Application of EEPD1 inhibitor in improvement of colorectal cancer immunotherapy sensitivity

Abstract

The invention relates to application of an EEPD1 inhibitor in improvement of colorectal cancer immunotherapy sensitivity. The invention determines that the expression of EEPD1 in advanced colorectal cancer is increased, and the EEPD1 is closely related to immune tolerance and poor prognosis of colorectal cancer; proliferation and metastasis of colorectal cancer cells can be inhibited in vivo and in vitro through targeting EEPD1, the microenvironment of colorectal cancer immune tolerance is improved, more immune cells are activated to participate in anti-tumor immunity, and then the immunotherapy curative effect of the PD-1 antibody is improved. According to the invention, the understanding of the prior art on the PD-1 treatment drug resistance potential mechanism of the colorectal cancer patient is deepened, a theoretical basis is provided for optimizing the colorectal cancer immunotherapy strategy, and a new direction is provided for potential treatment target mining and drug research and development of the colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of EEPD1 inhibitors in improving the sensitivity of colorectal cancer to immunotherapy. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors in humans and the second leading cause of cancer-related deaths worldwide, posing a heavy socioeconomic burden and significant public health challenges. Current clinical treatments for colorectal cancer include local and systemic therapies. Local therapies include endoscopic resection, surgical treatment, and local radiotherapy, while systemic therapies include targeted therapy and immunotherapy.

[0003] For targeted therapy regimens, especially for patients who may require multiple targeted drugs, the cost-effectiveness balance is not ideal. Secondly, targeted therapy may cause additional adverse events, particularly in combination with other targeted drugs, with grade 3 or 4 adverse events occasionally observed. Due to cross-linking and compensatory mechanisms between tumor cell pathways, resistance to targeted drugs often develops, and acquired resistance further increases the complexity of the disease and the difficulty of treatment after progression. Furthermore, the efficacy of targeted therapy varies greatly from person to person, leading to a sharp increase in the costs associated with precisely matching targeted therapy to patients and monitoring follow-up. These issues may limit the clinical application of targeted drugs and therefore urgently need to be addressed.

[0004] The most widely used immunotherapy in colorectal cancer is immune checkpoint inhibitor therapy (ICIs). ICIs target immune checkpoint proteins that act as receptors or ligands, such as programmed cell death 1 (PD-1), PD-1 ligand 1 (PD-L1), and cytotoxic T-lymphocyte antigen 4 (CTLA-4), thereby relieving the inhibitory effect of tumor cells on the body's anti-tumor immune response and overcoming tumor immune tolerance. However, ICI therapy alone for colorectal cancer also has its limitations: such as low immunotherapy response rates due to insufficient tumor antigenicity or antigen presentation; tumor ICI therapy may induce the activation of abnormal tumor pathways, thereby resisting the anti-tumor immune response; in addition, the tumor immune tolerance microenvironment can also greatly weaken the efficacy of immune checkpoint therapy. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the targeted therapy and immunotherapy for colorectal cancer in the prior art, thereby providing the application of EEPD1 inhibitors in improving the sensitivity of colorectal cancer to immunotherapy. By using EEPD1-based targeted therapy combined with immune checkpoint inhibition, it is hoped that targeted therapy can improve the immunogenicity of tumor cells, control the activation of abnormal pathways, and improve the tumor immune microenvironment, thereby significantly improving the efficacy of colorectal cancer immunotherapy.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0007] The first aspect of this invention provides the use of EEPD1 inhibitors in the preparation of medicaments that enhance the sensitivity of PD-1 inhibitors to colorectal cancer treatment.

[0008] Preferably, the EEPD1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the EEPD1 gene.

[0009] Preferably, the EEPD1 inhibitor is selected from shRNA designed based on the EEPD1 gene.

[0010] Preferably, the sequence of the shRNA designed based on the EEPD1 gene is selected from one or more of SEQ ID NO: 1 (5'-TCGTGGAGTACCGAGAGTATA-3'), SEQ ID NO: 2 (5'-GACAACATCTGGATCAGTAAA-3'), SEQ ID NO: 3 (5'-GCATCGTGGAATACCGAGAAT-3'), and SEQ ID NO: 4 (5'-AGATTCAAGGTGGGAAGTAAT-3').

[0011] Preferably, the PD-1 inhibitor is selected from one or more of pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, camrelizumab, penaprilimab, cepalimumab, fenolinimab, and slulimab.

[0012] The second aspect of this invention provides the use of EEPD1 inhibitors and PD-1 inhibitors in the preparation of medicaments for treating colorectal cancer.

[0013] Preferably, the EEPD1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the EEPD1 gene.

[0014] Preferably, the EEPD1 inhibitor is selected from shRNA designed based on the EEPD1 gene.

[0015] Preferably, the sequence of the shRNA designed based on the EEPD1 gene is selected from one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0016] Preferably, the PD-1 inhibitor is selected from one or more of pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, camrelizumab, penaprilimab, cepalimumab, fenolinimab, and slulimab.

[0017] A third aspect of the present invention provides a pharmaceutical composition for treating colorectal cancer, comprising an EEPD1 inhibitor and a PD-1 inhibitor.

[0018] Preferably, the EEPD1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the EEPD1 gene.

[0019] Preferably, the EEPD1 inhibitor is selected from shRNA designed based on the EEPD1 gene.

[0020] Preferably, the sequence of the shRNA designed based on the EEPD1 gene is selected from one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0021] Preferably, the PD-1 inhibitor is selected from one or more of pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, camrelizumab, penaprilimab, cepalimumab, fenolinimab, and slulimab.

[0022] The fourth aspect of the present invention provides the use of a reagent for detecting EEPD1 expression levels in the preparation of products for evaluating the sensitivity of PD-1 inhibitors to colorectal cancer treatment.

[0023] Preferably, the reagent for detecting EEPD1 expression level includes primers for detecting EEPD1 gene expression level and / or reagents for detecting EEPD1 protein content.

[0024] Preferably, the primers for detecting EEPD1 gene expression levels are selected from any one of the following primer pairs:

[0025] Primer pair 1: Upstream sequence as shown in SEQ ID NO: 5 (5'-CTGGGTATGCAGGATTCCTATGG-3'), downstream sequence as shown in SEQ ID NO: 6 (5'-GGTCGTGACTTCCCACCTTG-3');

[0026] Primer pair 2: The upstream sequence is shown in SEQ ID NO: 7 (5'-GGCTGCCATCGCTCTATCC-3'), and the downstream sequence is shown in SEQ ID NO: 8 (5'-TAGCCGCTCCTGATTCACCA-3').

