Application of tumor-associated macrophages highly expressing SLC16A10 in prognosis diagnosis and treatment of colorectal cancer

Abstract

The invention belongs to the field of biotechnology and medical technology, and discloses application of tumor-associated macrophages with high expression of SLC16A10 in prognosis diagnosis and treatment of colorectal cancer. According to the invention, colorectal cancer single-cell transcriptome sequencing data analysis before and after anti-PD-1 treatment is carried out in the earlier stage; the tumor-associated macrophage subgroup with high expression of the SLC16A10 is enriched in a response group after colorectal cancer anti-PD-1 treatment, and the prognosis of a colorectal cancer patient with high expression of the SLC16A10 is good. Knock-down of the SLC16A10 leads to reduction of expression of the macrophage M1 type marker, and activation and toxicity of co-cultured T cells are reduced. The SLC16A10 promotes T cell activation and weakens immunosuppression on T cells, so that colorectal cancer anti-PD-1 treatment response is caused. The research explains the influence and mechanism of the macrophage SLC16A10 on colorectal cancer anti-PD-1 treatment, and provides a new strategy and theoretical basis for immunotherapy of colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and medical technology, and more specifically, relates to the application of tumor-associated macrophages with high expression of SLC16A10 in the prognosis, diagnosis and treatment of colorectal cancer. Background Art

[0002] Colorectal cancer causes nearly 700,000 deaths annually, making it the fourth most deadly cancer worldwide after lung cancer, liver cancer, and gastric cancer. The pathogenesis of colorectal cancer is complex, with numerous risk factors, such as family history, inflammatory bowel disease, smoking, and excessive alcohol consumption. The disease often presents in an insidious onset and a complex course, often diagnosed in the advanced stages. In recent years, tumor immunotherapy has emerged as an emerging strategy, particularly the use of immunosuppressants, which have significantly improved the survival of many patients with advanced cancer. Among these, research on immunosuppressants targeting the PD-1 / PD-L1 signaling pathway has garnered significant attention, with related antibody drugs demonstrating significant efficacy in the treatment of malignancies such as lung cancer. However, 95% of CRC patients with a microsatellite stable (MSS) phenotype are poorly responsive to anti-PD-1 immunotherapy, rendering immunotherapy ineffective and leading to a poor overall prognosis for colorectal cancer patients.

[0003] Immunotherapies targeting the tumor microenvironment (TME) have shown promising results in various cancer types. Macrophages are a major immune cell type in the TME, and although tumor-associated macrophages (TAMs) can promote tumor growth, metastasis, immunosuppression, angiogenesis, and drug resistance, the heterogeneity and function of macrophages in colorectal cancer have not been fully characterized.

[0004] It is known that various immune cell types express single solute carriers (SLCs), and SLCs also play an important role in regulating macrophages. Tumor sites mount a highly metabolically active T cell response to cancer. While dysregulation of SLC family members has been implicated in the pathological development of certain cancers, a link between SLC family members and colorectal cancer has not been demonstrated to date. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the primary purpose of the present invention is to provide an application of tumor-associated macrophages that highly express SLC16A10 in the prognosis, diagnosis and treatment of colorectal cancer.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention analyzed the survival prognosis of colorectal cancer patients with high or low SLC16A10 expression using the GEPIA2 and Kaplan-Meier plotter databases. The results showed that patients with colon and rectal cancer who expressed high SLC16A10 had a favorable prognosis. In the data from PD-1 treatment, patients with high SLC16A10 expression had longer progression-free survival and overall survival. The applicant's team then collected tissue samples from 36 patients with colorectal cancer who were clinically treated with anti-PD-1 therapy and identified a total of 42,111 cells from all samples using scRNA-seq. Subsequently, we defined the types of each cell cluster using different cell type markers. Seven major cell types were identified. Myeloid cells were divided into 11 cell subsets based on specific surface markers, and macrophages were divided into five cell subsets: TAM_APOE, TAM_SPP1, TAM_SLC16A10, TAM_LYVE1, and TAM_IL1B. Next, we compared the enrichment of different macrophage subsets in the pre-treatment responder group, the pre-treatment non-responder group, the post-treatment responder group, and the post-treatment non-responder group. Among them, the macrophage subset with high SLC16A10 expression (TAM_SLC16A10) was enriched in the post-treatment responder group. These results suggest that SLC16A10+TAM may play an important regulatory role in the response to colorectal cancer immunotherapy.

