Application of calcium binding protein S100A1 as lung cancer immunotherapy biomarker

Abstract

The invention discloses an application of a calcium binding protein S100A1 as a lung cancer immunotherapy biomarker. The calcium binding protein S100A1 has a judgment value in lung cancer immunotherapy response and survival prognosis, and the reduction of the expression level of the calcium binding protein S100A1 can significantly influence the polarization of macrophage M1 and the anti-tumor immune function of T cells, remodel the tumor immune microenvironment and enhance the sensitivity of tumors to immunotherapy. The S100A1 / USP7 / p65 / GM-CSF regulatory axis of lung cancer cells is intervened, the problem of high heterogeneity of the current immunotherapy curative effect can be solved, and a new theoretical and experimental basis is provided for developing and improving a combined intervention strategy of immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of calcium-binding protein S100A1 as a biomarker for immunotherapy of lung cancer. Background Art

[0002] In recent years, immunotherapy represented by immune checkpoint inhibitors (ICIs) has made unprecedented breakthroughs in the comprehensive treatment of tumors, especially in solid tumors such as non-small cell lung cancer (NSCLC), melanoma, and breast cancer. Immunotherapy alone or in combination has become the standard regimen. However, its treatment effect shows obvious individual differences, and some patients still fail to respond effectively. The molecular mechanisms of tumor immunotherapy response and resistance are not yet fully understood. Studies have found that tumor-mediated immune escape plays an important role in immunotherapy response. Therefore, from the perspective of tumor-mediated immune escape, it is of great clinical significance to find key regulatory factors and biomarkers of immunotherapy response.

[0003] Calcium ions (Ca 2+ ) is an important second messenger in cells, playing a vital role in cell proliferation, differentiation, death and various cell signal transduction. 2+ Signaling regulation and its impact on tumorigenesis, progression, metastasis, and tumor microenvironment (TME) have attracted extensive attention. Increasing evidence shows that tumor cells can regulate the expression of Ca 2+ Calcium homeostasis regulation involves Ca 2+ There are many molecular regulatory mechanisms such as binding proteins, calcium pumps and calcium channels, among which Ca 2+ The binding protein is Ca 2 + It is worth mentioning that S100 calcium binding protein A1 (S100A1) plays an important role in regulating intracellular Ca 2+ Signaling and Ca 2+ S100A1 is composed of 94 amino acid residues, including two EF-hand type Ca 2+The binding domain can respond to changes in calcium concentration inside and outside the cell and interact with the target protein by changing its own conformation to exert a variety of biological functions. Recent studies have found that S100A1 is abnormally expressed in various types of tumors, involving biological processes such as cell cycle regulation, apoptosis, malignant invasion and metastasis, and immune escape, and is closely related to poor patient prognosis. However, the specific role and mechanism of S100A1 in the response to lung cancer immunotherapy has not yet been clarified and still needs to be explored. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an application of calcium binding protein S100A1 as a biomarker for immunotherapy of lung cancer.

[0005] In a first aspect, the present invention provides a first use of calcium binding protein S100A1, which is achieved by adopting the following technical solution.

[0006] A substance for detecting the expression level of calcium-binding protein S100A1 is used in the preparation of a product for evaluating the efficacy of lung cancer immunotherapy and / or lung cancer prognosis assessment.

[0007] Furthermore, the samples for detecting the expression level of calcium-binding protein S100A1 are derived from blood or cell supernatant.

[0008] Furthermore, the lung cancer includes non-small cell lung cancer and small cell lung cancer; non-small cell lung cancer includes lung adenocarcinoma and lung squamous cell carcinoma.

[0009] Furthermore, the substance for detecting the expression level of calcium-binding protein S100A1 includes reagents used in chemiluminescence, radioimmunoassay, fluorescence immunoassay, mass spectrometry, protein immunoblotting or enzyme-linked immunosorbent assay.

[0010] In a second aspect, the present invention provides a kit for evaluating the efficacy of immunotherapy for lung cancer, which is achieved by adopting the following technical scheme.

[0011] A kit for evaluating the efficacy of immunotherapy for lung cancer comprises a substance for detecting the expression level of calcium-binding protein S100A1.

[0012] Furthermore, the samples for detecting the expression level of calcium-binding protein S100A1 are derived from blood or cell supernatant.

[0013] Furthermore, the lung cancer includes non-small cell lung cancer and small cell lung cancer; non-small cell lung cancer includes lung adenocarcinoma and lung squamous cell carcinoma.

[0014] Furthermore, the substance for detecting the expression level of calcium-binding protein S100A1 includes reagents used in chemiluminescence, radioimmunoassay, fluorescence immunoassay, mass spectrometry, protein immunoblotting or enzyme-linked immunosorbent assay.

[0015] In a third aspect, the present invention provides a kit for evaluating the prognosis of lung cancer, which is achieved by adopting the following technical solution.

[0016] A kit for evaluating the prognosis of lung cancer comprises a substance for detecting the expression level of calcium-binding protein S100A1.

[0017] Furthermore, the samples for detecting the expression level of calcium-binding protein S100A1 are derived from blood or cell supernatant.

[0018] Furthermore, the lung cancer includes non-small cell lung cancer and small cell lung cancer; non-small cell lung cancer includes lung adenocarcinoma and lung squamous cell carcinoma.

[0019] Furthermore, the substance for detecting the expression level of calcium-binding protein S100A1 includes reagents used in chemiluminescence, radioimmunoassay, fluorescence immunoassay, mass spectrometry, protein immunoblotting or enzyme-linked immunosorbent assay.

[0020] In a fourth aspect, the present invention provides a second use of the calcium binding protein S100A1, which is achieved by adopting the following technical solution.

[0021] Use of a substance that reduces the expression level of calcium-binding protein S100A1 in the preparation of at least one of the following drugs:

[0022] a. Treatment of lung cancer;

[0023] b. Enhance the sensitivity of lung cancer patients to immunotherapy;

[0024] c. Reshape the tumor immune microenvironment in lung cancer patients;

[0025] d. Promote M1 polarization of macrophages and T cell anti-tumor immunity in patients with lung cancer.

[0026] Furthermore, substances that reduce the expression level of calcium-binding protein S100A1 include reagents used in CRISPR-Cas9, lentiviral infection, and siRNA transfection.

[0027] In a fifth aspect, the present invention provides a drug for treating lung cancer, which is achieved by adopting the following technical solution.

[0028] A drug for treating lung cancer, comprising a substance that reduces the expression level of calcium-binding protein S100A1.

[0029] Furthermore, the substance that reduces the expression level of calcium-binding protein S100A1 includes reagents used in lentiviral transfection.

[0030] In a sixth aspect, the present invention provides a drug for enhancing the sensitivity of lung cancer patients to immunotherapy, which is achieved by adopting the following technical solution.

[0031] A drug for enhancing the sensitivity of lung cancer patients to immunotherapy, comprising a substance that reduces the expression level of the calcium-binding protein S100A1.