[0027] Preferably, the reagent for detecting EEPD1 protein content is selected from anti-EEPD1 antibodies; for example, Proteintech's 24310-1-AP can be selected.

[0028] Preferably, the PD-1 inhibitor is selected from one or more of pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, camrelizumab, penaprilimab, cepalimumab, fenolinimab, and slulimab.

[0029] The fifth aspect of this invention provides the use of EEPD1 inhibitors in the preparation of medicaments that improve the immune microenvironment in patients with colorectal cancer.

[0030] Preferably, the immune microenvironment includes, but is not limited to, CD8. + One or more of the following: T cell infiltration and activation, macrophage M1 polarization, macrophage M2 polarization, DC cell maturation, and MHC-I expression level.

[0031] Preferably, the EEPD1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the EEPD1 gene.

[0032] Preferably, the EEPD1 inhibitor is selected from shRNA designed based on the EEPD1 gene.

[0033] Preferably, the sequence of the shRNA designed based on the EEPD1 gene is selected from one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0034] The sixth aspect of this invention provides the use of EEPD1 inhibitors in the preparation of medicaments for treating colorectal cancer.

[0035] Preferably, the EEPD1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the EEPD1 gene.

[0036] Preferably, the EEPD1 inhibitor is selected from shRNA designed based on the EEPD1 gene.

[0037] Preferably, the sequence of the shRNA designed based on the EEPD1 gene is selected from one or more of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0038] It should be understood that, unless otherwise specified, in the context of this invention, the EEPD1 inhibitor refers to a substance capable of specifically downregulating the expression level of EEPD1 and / or the transcriptional level of its mature mRNA and / or the expression level or activity of the EEPD1 protein. For example, methods such as antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA erasers, target masking, and / or multi-target methods can be used to downregulate the expression level and / or activity of EEPD1; any method that can reduce the level and / or activity of EEPD1 is acceptable. The primers and / or primer pairs refer to PCR primers used to synthesize the EEPD1 gene cDNA strand in PCR, thereby detecting the expression level of the EEPD1 gene mRNA. In addition to the primers and / or primers listed in this invention, those skilled in the art are fully capable of designing corresponding primers, primer pairs, and antibodies based on the EEPD1 gene sequence using conventional methods and techniques in the field, including but not limited to molecular biology, and screening the designed primers and / or primer pairs through conventional experimental methods, or obtaining commercially available primers, as long as they can specifically detect the EEPD1 expression level; other conventional reagents and methods in the field can also be used to detect the EEPD1 protein expression level.

[0039] EEPD1 (exonuclease / endonuclease / phosphatase family domain 1) has the function of a 5' nuclease. It can form a protein complex with EXO / BLM to cleave the residual ends of damaged DNA, promote the recruitment of homologous recombination repair-related proteins at the site of DNA damage, thereby initiating homologous recombination repair with higher accuracy, improving DNA replication fork damage and fork arrest caused by replication stress, and maintaining the stability of the cell genome. It is known as the "gatekeeper" of DNA replication fork repair under stress.

[0040] Due to the high degree of tumor heterogeneity, previous reports have shown that the role of EEPD1 is not entirely consistent across different tumor types. In esophageal cancer, EEPD1 can induce radioresistance in esophageal cancer cells by activating the EEPD1 / ATR / CHK1 signaling axis. In RAD52-deficient BRCA1-mutated breast cancer and elderly patients with acute myeloid leukemia, EEPD1 is considered to act as a protective factor promoting synergistic lethality in breast cancer or as a marker of low-risk groups in leukemia. Furthermore, EEPD1 has also been reported as a major biomarker in prostate cancer. These findings indicate that EEPD1 plays an important role in tumorigenesis and development, but no studies have yet discovered how EEPD1 mediates the formation of an immunosuppressive microenvironment in colorectal cancer.

[0041] Following targeted cancer therapy, tumor cells can activate compensatory signaling pathways, bypassing the original target and its downstream pathways, leading to adaptive resistance to the targeted therapy. In immunotherapy, tumor cells can activate immunogenic downregulation pathways, inhibiting the activation of anti-tumor immune cells in the tumor immune microenvironment, resulting in poor response to tumor immunotherapy. To address cancer's adaptation to targeted therapy and resistance to immunotherapy, and based on the complementary mechanisms and synergistic potential of the two therapies, this invention explores effective combinations of targeted cancer therapy and immunotherapy. The aim is to identify specific target molecules in colorectal cancer and, by specifically regulating the expression of these molecules, to both inhibit tumor growth and improve the tumor immune tolerance microenvironment, thereby enhancing the efficacy of immunotherapy for colorectal cancer.

[0042] To address this, this invention utilizes multiple tumor and immune databases to screen and identify the EEPD1 gene, which is associated with colorectal cancer progression and the formation of an immune tolerance microenvironment. Furthermore, the correlation between EEPD1 expression and immune cell infiltration was validated in clinical colorectal cancer patient specimens, revealing that elevated EEPD1 expression in tumor cells of advanced colorectal cancer patients is closely related to an immunosuppressive microenvironment and poor patient prognosis. Further, this invention constructs a colorectal cancer cell line with silenced EEPD1 expression. Based on this, cell function experiments were conducted using mouse subcutaneous xenograft models, mouse intestinal orthotopic models, mouse liver metastasis models, and lung metastasis models to demonstrate the effect of silencing EEPD1 expression on tumor cell proliferation and metastasis. At the molecular level, through flow cytometry and RNA transcriptomics analysis of mouse subcutaneous xenografts formed from EEPD1-silenced CT26 and MC38 cells, and immunofunctional analysis of extracted mouse primary immune cells, it was clarified that inhibition of EEPD1 expression in colorectal cancer cells enhances anti-tumor immune responses by activating type I interferon responses and improving the tumor immune tolerance microenvironment. Finally, mouse subcutaneous xenograft models constructed from EEPD1-silenced CT26 and MC38 cells were treated with PD-1 neutralizing antibody to verify the role of targeting EEPD1 in improving the efficacy of PD-1 antibody immunotherapy for colorectal cancer.

