Pharmaceutical application of EGR1 + neutrophil inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and relates to pharmaceutical application of an EGR1 < + > neutrophil inhibitor. The invention reveals that high expression of KIAA1199 induces EGR1 < + > neutrophils through a KIAA1199-PPAR gamma / SAA2-FPR2-EGR1 signal axis for the first time, so that angiogenesis is promoted, and then liver metastasis is accelerated. The FPR2 antagonist, the PPAR gamma agonist, the PI3K inhibitor or the EGR1 inhibitor can block the axis and inhibit formation of liver metastasis, and a new target and strategy are provided for prevention and treatment of colorectal cancer liver metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to EGR1. + Pharmaceutical applications of neutrophil inhibitors. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, ranking among the top cancers in both incidence and mortality. The leading cause of death in CRC patients is not the primary tumor itself, but rather distant metastasis, with the liver being the most prevalent target organ. Colorectal cancer liver metastases (CRLM) are a key factor affecting patient prognosis. Despite advancements in comprehensive treatments such as surgery, chemotherapy, and targeted therapy, the 5-year survival rate for CRLM patients remains low, at only 12%-30%. Therefore, elucidating the molecular and cellular mechanisms of CRLM development and identifying effective early intervention targets and novel treatment strategies are significant challenges currently facing clinical practice.

[0003] Successful metastasis and colonization is a complex, multi-step process. Recent studies have shown that before tumor cells spread, the primary tumor can remotely regulate the microenvironment of distant organs, forming a so-called "pre-metastatic microenvironment" (PMN), providing a suitable "soil" for incoming circulating tumor cells. PMN formation involves multiple biological processes, including immunosuppression, angiogenesis, and extracellular matrix remodeling. Among these, angiogenesis is a key event in the early stages of PMN formation; newly formed blood vessels not only provide nutrients to metastatic lesions but also promote the exudation and colonization of tumor cells.

[0004] Among the various cellular components of the PMN, myeloid immune cells, particularly neutrophils, have been identified as key coordinators. Neutrophils have traditionally been considered short-lived, terminally differentiated effector cells. However, recent research has revealed the high heterogeneity and plasticity of neutrophils, with "tumor-associated neutrophils" (TANs) significantly promoting tumor progression and metastasis through the secretion of matrix metalloproteinases (such as MMP-9) and vascular endothelial growth factor (VEGF), as well as the formation of extracellular neutrophil traps (NETs). However, the upstream signals and precise mechanisms driving neutrophil differentiation into pro-metastatic phenotypes, especially in the specific context of colorectal cancer liver metastasis, remain unclear.

[0005] In summary, current technologies still provide an incomplete understanding of the mechanisms of colorectal cancer liver metastasis, particularly regarding long-range communication between the primary tumor and distant organ microenvironments, where a clear molecular pathway map is lacking. This significantly limits the ability to provide early warning and effective intervention for colorectal liver metastasis (CRLM). Therefore, there is an urgent need in this field to uncover a new and complete signal transduction axis and, based on this, develop novel methods capable of predicting, preventing, or treating CRLM. Summary of the Invention

[0006] This invention provides EGR1 + The pharmaceutical use of neutrophil inhibitors aims to address the lack of effective interventions in existing technologies targeting key immune cell subsets in the pre-metastatic microenvironment of colorectal cancer liver metastases.

[0007] The inventors discovered that KIAA1199-overexpressing colorectal cancer cells remotely regulate the liver microenvironment, leading to the inhibition of the PPARγ signaling pathway in hepatocytes, thereby relieving the inhibition of SAA2 gene transcription and significantly upregulating SAA2 protein expression and secretion. SAA2, through its binding to its receptor FPR2, activates the PI3K / AKT signaling pathway in neutrophils, inducing EGR1. + The formation of neutrophil subsets; this specialized EGR1 + Neutrophils promote angiogenesis by secreting VEGFA, thereby accelerating liver metastasis in colorectal cancer. Intervention with one or more of the following: FPR2 antagonists, PPARγ agonists, PI3K inhibitors, and EGR1 inhibitors can effectively block the above signaling pathways and inhibit liver metastasis formation.

[0008] Based on the above findings, the present invention provides the following technical solution: In a first aspect, the present invention provides EGR1 + Application of neutrophil inhibitors in the preparation of drugs for the prevention or treatment of diseases driven by KIAA1199 high expression.

[0009] In conjunction with the first aspect, in some implementations, the EGR1 + Neutrophil inhibitors inhibit EGR1 + Neutrophil subset formation or inhibition of EGR1 + Substances that contribute to neutrophil function.

[0010] In conjunction with the first aspect, in some implementations, the EGR1 + The neutrophil inhibitor is one or more of the following: an FPR2 antagonist, a PPARγ agonist, an EGR1 inhibitor, and a PI3K inhibitor. Further, the FPR2 antagonist is WRW4 or a pharmaceutically acceptable derivative thereof; and / or, The PPARγ agonist is rosiglitazone or a pharmaceutically acceptable derivative thereof; and / or The EGR1 inhibitor is a substance that inhibits EGR1 expression or activity; and / or, The PI3K inhibitor is Wortmannin or a pharmaceutically acceptable derivative thereof.

[0011] In conjunction with the first aspect, in some embodiments, the high expression of KIAA1199 is manifested as a significant increase in the level of KIAA1199 protein or mRNA in lesions compared to healthy individuals.

[0012] In conjunction with the first aspect, in some embodiments, the disease driven by high expression of KIAA1199 is colorectal cancer liver metastasis. Further, the physiological characteristics of colorectal cancer liver metastasis include one or more of the following: 1) Changes in the liver microenvironment; 2) Patient survival time is significantly shortened; 3) Progressively increasing liver metastatic burden; 4) Significant pathological angiogenesis.

[0013] In a second aspect, the present invention provides a pharmaceutical composition for preventing and treating liver metastases of colorectal cancer.

[0014] In conjunction with the second aspect, in some embodiments, the pharmaceutical composition comprises at least two EGR1s selected from the group consisting of: + Neutrophil inhibitors: FPR2 antagonists, PPARγ agonists, EGR1 inhibitors, PI3K inhibitors.

[0015] Furthermore, in some embodiments, the FPR2 antagonist is WRW4 or a pharmaceutically acceptable derivative thereof; and / or, The PPARγ agonist is rosiglitazone or a pharmaceutically acceptable derivative thereof; and / or The EGR1 inhibitor is a substance that inhibits EGR1 expression or activity; and / or, The PI3K inhibitor is Wortmannin or a pharmaceutically acceptable derivative thereof.

[0016] In conjunction with the first or second aspect, in some embodiments, the drug or drug composition further comprises a pharmaceutically acceptable carrier and is formulated as an injection, tablet or capsule.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: 1. A novel, functionally specialized EGR1-positive neutrophil was identified. +The study identified the Neutrophil subpopulation and confirmed it as a core effector cell driving angiogenesis in the pre-metastatic liver microenvironment. Simultaneously, it identified FPR2 and hepatocyte PPARγ as two highly effective targets for intervention along this axis. The study demonstrated that clinically used PPARγ agonists (rosiglitazone) and known FPR2 antagonists (WRW4) and PI3K inhibitors (Wortmannin) can effectively block this signaling axis and inhibit liver metastasis. This significantly shortens the drug development cycle and reduces development risks.