[0032] Furthermore, the substance that reduces the expression level of calcium-binding protein S100A1 includes reagents used in lentiviral transfection.

[0033] In a seventh aspect, the present invention provides a drug for reshaping the tumor immune microenvironment of lung cancer patients, which is achieved by adopting the following technical solution.

[0034] A drug for reshaping the tumor immune microenvironment of lung cancer patients, including a substance that reduces the expression level of calcium-binding protein S100A1.

[0035] Furthermore, the substance that reduces the expression level of calcium-binding protein S100A1 includes reagents used in lentiviral transfection.

[0036] In an eighth aspect, the present invention provides a drug for promoting M1 polarization of macrophages and anti-tumor immunity of T cells in patients with lung cancer, which is achieved by adopting the following technical scheme.

[0037] A drug for promoting M1 polarization of macrophages and T cell anti-tumor immunity in patients with lung cancer, including a substance that reduces the expression level of calcium-binding protein S100A1.

[0038] Furthermore, the substance that reduces the expression level of calcium-binding protein S100A1 includes reagents used in lentiviral transfection.

[0039] In a ninth aspect, the present invention provides a use of a combined use of cytokines and immune checkpoint inhibitors, which is achieved by adopting the following technical solution.

[0040] A cytokine and an immune checkpoint inhibitor are used in combination to prepare a drug for treating tumors with high expression of calcium-binding protein S100A1.

[0041] Furthermore, immune checkpoint inhibitors are selected from one or more of anti-PD-1, anti-PD-L1 and anti-CTLA4; cytokines are selected from GM-CSF factor; tumors with high expression of calcium-binding protein S100A1 include lung cancer, melanoma, breast cancer, and urothelial carcinoma.

[0042] In a tenth aspect, the present invention provides a pharmaceutical composition for treating tumors with high expression of calcium-binding protein S100A1, which is achieved by adopting the following technical scheme.

[0043] A pharmaceutical composition for treating tumors with high expression of calcium-binding protein S100A1, comprising cytokines and immune checkpoint inhibitors.

[0044] Furthermore, the immune checkpoint inhibitor is selected from one or more of anti-PD-1, anti-PD-L1 and anti-CTLA4; the cytokine is selected from GM-CSF factor; and the tumors with high expression of calcium-binding protein S100A1 include lung cancer, melanoma, breast cancer, and urothelial carcinoma.

[0045] This application has the following beneficial effects.

[0046] In the present invention, S100A1 is closely related to the immunotherapy response and survival prognosis of lung cancer patients. Patients with high expression of S100A1 have a poor prognosis in immunotherapy. The expression level of S100A1 is closely related to macrophage M1 polarization and CD8 + T cell infiltration showed a significant negative correlation, suggesting that high expression of S100A1 may be associated with "cold" tumors; the present invention further proposed the S100A1 / USP7 / p65 / GM-CSF regulatory axis through functional and mechanism experiments, which can significantly regulate macrophage M1 polarization and T cell anti-tumor immune function, thereby affecting tumor immune escape and tumor immunotherapy response. In vivo model studies have shown that GM-CSF combined with anti-PD-1 can significantly inhibit the growth of tumors with high expression of S100A1, and S100A1 can be used as a potential marker for the treatment of "cold" tumors with GM-CSF combined with aPD-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 1 is a graph showing the correlation between the expression of S100A1 in the cell supernatant of the present invention and the mRNA and protein expression levels of S100A1 (wherein A. qRT-PCR detection of S100A1 in HEK293T cells OE The relationship between transfection dose and S100A1 mRNA expression; B. Western-blot detection of S100A1 in HEK293T cells OE Relationship between transfection dose and S100A1 protein expression; C. ELISA detection of S100A1 in HEK293T cells OE Relationship between transfection dose and S100A1 secretion level in cell supernatant; D. Correlation analysis between S100A1 mRNA expression in HEK293T cells and S100A1 secretion level in cell supernatant; E. Correlation analysis between S100A1 protein expression in HEK293T cells and S100A1 secretion level in cell supernatant);

[0048] Figure 2It is a result diagram of the correlation between plasma S100A1 expression and immunotherapy efficacy and survival prognosis of lung cancer patients of the present invention (wherein, A. lung cancer immunotherapy cohort analysis of plasma S100A1 expression differences in PR and SD / PD patients before and after treatment; B. imaging data of patients with high / low plasma S100A1 expression before and after immunotherapy; C. ROC curve evaluation of the efficacy of plasma S100A1 expression in predicting immunotherapy response; D. lung cancer immunotherapy cohort analysis of plasma S100A1 high / low expression group patients after immunotherapy efficacy difference; EK-M survival curve analysis of the relationship between plasma S100A1 expression in lung cancer immunotherapy cohort and PFS of patients after immunotherapy);

[0049] Figure 3 The result diagram of the correlation between S100A1 and tumor immunity of the present invention (wherein, A. the effect of knocking down S100a1 on the tumor-forming ability of LLC cell line in NCG immunodeficient mice; B. the effect of knocking down S100a1 on the tumor-forming ability of LLC cell line in nude mice; C. the effect of knocking down S100a1 on the tumor-forming ability of LLC cell line in C57 immune-competent mice; D. HE staining analysis of the effect of knocking down S100a1 on the in situ tumor-forming ability of LLC cell line in C57 immune-competent mice; E. comparison of the difference in lung weight between LLC cell line S100a1 knockdown group and control group of mice with orthotopic transplanted tumors; FK-M survival curve analysis of the difference in OS between LLC cell line S100a1 knockdown group and control group of mice with orthotopic transplanted tumors);

[0050] Figure 4 It is a result diagram showing that knocking down S100A1 of the present invention promotes the sensitivity of LLC subcutaneous transplanted tumors to anti-PD-1 treatment (wherein, A. compares the differences in subcutaneous transplanted tumor volumes among the four groups of control group + isotype control, S100a1 knockdown + isotype control, control group + anti-PD-1, and S100a1 knockdown + anti-PD-1; BK-M survival curve analyzes the differences in OS among the four groups of tumor-bearing mice among the control group + isotype control, S100a1 knockdown + isotype control, control group + anti-PD-1, and S100a1 knockdown + anti-PD-1; C. IHC detects the differences in the expression of CD8 in the four groups of subcutaneous transplanted tumors among the control group + isotype control, S100a1 knockdown + isotype control, control group + anti-PD-1, and S100a1 knockdown + anti-PD-1, scale bar = 50 μm);

[0051] Figure 5-1The figure is the result of the changes of various immune cell subsets in lung cancer TME after the intervention of S100A1 by the present invention (wherein, A. Flow cytometry analysis of the effect of knocking down S100a1 on the infiltration levels of MDSCs, NK cells, B cells, Tregs, TAMs and T cells in LLC subcutaneous transplanted tumors; B. Flow cytometry analysis of the effect of knocking down S100a1 on the expression of M1 polarization markers (iNOS and CD80) of macrophages in LLC subcutaneous transplanted tumors; C. Flow cytometry analysis of the effect of knocking down S100a1 on the expression of CD4 + T and CD8 + T cell infiltration level);