[0043] In summary, this invention clarifies that EEPD1 expression is elevated in advanced colorectal cancer and is closely related to immune tolerance and poor prognosis in colorectal cancer. Targeting EEPD1 can inhibit the proliferation and metastasis of colorectal cancer cells in vitro and in vivo, improve the microenvironment of immune tolerance in colorectal cancer, and activate more immune cells to participate in anti-tumor immunity, thereby improving the efficacy of PD-1 antibody immunotherapy. This invention deepens the understanding of the potential mechanisms of PD-1 therapy resistance in colorectal cancer patients, provides a theoretical basis for optimizing colorectal cancer immunotherapy strategies, and offers new directions for the discovery of potential therapeutic targets and drug development in colorectal cancer. Attached Figure Description

[0044] Figure 1 This is a schematic diagram showing the results of immunofluorescence assays to detect the expression levels of CD8a and GzmB in tumor tissues of patients with early (stage I / II) and late (stage IV) COAD.

[0045] Figure 2 CD8 of COAD patients in the TCGA-COAD database + The relationship between T-cell infiltration and overall patient survival; *p<0.05, **p<0.01.

[0046] Figure 3 Immunohistochemical staining results of EEPD1 and CD8a in serial paraffin sections of advanced colorectal cancer samples and CD8a+ Schematic diagram of T cell infiltration ratio analysis results; *p<0.05, **p<0.01.

[0047] Figure 4 This diagram illustrates the differences in EEPD1 expression levels between cancerous and adjacent clinical samples from colorectal cancer patients; the left image shows RNA results, the middle image shows protein results, and the right image shows paraffin section results.

[0048] Figure 5 This is a schematic diagram illustrating the correlation between EEPD1 expression and survival prognosis in COAD patients in the GEPIA database.

[0049] Figure 6 This is a schematic diagram showing the results of analyzing the average expression level of EEPD1 in different stages of colorectal cancer in the GEPIA database.

[0050] Figure 7 This diagram illustrates the immunohistochemical staining results and H-score statistics of EEPD1 in paraffin sections from patients with early (stage I / II) and advanced (stage IV) colorectal cancer.

[0051] Figure 8 This is a schematic diagram showing the immunohistochemical staining results and Allred score statistics of EEPD1 in primary lesions, lymph node metastases, and liver metastases of colorectal cancer patients; *p<0.05, **p<0.01.

[0052] Figure 9 This is a schematic diagram showing the results of qPCR detection of EEPD1 mRNA levels in different cells; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0053] Figure 10 This is a schematic diagram showing the results of Western blot analysis of EEPD1 protein levels in different cells; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0054] Figure 11 Schematic diagram of the colony formation experiment results and statistical analysis results of HCT116, HT29, MC38 and CT26 cell lines with silenced EEPD1; *p<0.05, **p<0.01.

[0055] Figure 12 A schematic diagram showing the MTT assay results and statistical analysis results for HCT116, HT29, MC38 and CT26 cell lines with silenced EEPD1; *p<0.05, **p<0.01.

[0056] Figure 13Schematic diagram of in vivo imaging results, ex vivo imaging results, and HE staining results of paraffin-embedded sections of an in situ intestinal model of silencing EEPD1 HCT116-luci-hygro cells.

[0057] Figure 14 Schematic diagrams of in vivo imaging results, liver metastases, and HE staining results of paraffin-embedded liver sections in a liver metastasis model induced by intrasplenic injection of silencing EEPD1 HCT116-luci-hygro cells; and schematic diagrams of in vivo imaging results, lung metastases, and HE staining results of paraffin-embedded lung sections in a lung metastasis model induced by tail vein injection of silencing EEPD1 HCT116-luci-hygro cells.

[0058] Figure 15 A schematic diagram showing the statistical analysis results of silencing EEPD1 CT26 cells in subcutaneous xenograft models in Balb / c (nu / nu) and Balb / c, including tumor growth curves, tumor volume, tumor weight, and tumor weight ratio; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0059] Figure 16 Schematic diagram of statistical analysis results of EEPD1-silenced MC38 cells in subcutaneous xenograft models in Balb / c(nu / nu) and C57BL / 6, including tumor growth curves, tumor volume, tumor weight, and tumor weight ratio; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0060] Figure 17 The infiltration levels of various immune cell populations and CD8 in subcutaneous xenografts of CT26-shNC and CT26-shEEPD1. + A schematic diagram illustrating the statistical analysis results of T cell infiltration levels, as well as the infiltration levels of various immune cell populations and CD8 in subcutaneous xenografts of MC38-shNC and MC38-shEEPD1. + A schematic diagram of the statistical analysis results of T cell infiltration levels; *p<0.05, **p<0.01.

[0061] Figure 18 CD8 in subcutaneous xenografts of CT26-shNC and CT26-shEEPD1 + A schematic diagram showing the expression levels and statistical analysis results of GzmB and IFN-γ on T cells, as well as CD8 expression in subcutaneous xenografts of MC38-shNC and MC38-shEEPD1. + Schematic diagram of the expression levels of GzmB and IFN-γ in T cells and the results of statistical analysis; *p<0.05, **p<0.01.

[0062] Figure 19 M1 and M2 polarization levels and proportions of macrophages in CT26 and MC38 subcutaneous tumors with silenced EEPD1; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0063] Figure 20 The infiltration level and maturity ratio of DC cells in CT26 and MC38 subcutaneous tumors with silenced EEPD1; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0064] Figure 21 Schematic diagram of the results and statistical analysis of tumor growth curves, tumor weight, and tumor weight ratio in a mouse subcutaneous xenograft model induced by injection of silencing EEPD1 combined with IFNAR monoclonal antibody; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0065] Figure 22 CD8 + Flow cytometry was used to detect CD8+ after co-culturing T cells with DC cells and EEPD1-silenced MC38-OVA cells. + A schematic diagram of the amplification ratio of T; *p<0.05.

[0066] Figure 23 CD8 + Schematic diagram of the killing effect of T cells on EEPD1-silenced MC38-OVA cells; ***p<0.001, ****p<0.0001.

[0067] Figure 24 Schematic diagram of the phagocytic effect of BMDM on silencing EEPD1 MC38 and CT26 cells detected by flow cytometry; *p<0.05, ****p<0.0001.

[0068] Figure 25 A schematic diagram showing the statistical analysis results of the phagocytic effect of BMDM on EEPD1-silenced MC38 and CT26 cells and the phagocytic index of BMDM on EEPD1-silenced MC38 and CT26 cells; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0069] Figure 26 This is a schematic diagram showing the pattern of targeted EEPD1 combined with anti-PD-1 immunotherapy and the results of subcutaneous tumor growth in CT26 and MC38 mouse subcutaneous xenograft models.