[0018] 2. For the first time, a complete and continuous signal regulation axis (KIAA1199-PPARγ / SAA2-FPR2-EGR1) from the primary tumor to the liver microenvironment was revealed, systematically elucidating the remote regulation mechanism of colorectal cancer liver metastasis. This provides a solid theoretical basis for clinical multi-node intervention strategies, increases the success rate of treatment and the flexibility in dealing with drug resistance. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 EGR1 provided in Example 1 + The results of the pre-metastatic microenvironment formation characterized by neutrophil subset infiltration are shown in the following figures: A is a schematic diagram of the pre-metastatic microenvironment study; B is a t-SNE diagram and proportion of liver immune cells in the vector group and the KIAA1199 overexpression group; C is single-cell RNA sequencing cluster analysis of liver neutrophil subsets (Neu-C0 to Neu-C4); D is pseudo-time trajectory analysis of neutrophil subsets; E is a heatmap of gene set enrichment analysis (GSVA) ​​along the pseudo-time trajectory; F is a Western blot analysis of EGR1 protein expression in mouse liver neutrophils; G is the flow cytometry quantification of intrahepatic EGR1. + Neutrophil percentage; H represents EGR1 in liver tissue sections. + Multiplex immunofluorescence staining of neutrophils; I shows t-SNE projections of liver neutrophil subsets under different conditions in an external dataset, with the first two images showing liver neutrophil status from patients with low and high KIAA1199 expression, respectively, and the following two images corresponding to tissue samples from adjacent liver tissue and liver metastases, respectively; J shows EGR1 in liver metastases of CRLM patients. +Spatial distribution and proximity analysis of neutrophils and angiogenesis regions; K represents EGR1 in mouse and human tumor tissues. + H&E staining and multiplex immunofluorescence images of neutrophils; L represents the effect of EGR1-overexpressing neutrophil conditioned medium on endothelial tubular formation; M represents EGR1. + A schematic diagram illustrating how neutrophils promote angiogenesis in the liver microenvironment.

[0021] Figure 2 The functionally reprogrammed hepatocytes provided in Example 2 coordinate EGR1 via the SAA2-FPR2 axis. + Localization of neutrophils at the tumor-liver interface. EGR1 is shown in different tissues. + Flow cytometry analysis of neutrophils; B represents intrahepatic EGR1 at different time points after orthotopic transplantation. + Dynamic changes in neutrophil proportion; C is the UMAP map of hepatocyte subsets in the vector and KIAA1199 overexpression groups; D is the functional enrichment pattern of hepatocyte subsets along pseudo-time trajectory; E is the spatial localization of functionally reprogrammed hepatocytes (Hepa-C11) and epithelial-mesenchymal transition (EMT) regions in CRLM samples; F is the SAA2 and EGR1 in mouse liver tissue. + Multiplex immunofluorescence colocalization of neutrophils; G represents EGR1 in the mouse CRLM model. + Spatial colocalization of neutrophils, SAA2, and the vascular system; H is a schematic diagram of the in vivo pharmacological intervention experimental protocol; I is a Western blot analysis of EGR1 protein expression in liver neutrophils of mice in different treatment groups; J is the intrahepatic EGR1 in each group. + Flow cytometry quantification of neutrophil proportion; K represents the experimental timeline of WRW4 intervention in vivo; L represents gross images of the liver of mice in each group and H&E staining; MN represents the quantitative analysis of the percentage of liver metastasis area and the number of metastatic nodules; O represents the Kaplan-Meier survival curves of mice in each group.

[0022] Figure 3The functionally reprogrammed hepatocytes provided in Example 3 promoted SAA2 expression through PPARγ-mediated metabolic reprogramming. A shows the gene set enrichment analysis (GSEA) of downregulated PPAR signaling pathway genes in the target group; B shows the expression heatmap of PPAR pathway-related genes in hepatocyte subsets; C shows the metabolic pathway activity heatmap of hepatocyte subsets; D shows the Western blot analysis of PPAR isotype protein expression in hepatocytes after co-culturing with different CRC cells; E shows the effect of rosiglitazone (ROG) treatment on PPARγ and SAA2 protein levels in mouse liver tissue; F shows the spatial transcriptome map of SAA2 and PPARγ gene expression in CRLM samples; G shows the predicted PPARγ binding motifs (P1 and P2) in the mouse SAA2 promoter region; H shows the detection of wild-type and mutant SAA2 promoter reporter gene activity; I shows the ChIP-qPCR verification of PPARγ enrichment in the hepatocyte SAA2 promoter region; J is a schematic diagram of the in vitro co-culture system experiment; K shows the ELISA detection of SAA2 secretion levels in the co-culture system. LM is a flow cytometry assay used to detect the effects of different treatments on EGR1. + The effect of neutrophil ratio; N is a schematic diagram of the in vivo rosiglitazone treatment experimental protocol; O is a gross image of the liver of mice in each group and H&E staining; PR is a quantitative analysis of the percentage of liver metastasis area, the number of metastatic nodules and liver weight; S is the Kaplan-Meier survival curve of mice in each group.

[0023] Figure 4 EGR1 from liver source provided in Example 4 + Neutrophils prolong their lifespan and secrete VEGFA through the PI3K / AKT / EGR1 signaling circuit, thereby promoting metastasis. Here, A represents EGR1. + Schematic diagram of the neutrophil adoptive transfer model experiment; B represents the control group and EGR1. + Representative liver images and H&E staining of mice in the neutrophil co-injection group; CD represents quantitative analysis of the number and area of ​​metastatic liver nodules; E represents Kaplan-Meier survival curves of the two groups of mice; F represents EGR1. + Gene set enrichment analysis of neutrophils (GSEA); G represents Western blot analysis and quantitative heatmap of phosphorylation levels of PI3K-AKT pathway proteins in neutrophils in a triple co-culture system; H represents EGR1 in in vitro time-course experiments. +Dynamic changes in neutrophil proportion; I represents the quantitative proportion of dead neutrophils at different time points; J represents the flow cytometry quantitative proportion of Ki67-positive cells in neutrophils; KL represents the ELISA analysis of VEGFA protein levels in the co-culture supernatant; M represents the Western blot analysis and quantitative heatmap of VEGFA protein expression in neutrophils under different treatments; N represents the ELISA analysis of VEGFA secretion levels in the cell culture supernatant; O represents the effect of Wortmannin treatment on p-AKT and EGR1 protein expression in dHL-60 cells; P represents the ELISA analysis of VEGFA levels in the cell supernatant under different treatments; Q represents the effect of Western blot analysis on cleaved Caspase-3 protein expression; R represents the flow cytometry quantitative proportion of dead neutrophils under different treatments.