[0052] Figure 5-2 The present invention analyzed the effect of knocking down S100a1 on LLC subcutaneous transplanted tumor CD8 + The effect on the secretion capacity of IFN-γ, TNF-α and GZMB of T cells;

[0053] Figure 6 : This is a diagram of the results of multiple immunofluorescence experiments on postoperative tissue specimens of NSCLC patients of the present invention (wherein, A. Multiple immunofluorescence experiments analyze the differences in the expression of CD8 and macrophage M1 polarization marker CD86 in NSCLC patients with high / low S100A1 expression; B. IHC detection of the differences in the expression of CD8 and macrophage M1 polarization marker CD86 in NSCLC patients with high / low S100A1 expression, scale = 200 μm; C. IHC analysis of the relationship between S100A1 expression and CD8 expression; D. IHC analysis of the relationship between S100A1 expression and macrophage M1 polarization marker CD86);

[0054] Figure 7 The diagram of the effect and mechanism of lung cancer cell S100A1 on TIME remodeling of the present invention (wherein, A. UMAP-based dimensionality reduction clustering and annotation of each TME cell subpopulation; B. Analysis of the proportion changes of each TME cell subpopulation after intervention of tumor cell S100A1; C. Density map analysis of the changes of each TME cell subpopulation after intervention of tumor cell S100A1; D. tSNE-based dimensionality reduction clustering and annotation of macrophage subpopulations; E. GSVA-based analysis of the changes in phagocytosis, angiogenesis, M1 polarization and M2 polarization characteristics of macrophage subpopulations after intervention of tumor cell S100A1; F. Contour map analysis of Spp1 after intervention of tumor cell S100A1 + TAM and C1q + Changes in inflammatory response pathways and TNF-α / NF-κβ signaling pathways in TAM subpopulations; G. Contour map analysis of changes in T cell activation and active cytokine-related pathways after intervention of tumor cell S100A1);

[0055] Figure 8It is a spatiotemporal specific result diagram of TME gene expression after intervention of lung cancer cell S100A1 by the present invention (wherein, A. the effect of intervention of tumor cell S100A1 on TME based on spatial transcriptome sequencing celltrack annotation analysis; B. the effect of intervention of tumor cell S100A1 on M1 polarization and T cell inflammatory pathways evaluated based on ssGSEA);

[0056] Figure 9-1 The results of the intervention of tumor cell S100A1 on the anti-tumor immune function of T cells in the present invention are shown in Figure 1 (A. The intervention of tumor cell S100A1 on CD8 + Effects of S100A1 intervention on CD8 T cells secretion capacity of IFN-γ, TNF-α and GZMB; B. Schematic diagram of tumor cell-macrophage-T cell co-culture model; C. Analysis of the effect of S100A1 intervention on CD8 T cells based on the tumor cell-macrophage-T cell co-culture model + Effects on the secretion of IFN-γ, TNF-α and GZMB by T cells; D. Schematic diagram of CD45.1 / CD45.2 heterozygous mouse model and T cell adoptive transfer experiment);

[0057] Figure 9-2 The results of the intervention of tumor cell S100A1 on the anti-tumor immune function of T cells according to the present invention (wherein, E. The intervention of tumor cell S100A1 on CD8 based on CD45.1 / CD45.2 heterozygous mouse model and T cell adoptive analysis + Effects of S100A1 intervention on the secretion of tumor-specific CD8 + Differences in T cell infiltration levels and IFN-γ and TNF-α secretion capacity);

[0058] Fig.10The results of the intervention of tumor cell S100A1 on macrophage function of the present invention are shown in Figure 1 (A. The effect of knocking down LLC cell line S100a1 on the expression of macrophage M1 polarization marker iNOS was analyzed based on the tumor cell-BMDMs co-culture model; B. The effect of overexpressing B16-OVA cell line S100a1 on the expression of macrophage M1 polarization marker iNOS was analyzed based on the tumor cell-BMDMs co-culture model; C. The effect of knocking down LLC cell line S100a1 on the expression of macrophage M1 polarization marker iNOS was analyzed based on the tumor cell-RAW264.7 co-culture model; D. Western-blot detection of knocking down LLC cell line S100a1 on the expression of macrophage M1 polarization marker iNOS. E. The effect of S100a1 on the expression of iNOS and CD86, the polarization markers of macrophage M1; F. The effect of knocking down S100a1 in LLC cell line on the expression of iNOS, the polarization marker of macrophage M1, was evaluated based on the culture supernatant of tumor cells; G. The effect of overexpressing S100a1 in B16-OVA cell line on the expression of iNOS, the polarization marker of macrophage M1, was evaluated based on the culture supernatant of tumor cells; H. The effect of knocking down S100a1 in LLC cell line on the expression of iNOS, the polarization marker of macrophage M1, was evaluated based on the culture supernatant of tumor cells and S100A1 recombinant protein);

[0059] Fig.11 The results of the present invention intervening the regulation mechanism of lung cancer cell S100A1 on macrophage M1 polarization (wherein, A. Based on ProcartaPlex TM A. Detect the differential expression profiles of Th1 / Th2 cytokines in the cell culture supernatants of LLC S100a1 knockdown group and control group; B. qRT-PCR was used to detect the changes in Csf2 mRNA expression levels after knockdown / overexpression of S100a1; C. Western-blot was used to detect the changes in GM-CSF protein levels after knockdown / overexpression of S100a1; D. ELISA was used to detect the changes in GM-CSF secretion levels after knockdown / overexpression of S100a1; E. Based on tumor cell culture supernatant and anti-GM-CSF active monoclonal antibody, the effect of neutralizing GM-CSF on the expression of iNOS, a macrophage M1 polarization marker, was evaluated; F. Based on anti-GM-CSF active monoclonal antibody, the effect of neutralizing GM-CSF on the tumorigenic ability of LLC cell line in vivo was evaluated);

[0060] Figure 12-1 The molecular mechanism of S100A1 regulating GM-CSF transcription in the present invention is shown in Figure 1 (A. LC-MS analysis suggests that USP7 is a potential interacting protein of S100A1; B. CoIP-based detection of LLC cell line and MC38 S100a1 OEInteraction between S100A1 protein and USP7 protein in cell lines; C. Detection of co-localization between S100A1 protein and USP7 protein based on immunofluorescence experiment; D. Western-blot detection of changes in USP7 and p65 protein levels after knockdown / overexpression of S100a1);

[0061] Figure 12-2 It is a result diagram of the molecular mechanism of S100A1 regulating GM-CSF transcription of the present invention (wherein, E. Schematic diagram of USP7 truncation mutant; F. CoIP-based detection of the interaction between S100A1 protein and USP FL, USP7ΔMATH, USP7ΔUBL and MATH and other USP7 truncation mutants; G. CoIP-based detection of the interaction changes between USP7 protein and p65 protein after knockdown / overexpression of S100a1);