[0070] Figure 27This is a schematic diagram showing the tumor growth curves, tumor weight, and tumor weight ratio statistical analysis results of targeted EEPD1 combined with anti-PD-1 immunotherapy in CT26 and MC38 mouse subcutaneous xenograft models; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0071] Figure 28 This is a schematic diagram showing the infiltration of major immune cells in CT26 and MC38 mouse subcutaneous xenograft models treated with EEPD1 combined with anti-PD-1 therapy by spectroscopic flow cytometry.

[0072] Figure 29 CD8 + T cells, GzmB + CD8 + T cells and IFN-γ + CD8 + The proportion of T cells infiltrating subcutaneous tumors in CT26 and MC38 mice treated with EEPD1 combined with anti-PD-1 therapy; *p<0.05, **p<0.01, ***p<0.001.

[0073] Figure 30 This study aims to develop subcutaneous xenograft models of CT26 and MC38 tumors treated with EEPD1 combined with CD8 monoclonal antibody.

[0074] Figure 31 A schematic diagram showing the growth curves, tumor weight, and statistical analysis results of CT26 and MC38 subcutaneous xenografts treated with EEPD1 combined with CD8 monoclonal antibody; *p<0.05, **p<0.01. Detailed Implementation

[0075] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0076] The cell lines listed in this invention, including MC38, CT26, HCT116, and HT29, were all procured through legitimate channels from the American Type Culture Collection (ATCC) or the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and cultured according to existing techniques. All cell lines were identified by short tandem repeat analysis at the Forensic Identification Center of Sun Yat-sen University, and the presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci). They were also cryopreserved in liquid nitrogen for subsequent experiments. All reagents used in this invention were commercially available. Informed consent was obtained from patients for all clinical specimens used, and the relevant procedures and methods complied with medical ethics requirements and Good Clinical Practice (GCP) guidelines for drug clinical trials. The experimental methods used in this invention, such as molecular biology experiments, cell experiments, animal experiments, and immunohistochemistry, are all conventional methods and techniques in the field. The expression level of EEPD1 in vitro and in vivo can be detected using conventional methods in the art (e.g., PCR, Western blot, etc.). This invention has verified the specificity of the primer pairs (primer pair 1-primer pair 2) and antibody 24310-1-AP (Proteintech) described in this invention for detecting EEPD1 expression levels through specific experiments. Regarding the inhibition of EEPD1, this invention has also verified the specific inhibition of EEPD1 expression levels using the designed shRNAs (#1-#4), all of which can specifically inhibit EEPD1 expression levels. Representative experimental results are selected and presented in this invention. However, given that the detection and inhibition of specific gene / protein expression levels are conventional methods in the art and not the main focus of this invention, some detection results are not specifically presented in this invention. Those skilled in the art can perform detection and verification according to the experimental methods described in this invention or other conventional methods in the prior art as needed. In addition to the primer pairs, antibodies, and shRNAs listed in this invention, those skilled in the art can also design their own primer pairs / antibodies and shRNAs based on the EEPD1 gene sequence and / or protein structure, or obtain commercially available reagents to detect or inhibit EEPD1 expression levels. Therefore, the specific information on primer pairs, antibodies, shRNAs, etc., listed in the context of this invention does not constitute a limitation on the actual scope of protection of this invention. Representative results from biological experimental replicates are presented in the accompanying figures, with data displayed as mean ± SD and mean ± SEM as specified in the figures. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. Data were analyzed using GraphPadPrism 8.0 software. Conventional medical statistical methods such as t-tests, chi-square tests, and analysis of variance were used to compare the differences in means between two or more groups. *p < 0.05 was considered a significant difference.

[0077] Example 1

[0078] Using the CIBRESORT computational method, and based on sequencing data from the TCGA colorectal adenocarcinoma (TCGA COAD) database and patient information, the relative abundance of immune cell infiltration in tumor tissues of stage I and IV colorectal adenocarcinoma patients was assessed and compared. Results showed that, compared to stage I patients, stage IV COAD patients had significantly higher levels of CD8+ in their tumor tissues. + T cells, activated CD4 + The infiltration abundance of immune cells with anti-tumor effects, such as memory T cells and M1 macrophages, was significantly reduced. Meanwhile, differential analysis of immune pathway-related genes showed that, compared to stage I patients, the expression levels of genes involved in anti-tumor immune signaling pathways, such as T cell activation, macrophage function, immune cell interaction, and antigen processing and presentation, were significantly downregulated in the tumor tissue of stage IV patients.

[0079] To validate these findings, pathological sections from patients with early-stage (stage I / II) and late-stage (stage IV) colorectal cancer were collected from the First Affiliated Hospital of Sun Yat-sen University. Immunofluorescence staining experiments with human CD8a and GzmB were performed, consistently revealing that CD8a... + The infiltration rate and activation level of T cells were significantly lower than in early-stage patients, indicating that advanced colorectal cancer is in a state of immune tolerance (see [link]). Figure 1 In addition, the overall survival and CD8 counts of patients in the TCGA-COAD database were compared. + Correlation analysis of T infiltration levels revealed low CD8 + T-cell infiltration levels are closely associated with poor prognosis in COAD patients (see [link]). Figure 2 The above data indicate that, compared with early-stage colorectal cancer, late-stage colorectal cancer lesions exhibit more significant immune tolerance characteristics.