[0024] Figure 5 EGR1 provided for Example 5 + Neutrophil-driven angiogenesis accelerates KIAA1199-mediated liver metastasis in colorectal cancer. In this study, A represents co-injection of EGR1. + The effect of neutrophils on the growth of subcutaneous tumors in mice (tumor volume curve and representative endpoint images); B represents CD31 in tumor tissue. + Immunofluorescence staining and quantitative analysis of blood vessel density; C represents EGR1 in mouse liver tissue. + Neutrophils (LY6G) + EGR1 + ) and blood vessels (CD31) + Multiple immunohistochemical staining and colocalization analysis; D represents EGR1 in mouse CRLM samples. + Spatial relationship between neutrophils and the vascular system (H&E and multiplex immunofluorescence); E represents EGR1 in human CRLM samples. + Colocalization of neutrophils and the vascular system; F and I represent the effects of different conditioned media on the survival rate of SVEC4-10 cells; G and K represent the effects of different conditioned media on endothelial cell migration; G and J represent scratch assays, and H and K represent Transwell assays; LN represents the effects of different conditioned media on the tube-forming ability of endothelial cells (representative images and the number of vascular segments and quantification of branching points); M represents the quantitative analysis of the relative number of vessels per field of view among the treatment groups; N represents the quantitative analysis of the number of junctions (vascular branching intersections) formed in each group. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0026] This invention, based on systematic mechanistic research, comprehensively utilizes single-cell RNA sequencing, spatial transcriptomics, multiplex immunofluorescence, proteomics, metabolomics, and in vitro and in vivo functional validation techniques to elucidate for the first time a complete "KIAA1199-PPARγ / SAA2-FPR2-EGR1" signaling axis driving colorectal cancer liver metastasis. The inventors discovered that colorectal cancer cells with high KIAA1199 expression remotely inhibit hepatocyte PPARγ signaling, leading to metabolic reprogramming and excessive secretion of SAA2. Hepatocyte-derived SAA2 activates the FPR2 receptor on the surface of neutrophils, inducing their differentiation into EGR1, which has pro-angiogenic functions. + Neutrophil subsets, in turn, promote angiogenesis through the PI3K / AKT pathway, ultimately driving liver metastasis.

[0027] This invention provides EGR1 + Application of neutrophil inhibitors in the preparation of drugs for the prevention or treatment of diseases driven by KIAA1199 high expression.

[0028] In some embodiments, the EGR1 + Neutrophil inhibitors inhibit EGR1 + Neutrophil subset formation or inhibition of EGR1 + Substances that contribute to neutrophil function.

[0029] In some embodiments, the EGR1 + The neutrophil inhibitor is one or more of the following: an FPR2 antagonist, a PPARγ agonist, an EGR1 inhibitor, and a PI3K inhibitor. Further, the FPR2 antagonist is WRW4 or a pharmaceutically acceptable derivative thereof; and / or, The PPARγ agonist is rosiglitazone or a pharmaceutically acceptable derivative thereof; and / or The EGR1 inhibitor is a substance that inhibits EGR1 expression or activity; and / or, The PI3K inhibitor is Wortmannin or a pharmaceutically acceptable derivative thereof.

[0030] In Example 2, EGR1 in the liver of mice in the WRW4 treatment group + The proportion of neutrophils was significantly reduced ( Figure 2 J), EGR1 protein levels decreased (2I), WRW4, as an FPR2 antagonist, blocked SAA2-FPR2 binding, preventing neutrophils from converting to EGR1 at the source. + Phenotypic differentiation significantly reduces liver transplant burden. Figure 2 LN), and significantly prolonged the overall survival of mice (20); In Example 3, rosiglitazone, as a PPARγ agonist, significantly reduced SAA2 secretion ( Figure 3 K), and reduced EGR1 + The proportion of neutrophils ( Figure 3 LM). In animal models, rosiglitazone treatment also significantly reduced KIAA1199-driven liver metastasis burden in mice (LM). Figure 3 OR), and prolonged the overall survival of tumor-bearing mice ( Figure 3 S); In Example 4, Wortmannin inhibited the PI3K-AKT pathway while simultaneously reducing EGR1 protein expression (4O) and increasing EGR1. + The number of neutrophil apoptosis was also affected; in addition, Wortmannin significantly reduced EGR1. + Angiogenic activity of neutrophil conditioned medium ( Figure 4 P); In Example 5, the use of WRW4 and rosiglitazone effectively weakened EGR1. + The pro-angiogenic function of neutrophils; The above examples demonstrate targeted inhibition of EGR1 + FPR2 antagonists, PPARγ agonists, EGR1 inhibitors, and PI3K inhibitors, which affect neutrophil production or function, all have potential applications in treating liver metastases from colorectal cancer.

[0031] In some embodiments, high KIAA1199 expression is manifested as a significantly higher level of KIAA1199 protein or mRNA in the lesion compared to healthy individuals. The disease driven by this high KIAA1199 expression is colorectal cancer liver metastasis. Further, the physiological characteristics of this colorectal cancer liver metastasis include one or more of the following: (1) Changes in the liver microenvironment: like Figure 1 t-SNE analysis of BC showed that KIAA1199 overexpression significantly altered the composition of hepatic immune cells, inducing the formation of EGR1-based immune cells. + A microenvironment characterized by specific enrichment of the neutrophil subset (Neu-C2); Figure 2 FG's multiplex immunofluorescence further revealed that EGR1 + Neutrophils and SAA2+ Hepatocytes exhibit spatial co-localization at the tumor-liver interface, indicating that EGR1... + Neutrophil infiltration led to a significant reprogramming of the liver microenvironment.

[0032] (2) Patient survival time is significantly shortened: Figure 2 O and Figure 3 Survival analysis of S confirmed that inhibition of EGR1 through pharmacological intervention (WRW4 or rosiglitazone) + Neutrophil activity significantly prolonged the survival of tumor-bearing mice, indicating that EGR1 + The presence of neutrophils is directly associated with poor prognosis.

[0033] (3) Progressively increasing liver metastatic burden: Quantitative analyses in Examples 2 and 3 showed that the liver weight, number of liver metastatic nodules, and percentage of metastatic area were significantly increased in mice with high KIAA1199 expression. Intervention with FPR2 antagonists or PPARγ agonists significantly reduced the liver metastatic burden, indicating that EGR1... + Neutrophils drive the progressive aggravation of metastasis. Figure 2 LN and Figure 3 OR); (4) Significant pathological angiogenesis: like Figure 5 Immunofluorescence quantitative analysis of B showed that it was related to EGR1. + CD31 in tumor tissue co-injected with neutrophils + Significantly increased vascular density; Figure 5 In vitro angiogenesis experiments of LN showed that EGR1 + Neutrophil conditioned medium significantly enhances endothelial cell lumen formation ability; when upstream signaling is intervened using the PPARγ agonist ROG or the FPR2 antagonist WRW4, EGR1... + The angiogenesis-promoting ability of neutrophil conditioned medium was significantly weakened, specifically, its promoting effect on endothelial cell angiogenesis was significantly reversed. Figure 5 LN). This indicates that EGR1 + Neutrophils are a key driver of pathological angiogenesis.

[0034] This invention provides a pharmaceutical composition for preventing and treating liver metastases from colorectal cancer, the pharmaceutical composition comprising at least two EGR1s selected from the group consisting of: + Neutrophil inhibitors: FPR2 antagonists, PPARγ agonists, EGR1 inhibitors, PI3K inhibitors.