[0062] Fig.13 The result diagram of the present invention S100A1 regulating the ubiquitination level of p65 (wherein A. Western-blot detection of the effect of knocking down S100a1 on the half-life of p65 protein; B. Western-blot detection of the effect of overexpressing S100a1 on the half-life of p65 protein; immunofluorescence experiment detection of the co-localization between S100A1 protein and USP7 protein; C. CoIP detection of the effect of knocking down / overexpressing S100a1 on the ubiquitination level of p65 protein);

[0063] Fig.14 It is a diagram showing the changes in transcription factor p65 and its binding in the CSF2 promoter region after the intervention of S100A1 by the present invention (wherein A. the effect of knockdown / overexpression of S100a1 on the nuclear entry of p65 was detected based on immunofluorescence experiments; B. the effect of low / overexpression of S100a1 on the enrichment of p65 in the Csf2 promoter region was detected by CUT&Tag);

[0064] Fig.15 The results of the treatment of tumors with high expression of S100A1 by GM-CSF combined with anti-PD-1 of the present invention (A. Evaluation of the therapeutic effect of GM-CSF combined with anti-PD-1 based on LLC cell line subcutaneous transplanted tumors; B. Evaluation of the therapeutic effect of GM-CSF combined with anti-PD-1 based on MC38S100a1 OE The therapeutic effect of GM-CSF combined with anti-PD-1 was evaluated in subcutaneous transplanted tumors of cell lines). DETAILED DESCRIPTION

[0065] The present invention will be further described below in conjunction with the embodiments.

[0066] 1. Experimental Methods

[0067] 1. DNA transfection: HEK293T cells with good growth status were selected and cultured in 12-well culture plates. 250 μL serum-free Opti-MEM medium, 5 μg pCMV3-S100A1-Flag and 2 μL P3000 were added to each well. TM Enhanced transfection reagent with 250 μL serum-free Opti-MEM medium and 1.5 μL Lipofectamine TM 3000 reagents were fully mixed and transfected into HEK293T cells, and downstream analysis was performed 72 hours later.

[0068] 2. Lentivirus infection: dilute the virus stock solution with culture medium and add it to mouse tumor cells, and culture it in a 37°C, 5% CO2 incubator; after 6 hours of culture, replace the medium with fresh culture medium; 48 hours after the end of infection, use Puromycin to screen resistant cells, which are mouse tumor cell lines with overexpression or knockdown of S100a1.

[0069] 3. Quantitative real-time PCR (qRT-PCR): Collect cells and extract total RNA, measure RNA concentration and reverse transcribe, and use real-time fluorescence qPCR to detect S100A1 mRNA expression level.

[0070] 4. Western-blot experiment: extract total cell protein, determine protein concentration, perform electrophoresis, transfer, block and then incubate with antibodies, and use a chemiluminescence imager to expose and image to detect the expression level of the target protein.

[0071] 5. Enzyme linked immunosorbent assay (ELISA): ELISA was used to detect the content of S100A1 in the supernatant of HEK293T cell lines and plasma samples of lung cancer patients. The operation steps were carried out according to the instructions of the Human S100A1 ELISA kit of Invitrogen.

[0072] 6. Mouse transplant tumor model: S100a1 knockdown or overexpression mouse tumor cells and control group cells in the logarithmic growth phase were collected to construct a subcutaneous transplant tumor model of mouse tumor cell line; tumor cells were injected into the tail vein to construct an orthotopic transplant tumor experiment of mouse lung cancer.

[0073] 7. HE staining: After the sample sections are dewaxed and hydrated, they are stained with hematoxylin for 20 minutes to dye the cell nuclei blue or purple, and eosin for 30 seconds to dye the cytoplasm, fibers and other cell structures red or pink; then they are dehydrated in a gradient manner, dried in a 70°C oven for about 5 minutes, and sealed with neutral gum.

[0074] 8. Immunohistochemistry (IHC): After the sample sections were dewaxed and hydrated, antigen repair was performed on the sections using citric acid repair solution (pH 6.0). The sections were incubated with peroxidase blocking solution at room temperature for 15 minutes to block endogenous catalase, blocked with goat serum at room temperature for 1 hour, and incubated with primary antibody overnight; the secondary antibody was incubated at room temperature for 1 hour; then DAB color development was performed, hematoxylin was used to stain the nucleus for 30 seconds, 1% hydrochloric acid alcohol was used for differentiation for 2 seconds, and the sections were rinsed under running water for 10 minutes for anti-blueing; after gradient dehydration, the sections were placed in a 70°C oven and dried for about 5 minutes, and then sealed with neutral gum, and the Image-pro-plus 6.0 software was used to quantitatively analyze the cumulative optical density value of the target protein as its protein expression.

[0075] 9. Flow cytometry analysis of mouse subcutaneous transplanted tumors: Prepare single-cell suspension of mouse tumor tissue, block with Purified Anti-Mouse CD16 / CD32 at room temperature for 10 minutes, then perform surface staining, and then perform intracellular cytokine staining.

[0076] 10. Multiplex immunofluorescence (mIF): dewaxing, hydration and fixation, antigen repair and blocking, primary and secondary antibody incubation, fluorescent staining to amplify the signal, and sealing

[0077] 11. Single-cell transcriptome sequencing and analysis: Prepare single-cell suspension of mouse tumor tissue, use Illumina technology platform to sequence the library, process raw data and identify cell subpopulations, and use gene set variation analysis (GSVA) ​​to calculate the M1 / M2 score of TAMs and the antigen processing, presentation and immune regulation score of T cells

[0078] 12. Spatial transcriptome sequencing: After tissue embedding and quality inspection, tissue permeabilization is performed, followed by sectioning, HE staining imaging, cDNA synthesis and amplification, library construction, and sequencing of the Visium spatial gene expression library using the IlluminaNovaSeq 6000 platform. Spatial transcriptome data processing, integration, and visualization, ssGSEA scoring is performed on the sections based on the provided M1 and M2 macrophage subset functional gene sets, and the scores are displayed on the spots. scRNA is combined with idle data for deconvolution analysis, and three deconvolution methods, Seurat, RCTD, and SPOTlight, are provided to annotate spots. scRNA is combined with idle data for cell mapping analysis, and single-cell data are mapped to sections.

[0079] 13. Construction of co-culture model of B16-0VA mouse melanoma cell line and BMDMs: mature BMDMs were seeded on the bottom layer of a 24-well plate, placed in a Transwell chamber with a pore size of 0.4 μm, and B16-0VA melanoma cell line was seeded on the upper layer of the Transwell chamber. Sufficient culture medium was added to cover the upper layer of the chamber, and cultured for 48 h. BMDMs and OT-1CD8 + T cells were co-cultured for 24 h.