[0080] To explore targets associated with the immunosuppressive microenvironment and poor prognosis in advanced colorectal cancer, differential analysis was used to screen 200 highly expressed genes in advanced colorectal cancer from the TCGA database as one gene set, and the GEPIA database was used to screen the 200 genes most highly associated with poor prognosis in colorectal cancer as another gene set. The intersection of the two gene sets yielded eight candidate molecules. To select target molecules related to immune tolerance, the correlation between these eight candidate molecules and immune cell infiltration was analyzed one by one in the TIMER database. The results showed that only EEPD1 expression was associated with CD8+ expression in colorectal cancer. + T cell infiltration was negatively correlated; further validation using the CIBRESORT algorithm showed that high expression of EEPD1 was associated with CD8+ in colon cancer. +Reduced T-cell infiltration was associated with this finding. Furthermore, the TIDE score was used to predict immunotherapy response in patients with high and low EEPD1 expression. The results showed that patients with high EEPD1 expression had lower immunotherapy response rates and higher TIDE scores, suggesting that EEPD1 expression is associated with anti-tumor immune tolerance. To verify the consistency between the EEPD1-related bioinformatics analysis results and clinical sample data, serial paraffin sections of advanced colorectal cancer samples obtained from the First Affiliated Hospital of Sun Yat-sen University were subjected to immunohistochemical staining for EEPD1 and CD8a. The samples were then divided into high-EEPD1 expression and low-EEPD1 expression groups based on the H-score, and the CD8a expression levels of the two groups were compared. + The proportion of T-cell infiltration was found to be low, with patients expressing high levels of EEPD1 having lower CD8 counts. + T-cell infiltration ratio suggests a lower level of antitumor immunity (see [link to relevant documentation]). Figure 3 ).

[0081] To further elucidate the potential and basis of EEPD1 as a therapeutic target in colorectal cancer, the expression level of EEPD1 in colorectal cancer lesions and normal tissues was analyzed using public databases. The results showed that EEPD1 was highly expressed in cancerous tissues, meeting the prerequisites for its use as a therapeutic target in colorectal cancer. Furthermore, RNA from paired cancerous and adjacent normal tissues obtained from the First Affiliated Hospital of Sun Yat-sen University was detected by fluorescent semi-quantitative PCR, and the corresponding proteins were detected by Western blotting. Immunohistochemical staining and Allred scoring of paraffin sections for EEPD1 were also performed. All results indicated that the mRNA and protein levels of EEPD1 in colorectal cancer were significantly higher than those in adjacent normal tissues (see [link to study]. Figure 4 Furthermore, a survival analysis of colorectal cancer patients using the GEPIA database related to EEPD1 showed that high expression of EEPD1 was associated with poor overall survival (OS) and disease-free survival (PFS), indicating a poor prognosis (see [link to study]). Figure 5 ).

[0082] Because patients with advanced and late-stage colorectal cancer rely more heavily on systemic therapies such as targeted therapy, high expression of the target in advanced lesions and metastases is more conducive to precise targeting. To investigate the expression of EEPD1 in advanced colorectal cancer and metastases, analysis of the GEPIA database revealed high expression of EEPD1 in advanced colorectal cancer (see...). Figure 6 Further immunohistochemical staining in clinical samples also revealed significantly elevated EEPD1 protein levels in patients with stage IV colorectal cancer (see [link to study]). Figure 7Furthermore, immunohistochemical staining of EEPD1 in paired primary lesions, lymph node metastases, and liver metastases from colorectal cancer patients obtained from the First Affiliated Hospital of Sun Yat-sen University revealed that EEPD1 expression in lymph node metastases and liver metastases was significantly higher than that in the primary lesion (see [link to study]). Figure 8 This aligns with the characteristics of EEPD1 as a cancer-promoting molecule and is also beneficial for EEPD1-targeted therapy in advanced patients.

[0083] Example 2

[0084] To investigate the regulatory role of EEPD1 in colorectal cancer, EEPD1-silencing cell lines were constructed using shRNA technology (shEEPD1#1 (5'-TCGTGGAGTACCGAGAGTATA-3'), shEEPD1#2 (5'-GACAACATCTGGATCAGTAAA-3'), shEEPD1#3 (5'-GCATCGTGGAATACCGAGAAT-3'), and shEEPD1#4 (5'-AGATTCAAGGTGGGAAGTAAT-3')) in human colorectal cancer cell lines HCT116 and HT29, and mouse colon cancer cell lines MC38 and CT26, respectively. RNA and protein were extracted and analyzed by real-time quantitative qPCR and Western blotting to determine the expression level and silencing efficiency of EEPD1. Real-time quantitative qPCR results confirmed the effect of EEPD1 silencing at the mRNA level (see [link to shRNA analysis]). Figure 9 Western blot results confirmed the efficiency of silencing EEPD1 at the protein level (see...). Figure 10 This meets the requirements for conducting subsequent functional experiments.

[0085] Subsequently, colony formation experiments were conducted using stably silenced EEPD1 cell lines HCT116, HT29, MC38, and CT26, and the number of colonies was statistically analyzed. The results showed that the colony formation ability of EEPD1-silenced cell lines was significantly weaker than that of the control group (see [link to study]). Figure 11 This suggests that downregulation of EEPPD1 expression can weaken the proliferative capacity of colorectal cancer cells. Furthermore, the effect of silencing EEPD1 on the growth rate of colorectal cancer cells was examined using the MTT assay. The results were consistent with the clonogenic assay; cell lines with silenced EEPD1 all showed decreased proliferative capacity and prolonged cell doubling time (see [link to study].) Figure 12 ).

[0086] To investigate the effect of EEPD1-silenced cell lines on tumor proliferation and metastasis in vivo, an HCT116-luci-hygro cell line was constructed, and EEPD1 was silenced on this cell line. Subsequently, control HCT116 cells and EEPD1-silenced (shEEPD1#2) HCT116 cells were used at a rate of 1×10⁻⁶. 6 A mouse intestinal orthotopic model was established by subserosa injection of EEPD1 cells into the terminal cecum of Balb / c (nu / nu) nude mice to better simulate the in situ environment of colon cancer. The growth of the in situ intestinal tumors was rigorously monitored using in vivo imaging techniques, and fluorescence intensity was recorded. Two weeks after inoculation, mice were sacrificed to obtain the intestinal tract and in situ tumor foci for in vitro fluorescence intensity detection and recording. Simultaneously, samples were fixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). Results showed that the tumorigenicity of the EEPD1-silenced cell line was significantly reduced in vivo, with both tumor volume and number lower than the control group (see [link to study]). Figure 13 This suggests that downregulation of EEPD1 expression can weaken the proliferation and tumorigenicity of colorectal cancer in vivo.