[0008] Therefore, the present invention firstly protects the application of tumor-associated macrophages that highly express SLC16A10 in the development and screening of functional products for colorectal cancer.

[0009] The present invention examined the expression of M1 or M2 subtype polarization marker genes in five macrophage subsets in single-cell data. In the SLC16A10+TAM subgroup, more M1 subtype marker genes were highly expressed. After knocking down SLC16A10 in THP-1 cells in vitro, the expression of macrophage M1 marker genes including TNFA, CD86, and IL1B decreased, while the expression of macrophage M2 marker molecules including IL10, CD36, and CD206 increased, indicating that SLC16A10 can regulate macrophage polarization.

[0010] Therefore, preferably, in the above application, the tumor-associated macrophages that highly express SLC16A10 are of M1 type.

[0011] The present invention found that patients with colon cancer and rectal cancer with high expression of SLC16A10 have a good prognosis, and in the data of PD-1 treatment, patients with high expression of SLC16A10 have longer progression-free survival and overall survival.

[0012] Therefore, the present invention also protects the use of a product for detecting the expression level of the SLC16A10 gene in preparing a kit for predicting the prognosis of colorectal cancer patients.

[0013] Preferably, in the above application, the kit performs diagnosis by detecting the expression level of the SLC16A10 gene in a patient tissue sample.

[0014] As another preferred embodiment, the kit of the present invention performs diagnosis by detecting the expression level of the SLC16A10 gene in tissue samples of patients after anti-PD-1 treatment.

[0015] The present invention also studied whether SLC16A10+TAM affects the state changes of T cells and thus affects the efficacy of anti-PD-1 treatment. Using Cell Chat interaction analysis, it was found that in the post-treatment response group, SLC16A10+TAM and its precursor IL-1β+TAM subpopulation had extensive interactions with CD8+T cells, while in the post-treatment non-responder group, SLC16A10+TAM and its precursor IL-1β+TAM had no interaction with T cells. T cell activation and killing decreased in the SLC16A10 knockdown co-culture group. This shows that macrophage SLC16A10 can enhance T cell toxicity and activation ability.

[0016] Therefore, the present invention also protects the use of tumor-associated macrophages that highly express SLC16A10 in the preparation of products that enhance the toxicity and activation ability of T cells.

[0017] The present invention also protects the use of a substance that has a promoting effect on the SLC16A10 gene or its expression product in the preparation of a product that enhances the toxicity and activation ability of T cells.

[0018] The present invention also protects the use of the SLC16A10 gene or its expression product in the development and screening of products that enhance T cell toxicity and activation ability, wherein the functional product has a promoting effect on the SLC16A10 gene or its expression product.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Previous analysis of single-cell transcriptome sequencing data from colorectal cancer cells before and after anti-PD-1 treatment revealed that a subset of tumor-associated macrophages with high expression of the amino acid transporter SLC16A10 was enriched in the responder group after anti-PD-1 treatment. In this responder group, there was a strong interaction between SLC16A10+ TAMs and CD8+ T cells. Colorectal cancer patients with high SLC16A10 expression had a favorable prognosis. Knockdown of SLC16A10 reduced the expression of M1 macrophage markers and increased the expression of M2 macrophage markers, reducing the activation and toxicity of co-cultured T cells. Based on these findings, SLC16A10 promotes T cell activation and reduces immunosuppression of T cells, leading to response to anti-PD-1 treatment in colorectal cancer. This study, combined with single-cell omics, aims to elucidate the impact and mechanism of macrophage SLC16A10 on anti-PD-1 treatment in colorectal cancer at the cellular and animal levels, providing new strategies and theoretical basis for the immunotherapy of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 showed that SLC16A10 expression is associated with a good prognosis in patients with colorectal cancer; Figure 1 A: GEPIA2 database analysis shows the overall survival time of colon cancer patients with high / low SLC16A10 expression; Figure 1 B: GEPIA2 database analysis shows the overall survival time of rectal cancer patients with high / low SLC16A10 expression; Figure 1 C: Kaplan-Meierplotter database analysis shows the progression-free survival time of PD-1-treated patients with high / low SLC16A10 expression; Figure 1 D: Kaplan-Meierplotter database analysis shows the overall survival time of PD-1-treated patients with high / low SLC16A10 expression;