[0035] The aforementioned FPR2 antagonists are WRW4 or pharmaceutically acceptable derivatives thereof; the aforementioned PPARγ agonists are rosiglitazone or pharmaceutically acceptable derivatives thereof; the aforementioned EGR1 inhibitors are substances that inhibit EGR1 expression or activity, including shRNA, siRNA, or neutralizing antibodies against the EGR1 protein; the aforementioned PI3K inhibitors are Wortmannin or pharmaceutically acceptable derivatives thereof. The combined use of these drugs can increase treatment success rates and flexibility in managing drug resistance.

[0036] In some embodiments, the above-mentioned drugs or drug combinations further comprise pharmaceutically acceptable carriers, and are formulated as injections, tablets, or capsules. WRW4 was administered to mice via intraperitoneal injection in Example 3 at an effective dose of 2 mg / kg, using a PBS solution containing 1% DMSO as the solvent; in Example 5, it was administered via in vitro cell administration as a suspension drip at a concentration of 10 μM. ROG was administered to mice via intraperitoneal injection in Example 4 at an effective dose of 10 μg / kg, using a PBS solution containing 1% DMSO as the solvent; in Example 4, it was administered via in vitro cell administration as a suspension drip at a concentration of 10 μM (dissolved in culture medium ≤0.1% DMSO); Wortmannin was administered via in vitro cell administration as a suspension drip at a concentration of 100 nM in Example 4.

[0037] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0038] Unless otherwise specified, the experimental procedures described in the following examples are all conventional techniques in the art, including but not limited to cell culture and transfection, total mRNA and total protein extraction, protein sample preparation, molecular biological detection (such as ELISA, Western blot, immunofluorescence / immunohistochemistry), cell function experiments (such as cell proliferation and migration), H&E staining, animal model construction, data collection, bioinformatics analysis, and statistical methods. All experimental reagents used were purchased commercially and met general quality standards in the art. Experimental animals were purchased commercially and then fed and modeled according to standard operating procedures in the art. The implementation methods of the above-mentioned conventional techniques are common knowledge to those skilled in the art, and specific operations can be referred to relevant authoritative literature. Experimental and control groups were set up according to the grouping methods commonly used in the art.

[0039] The mice used in the following examples were all from Liaoning Changsheng Biotechnology Co., Ltd., and were housed in an SPF-grade environment. All human studies were conducted in accordance with the protocols approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Approval No.: UHCT20210271-01), and all animal experiments were approved by the Ethics Committee of the Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Approval No.: 202410259).

[0040] Table 1 Information on Cell Line Materials

[0041] Table 2 Animal Material Information Sheet

[0042] Table 3. Information on Main Reagents

[0043] All statistical analyses in this invention were performed using R software (version 4.2.2) and GraphPad Prism (version 9.0). All experiments included at least three independent biological replicates. Continuous data are expressed as mean ± standard deviation unless otherwise specified. Statistical significance was defined as P < 0.05, and the following notation was used: P* < 0.05, P** < 0.01, P*** < 0.001. The technical solution provided by this invention will be described in detail below with reference to embodiments.

[0044] Example 1: EGR1 + Identification and functional characterization of neutrophils in the KIAA1199-driven prehepatic metastatic microenvironment 1. Purpose In the liver microenvironment remodeled by KIAA1199, key pro-metastatic immune cell subsets were identified and their characteristics were analyzed.

[0045] 2. Experimental Methods: 2.1 Establishment of animal models Six-week-old C57BL / 6 or C57 mice were used to establish an orthotopic colorectal cancer model by injecting stably KIAA1199-overexpressing colon cancer cells (MC38 or CT26 cells) or control vector colon cancer cells into the submucosa of the cecum. On day 14 post-inoculation, before any visible liver metastases appeared, the mice were sacrificed, and liver tissue was obtained.

[0046] 2.2 Single-cell RNA sequencing (scRNA-seq) Liver cells from mice on day 14 were collected and single-cell suspensions were prepared. scRNA-seq was performed using the BD Rhapsody platform. Quality control, standardization, dimensionality reduction (UMAP / t-SNE), and cluster analysis were performed using Seurat software.

[0047] 2.3 Bioinformatics Analysis We used Monocle for pseudo-time trajectory analysis to infer cell differentiation status; we used Gene Set Enrichment Analysis (GSEA) to identify the function of differentially expressed genes; and we used pySCENIC to infer transcription factor regulatory networks.

[0048] 2.4 Experimental Verification EGR1 was confirmed at the protein level using flow cytometry and multiplex immunofluorescence staining of liver tissue. + The presence, proportion, and spatial location of neutrophils.

[0049] 2.5 Validation of in vitro angiogenesis function: Induced HL 60 cells were used to obtain neutrophil-like dHL 60 cells; EGR1-overexpressing dHL was constructed via lentiviral transfection. The 60 cell lines and control cell lines were cultured for 24 hours, and the conditioned medium was collected. After adding the human umbilical vein endothelial cell (HUVECs) culture system, scratch assay and Matrigel lumen formation assay were performed.

[0050] 3. Experimental Results like Figure 1 As shown in B-1M, scRNA-seq successfully identified five neutrophil subsets, among which the Neu-C2 cluster was specifically enriched in the KIAA1199 high-expression group. Figure 2 C). The characteristics of this subgroup are: 1) Transcriptional characteristics: High expression of EGR1 ( Figure 1 F), is in the terminal differentiation state ( Figure 2 D), and the gene set is enriched in pathways such as cell adhesion and angiogenesis. Figure 2 E).

[0051] 2) Spatial characteristics: Flow cytometry ( Figure 2 G,2I) and immunofluorescence (G,2I) Figure 2 H,2J-K) confirmed that this cell group had a significantly increased proportion in the liver and was located in the peritumoral region.

[0052] 3) Functional characteristics: When conditioned medium overexpressing EGR1 in dHL-60 cells was added to the human umbilical vein endothelial cell culture system, compared with the control group, the endothelial cells formed more and more complex tubular structures. Figure 2 L).

[0053] Figure 1 A is a schematic diagram of the pre-transfer microenvironment study experiment in this embodiment. Figure 1 M describes EGR1 + Neutrophils were enriched in the pre-metastatic liver microenvironment induced by CRC-induced KIAA1199 high expression and supported the overall model of metastatic colonization by promoting angiogenesis.

[0054] 4. Conclusion: This embodiment discovered and identified a novel EGR1-positive neutrophil subset with pro-angiogenic potential, laying the foundation for further in-depth research on its induction mechanism and functional execution mechanism.

[0055] Example 2: Hepatocytes regulate EGR1 via the SAA2-FPR2 axis + Mechanisms of neutrophils 1. Purpose This embodiment aims to elucidate the intercellular communication mechanism between hepatocytes and neutrophils in the pre-metastatic liver microenvironment induced by KIAA1199-overexpressing colorectal cancer.

[0056] 2. Experimental Methods "In situ-splenic dual tumor model": CT26 cells or MC38 cells stably overexpressing KIAA1199 or empty vector control were orally transplanted into the submucosal cecum of mice. On day 7, wild-type CT26 cells were injected intrasplenically, and splenectomy was performed 15 minutes later.