[0087] Furthermore, the effect of targeting EEPD1 on the in vivo metastatic ability of tumors was investigated using the HCT116-luci-hygro cell line with silenced EEPD1 (shEEPD1#2). Specifically, at a concentration of 5 × 10⁻⁶ cells... 5 Cells were used to construct liver and lung metastases of colon cancer via intrasplenic and tail vein injection. Simultaneously, the fluorescence intensity of metastatic lesions and the mouse status were closely monitored using in vivo imaging techniques. Results showed that EEPD1, targeting colon cancer cells, could inhibit the formation of liver and lung metastases. Subsequently, mice were sacrificed, and the livers of the liver metastasis model and the lungs of the lung metastasis model were removed, photographed, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and finally stained with hematoxylin and eosin (HE). The formation of metastatic lesions was observed under a microscope (see [link to article]). Figure 14 The results were consistent with in vivo imaging; targeting the expression of EEPD1 in colon cancer cells significantly reduced the size and number of liver and lung metastases, indicating that silencing EEPD1 can inhibit the in vivo metastatic ability of tumor cells.

[0088] Example 3

[0089] To investigate whether targeting EEPD1 in colorectal cancer would affect the body's anti-tumor immune response, CT26 cell lines with silenced EEPD1 (shEEPD1#3) were used, and the cells were administered at a rate of 1×10⁻⁶. 6We used a cell count of [number] cells per mouse to construct subcutaneous xenograft models in both immunodeficient Balb / c (nu / nu) and immunocompetent Balb / c mice, and closely monitored tumor growth. Results showed that in immunocompetent mice, targeting EEPD1 induced a stronger antitumor effect, with significantly greater reductions in tumor volume and weight compared to the immunodeficient mouse group (see [link]). Figure 15 The results suggest that downregulation of EEPD1 expression not only directly inhibits tumor proliferation and growth but also activates anti-tumor immune responses in vivo, further limiting the in vivo progression of colorectal cancer. To make the validation more comprehensive, MC38-shEEPD1 (shEEPD1#3) cell lines with a C57BL / 6 background were used, and the cells were also cultured at 1×10⁻⁶ cells per cell line. 6 The cell count was [number] cells / mouse, and subcutaneous xenograft models were constructed in both immunodeficient Balb / c (nu / nu) mice and immunocompetent C57BL / 6 mice. Results were consistent with CT26; MC38 cells targeting EEPD1 also activated the anti-tumor immune response in mice, enhancing the anti-tumor effect induced by silencing EEPD1. Tumor volume and weight were lower in the MC38 group than in the immunodeficient mouse group (see [link]). Figure 16 ).

[0090] Since the body's anti-tumor immunity ultimately requires immune cells to function within the tumor microenvironment, this study investigated the impact of EEPD1 expression targeting colon cancer cells on the tumor immune microenvironment. Single-cell suspensions of subcutaneous tumors from immunocompetent mice were prepared and analyzed by flow cytometry. The results showed that the most significant increase in infiltration of CD8 cells into the immune microenvironment of CT26 and MC38 subcutaneous tumors with silenced EEPD1 (shEEPD1#3 and shEEPD1#4) was observed. + T cell population, which is likely the most important cell type for targeting EEPD1 to exert anti-tumor immunity (see [link]). Figure 17 Meanwhile, studies generally agree that the expression of GzmB and IFN-γ, which have tumor-killing effects, is a key characteristic of CD8. + T cell activation and anti-tumor effects are markers of T cell activation; therefore, CD8+ was also detected in a subcutaneous tumor model. + The expression levels of GzmB and IFN-γ on T cells were also examined. The results indicated that, regardless of whether it was CT26 or MC38 cells, silencing EEPD1 resulted in the expression and secretion of GzmB and IFN-γ by CD8+ cells in the immune microenvironment. + A higher proportion of T cells indicates activated CD8+. + With a higher number of T cells, it possesses a stronger tumor-killing effect (participating in...). Figure 18 ).

[0091] To gain a more comprehensive and in-depth understanding of the subcutaneous tumor microenvironment of silenced EEPD1, other infiltrating immune cells were also analyzed. Macrophage polarization is important for assessing their anti-tumor immune status. It is generally believed that M1-polarized macrophages play a pro-inflammatory role in immune responses, promoting macrophage-mediated anti-tumor immune responses, while M2-polarized macrophages tend to have anti-inflammatory immune responses, promoting macrophage-mediated immune tolerance to tumors. The results showed that the number of infiltrating macrophages did not differ significantly between the silenced EEPD1 groups, but the proportion of infiltrating M1-type macrophages (highly expressing CD86 and lowly expressing CD206) was significantly increased, while the proportion of infiltrating M2-type macrophages (highly expressing CD206 and lowly expressing CD86) was significantly decreased. The ratio of M1-type macrophages to M2-type macrophages was significantly higher in the silenced EEPD1 groups (shEEPD1#3 and shEEPD1#4) than in the control group (see [link to relevant documentation]). Figure 19 The results suggest that silencing EEPD1 can promote M1 polarization of BMDM, inhibit M2 polarization that induces immune tolerance, increase the M1 / M2 ratio, promote macrophage polarization towards anti-tumor immunity, and enhance the anti-tumor immune response of the tumor immune microenvironment.

[0092] Previous studies have shown that mature dendritic cells (DCs) play an important synergistic role in antigen presentation and T cell activation; therefore, DC maturation is also a marker of anti-tumor response in the tumor immune microenvironment. To further verify this conclusion, bone marrow-derived DCs (BMDCs) were extracted from 6-8 week old C57BL / 6 and Balb / c mice and co-cultured with EEPD1-silenced MC38 cells and CT26 cells (shEEPD1#3 and shEEPD1#4), respectively. Cells were harvested and analyzed after 48 hours. The results showed that the mean infiltration and maturity of DCs in CT26-shEEPD1 subcutaneous tumors were higher than those in the control group, but the difference was not statistically significant. However, the number and maturity of DCs in MC38-shEEPD1 subcutaneous tumors were significantly higher than those in the control group (see [link to study]. Figure 20 This indicates that targeting EEPD1 expression can, to some extent, promote the maturation of dendritic cells (DCs) and participate in anti-tumor immunity, resisting the tumor microenvironment of immune tolerance. In summary, these results suggest that targeting EEPD1 can promote CD8 expression. + T cell infiltration and activation, promoting M1 polarization of macrophages and inhibiting M2 polarization, and promoting DC cell maturation are among the pathways that improve the microenvironment of immune tolerance in colorectal cancer and activate the body's anti-tumor immune response.

