healthy mice (2), increase slightly under mouse intestinal inflammation, and significantly increase in the intestinal tissue of mouse in situ colorectal cancer (3,4). The CD68 antibody-positive and C1Q antibody-positive cells are collected by flow cytometry collection tubes to obtain the C1Q-positive tumor-associated macrophage subpopulation of tissue-resident differentiation. In this group of samples, approximately 1.5×10 7 cells, and 17.9% of them were tissue-resident C1Q-positive tumor-associated macrophages, which means that a maximum of 2.68×10 6 C1Q-positive tumor-associated macrophage subsets of tissue-resident origin.
[0054] The present invention provides a model and conditions for further studying colorectal cancer tumor immunity, tumor immunotherapy, and the regulation and application of tumor-associated macrophages on tumor immunity. The reagent and antibody combination provided by the present invention can quickly and conveniently identify or sort C1Q-positive tumor-associated macrophages in relatively simplified steps. The present invention can significantly improve the understanding of innate immune cell subpopulations in the colorectal tumor immune microenvironment, provide a new target for the diagnosis and treatment of colorectal cancer, and is of great significance to the research in related fields.
Claims
1. A kit for identifying mouse tumor-associated macrophage subsets, characterized in that: The identification kit comprises a packaging box and a PBS phosphate buffer reagent bottle, a cell digestion solution reagent bottle, a red blood cell lysis solution reagent bottle, an antigen blocking solution reagent bottle, a phycoerythrin (PE)-labeled F4 / 80 antibody A reagent bottle, a peridinin-chlorophyll-protein complex (PerCP)-labeled CD68 antibody B reagent bottle, and a fluorescein isothiocyanate (FITC)-labeled C1Q antibody C reagent bottle.
2. The identification kit for mouse tumor-associated macrophage subpopulations according to claim 1, characterized in that: The PBS phosphate buffer reagent bottle contains 50 ml of 10-fold concentrated PBS phosphate buffer, which is stored at 4°C; the cell digestion solution reagent bottle has a specification of 40 ml and is stored at 4°C; the red blood cell lysis solution reagent bottle has a specification of 50 ml and is stored at 4°C; the antigen blocking solution reagent bottle has a specification of 10 ml, which is prepared into a liquid with a mass concentration of 1% using bovine serum albumin and PBS, and is filtered with a 0.22-micron filter membrane and then packaged into 2 ml per bottle, which is stored at -20°C; the volume of the phycoerythrin (PE) labeled F4 / 80 antibody A reagent bottle is 30 μL, which is sealed and stored at -20°C in the dark; the volume of the CD68 antibody B reagent bottle labeled with polydinin-chlorophyll-protein complex (PerCP) is 30 μL, which is sealed and stored at -20°C in the dark; the volume of the C1Q antibody C reagent bottle labeled with fluorescein isothiocyanate (FITC) is 30 μL, which is sealed and stored at -20°C in the dark.
3. A method for identifying a mouse tumor-associated macrophage subpopulation, using the identification kit according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1: retrieve data, retrieve colorectal cancer single-cell transcriptomics data through the NCBI-GEO database, and analyze the innate immune cell subsets specifically expressed in the colorectal tumor immune microenvironment; S2: Cell clustering, labeling and annotating each cell, and classifying cells into various cell groups, including macrophages, according to existing marker genes; S3: Perform differential analysis on genes in each cell population in the sample, define genes with differential fold FC>2 times or FC<0.5 times and pvalue<0.05 as differential genes, and find new marker genes specifically in each cell population through differential genes, as well as the cell subpopulation represented by the marker gene; S4: Targeting tumor-associated macrophages, we discovered the tumor-associated macrophage marker gene C1Q and the cell population that expressed this marker gene positively, namely the C1Q-positive macrophage subpopulation; S5: Use the identification kit to identify or sort the C1Q-positive macrophage subpopulation in colorectal tumor tissue in colorectal tumor-bearing mice, and identify and sort whether the subpopulation is derived from bone marrow differentiation or tissue-resident macrophage differentiation.
4. The method for identifying mouse tumor-associated macrophage subpopulations according to claim 3, characterized in that: The S5 is specifically: S51: Establish an orthotopic colorectal cancer mouse model, dissect the mouse, obtain colorectal tumor tissue, and rinse the tissue surface with PBS; S52: Cut the colorectal tumor tissue into pieces with dissecting scissors, immerse the colorectal tumor tissue in 1 mL of cell digestion solution, prepare a 100-mesh cell sieve, grind the tissue with a 2 mL syringe stopper for 3-5 minutes, and place the ground tissue in a 15 mL centrifuge tube to form a cell suspension; S53: Add 2 mL of cell digestion solution to the cell suspension and digest for 45 min at 37°C and 40 rpm on a shaker. S54: Centrifuge the cell suspension at 1000-2000g for 10 minutes at room temperature, collect the cell pellet, and wash once with 2 mL of phosphate buffered saline (PBS); S55: discard the supernatant, add 2 mL of red blood cell lysis buffer, lyse the red blood cells at 4°C for 15 minutes, collect the cells by centrifugation, and resuspend in PBS; S56: Take some cells and place them in a 1.5 mL EP tube, block with 150 μL 1% BSA at room temperature for 30 minutes, and mix by inverting every 10 minutes; S57: Use 1% BSA to prepare the antibody. Each sample requires 150 μL of antibody working solution. Add the prepared working solution into the EP tube containing the cell suspension and stain at 4°C for 1 hour in the dark. S58: Centrifuge the EP tube in S57 at 2000 rpm for 10 minutes using a conditioned shaker, discard the supernatant, wash once with PBS, and finally resuspend with 1 mL of PBS and analyze by flow cytometry. Use phycoerythrin (PE)-labeled F4 / 80 antibody A as a bone marrow-derived macrophage marker, use peridinocyanin-chlorophyll-protein complex (PerCP)-labeled CD68 antibody B as a tissue-resident macrophage marker, and use fluorescein isothiocyanate (FITC)-labeled FITC antibody C as a C1Q-positive macrophage marker.
5. The method for identifying mouse tumor-associated macrophage subpopulations according to claim 4, characterized in that: In S57, the antibody working solution includes a bone marrow-derived macrophage marker flow cytometry antibody, a tissue-resident macrophage marker flow cytometry antibody and a C1Q macrophage subpopulation marker flow cytometry antibody. The combination of antibody A and antibody C can identify and sort bone marrow-derived C1Q-positive macrophage subpopulations, and the combination of antibody B and antibody C can identify and sort tissue-resident C1Q-positive macrophage subpopulations.