[0057] Histological analysis: Mice were sacrificed at specified time points, and liver tissue was collected. Some tissues were digested with collagenase IV to prepare single-cell suspensions for flow cytometry analysis; other tissues were embedded in OCT and then frozen sections for immunofluorescence staining.

[0058] Flow cytometry: Liver single-cell suspensions were stained with anti-LY6G and anti-EGR1 antibodies, and LY6G was detected by flow cytometry. + EGR1 + Cells (i.e., EGR1) + The proportion of neutrophils.

[0059] Immunofluorescence / immunohistochemistry: Multiplex fluorescence staining of frozen liver sections with LY6G, EGR1, and SAA2 was performed. EGR1 was observed and analyzed using confocal microscopy. + Spatial colocalization relationship between neutrophils and SAA2 signaling.

[0060] In vivo intervention experiment: The experimental procedure is as follows Figure 2 As shown in H, based on the dual tumor model, starting from day 6, mice were injected intraperitoneally daily with either the FPR2 antagonist WRW4 (2 mg / kg) or recombinant SAA2 protein (1 μg / kg) until the experimental endpoint (day 21) to assess liver metastasis burden (number of nodules, metastatic area) and mouse survival.

[0061] 3. Experimental Results 3.1 EGR1 + Neutrophils are specifically enriched in the liver like Figure 2As shown in Figure A, flow cytometry analysis revealed that in mice carrying KIAA1199-overexpressing orthotopic tumors, EGR1... + Neutrophils (LY6G) + EGR1 + The proportion of it in the liver is significantly higher than that in other tissues such as tumors, lymph nodes, blood, and spleen. Figure 2 Time-series analysis of B further revealed that the enrichment of this cell subset in the liver was a dynamic process that gradually increased with the progression of the primary tumor. These results indicate that EGR1 + The accumulation of neutrophils is liver-specific, suggesting that it may be induced by the local liver microenvironment.

[0062] 3.2 Identification of functionally reprogrammed hepatocyte subsets Single-cell RNA sequencing analysis of liver cells, such as Figure 2 The UMAP plot of C showed that hepatocytes could be divided into 13 subsets (Hepa-C0 to C12). Among them, the Hepa-C11 subset was specifically enriched in the KIAA1199 high-expression group ( Figure 2 Figure C (right). Pseudo-time trajectory enrichment analysis ( Figure 2 D) indicates that this subpopulation is significantly enriched in pathways related to tumor progression, such as angiogenesis and cell adhesion, and is therefore defined as "functionally reprogrammed hepatocytes".

[0063] 3.3 Functional reprogramming of hepatocytes and EGR1 + Neutrophil colocalization at the tumor-liver interface Spatial transcriptome analysis ( Figure 2 E) showed that in human CRLM samples, the inferred Hepa-C11 hepatocytes and the epithelial-mesenchymal transition (EMT) active region co-localized at the tumor-liver interface. Multiplex immunofluorescence staining ( Figure 2 In both mouse and human samples, FG clearly demonstrated that SAA2 protein signaling is related to EGR1. + Neutrophils are spatially close together, especially at the forefront of tumor invasion.

[0064] 3.4 The SAA2-FPR2 axis is the induced EGR1 + Key phenotypes In vivo functional experiments confirmed that exogenous administration of recombinant SAA2 protein significantly increased EGR1 in mouse liver. + The proportion of neutrophils, and treatment with the FPR2 antagonist WRW4 can effectively block this effect ( Figure 2 J). Western Blot analysis ( Figure 2I) WRW4 also showed that it reduced EGR1 protein levels in hepatic neutrophils. This suggests that SAA2-FPR2 binding is crucial for inducing EGR1. + Phenotype is both necessary and sufficient.

[0065] 3.5 Inhibition of the SAA2-FPR2 axis can significantly inhibit liver metastasis. like Figure 2 As shown in KO, in a dual-tumor model with high KIAA1199 expression, the number of liver metastatic nodules in mice treated with WRW4 ( Figure 2 N) and percentage of transfer area ( Figure 2 M) were significantly lower than in the control group. More importantly, WRW4 treatment significantly prolonged the overall survival of mice ( Figure 2 O).

[0066] 4. Conclusion This embodiment demonstrates through rigorous in vitro and in vivo experiments that: 1) EGR1 + Neutrophils are specifically enriched in the liver in the context of high KIAA1199 expression.

[0067] 2) A subset of hepatocytes (Hepa-C11) that is functionally reprogrammed by KIAA1199 exists in the liver, which is related to EGR1. + Neutrophils are spatially co-localized at the tumor-liver interface.

[0068] 3) Functional reprogramming of hepatocyte-derived SAA2, through binding to its receptor FPR2, induces neutrophils to transform into EGR1. + A key intercellular signaling axis that promotes metastatic phenotypic differentiation.

[0069] 4) Pharmacological inhibition of FPR2 (WRW4) can effectively block EGR1. + It promotes neutrophil production, significantly inhibits liver metastasis of colorectal cancer, and prolongs host survival.

[0070] 5. Summary The SAA2-FPR2 signaling axis is a core link connecting hepatocyte functional reprogramming and neutrophil phenotype activation, providing a new and effective target for the prevention and treatment of colorectal cancer liver metastases.

[0071] Example 3: Functionally reprogrammed hepatocytes promote SAA2 expression through PPARγ-mediated metabolic reprogramming. 1. Purpose This embodiment aims to reveal the precise molecular pathway by which KIAA1199-overexpressing colorectal cancer cells remotely regulate hepatocyte functional reprogramming.

[0072] 2. Experimental Methods Bioinformatics and Omics Analysis: 1) Gene set enrichment analysis Gene set enrichment analysis (GSEA) was performed on the transcriptome data of hepatocytes in the KIAA1199 co-culture system.

[0073] 2) Single-cell RNA sequencing data analysis Create a heatmap of normalized expression of key PPAR pathway-related genes in hepatocyte subsets (Hepa-C1 to Hepa-C12).

[0074] 3) Spatial transcriptome analysis Spatial transcriptome sequencing was performed on samples of colorectal cancer liver metastases (CRLM).

[0075] Molecular biology experiments: 1) Dual-luciferase reporter gene assay: The promoter region of the mouse SAA2 gene (containing the predicted PPARγ response element) was cloned into the pGL3 vector, and co-transfected with the PPARγ expression plasmid into AML12 cells to detect changes in luciferase activity.

[0076] 2) Chromatin immunoprecipitation (ChIP-qPCR): ChIP experiments were performed on AML12 cells (co-cultured with CRC cells) using anti-PPARγ antibody, and the enrichment of the SAA2 promoter region was detected by qPCR.

[0077] 3) Western blotting: a. In vitro co-culture model: AML12 hepatocytes were indirectly co-cultured with control cells or KIAA1199-overexpressing CT26 / MC38 colorectal cancer cells in a Transwell chamber for 48 hours. Hepatocytes were collected, and total protein, cytoplasmic protein, and nuclear protein were separated. The protein levels of PPARα, PPARβ / δ, and PPARγ were detected.

[0078] b. In vivo model: Liver tissue proteins were extracted from mice with orthotopic colorectal cancer (CT26 empty vector or KIAA1199 overexpression), and the protein levels of PPARγ and SAA2 were detected. Some mice were treated with rosiglitazone (10 μg / kg / day, intraperitoneal injection).