[0093] Example 4

[0094] GSEA pathway enrichment analysis of transcriptome sequencing results from EEPD1-silenced cell lines showed that the type I interferon response pathway was enriched in EEPD1-silenced colon cancer cell lines. Type I interferon response is widely considered to play an important role in anti-tumor immunity, and it has been reported that activation of the type I interferon pathway in tumor cells can promote MHC-I expression and enhance the ability of tumor cells to process and present antigens. Based on this, it is hypothesized that targeting EEPD1 may promote MHC-I expression by activating the cellular type I interferon response. To verify this conclusion, a monoclonal antibody against the type I interferon receptor (IFNAR) was added to EEPD1-silenced cell lines to inhibit the type I interferon response in tumor cells. Flow cytometry analysis of MHC-I expression on the surface of tumor cells showed that inhibiting the activation of the type I interferon pathway could reverse the increase in cellular MHC-I expression induced by EEPD1 silencing. To further verify the function of type I interferon pathway activation in silencing EEPD1 tumor cells, MC38-shNC and MC38-shEEPD1 tumor cells (shEEPD1#4) were subjected to 1×10⁻⁶ ion exchange assays. 6 Subcutaneous xenograft tumor models were established in immunocompetent C57BL / 6 mice using a cell count of 200 μg / mouse. Simultaneously, mice were intraperitoneally injected with anti-IFNAR monoclonal antibody. Subcutaneous tumor growth was then closely monitored. The results were consistent with in vitro experiments; the anti-IFNAR monoclonal antibody could partially reverse the anti-tumor effect induced by EEPD1 targeting, and tumor growth rate, volume, and weight were all increased compared to the group with EEPD1 silencing alone (see [link to relevant documentation]). Figure 21 The above results collectively indicate that the increased MHC-I expression and enhanced antigen processing and presentation capacity induced by targeting EEPD1 are due to the activation of the type I interferon response in colon cancer cells.

[0095] The aforementioned results indicate that silencing EEPD1 expression in colon cancer cells can promote antigen processing and presentation, as well as MHC-I expression, by activating type I interferon responses. Previous studies have generally suggested that the antigen-presenting capacity and MHC-I expression of tumor cells are related to CD8+ in the tumor immune microenvironment. + T cell proliferation, activation, and killing are closely related. To verify this conclusion, a colon cancer cell line MC38-OVA with stable high expression of ovalbumin (OVA) was constructed, and EEPD1 was silenced. Simultaneously, the expression of the OVA antigen peptide sequence SIINFEKL on the cell membrane surface was detected by flow cytometry. The results were consistent with the previous findings, indicating that silencing EEPD1 in tumor cells increased the expression of the OVA antigen peptide sequence SIINFEKL on the colon cancer cell surface, suggesting enhanced antigen processing and presentation functions of tumor cells.

[0096] CD8 was then extracted from the spleen of 6-8 week old OT-1 mice. + T cells and DC cells, including CD8 + T cells via CD3 + / CD28 + 72 hours after activation with antibody-conjugated magnetic beads, activated CD8 were analyzed using Cell trace violet. + T cells were live-cell stained and labeled, and then MC38-OVA cells, DC cells, and activated CD8+ were mixed at an effector-target ratio of 1:1:5. + T cells were co-cultured for 24 hours, and the expression of cell trace violet was detected by flow cytometry, reflecting the expression of CD8+. + The proliferation rate of T cells after stimulation with OVA antigen peptides was significantly increased. Results showed that CD8+ cells co-cultured with EEPD1-silenced (shEEPD1#4) MC38-OVA cells... + The significantly increased proportion of T cells indicates that EEPD1, which targets tumor cells, can indeed promote CD8+ in the immune microenvironment. + T cell expansion enhances anti-tumor immune responses (see...) Figure 22 ).

[0097] To investigate the aforementioned activated CD8 + T cells kill tumor cells by inhibiting CD8. + T cells were co-cultured with MC38-OVA cells that had silenced EEPD1 (shEEPD1#4) at effector-to-target ratios (effector cells to tumor cells) of 0:1, 1:1, and 5:1 for 48 hours. MC38 cell death was then detected by flow cytometry to reflect CD8+ cell death. + The killing effect of T. Results showed that after targeting EEPD1 expression in MC38-OVA, co-cultured CD8... + T cell activation was significantly enhanced, its killing effect on tumor cells was significantly increased, and the proportion of dead colon cancer cells was significantly increased (see [link to relevant documentation]). Figure 23 In summary, EEPD1 targeting tumor cells can promote the presentation of more antigens, thereby enhancing the CD8+ expression in the tumor immune microenvironment. + T cell activation and killing of tumor cells enhance anti-tumor immune responses.

[0098] The aforementioned results indicate that targeting EEPD1 in tumor cells, in addition to promoting CD8... +In addition to T cell proliferation and activation, macrophage polarization was also regulated. To further elucidate the effect of silencing tumor cell EEPD1 expression on macrophage function, bone marrow-derived macrophages (BMDM) were extracted from the bone marrow of 6-8 week old C57BL / 6 and Balb / c mice and directly co-cultured with CFSE-stained MC38-shEEPD1 and CT26-shEEPD1 tumor cells (shEEPD1#3 and shEEPD1#4) for 48 h. Subsequently, the fluorescence intensity of macrophage marker F4 / 80 and tumor cell marker CFSE was detected by flow cytometry, and F4 / 80 cells were circled. + CFSE + The cell population, namely BMDM that has phagocytosed tumor cells, was observed. Results showed that the phagocytosis rate of MC38-shEEPD1 and CT26-shEEPD1 cells by BMDM was significantly higher than that of MC38-shNC and CT26-shNC, indicating that targeting EEPD1 expression in colorectal cancer can promote the phagocytic capacity of BMDM and enhance the anti-tumor immune response of the tumor immune microenvironment (see [link to study]). Figure 24 To more intuitively observe the phagocytosis of tumor cells by macrophages and calculate the corresponding phagocytic index, directly co-cultured BMDM and CFSE-labeled (green) MC38-shEEPD1 cells or CT26-shEEPD1 cells (shEEPD1#3 and shEEPD1#4) were fixed with 4% paraformaldehyde, then stained with F4 / 80 specific fluorescent antibody (red), and the phagocytic effect of BMDM on tumor cells was observed under a microscope. The results showed that cell lines with silenced EEPD1 promoted the phagocytosis of BMDM, with a significantly higher phagocytic index than the control group cells, exhibiting higher anti-tumor immune activity. This facilitated the subsequent processing and presentation of tumor cell antigens by macrophages and activation of CD8. + T cells provide the necessary preconditions (see...) Figure 25 ).