[0080] 14.OT-1CD8 + T cell killing experiment in vitro: The activated OT-1T cells or the OT-1T cells co-cultured with BMDMs were aspirated, washed with PBS, and the OT-1T cells were co-cultured with the B16-OVA cell line in a certain ratio. After 24 hours of culture, flow cytometry analysis was performed to detect the level of secreted cytokines.

[0081] 15.T cell adoptive transfer experiment: sorted OT-1naive CD45.2 + CD8 + T(1×10 6 / mouse) were injected into tumor-bearing mice through the tail vein. The mice were killed 7 days later, and the tumors were digested and analyzed by flow cytometry. + CD8 + T cell activation.

[0082] 16. Th1 / Th2 Cytokine 11-Plex Mouse ProcartaPlex TM Detection: dilute the standard, wash the microspheres, add the sample for incubation, wash the plate, incubate with the antibody, add streptavidin-PE (SA-PE), and test on the machine

[0083] 17. Liquid chromatography-tandem mass spectrometry (LC-MS) analysis: First, an immunoprecipitation experiment was performed. The reaction solution (1% SDC; 100mM Tris-HCL pH=8.5; 10mM TCEP; 40mM CAA) was added to the sample and incubated at 60°C for 1h for one-step protein denaturation, reduction and alkylation. After adding an equal volume of ultrapure water for dilution, trypsin was added at a mass ratio of 1:50 between enzyme and protein, and the enzyme digestion was performed at 37°C with shaking overnight. On the second day, TFA was added to terminate the digestion, and the supernatant was centrifuged at 16000g and desalted with a homemade SDB desalting column; then mass spectrometry detection was performed, and the mass spectrometry data was collected using a Q Exactive HF-X mass spectrometer connected to an EASY-nLC 1200 liquid phase liquid-mass spectrometry system. The mass spectrometry data was retrieved using MaxQuant (V1.6.6) software, and the database retrieval algorithm used was Andromeda. The database used for the search was the proteome reference database of Tomato in Uniprot (containing 34,655 protein sequences); the search results were screened based on a false discovery rate (FDR) of 1% at the protein and peptide levels, and the entries of anti-library proteins, contaminating proteins, and proteins with only one modified peptide were deleted, and the remaining identification information was used for subsequent analysis.

[0084] 18. Immunofluorescence confocal experiment: seed cells into μ-Slide V1 wells, 1-3×10 4 When the cells adhered to the wall, the culture medium was removed, and 60 μL of pre-cooled 4% paraformaldehyde was added to fix at room temperature for 15-30 min. 60 μL of PBS containing 5% Triton X-100 was added to permeabilize at room temperature for 15 min to increase the permeability of the cells to facilitate the entry of subsequent antibodies. TBST containing 5% BSA was added to block the cells at room temperature on a shaker at 70 rpm for 1 h. After the primary antibody was added to the cells, the cells were incubated on a shaker at 4°C overnight, and the fluorescent secondary antibody was incubated on a shaker at room temperature for 2 h. After adding 50 μL of anti-fade mounting medium, the cells were observed and photographed under a confocal microscope.

[0085] 19. Co-immunoprecipitation (CoIP): Add 500 μL of IP lysis buffer containing protease inhibitors to the culture dish, gently scrape the cells in the same direction with a scraper, lyse on a shaker at 4°C for 30 minutes, and further lyse the cells using low-intensity ultrasound; centrifuge at 15,000 g for 15 minutes to remove the cell precipitate, collect the supernatant, add the washed protein A / G magnetic beads and the target protein antibody, and incubate on a shaker at 4°C for 12 hours; place the tube on a magnetic rack, discard the supernatant after the magnetic beads are adsorbed, wash the magnetic beads 4-6 times with pre-cooled PBST to remove unbound proteins; resuspend the magnetic beads with 1×SDS PAGE loading buffer, boil the sample on a 99°C metal bath for 10 minutes, and verify it by subsequent mass spectrometry or protein immunoblotting experiments.

[0086] 20. Construction of USP7 truncation mutants: Use the kit and follow the product instructions to extract the genome of HEK293T cells, determine the DNA concentration, and store at -20°C; use the HEK293T cell genome and USP7 truncation mutant fragment amplification primers to amplify each truncation mutant fragment, set a temperature gradient for PCR, and recover the fragments on the gel after electrophoresis; connect the recovered products, react at 22°C for 2h, transform the ligation products into DH5α competent cells and plate them, identify the length of the inserted fragment in the positive clone by PCR, and sequence them.

[0087] 21. Chromosome targeted cutting and tagging (CUT&Tag) experiment: Collect cells, add 500μL Washbuffer, centrifuge at 2500rpm for 5min, and resuspend cells with 100μL Wash buffer; transfer 100μL cells to a tube containing activated ConA Beads Pro, mix well, incubate at room temperature for 10min, and remove supernatant by magnetic adsorption after incubation; add 50μL pre-cooled Antibodybuffer to each sample, add different primary antibodies according to concentration, and stand at 4℃ overnight; remove primary antibody by magnetic adsorption, add diluted secondary antibody, incubate at room temperature for 60min, remove secondary antibody by magnetic adsorption, and wash cells three times with Dig-wash Buffer; add 100μL diluted pA / G-Tnp Pro transposon to each tube, incubate at room temperature for 60min, and use Dig-300 Wash cells three times with B&W Buffer; add 50 μL fragmentation reagent to each tube, incubate at 37°C in a PCR instrument for 60 min, add 2 μL 10% SDS and 1 pg DNA Spike-in to the fragmentation product, incubate at 55°C for 10 min, adsorb magnetically, and transfer the supernatant to a clean tube; DNA extraction: add 50 μL treated DNAExtract Beads Pro magnetic beads to the supernatant, mix well, incubate at room temperature for 20 min, remove the supernatant by magnetic adsorption, wash cells three times with B&W Buffer, keep the PCR tube moderately on the magnetic rack during this period, open the lid to dry the magnetic beads until there is no reflection on the surface, add 15 μL ddH20 to resuspend the magnetic beads, add 5 μL Stop Buffer, incubate at 95°C for 5 min, adsorb magnetically, transfer the supernatant to a clean tube, and finally perform qPCR detection.

[0088] 22. Statistical analysis: SPSS 22.0 statistical software and Graphics Prism 6.0 statistical software were used for processing. When comparing the means between the two groups of data, the independent-sample T test (Independent-sample T test) and the independent sample nonparametric test (Mann Whitney test) were used; the contingency table analysis was performed using the χ 2 Test; Univariate / multivariate analysis of variance of prognostic predictors was performed using the Cox proportional hazards regression model: the model test was tested using the likelihood ratio test, the parameter estimation was done using the maximum likelihood method, and the parameter test was done using the Wald test. P < 0.05 was considered statistically significant.