[0079] Cell biology experiments: 1) Triple co-cultivation system CT26 or MC38 colorectal cancer cells (empty vector control or KIAA1199 overexpression) were cultured in the lower chamber for 24 hours, followed by AML12 hepatocytes added to the upper chamber and cultured for another 24 hours. Then, bone marrow-derived neutrophils from Balb / c or C57 mice were added to the lower chamber with or without rosiglitazone (10 μM) and cultured for 24 hours.

[0080] 2) Flow cytometry EGR1 was identified and quantified using staining with anti-LY6G and anti-EGR1 antibodies. + The proportion of neutrophils.

[0081] In vivo animal experiments: Based on the dual-tumor model in Example 2, the PPARγ agonist rosiglitazone (10 μg / kg) was injected intraperitoneally daily starting on day 6 and continued until the experimental endpoint (day 21). Liver metastasis burden and survival were measured.

[0082] 3. Experimental Results 3.1 Functional reprogramming of hepatocytes showed inhibition of the PPARγ signaling pathway and significant metabolic reprogramming. like Figure 3 As shown in Figure A, gene set enrichment analysis (GSEA) revealed that the PPAR signaling pathway was significantly inhibited in hepatocytes co-cultured in the KIAA1199 system. Further heatmap analysis showed that the expression of PPARγ and its downstream target genes was downregulated in the functionally reprogrammed hepatocyte subset (Hepa-C11). Figure 3 B). Metabolomics analysis ( Figure 3 C) indicates that these cells underwent a significant shift from lipid synthesis / storage to malignantly related metabolic pathways such as glycolysis and glutamine breakdown, with PPARγ being the most significantly downregulated member of the PPAR family in functionally reprogrammed hepatocytes. Figure 3 D). Western blot confirmed that co-culturing with CRC cells overexpressing KIAA1199 reduced the level of PPARγ protein in the hepatocyte nucleus. Figure 3 D).

[0083] 3.2 The PPARγ agonist rosiglitazone can reverse KIAA1199-induced SAA2 upregulation In an in vitro co-culture system, the addition of the PPARγ agonist rosiglitazone (ROG) significantly inhibited the increase in SAA2 expression and secretion induced by KIAA1199. Figure 3 K). In vivo, rosiglitazone treatment also significantly reduced the protein level of SAA2 in the liver of KIAA1199-overexpressing tumor-bearing mice (K). Figure 3 E). Spatial transcriptomic analysis showed that in CRLM patient samples, regions with low PPARγ expression were associated with high SAA2 expression (E). Figure 3 F) further supports the negative correlation between the two.

[0084] 3.3 PPARγ directly binds to the SAA2 promoter and inhibits its transcription. Bioinformatics analysis identified two evolutionarily conserved PPARγ binding motifs (P1 and P2) within the -1000bp to +200bp region of the mouse SAA2 gene promoter region. Figure 3 G). Dual-luciferase reporter gene assays confirmed that overexpression of PPARγ inhibited the activity of the wild-type SAA2 promoter, while the mutant P2 site (SAA2-MT2) completely eliminated this inhibitory effect, indicating that the P2 site is crucial for the transcriptional repressive function of PPARγ. Figure 3 H). Chromatin immunoprecipitation (ChIP-qPCR) experiments further confirmed that PPARγ protein binds directly to the promoter region of the Saa2 gene in vivo. Figure 3 I).

[0085] 3.4 Activation of PPARγ can inhibit EGR1 + Neutrophil production and liver metastasis In an in vitro triple co-culture system (CRC cells-hepatocytes-neutrophils), rosiglitazone treatment significantly reduced SAA2 secretion. Figure 3 K), and reduced EGR1 + The proportion of neutrophils ( Figure 3 LM). In animal models, rosiglitazone treatment significantly reduced KIAA1199-driven liver metastasis burden in mice ( ). Figure 3 OR), and prolonged the overall survival of tumor-bearing mice ( Figure 3 S).

[0086] 4. Conclusion This implementation 4, through the aforementioned multi-level and multi-technical experimental methods, fully elucidates the upstream mechanism by which KIAA1199 regulates SAA2 expression in hepatocytes: Key molecular event: CRC cells “educate” hepatocytes via KIAA1199, the core feature of which is the significant inhibition of the PPARγ signaling pathway within the “educated” hepatocytes and the resulting metabolic reprogramming.

[0087] Direct transcriptional regulation: PPARγ, as a transcriptional repressor, directly binds to a specific site (P2) of the SAA2 gene promoter, thereby inhibiting its transcription. After PPARγ signaling is inhibited, the braking effect on SAA2 is released, leading to a significant upregulation of its expression and secretion.

[0088] Therapeutic validation: Pharmacological activation of PPARγ (using rosiglitazone) can effectively reverse the above process, inhibiting SAA2 production and EGR1. + It promotes neutrophil production and ultimately significantly inhibits liver metastasis of colorectal cancer.

[0089] 5. Summary The “KIAA1199-hepatocyte PPARγ inhibition-SAA2 transcriptional desuppression” is a complete and targetable upstream and downstream signaling axis, providing a solid theoretical basis and experimental support for preventing liver metastasis through metabolic normalization (such as the use of PPARγ agonists).

[0090] Example 4: EGR1 from the liver + Neutrophils promote metastasis through the PI3K / AKT / EGR1 pathway. 1. Purpose This embodiment aims to elucidate the specific molecular mechanism by which EGR1-positive neutrophils are activated internally to perform metastasis-promoting functions after receiving upstream SAA2-FPR2 signals.

[0091] 2. Experimental Methods 2.1 Neutrophil isolation and induction: Mouse bone marrow cells were collected, and neutrophils were separated using the MojoSort™ Mouse Neutrophil Isolation Kit. EGR1 was induced in vitro by stimulation with rSAA2 (20 μg / mL) for 12–16 hours. + Phenotype.

[0092] The grouping is as follows: Negative control group (NC): Neutrophils without any active stimulants added; Treatment group 1 (rSAA2-EGR1) - Neutrophils: Neutrophils that do not express or express low levels of the EGR1 transcription factor after stimulation with rSAA2; Treatment group 2 (rSAA2-EGR1) + Neu): After stimulation with rSAA2, EGR1 cells become positive neutrophils; 2.2 Adoption Transfer Model: Induced EGR1 + Neutrophils or control neutrophils were mixed with CT26 cells at a 1:1 ratio and injected intrasplenically into recipient mice (5 × 10⁻⁶ cells). 5 (Cells / type), splenectomy was performed 15 minutes later. Mice were sacrificed on day 14 for endpoint analysis. The experimental procedure is as follows: Figure 4 As shown in A: The grouping is as follows: Control group (Neu group): Control neutrophils (pretreated with PBS) + CT26 tumor cells → co-injected into mouse spleen.

[0093] Experimental group (EGR1) + Neu group): EGR1 + Neutrophils (pretreated with SAA2) + CT26 tumor cells were co-injected into the spleen of mice.

[0094] Observation indicators: number of liver metastatic nodules, metastatic area, and mouse survival time.