[0099] Example 5

[0100] To investigate the effect of EEPD1 targeting in colorectal cancer on anti-PD-1 immunotherapy, EEPD1-silenced CT26 and MC38 cell lines (shEEPD1#3 and shEEPD1#4) were subcutaneously injected into Balb / c and C57BL / 6 mice at a dose of 1×10⁻⁶. 6A mouse subcutaneous xenograft model was constructed using cells from a sample size of / mouse. Simultaneously, mice were treated with an anti-PD-1 monoclonal antibody (Selleck's A2122 mouse PD-1 antibody, 200 μg / mouse, administered intraperitoneally twice weekly). The growth of the subcutaneous tumors was closely monitored. Results showed that the combined treatment targeting EEPD1 expression in CT26 and MC38 cells with anti-PD-1 therapy was significantly more effective than anti-PD-1 monoclonal antibody therapy alone. The combined treatment group showed lower growth rate, tumor volume, and tumor weight compared to the anti-PD-1 treatment group alone (see [link to relevant documentation]). Figure 26-27 This suggests that targeting EEPD1 expression in colorectal cancer can improve the efficacy of anti-PD-1 immunotherapy. Furthermore, we found that MC38, as a microsatellite unstable colorectal cancer cell line, is more sensitive to the combined effects of EEPD1-targeted therapy and anti-PD-1 therapy.

[0101] Since the aforementioned results indicate that targeting EEPD1 can improve the microenvironment of immune tolerance in colorectal cancer, it is presumed that this change enhances the efficacy of anti-PD-1 immunotherapy. To verify this conclusion, subcutaneous tumor tissues from mice treated with the above-mentioned EEPD1-targeted combined anti-PD-1 therapy were obtained and single-cell suspensions were prepared. Spectroscopic flow cytometry analysis was used to analyze the infiltration and activation of major immune cells in the tumor immune microenvironment. The results showed that CD8+ cells... + The changes in T cell infiltration and activation were most pronounced in the group receiving targeted EEPD1 combined with anti-PD-1 immunotherapy, with CD8+ in mouse tumors. + The proportion of T-cell infiltration was significantly increased in the combination therapy group compared to the single anti-PD-1 immunotherapy group. Furthermore, the CD8+ infiltration rate was significantly higher in the combination therapy group. + The expression levels of GzmB and IFN-γ on T cells were higher than those in the anti-PD-1 immunotherapy group (see [link]). Figures 28-29 This suggests that targeting EEPD1 combined with anti-PD-1 therapy can significantly increase CD8 levels. + The activation and killing ability of T cells enhance the body's anti-tumor immune response.

[0102] The aforementioned results indicate that the improvement of the immune microenvironment in colorectal cancer and the enhancement of the efficacy of anti-PD-1 immunotherapy by silencing EEPD1 mainly depend on CD8. + T infiltration and activation. To further observe CD8 +The role of T cells was investigated. Subcutaneous xenograft models of EEPD1-silenced CT26 and MC38 mice (shEEPD1#3 and shEEPD1#4) were constructed. Simultaneously, mice were administered a CD8 monoclonal antibody (Selleck's CD8α in vivo antibody, catalog number A2102, administered at a dose of 200 μg / mouse via intraperitoneal injection twice weekly). The growth of the subcutaneous tumors was then closely monitored. Results showed that the CD8 monoclonal antibody could indeed partially counteract the anti-tumor immune response induced by EEPD1 targeting. The tumor volume and weight in the EEPD1-targeted combined CD8 monoclonal antibody treatment group were both higher than those treated with EEPD1-silenced antibodies alone. The tumor weight ratio of the EEPD1-targeted group to the control group also increased after the combined use of the CD8 monoclonal antibody (see [link to study]). Figures 30-31 The above results further validate CD8. + T cells are the main cells that play a role in the anti-tumor immune response activated by targeting EEPD1.

[0103] In summary, this invention, through bioinformatics analysis of public databases such as human tumor databases and immune databases, has revealed that EEPD1 is highly expressed in colorectal cancer, especially advanced colorectal cancer, and is closely related to the immunosuppressive microenvironment and poor prognosis of colorectal cancer. Simultaneously, this invention has constructed a stable EEPD1 knockdown cell line model and conducted related functional experiments, verifying in multiple animal models that EEPD1 downregulation inhibits the proliferation and metastasis of colorectal cancer in vitro and in vivo. This invention focuses on the molecular mechanism and biological effects of targeting EEPD1 to inhibit colorectal cancer progression. Through experiments such as mouse subcutaneous tumor models and flow cytometry, it has for the first time revealed the genomic instability caused by targeting EEPD1, directly limiting the proliferation and metastasis of colorectal cancer cells and promoting CD8+ in the tumor immune microenvironment. + The proliferation and activation of T cells enhance the phagocytic capacity and M1 polarization of macrophages, induce the maturation of DC cells, and improve the antigen processing and presentation capacity and MHC-I expression of colorectal cancer cells, ultimately improving the immune tolerance microenvironment of colorectal cancer and thus enhancing the efficacy of PD-1 antibody immunotherapy.

[0104] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. Application of EEPD1 inhibitors in the preparation of drugs that enhance the sensitivity of PD-1 inhibitors to colorectal cancer treatment.

2. The application according to claim 1, characterized in that, The EEPD1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the EEPD1 gene.

3. Application of EEPD1 inhibitors and PD-1 inhibitors in the preparation of drugs for the treatment of colorectal cancer.

4. The application according to claim 3, characterized in that, The EEPD1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the EEPD1 gene.

5. A pharmaceutical composition for treating colorectal cancer, characterized in that, This includes EEPD1 inhibitors and PD-1 inhibitors.

6. The pharmaceutical composition according to claim 5, characterized in that, The EEPD1 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the EEPD1 gene.

7. The pharmaceutical composition according to claim 5, characterized in that, The PD-1 inhibitor is selected from one or more of pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, camrelizumab, penaprilimab, cepalimumab, fenolinimab, and slulimab.

8. Application of reagents for detecting EEPD1 expression levels in the preparation of products for evaluating the sensitivity of PD-1 inhibitors to colorectal cancer treatment.

9. Application of EEPD1 inhibitors in the preparation of drugs that improve the immune microenvironment in patients with colorectal cancer.

10. The application according to claim 9, characterized in that, The immune microenvironment includes CD8 + One or more of the following: T cell infiltration and activation, macrophage M1 polarization, macrophage M2 polarization, DC cell maturation, and MHC-I expression level.

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