[0022] Figure 2 Single-cell atlas for anti-PD-1 therapy in colorectal cancer; Figure 2 A: Flowchart of single-cell sequencing of colorectal cancer tissues treated with anti-PD-1 therapy; Figure 2 B: UMAP image shows that 42,111 cells from 36 samples are clustered into seven major cell types. Each dot represents a cell, colored according to its cell type. Figure 2 C: Expression of typical marker genes that define major cell types. Each dot represents a cell, and the color depth from light gray to dark purple represents the expression of marker genes from low to high. Figure 2 D: Violin plots showing the expression of typical marker genes for major myeloid cell types;

[0023] Figure 3It showed that the proportion of SLC16A10+TAM increased in the response group after colorectal cancer immunotherapy; Figure 3 A: UMAP image shows the clustering and annotation of myeloid cells in colorectal cancer samples treated with anti-PD-1. Different colors represent different cell subsets, and each dot represents a single cell. Figure 3 B: Dot plots showing the characteristic gene expression of different myeloid cells; Figure 3 C: Heat map showing the enrichment of 11 different myeloid cell subsets in the responder and non-responder groups before and after treatment; Figure 3 D: Characteristic gene expressions of 5 different macrophage subsets;

[0024] Figure 4 showed that macrophage SLC16A10 enhances T cell activation and cytotoxicity; Figure 4 A: GO enrichment analysis showed the functional enrichment pathway of SLC16A10+TAM; Figure 4 B: CellChat ring diagram showing the interaction strength between five macrophage subsets and CD8+ T cell subsets in the post-treatment response group, where each color represents a cell type and the line thickness represents the interaction strength; Figure 4 C: CellChat ring diagram showing the interaction intensity between five macrophage subsets and CD8+ T cell subsets in the non-responder group after treatment; Figure 4 D: Flow cytometry shows the changes in CD69 expression on T cells after 24 hours of co-culture with SLC16A10 knockdown THP-1 cells;

[0025] Figure 5 It showed that SLC16A10+TAMs tended to be polarized to the M1 subtype; Figure 5 A heat map shows the expression of marker genes of the five different macrophage subsets M1 / M2 polarization subtypes; Figure 5 B: qPCR results show the expression of M1 subtype marker genes after knockdown of SLC16A10 in THP-1 cells; Figure 5 C: qPCR results show the expression of M2 subtype marker genes after knockdown of SLC16A10 in THP-1 cells; Figure 5 D: Each myeloid cell subset was scored using the AddModule Score according to the inflammatory macrophage marker gene set. The violin plot shows the expression score of each subtype of myeloid cells.

[0026] Figure 6 It showed that the expression of macrophage SLC16A10 was negatively correlated with Galectin9 / PD-L1; Figure 6 A: Schematic diagram of T cell activating and inhibitory receptors; Figure 6B: Heat map showing the expression of ligands for T cell activating and inhibitory receptors corresponding to the five macrophage subsets; blue to red indicates low to high expression of ligands after data normalization; Figure 6 C: Spearman correlation analysis results show the correlation between LGALS9 and SLC16A10 expression in colorectal cancer tissues; Figure 6 D: qPCR results showed the expression of LGALS9 and CD274 after knockdown of SLC16A10 in THP-1 cells. DETAILED DESCRIPTION

[0027] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.

[0028] Example 1 Colorectal cancer patients with high expression of SLC16A10 have a good prognosis

[0029] To explore the potential role of SLC16A10 in the development and progression of colorectal cancer, the survival prognosis of colorectal cancer patients with high or low SLC16A10 expression was analyzed in the GEPIA2 and Kaplan-Meier plotter databases. The results showed that patients with colon and rectal cancer with high SLC16A10 expression had a good prognosis ( Figure 1 A, Figure 1 B). In addition, in the data of PD-1 treatment, patients with high expression of SLC16A10 had longer progression-free survival and overall survival ( Figure 1 C, Figure 1 D). This suggests that SLC16A10 may play a key role in inhibiting the occurrence and development of colorectal cancer, and also exhibits significant anti-tumor effects in PD-1 immunotherapy.