[0095] 2.3 Triple Co-culture: Similar to Example 2, neutrophils or supernatant were collected after co-culture; the VEGFA protein content in the neutrophil culture supernatant was detected by ELISA, and the PI3K-AKT pathway protein level in neutrophils in the triple co-culture system was detected by Western blot.

[0096] 2.4 Cell survival and apoptosis: The apoptosis of neutrophils under different treatment conditions was detected by flow cytometry (Annexin V / PI staining) and Western blotting (cleaved Caspase-3).

[0097] 2.5 Pharmacological inhibition experiments: In in vitro culture or in vivo experiments, the effects of adding the PI3K inhibitor Wortmannin (100 nM) or the FPR2 receptor antagonist WRW4 (10 μM) on reversing the above phenotypes were observed. Grouping details are as follows: Positive control group: Neutrophils + SAA2; FPR2 receptor antagonist group: neutrophils + SAA2 + WRW4; PI3K-AKT pathway inhibitor group: neutrophils + SAA2 + Wortmannin.

[0098] 2.6 Gene set enrichment analysis: Transcriptome data from Egr1+ neutrophils were analyzed using GSEA, following the same method as in step 3.

[0099] 3. Experimental Results 3.1 EGR1 + Neutrophils have a strong ability to promote liver metastasis in vivo. To directly verify EGR1 + The function of neutrophils was investigated through adoptive transfer experiments in this embodiment. Figure 4 As shown in Figure A, EGR1 pretreated with SAA2 + Neutrophils and CT26 cancer cells were co-injected into the spleen of mice. The results showed that, compared with the control group, EGR1... + Neutrophils can significantly accelerate the formation of liver metastases, as evidenced by the increased number of metastatic liver nodules. Figure 4 C) and percentage of transferred area ( Figure 4 A significant increase in D) led to a significant shortening of mouse lifespan ( Figure 4 E).

[0100] 3.2 EGR1 + The PI3K-AKT signaling pathway in neutrophils is specifically activated. To elucidate its internal activation mechanism, gene set enrichment analysis (GSEA) was performed in this embodiment. The results showed that, compared with the control group, EGR1... + The PI3K-AKT signaling pathway is significantly activated in neutrophils. Figure 4 F). Western blot analysis further confirmed the effectiveness of the in vitro co-culture system ( Figure 4 G) and EGR1 in liver-infiltrating neutrophils isolated in vivo. + The population level of p-AKT (phosphorylated AKT) protein was significantly elevated, indicating that the PI3K-AKT pathway is the main downstream response pathway of SAA2-FPR2 signaling.

[0101] 3.3 PI3K-AKT pathway activation prolongs EGR1 + Neutrophil survival time Flow cytometry analysis showed that SAA2-induced EGR1 + During in vitro culture, the proportion of apoptotic cells in neutrophils was significantly lower than that in the control group over time. Figure 4 I). Western blot analysis revealed a corresponding decrease in the expression level of cleaved caspase-3, a key protein in apoptosis. Figure 4 Q). Treatment with the PI3K inhibitor Wortmannin effectively reversed this survival advantage and significantly increased EGR1. + The proportion of neutrophils undergoing apoptosis ( Figure 4 R). This indicates that activation of the PI3K-AKT pathway significantly prolongs EGR1 by inhibiting apoptosis. + The lifespan of neutrophils allows them to continue functioning in the microenvironment.

[0102] 3.4 PI3K-AKT-EGR1 axis drive EGR1 + Neutrophils secrete VEGFA to promote angiogenesis Mechanistically, the inventors discovered that SAA2 stimulation can induce increased expression and secretion of VEGFA in neutrophils, while inhibiting PI3K-AKT signaling or blocking FPR2 can effectively block this effect. Figure 4 Importantly, Western blot showed that Wortmannin, while inhibiting PI3K-AKT, also reduced EGR1 protein levels (KN). Figure 4The presence of O indicates that EGR1 is a downstream target of the PI3K-AKT pathway. Functionally, the use of Wortmannin significantly attenuates EGR1. + Angiogenic activity of neutrophil conditioned medium ( Figure 4 This indicates that the PI3K-AKT pathway, by upregulating EGR1 and driving VEGFA secretion, is a key mechanism for its pro-angiogenic function.

[0103] 4. Conclusion This embodiment systematically elucidates the function of EGR1 through functional acquisition, mechanism exploration, and pharmacological intervention experiments. + Downstream mechanisms of action of neutrophils: 1) Direct metastasis-promoting function: EGR1 + Neutrophils have a significantly enhanced ability to promote liver metastasis of colorectal cancer in vivo.

[0104] 2) Core signal path: Its function depends on the downstream PI3K-AKT signal path activated by the SAA2-FPR2 axis.

[0105] 3) Molecular basis of functional advantages: Activation of the PI3K-AKT pathway works through a dual mechanism: 4) Prolonged survival: By inhibiting caspase-3-mediated apoptosis, this cell subpopulation is given a survival advantage in the metastasis microenvironment.

[0106] 5) Enhanced secretion: By stabilizing or upregulating transcription factor EGR1, it drives the secretion of large amounts of the pro-angiogenic factor VEGFA, thereby efficiently promoting angiogenesis.

[0107] 6) Therapeutic potential: Pharmacological inhibition of the PI3K-AKT pathway can simultaneously eliminate EGR1 + Neutrophil survival advantage and angiogenesis function.

[0108] 5. Summary The PI3K-AKT-EGR1 signal axis is EGR1. + Neutrophils perform metastatic functions through a core downstream mechanism, and targeting this pathway (e.g., using PI3K inhibitors) is a potentially effective strategy to eliminate their metastatic activity.

[0109] Example 5: EGR1 + Neutrophil-driven angiogenesis accelerates KIAA1199-mediated liver metastasis in colorectal cancer. 1. Purpose This embodiment aims to provide conclusive evidence of the core function of EGR1-positive neutrophils in colorectal cancer liver metastases.

[0110] 2. Experimental Methods In vivo functional validation model (adoptive transfer): Same as Example 4. Observation indicators: tumor volume is measured regularly, and samples are taken at the endpoint. The tumor tissue is weighed, H&E stained, and CD31 immunofluorescence stained to quantify tumor vascular density.

[0111] In vitro angiogenesis function analysis: specifically including 1) Preparation of conditioned medium: Conditioned medium was collected from neutrophils or co-culture systems of different treatment groups (control, KIAA1199 overexpression, KIAA1199 overexpression + ROG / WRW4); 2) Endothelial cell function testing: Proliferation assay: CCK-8 assay was used to detect the survival / proliferation rate of endothelial cells under different culture conditions; Migration experiment: scratch healing experiment, measuring scratch closure rate; Angiogenesis experiment: Endothelial cells were seeded onto Matrigel, conditioned medium was added, and after incubation for 4-6 hours, the total length, branching points, and grid number of tubular structures were observed and quantified under a microscope.

[0112] Histological and spatial analysis: Frozen sections of mouse liver metastases or subcutaneous tumors were subjected to LY6G / EGR1 / CD31 multiplex immunofluorescence staining, and EGR1 was analyzed using confocal microscopy. + Neutrophils and CD31 + The spatial relationship of blood vessels.