[0089] 2. Experimental Results

[0090] 1. Correlation between S100A1 expression in cell supernatant and mRNA and protein expression levels of S100A1

[0091] Since S100 family members can be secreted or released from cells, this application detected the supernatant of HEK293T cells by enzyme linked immunosorbent assay (ELISA), and found that the expression of S100A1 in the cell supernatant was positively correlated with the mRNA and protein expression levels of S100A1. The above results reveal that S100A1 expression can be detected in blood samples of tumor patients receiving immunotherapy to evaluate its value in judging the response to immunotherapy and survival prognosis ( Figure 1 ).

[0092] 2. Plasma S100A1 expression is closely related to the efficacy of immunotherapy and survival prognosis in lung cancer patients

[0093] This application collected a clinical cohort of 77 lung cancer patients with paired blood samples before and after immunotherapy from the National Cancer Center / National Clinical Research Center for Cancer / Cancer Hospital of Peking Union Medical College, Chinese Academy of Medical Sciences, including 34 cases of lung adenocarcinoma, 26 cases of lung squamous cell carcinoma, 11 cases of small cell lung cancer and 6 cases of other pathological types of lung cancer, to explore the correlation between S100A1 expression and immunotherapy response and survival prognosis. The results showed that compared with PR (partial response) patients, the plasma S100A1 expression of SD (stable disease) / PD (progressive disease) patients before immunotherapy was significantly higher, and increased significantly after immunotherapy; the prognostic value of plasma S100A1 expression was evaluated by calculating the area under the ROC curve (AUC) based on the receiver operating characteristic curve (ROC). The results showed that the AUC of plasma S100A1 expression level in lung cancer patients in judging immunotherapy response was 0.706; KM survival analysis was performed on the progression-free survival (PFS) of the patients followed up. The plasma S100A1 expression level before immunotherapy was closely related to PFS. The PFS of patients with higher plasma S100A1 expression before immunotherapy was significantly shortened, while the overall prognosis of patients with lower plasma S100A1 expression was better. In addition, the contingency table analysis showed that there was no statistical difference in age, gender, smoking status, histological type, tumor stage, etc. between patients with high / low plasma S100A1 expression; multivariate Cox regression analysis showed that S100A1 expression was a prognostic factor for PFS in lung cancer patients after immunotherapy. The above results reflect the value of plasma S100A1 expression level in evaluating the efficacy and prognosis of immunotherapy in lung cancer patients, and reveal the potential of S100A1 as a biomarker for predicting the efficacy of immunotherapy in lung cancer patients ( Figure 2 ).

[0094] 3. Intervention of S100A1 expression in lung cancer cells affects tumor immune escape and anti-PD-1 efficacy

[0095] In order to clarify the function of S100A1 in lung cancer, the present invention used mouse S100a1 shRNA lentiviral vector to knock down endogenous S100a1 in mouse lung cancer cell line Lewis Lung Carcinoma (LLC). The results showed that knocking down S100A1 did not significantly change the proliferation and migration ability of LLC cells. The LLC cell line after S100A1 knockdown was further inoculated into NOD-Prkdc em26Cd52 Iqem26Cd22 / NjuCrl (NCG) triple immune-deficient mice and BALB / c-nude mice subcutaneously, and found that compared with the control group, knocking down S100A1 did not significantly affect the tumorigenicity of LLC cell lines in vivo, indicating that in an environment lacking immune function or T cell immunity, S100A1 is not necessary for the intrinsic adaptability of tumor cells. It is worth noting that after the above cell lines were inoculated subcutaneously into immune-competent C57 mice, the growth of LLC cell line subcutaneous transplanted tumors was significantly slowed down after knocking down S100A1, and the survival time of tumor-bearing mice was prolonged, suggesting that S100A1 is related to tumor immunity ( Figure 3 ).

[0096] In C57 mice, an orthotopic transplanted tumor model of lung cancer was established by injecting LLC cell line into the tail vein. It was found that compared with the control group, the tumorigenic ability of LLC was significantly reduced after knocking down S100A1, and the survival time of tumor-bearing mice was significantly prolonged, indicating that intervention of S100A1 can inhibit the tumorigenic ability of lung cancer cells, suggesting that S100A1 may be involved in the immune escape of lung cancer and thus affect tumor growth ( Figure 3 ).

[0097] In addition, in the anti-PD-1 treatment model, the present application observed that knocking down S100A1 could significantly promote the sensitivity of LLC subcutaneous transplanted tumors to anti-PD-1 treatment and increase CD8 + T cell infiltration indicates that intervening S100A1 in tumor cells can affect T cell-mediated adaptive immune responses, thereby enhancing the efficacy of anti-PD-1 therapy. In summary, this application shows that S100A1 may be involved in lung cancer immune escape and immunosuppressive TME remodeling, and is expected to provide an important theoretical basis and experimental basis for S100A1 as a combined target for lung cancer immunotherapy ( Figure 4 ).

[0098] 4. Intervention of S100A1 remodeling TIME in lung cancer cells

[0099] Flow cytometry was used to characterize the immune cell subsets in the TME of lung cancer after S100A1 intervention. It was found that the proportion of macrophages and T cells in the TME changed significantly after knocking down the expression of S100A1 in lung cancer cells, while the other immune cells, such as myeloid-derived suppressor cells (MDSCs), NK cells, B cells and Treg cells, did not change significantly. Further analysis of macrophage subsets and T cell subsets showed that compared with the control group, the expression of macrophage M1 polarization markers inducible nitric oxide synthase (iNOS) and CD80 in the tumors of the S100A1 knockdown group was significantly higher, indicating that the proportion of M1 macrophages in the tumors of the S100A1 knockdown group was significantly increased. At the same time, compared with the control group, the CD4 + T and CD8 + T cell infiltration increased significantly, CD8 + The ability of T cells to secrete killer factors such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and granzyme B (Gzm B) was significantly enhanced in the knockdown group ( Figure 5-1 , Figure 5-2 ).

[0100] Through multiple immunofluorescence experiments on postoperative tissue specimens of NSCLC patients, this application found that tumors with high S100A1 expression had lower levels of CD8 and M1 macrophage polarization marker CD86 expression, while tumors with low S100A1 expression had significantly higher CD8 and CD86 expression; at the same time, based on immunohistochemistry (IHC) experiments, it was also shown that the S100A1 expression level was significantly negatively correlated with CD8 and CD86 expression. Previous studies have found that macrophage M1 polarization can promote T cell anti-tumor immunity and enhance the sensitivity of tumors to immunotherapy. Therefore, the above results suggest that tumor cell S100A1 may affect CD8 + T cell infiltration and macrophage M1 polarization reshape TIME( Figure 6 ).