[0030] 1. SLC16A10+TAMs are associated with response to anti-PD-1 therapy in colorectal cancer

[0031] Single-cell transcriptome sequencing: Tissue specimens from patients with varying responses to anti-PD-1 therapy were collected from the clinic. Tumors were transferred to 1.5 mL EP tubes and thoroughly minced with curved scissors. Culture medium was added, and the cell pellet was resuspended and transferred to a culture dish. Tissue digestion enzyme solution was added and gently pipetted to mix thoroughly. The pellet was then transferred to a 37°C water bath and incubated for 1-2 hours. After digestion, the suspension was diluted with culture medium and filtered to obtain a single-cell suspension. This suspension was then subjected to droplet-based single-cell cDNA library construction using the Chromium Single Cell 5' Library Construction Kit. Specifically, 7,000 cells, a mixture of barcoded gel beads, enzymes, and oil were placed in a microfluidic cross-link system to form a single-cell oil-in-water structure. Total cellular mRNA was then isolated. A poly dT reverse transcription primer and dNTP substrate were combined and reverse transcriptase was used to generate cDNA with 10X barcodes and UMI information. The cDNA was then amplified on a thermal cycler, fragmented by enzyme digestion, and fragments of appropriate length were screened. End repair and adapter connection with Read2 sequencing primers were performed to construct a 3'-end expression spectrum library containing P5 and P7 adapters and double-ended indexes. Finally, the library was sequenced at 150 bp using the Illumina HiSeq 2000 instrument. After sequencing on the machine, the barcode and UMI information of the cells were extracted using Cell Ranger, aligned to the reference genome using STAR, the barcode was corrected, the UMI was filtered and corrected, and counted to finally obtain the gene expression matrix of each cell. Subsequently, the Seurat package in R language was used for further cell filtering and standardization, and UMAP dimensionality reduction was performed to obtain cell clustering typing results and differentially expressed gene information for subsequent data mining.

[0032] The applicant team collected tissue samples from 36 patients with colorectal cancer who were receiving anti-PD-1 treatment in the clinic, including 14 pre-treatment samples (3 non-responders and 11 responders) and 22 post-treatment samples (6 non-responders and 16 responders), and completed the single-cell sequencing library construction ( Figure 2 A). After rigorous quality control and removal of low-quality cells and doublets, we identified a total of 42,111 cells from all scRNA-seq samples. We then used different cell type markers to define the type of each cell cluster. Seven major cell types were identified, including endothelial cells, epithelial cells, fibroblasts, T cells, B cells, myeloid cells, and plasma cells ( Figure 2 B- Figure 2 D).

[0033] Next, we divided myeloid cells into 11 cell subsets based on specific surface markers, among which macrophages were divided into five cell subsets: TAM_APOE, TAM_SPP1, TAM_SLC16A10, TAM_LYVE1, and TAM_IL1B ( Figure 3 A-3B). Next, we compared the differences in the enrichment of different macrophage subsets in the pre-treatment response group, pre-treatment non-response group, post-treatment response group, and post-treatment non-response group. Among them, the macrophage subset with high expression of SLC16A10 (TAM_SLC16A10) was enriched in the post-treatment response group ( Figure 3 These results suggest that SLC16A10+TAM may play an important regulatory role in the response to immunotherapy in colorectal cancer.

[0034] Example 2 SLC16A10+TAM promotes CD8+ T cell activation and cytotoxicity

[0035] Human THP-1 cell lines were plated in 6-well culture plates and transfected with small interfering RNA (siRNA) against the human SLC16A10 gene (siSLC16A10, GenePharma) using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, MA, USA).

[0036] The siRNA targeting the SLC16A10 gene (siSLC16A10) has the following nucleotide sequence:

[0037] Article 1:

[0038] Sense strand (SEQ ID NO. 1): 5'-GCUCCAAAGACGAUGACAATT-3';

[0039] Antisense strand (SEQ ID NO. 2): 5'-UUGUCAUCGUCUUUGGAGCTT-3'.

[0040] Article 2:

[0041] Sense strand (SEQ ID NO. 1): 5'-CCAGUACCAAAGAUAAAGATT-3';

[0042] Antisense strand (SEQ ID NO. 2): 5'-UCUUUAUCUUUGGUACUGGUA-3'.