[0113] 3. Experimental Results 3.1 EGR1 + Neutrophils significantly promote tumor growth and angiogenesis in vivo. To directly verify its functionality, an adoption transfer experiment was conducted in this embodiment. For example... Figure 5 As shown in A, EGR1 + Neutrophils (EGR1) + Neutrophils (neutrophils) or control neutrophils (Ctrl Neutrophils) were co-injected subcutaneously with CT26 tumor cells in mice. Results showed that, compared to the control group, CT26+EGR1... + The Neu group significantly promoted tumor growth, as evidenced by a significant increase in tumor volume and weight. More importantly, immunofluorescence staining showed that EGR1... + CD31 in tumor tissue from the co-injection group of neutrophils and CT26 tumor cells + The blood vessel density was significantly higher than that of the control group. Figure 5 B). This indicates that EGR1 + Neutrophils have a strong ability to promote tumor angiogenesis.

[0114] 3.2 EGR1+ Neutrophils co-localize with neovascularization within liver metastases. To system verify EGR1 + The inventors analyzed the spatial localization of neutrophils in tissue samples from different model systems using multiplex immunofluorescence staining techniques to investigate the distribution characteristics of neutrophils at the primary disease site and their association with angiogenesis. The results showed that in a mouse liver metastasis model induced by KIAA1199 high expression, EGR1... + Neutrophils (LY6G) + EGR1 + ) and CD31 + Vascular endothelial cells exhibit significant spatial colocalization characteristics in the tumor-liver junction area. Figure 5 C). Notably, this phenomenon exhibits a clear pathological tissue regional preference, with co-localization primarily concentrated in specific microenvironment niches at the tumor invasion front. This phenomenon is not limited to liver metastasis models; it also exists at the invasion front of primary colorectal cancer in mice and humans. Figure 5 DE). This confirms EGR1 from a pathological perspective. + Neutrophils are key participants in angiogenesis in the pre-metastatic microenvironment.

[0115] 3.3 EGR1 + Neutrophils induce full activation of endothelial cell function through secretion of factors. The inventors further verified its angiogenesis-promoting mechanism in vitro. For example... Figure 5 As shown in Figure FN, endothelial cells were treated with conditioned media from different sources. Functional experiments showed that, compared with the control group, cells from EGR1... + Conditioned culture medium for neutrophils can significantly enhance the proliferation of endothelial cells. Figure 5 FI), migration ( Figure 5 GK) and in vitro angiogenesis capacity ( Figure 5 LN). Among them, quantitative angiogenesis experiments showed that EGR1 + The total length and number of branch points of the tubular structures formed in the conditioned medium group were significantly increased. Figure 5 MN).

[0116] 3.4 EGR1 + The pro-angiogenic function of neutrophils depends on the upstream KIAA1199 / SAA2 signaling axis and can be pharmacologically inhibited. Crucially, the inventors verified the operability of this function. When upstream signaling was intervened using the PPARγ agonist ROG or the FPR2 antagonist WRW4, EGR1... +The angiogenesis-promoting ability of neutrophil conditioned medium was significantly weakened. Specifically, its promoting effect on endothelial cell angiogenesis was significantly reversed. Figure 5 LN). This indicates that targeting the KIAA1199 / SAA2 signal axis can effectively eliminate EGR1. + Pathological function of neutrophils.

[0117] 4. Conclusion This embodiment systematically confirms, through the integration of in vivo and in vitro functional experiments, that: 1) Direct metastasis-promoting function: EGR1 + Neutrophils have the function of directly promoting tumor angiogenesis and accelerating the progression of colorectal cancer in vivo.

[0118] 2) Core mechanism of action: It fully activates the function (proliferation and migration) of endothelial cells by secreting soluble factors (such as VEGFA), thereby driving angiogenesis.

[0119] 3) Signaling pathway dependence: This pro-angiogenic function is upstream of KIAA1199-PPARγ / SAA2-FPR2-EGR1 Downstream events activated by the signal axis.

[0120] 4) Therapeutic target potential: Pharmacological intervention of upstream signals (such as using ROG or WRW4) can effectively reverse EGR1. + The pro-angiogenic function of neutrophils suggests that this signaling axis and its effector cells are potential effective targets for the prevention and treatment of colorectal cancer liver metastases.

[0121] 5. Summary EGR1 + Neutrophils are key effector cells in the liver metastasis microenvironment driven by KIAA1199 high expression in colorectal cancer. Targeting these cells or their activation pathways provides a new strategy for inhibiting metastasis.

[0122] The above embodiments together constitute a complete innovative system from target discovery and mechanism elucidation to the development of treatment strategies, providing new targets and strategies for the prevention and treatment of colorectal cancer liver metastasis.

[0123] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. EGR1 + Application of neutrophil inhibitors in the preparation of drugs for the prevention or treatment of diseases driven by KIAA1199 high expression.

2. The application according to claim 1, characterized in that: The EGR1 + Neutrophil inhibitors inhibit EGR1 + Neutrophil subset formation or inhibition of EGR1 + Substances that contribute to neutrophil function.

3. The application according to claim 1, characterized in that: The EGR1 + Neutrophil inhibitors are one or more of the following: FPR2 antagonists, PPARγ agonists, EGR1 inhibitors, and PI3K inhibitors.

4. The application according to claim 3, characterized in that: The FPR2 antagonist is WRW4 or a pharmaceutically acceptable derivative thereof; and / or, The PPARγ agonist is rosiglitazone or a pharmaceutically acceptable derivative thereof; and / or The EGR1 inhibitor is a substance that inhibits EGR1 expression or activity; and / or, The PI3K inhibitor is Wortmannin or a pharmaceutically acceptable derivative thereof.

5. The application according to claim 1, characterized in that: The high expression of KIAA1199 is manifested by a significant increase in the level of KIAA1199 protein or mRNA in lesions compared to healthy individuals.

6. The application according to claim 1, characterized in that: The disease driven by the high expression of KIAA1199 is colorectal cancer with liver metastasis.

7. The application according to claim 6, characterized in that: The physiological characteristics of colorectal cancer liver metastasis include one or more of the following: 1) Changes in the liver microenvironment; 2) Patient survival time is significantly shortened; 3) Progressively increasing liver metastatic burden; 4) Significant pathological angiogenesis.

8. A pharmaceutical composition for preventing and treating liver metastasis of colorectal cancer, characterized in that: The pharmaceutical composition comprises at least two EGR1s selected from the group consisting of: + Neutrophil inhibitors: FPR2 antagonists, PPARγ agonists, EGR1 inhibitors, PI3K inhibitors.

9. The pharmaceutical composition for preventing and treating liver metastasis of colorectal cancer according to claim 8, characterized in that: The FPR2 antagonist is WRW4 or a pharmaceutically acceptable derivative thereof; and / or, The PPARγ agonist is rosiglitazone or a pharmaceutically acceptable derivative thereof; and / or The EGR1 inhibitor is a substance that inhibits EGR1 expression or activity; and / or, The PI3K inhibitor is Wortmannin or a pharmaceutically acceptable derivative thereof.

10. The application according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8 or 9, characterized in that: The drug or drug composition further comprises a pharmaceutically acceptable carrier and is formulated as an injection, tablet or capsule.

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