[0101] In order to analyze the role and mechanism of S100A1 in lung cancer cells on TIME remodeling, this application collected the control group and S100A1 knockdown group of mouse lung cancer orthotopic transplanted tumors for single-cell RNA sequencing (scRNA-seq). The results showed that the proportion of macrophages and T cell subsets changed most significantly after S100A1 intervention. Compared with the control group, the proportion of T cells in the knockdown group increased significantly, and CD8 +T cell infiltration increased significantly. A total of 7 macrophage subsets were identified. + TAM and C1q + TAM subsets all showed classic pro-inflammatory macrophage M1 polarization characteristics, while the expression levels of genes related to immunosuppressive macrophage M2 polarization did not change significantly, suggesting that lung cancer cell S100A1 promotes TAMs reprogramming to an M1 polarization phenotype. In addition, the TNF-α / NF-κβ inflammatory pathway was significantly activated in the tumor macrophage subsets in the knockdown group. The above results indicate that intervention with lung cancer cell S100A1 can significantly change the functional phenotype of macrophages in the TME, promote macrophage M1 polarization, and reshape TIME. This study annotated T cell subsets and identified a total of 10 subsets. Compared with the control group, intervention with lung cancer cell S100A1 significantly affected T cell infiltration in the TME, as shown by CD8 + T cell subsets decreased in number, while memroy CD8 + T, effector CD8 + The above analysis results show that intervention of S100A1 in lung cancer cells can induce T cell-mediated anti-tumor immune response and reshape the inhibitory TIME into an immune activation state ( Figure 7 ).

[0102] In order to further illustrate the spatiotemporal specificity of TME gene expression after intervention of S100A1 in lung cancer cells, this application collected orthotopic transplanted tumors of lung cancer in mice in the control group and S100A1 knockdown group for spatial transcriptome sequencing (spatialtranscriptomics). The results showed that there were more macrophages and T cell infiltrations around the tumor cells in the S100A1 knockdown group, and the M1 polarization and T cell inflammatory signal scores were significantly higher than those in the control group tumors, indicating that knocking down S100A1 can affect the M1 polarization of macrophages around lung cancer cells and the anti-tumor immune function of T cells. The above results further show that intervention of S100A1 in lung cancer cells can reshape the tumor immune microenvironment and affect tumor immune escape ( Figure 8 ).

[0103] 5. S100A1 regulates macrophage M1 polarization and T cell anti-tumor immunity via the USP7 / p65 / GM-CSF axis

[0104] The present invention constructs an in vitro co-culture model of mouse melanoma cell line B16-OVA and OT-1 cells to explore whether intervention of tumor cell S100A1 can directly affect the anti-tumor immune function of T cells. The results show that when B16-OVA melanoma cells overexpressing S100A1 are co-cultured with OT-1 cells, the secretion capacity of cytokines such as IFN-γ, TNF-α and GZMB of OT-1 cells is not significantly changed; at the same time, the killing ability of OT-1 cells to tumor cells is not significantly changed. However, after tumor cells and bone-marrow derived macrophages (BMDMs) are co-cultured, the latter are then co-cultured with OT-1 cells, and the function of the latter is significantly changed: compared with the control group, BMDMs co-cultured with B16-OVA melanoma cells overexpressing S100A1 can significantly inhibit the secretion capacity of cytokines such as IFN-γ, TNF-α and GZMB of OT-1 cells and the killing ability to tumor cells. Using CD45.1 / CD45.2 heterozygous mouse model and T cell adoptive transfer experiments, it was also shown in vivo that intervention of tumor cell S100A1 can significantly affect tumor-specific CD8 + The above results show that although intervention of tumor cell S100A1 cannot directly affect OT-1 cell function, it can indirectly affect OT-1 cell function through BMDMs, suggesting that the regulation of tumor cell S100A1 on T cell anti-tumor immune function depends on the presence of BMDMs ( Figure 9-1 , Figure 9-2 ).

[0105] In order to clarify the effect of intervention of tumor cell S100A1 on macrophage function, LLC cell lines in the control group and S100A1 knockdown group, and B16-OVA cell lines in the control group and S100A1 overexpression group were co-cultured with BMDMs. The results showed that the expression of iNOS in BMDMs after co-culture with the S100A1 knockdown group cell line was significantly increased, while the expression of iNOS in BMDMs after co-culture with the S100A1 overexpression group cell line was significantly decreased. This shows that tumor cell S100A1 can induce macrophage M1 polarization and affect the latter's immunoregulatory function. Previous studies have reported that according to the difference in the site of action, S100 protein can be divided into three subgroups: those that only play a role in cells, those that have both intracellular and extracellular regulatory effects, and those that mainly produce effects outside cells. This application also confirmed in the early stage that S100A1 is present in cell supernatants and patient plasma, and is positively correlated with intracellular mRNA and protein expression levels. The present invention intends to explore whether extracellular secretory S100A1 can directly affect macrophage M1 polarization by exogenously adding S100A1 recombinant active protein. The present application confirms that the supernatant of the S100A1 knockdown group cell line and the supernatant of the overexpression group cell line have significant promoting and inhibiting effects on macrophage M1 polarization, respectively; however, after adding the S100A1 recombinant active protein to the supernatant of the S100A1 knockdown group cell line, there was no inhibitory effect on the polarization induction of M1 macrophages, indicating that the regulation of macrophage M1 polarization by intervening tumor cell S100A1 is not directly mediated by extracellular S100A1, suggesting that tumor cell S100A1 may play a regulatory role in the cell, indirectly affecting the expression and secretion of immune cell chemokines or regulatory factors by interacting with target proteins, and the latter may participate in macrophage M1 polarization in a paracrine manner, thereby inducing the latter's anti-tumor immune function ( Fig.10 ).

[0106] Based on the above, the present application uses the ProcartaPlex multiple immunoassay kit to detect the differential expression profiles of 11 cytokines in the supernatant of LLC cell lines in the control group and the S100A1 knockdown group to explore the regulatory mechanism of intervention of lung cancer cell S100A1 on macrophage M1 polarization. The results show that the S100A1 knockdown cell line can significantly affect the GM-CSF level in the supernatant, suggesting that the latter may play a key role in the regulation of macrophage M1 polarization by lung cancer cell S100A1. Lung cancer cells can secrete GM-CSF to promote inflammatory neutrophil infiltration and regulate macrophage M1 polarization, thereby promoting its anti-tumor immune function. After adding GM-CSF neutralizing antibody (anti-GM-CSF) to the supernatant of the S100A1 knockdown group cell line in the present invention, its induction effect on macrophage M1 polarization is significantly weakened; at the same time, intraperitoneal injection of anti-GM-CSF in C57 mice can significantly restore the inhibitory effect of knockdown tumor cell S100A1 on tumor growth. The above results indicate that tumor cell S100A1 regulates macrophage M1 polarization by affecting GM-CSF transcription, thereby regulating T cell anti-tumor immune function and reshaping the immunosuppressive TME ( Fig.11 ).