[0043] Non-targeting control siRNA (siNC, GenePharma) was used as a negative control. 48 hours after siRNA transfection, total RNA or total protein was collected from cells, and knockdown efficiency was verified using real-time quantitative reverse transcription PCR (RT-PCR) and Western blot assays.

[0044] Cell Line Co-culture: After 24 hours of adherent differentiation of THP-1 cells stimulated with phorbol methyl parathionate (PMA), the medium was aspirated and adherent cells were gently rinsed to remove non-adherent cells and residual PMA to prevent activation of Jurkat cells in the subsequent co-culture. Jurkat and THP-1 cells were co-cultured for one day. Non-adherent Jurkat cells were removed by gently pipetting the bottom of the plate three times with pre-chilled PBS. The supernatant was collected and the Jurkat cells were harvested by centrifugation for subsequent experiments.

[0045] T cell activation and toxicity assay: collect the stimulated T cells and add PBS to adjust the cell density to 5×10 6 / ml, aliquot 100μl of cell suspension into each flow cytometry tube. Add fluorescently labeled CD69 T cell surface marker antibody and stain for 15 minutes. Wash away excess antibody. Add 150μl of Cyto-Fast™ Fix Perm Buffer to each tube and incubate at room temperature in the dark for 20 minutes. Add 1ml of 1× Cyto-Fast™ Perm Wash Solution to each tube, centrifuge at 350×g for 5 minutes, and discard the supernatant. Resuspend the cells in 100μl of 1× Cyto-Fast™ Perm Wash Solution, add IFN-γ cytokine flow cytometry antibody, mix well, and incubate at room temperature in the dark for 20 minutes. Wash away excess permeabilization reagent and antibody, resuspend the cells in 300μl of PBS, and prepare for data acquisition.

[0046] The experimental method described above involves overexpressing SLC16A10 in a human THP-1 cell line using plasmid transfection or knocking down SLC16A10 using siRNA transfection. The efficiency of overexpression or knockdown was then assessed using qPCR and western blot. The cells were then co-cultured with human Jurkat T cells, and flow cytometry was used to measure the expression of CD69 and IFN-γ cytokines on the T cells.

[0047] To explore the function of SLC16A10+TAM. First, we extracted the top 100 specifically expressed genes that distinguished the SLC16A10+TAM subgroup from other subgroups, and used the human whole genome gene annotation as a control. The GO annotation data or Reactome annotation data of the genes were obtained from the GO or Reactome public database, including the correspondence between genes and molecular functions, cellular components, and biological processes. The hypergeometric test was used to calculate the enrichment of the top 100 specifically expressed gene sets in specific GO or Reactome terms, with FDR < 0.05 or p < 0.01 as the threshold for significant enrichment. The bar chart is sorted by the number of genes and displays the top 20 significantly enriched pathways. It was found that the two pathways with priority enrichment were the vesicle-mediated transport regulation pathway and the immune system cytokine signaling pathway ( Figure 4 A), which suggests that SLC16A10+TAM may play an important role in the immune regulation process, and the vesicle-mediated transport regulation pathway is closely related to the immune system cytokine signaling pathway.

[0048] It is well known that CD8+ cytotoxic T lymphocytes (CTLs) are key effector cells in tumor immunotherapy responses. Effective immunotherapy primarily depends on the successful activation of CD8+ T cells. To explore whether SLC16A10+ TAMs influence T cell status changes and thus the efficacy of anti-PD-1 therapy, we used Cell Chat analysis to integrate human ligand-receptor interaction information from the CellChatDB database, identifying differentially overexpressed ligands and receptors in each cell group. Gene expression data were mapped onto protein-protein interaction networks, confirming that ligands and receptors may have important communication functions in macrophages and T cells. Based on the law of mass action, the probability of intercellular communication was modeled. By summarizing the communication probabilities of all ligand-receptor interactions associated with each signaling pathway, the communication probability at the signaling pathway level was calculated, and a network diagram of intercellular macrophages and T cells was constructed to illustrate the interaction patterns between them. Interaction analysis revealed that in the post-treatment responder group, SLC16A10+ TAMs and their precursor IL-1β+ TAM subsets had extensive interactions with CD8+ T cells ( Figure 4 B), whereas in the non-responder group after treatment, SLC16A10+TAM and its precursor IL-1β+TAM had no interaction with T cells ( Figure 4 C). This suggests that the interaction between SLC16A10+TAMs and T cells may have an important impact on immunotherapy response.