[0107] This study suggests that tumor cell S100A1 plays a role in cells by interacting with target proteins. Liquid chromatography-tandem mass spectrometry (LC-MS) was used to screen and identify S100A1's interacting molecules and explore the molecular mechanism of S100A1 regulating GM-CSF transcription. Co-immunoprecipitation (CoIP) and immunofluorescence analysis showed that S100A1 interacted with USP7 and was mainly co-localized in the cytoplasm. The transcriptional activation of GM-CSF encoding gene CSF2 is regulated by transcription factor p65, suggesting that tumor cell S100A1 may affect p65 protein stability by regulating USP7 deubiquitination activity, thereby regulating GM-CSF transcription. WB experiments showed that knockdown and overexpression of S100A1 could upregulate and downregulate p65 protein levels, respectively, while USP7 protein levels did not change significantly, indicating that although S100A1 did not change USP7 protein levels, it might affect p65 protein stability by regulating USP7 deubiquitination activity, thereby regulating GM-CSF expression. CoIP experiments showed that S100A1 interacted with USP7 FL and USP7ΔMATH, but not with USP7ΔUBL and MATH, indicating that S100A1 binds to USP7 only when the C-terminal ubiquitin-like domain (UbL) is intact, and the UbL domain mediates the interaction between S100A1 and USP7. More importantly, knocking down S100a1 expression in LLC cell lines increased the interaction between USP7 and p65; while overexpressing S100a1 reduced the interaction between USP7 and p65, indicating that tumor cell S100A1 can target the interaction between USP7 and p65 to regulate p65 deubiquitination. This application proposes that the S100A1 / USP7 / p65 regulatory axis regulates GM-CSF expression, changes the polarization state of macrophages M1 and the anti-tumor immune function of T cells, and reshapes the TME ( Figure 12-1 , Figure 12-2 ).

[0108] This application uses CHX tracking experiments to detect changes in the half-life of p65 protein after intervention with S100A1, in order to clarify the mechanism of action of the S100A1 / USP7 / p65 / GM-CSF regulatory axis. The results showed that after knocking down S100a1 in the LLC cell line, the half-life of p65 protein was shortened and the protein stability was reduced; while after overexpressing S100a1, the half-life of p65 protein was prolonged and the protein stability was increased. Further ubiquitination experiments confirmed that after knocking down S100a1 in the LLC cell line, the ubiquitination level of p65 was reduced; while after overexpressing S100a1, the ubiquitination level of p65 was increased. This application proposes that intervening in tumor cell S100A1 can change the level of p65 ubiquitination degradation and affect its protein stability ( Fig.13 ).

[0109] Since p65, as a transcription factor, must enter the nucleus and bind to the promoter region of a specific target gene to exert a transcriptional regulatory effect, this application uses immunofluorescence experiments to detect changes in the nuclear entry of transcription factor p65 after intervention with S100A1. The results showed that after knocking down S100A1 in LLC cell lines, the nuclear entry of transcription factor p65 increased; while after overexpressing S100A1 in MC38 cell lines, the nuclear entry of transcription factor p65 decreased. CUT&Tag experiments were further used to clarify the changes in the specific binding level of p65 in the CSF2 promoter region after intervention with S100A1. It was found that after knocking down S100A1 in LLC cell lines, the enrichment of transcription factor p65 in the CSF2 promoter region was significantly increased; while after overexpressing S100A1 in MC38 cell lines, the enrichment of transcription factor p65 in the CSF2 promoter region was significantly reduced. The above trend of changes is consistent with the changes in the mRNA and protein levels of CSF2 after knocking down / overexpressing S100A1. The above results indicate that intervention of S100A1 in tumor cells can affect the deubiquitination regulation of USP7 on p65 by interacting with the UbL domain of USP7, changing the nuclear entry of p65 and its binding to the CSF2 promoter region, thereby regulating GM-CSF expression ( Fig.14 ).

[0110] 6. S100A1 can be used as a potential marker for the treatment of "cold tumors" with GM-CSF combined with aPD-1

[0111] The present invention uses the LLC cell line and the in vivo transplanted tumor model of the MC38 cell line overexpressing S100A1 to explore the therapeutic value of GM-CSF combined with anti-PD-1 for tumors with high S100A1 expression. The results showed that GM-CSF can effectively inhibit the growth of transplanted tumors of the LLC cell line and the MC38 cell line overexpressing S100A1; on this basis, GM-CSF can enhance the sensitivity of LLC cell line and MC38 cell line transplanted tumors overexpressing S100A1 to anti-PD-1, and GM-CSF combined with anti-PD-1 can effectively treat LLC cell line and MC38 cell line transplanted tumors overexpressing S100A1 (the anti-PD-1 dosage is twice the GM-CSF dosage, specifically, the anti-PD-1 dosage for mice is 200 μg / mouse and the GM-CSF dosage is 100 μg / mouse). The above results suggest that S100A1 can be used as a potential marker for the treatment of "cold" tumors with GM-CSF combined with aPD-1 ( Fig.15 ).

[0112] The embodiments of this specific implementation method are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore, all equivalent changes made based on the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of a substance for detecting the expression level of calcium-binding protein S100A1 in the preparation of a product for evaluating the efficacy of lung cancer immunotherapy and / or lung cancer prognosis assessment.

2. A kit for evaluating the efficacy of immunotherapy for lung cancer, characterized in that: The invention comprises a substance for detecting the expression level of calcium binding protein S100A1.

3. A kit for evaluating the prognosis of lung cancer, characterized in that: The invention comprises a substance for detecting the expression level of calcium binding protein S100A1.

4. Use of a substance for reducing the expression level of calcium-binding protein S100A1 in the preparation of at least one of the following drugs: a. Treatment of lung cancer; b. Enhance the sensitivity of lung cancer patients to immunotherapy; c. Reshape the tumor immune microenvironment in lung cancer patients; d. Promote M1 polarization of macrophages and T cell anti-tumor immunity in patients with lung cancer.

5. A drug for treating lung cancer, characterized in that: This includes substances that reduce the expression level of calcium binding protein S100A1.

6. A drug for enhancing the sensitivity of lung cancer patients to immunotherapy, characterized in that: This includes substances that reduce the expression level of calcium binding protein S100A1.

7. Application of cytokines and immune checkpoint inhibitors in combination for the preparation of drugs for treating tumors with high expression of calcium-binding protein S100A1.

8. The use according to claim 7, characterized in that: Immune checkpoint inhibitors include one or more of anti-PD-1, anti-PD-L1 and anti-CTLA4; cytokines include GM-CSF factor; tumors with high expression of calcium-binding protein S100A1 include lung cancer, melanoma, breast cancer and urothelial carcinoma.

9. A pharmaceutical composition for treating tumors with high expression of calcium-binding protein S100A1, characterized in that: Including cytokines and immune checkpoint inhibitors.

10. The pharmaceutical composition according to claim 9, characterized in that: The immune checkpoint inhibitor is selected from one or more of anti-PD-1, anti-PD-L1 and anti-CTLA4; the cytokine is selected from GM-CSF factor; the tumors with high expression of calcium-binding protein S100A1 include lung cancer, melanoma, breast cancer and urothelial carcinoma.

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