[0049] Next, we added PMA to stimulate THP-1 cells in vitro for 24 hours to induce cell adhesion. We then co-cultured SLC16A10-knockdown THP-1 cells with Jurkat T cells. After 24 hours, we detected the expression of T cell factor IFN-γ and T cell surface activation molecule CD69. Flow cytometry results showed that T cell activation and killing were reduced in the SLC16A10-knockdown co-culture group ( Figure 4 D, Figure 4 E). This indicates that macrophage SLC16A10 can enhance T cell cytotoxicity and activation.

[0050] Example 3 SLC16A10+TAM tends to be polarized to the M1 subtype

[0051] Since SLC16A10 and its family members can regulate the polarization fate of macrophages by affecting the transport of amino acids, we wanted to clarify the polarization status of the SLC16A10+TAM subpopulation. First, we examined the expression of M1 or M2 subtype polarization marker genes in the five macrophage subpopulations in the single-cell data. In the SLC16A10+TAM subpopulation, more M1 subtype marker genes were highly expressed ( Figure 5 A). After knocking down SLC16A10 in THP-1 cells in vitro, the expression of macrophage M1 marker genes including TNFA, CD86, and IL1B decreased, while the expression of macrophage M2 marker molecules including IL10, CD36, and CD206 increased ( Figure 5 B, Figure 5 C), which indicates that SLC16A10 plays a certain role in regulating macrophage polarization.

[0052] We used Add Module Score to score SLC16A10+TAM with reference to the inflammatory macrophage-specific gene set and found that the two subpopulations, SLC16A10+TAM and TAM_IL1B, showed inflammatory macrophage characteristics ( Figure 5 D). Based on the above results, it is reasonable to speculate that SLC16A10 promotes the polarization of macrophages to the M1 subtype.

[0053] Example 4 SLC16A10 inhibits the expression of Galectin9 / PD-L1 in macrophages

[0054] Next, we continue to explore how SLC16A10+TAMs affect the status of T cells. T cell activation is inseparable from its function and the receptors on its surface. T cell exhaustion in tumors may be potentially related to TIM3 and PD-L1, which are abundant in the tumor microenvironment ( Figure 6 A). We found in single-cell data that SLC16A10+TAMs significantly lower expressed LGALS9 and CD274 compared with other macrophage subsets ( Figure 6 B). In the bulk RNA-seq data of colorectal cancer tissues, we found that the expression of SLC16A10 was negatively correlated with that of LGALS9 ( Figure 6 C). To verify that SLC16A10 can regulate the expression of LGALS9 and CD274 in macrophages, we transiently transfected SLC16A10 siRNA into THP-1 cells. qPCR results showed that the expression of LGALS9 and CD274 in macrophages of the knockdown group increased ( Figure 6 D). The above results preliminarily suggest that macrophage SLC16A10 may alter T cell activation and cytotoxicity by inhibiting Galectin9 / PD-L1.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of tumor-associated macrophages with high expression of SLC16A10 in the development and screening of functional products for colorectal cancer.

2. The use according to claim 1, characterized in that The tumor-associated macrophages that highly express SLC16A10 have an M1 type.

3. Use of a product for detecting the expression level of the SLC16A10 gene in the preparation of a kit for predicting the prognosis of colorectal cancer patients.

4. The use according to claim 3, characterized in that The kit performs diagnosis by detecting the expression level of the SLC16A10 gene in a patient's tissue sample.

5. The use according to claim 3, characterized in that The kit performs diagnosis by detecting the expression level of the SLC16A10 gene in patient tissue samples after anti-PD-1 treatment.

6. Application of tumor-associated macrophages with high expression of SLC16A10 in the preparation of products that enhance T cell toxicity and activation ability.

7. Use of a substance that promotes the SLC16A10 gene or its expression product in the preparation of a product that enhances the toxicity and activation ability of T cells.

8. Use of the SLC16A10 gene or its expression product in developing and screening products that enhance T cell toxicity and activation, characterized in that: The functional product has a promoting effect on the SLC16A10 gene or its expression product.

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