Application, method and drug of crept in regulating macrophage infiltration related to tumor or inflammatory disease

Abstract

The present application provides the application, method and drug related to the regulation of tumor or inflammatory disease related macrophage infiltration by CREPT. The present application also provides the application of the inhibition of CREPT positive tumor associated macrophage (TAM) in the preparation of the preparation for regulating the tumor microenvironment, and the application in the preparation of the drug for treating tumor or promoting the action of T lymphocyte. The inhibition of the CREPT positive tumor associated macrophage (TAM) can provide a technical means for controlling tumor proliferation, invasion and metastasis. The CREPT positive tumor associated macrophage (TAM) can also be used as a biomarker for the risk of an individual suffering from tumor, or a biomarker for the development stage of the tumor of an individual, to judge the risk of an individual suffering from tumor or the stage and progress of the tumor of a tumor patient.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to the application of inhibition of CREPT-positive tumor-associated macrophages (TAMs) in regulating tumor / inflammatory diseases. Background Technology

[0002] The proliferation, invasion, and metastasis of tumors are complex processes resulting from the interaction of multiple cellular components. Recent research has revealed that the tumor-associated microenvironment (MAM) influences tumor development. As a highly complex system for tumor survival, the MLM is primarily composed of blood vessels, lymphatic vessels, endothelial cells, cancer-associated fibroblasts (CAFs), extracellular matrix (ECM), and various immune cells. The interactions among these components are closely related to tumor biological behavior. MLM-associated immune cells are key components of anti-tumor immune responses, potentially leading to tumor eradication or control. However, some MLM-associated immune cells can promote tumor growth and angiogenesis, and contribute to drug resistance and metastasis. Studies have found that tumor-associated macrophages, as important components of the MLM, can promote tumor growth, invasion, metastasis, and drug resistance.

[0003] As the main cells mediating adaptive immunity, T lymphocytes and B lymphocytes, as well as innate immune cells such as natural killer (NK) cells and tumor-associated macrophages (TAMs) that play important roles in the immune system, have been shown in multiple studies to be closely related to patient prognosis.

[0004] Macrophages undergo activation of different characteristics under various tumor microenvironment stimuli, including two types: ① classically activated macrophages, also known as M1 macrophages; ② alternatively activated macrophages, also known as M2 macrophages. M1 macrophages typically play an anti-tumor role, while M2 macrophages can promote tumor development and progression. CD68 is now clearly a macrophage-specific molecular marker, and studies have shown that CD163 is expressed in M2 macrophages. Clinical studies have demonstrated the efficacy of CD163 in breast cancer, rectal cancer, and leiomyosarcoma. + Macrophage infiltration density is closely related to survival and is a predictor of disease progression, early recurrence, and metastasis. For example, immunohistochemistry was used to detect CD68 in cancer tissues from 117 esophageal cancer patients and adjacent normal tissues from 30 patients. +Macrophage infiltration, combined with the patient's clinicopathological features and follow-up data analysis, confirmed that the density of CD68-positive macrophage infiltration in esophageal cancer tissue was significantly increased compared to adjacent normal tissue. + Macrophage infiltration density was negatively correlated with patient survival time.

[0005] The inflammatory response is a complex physiological reaction of the body's immune system to external stimuli such as infection and tissue damage, involving chemokines, cytokines, and lipid inflammatory mediators. Macrophages are the main effector cells in the inflammatory response. Activated macrophages rapidly reach the site of inflammation after the onset of inflammation, participating in the phagocytosis of pathogens and the secretion of various mediators and cytokines involved in the inflammatory response. While effectively clearing pathogens, the large number of inflammatory mediators produced during the inflammatory response can also cause pathological damage to normal cells and tissues.

[0006] Inflammatory diseases, such as polymyositis (PM), are independent autoimmune connective tissue diseases that primarily affect proximal muscles and other body parts, such as skeletal muscle, esophagus, heart, and joints. Studies have shown that macrophage infiltration and the release of inflammatory factors are the main mechanisms causing PM. Research also indicates that inhibiting the inflammatory infiltration capacity of macrophages can effectively alleviate PM.

[0007] Diabetic nephropathy is a chronic inflammatory disease characterized by inflammatory cell infiltration and overexpression of pro-inflammatory factors. Macrophages are key inflammatory cells, playing a crucial role in the development and progression of diabetic nephropathy. Macrophage infiltration of the glomeruli and tubulointerstitium is the main pathological feature of most kidney injuries. Over-infiltrating macrophages are activated by abnormally elevated pathogenic factors in the renal environment, such as high glucose, advanced glycation end products (AGEs), and renin. After activation, macrophages can secrete inflammatory cytokines and chemokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1), leading to damage to glomerular cells and the basement membrane, resulting in interstitial fibrosis. Studies have found that removing recruited macrophages from the kidneys can effectively reduce the process of kidney injury.

[0008] Therefore, by targeting and regulating the infiltration capacity of macrophages associated with tumors or inflammatory diseases, new ideas can be provided for controlling tumor proliferation, invasion, and metastasis, determining the stage, progression, and prognosis of cancer patients, and alleviating inflammatory diseases. This will provide research directions for developing drugs to control tumor proliferation, invasion, and metastasis or alleviate inflammatory diseases.

[0009] The CREPT gene is known to be expressed significantly higher in tissues of various cancers than in non-tumor cells and adjacent normal tissues. Its expression is associated with cancer development and also directly or indirectly promotes cell proliferation. However, the role of CREPT in macrophages has not yet been studied, and how CREPT exerts its effects by regulating tumor-associated macrophages has not been reported.

[0010] Therefore, studying the regulatory mechanism and theoretical issues of CREPT in macrophages, and then proposing technical means to control the occurrence and development of tumors and inflammation-related diseases, is a problem that needs to be solved in the current technology. Summary of the Invention

[0011] Tumor-associated macrophages (TAMs) have become an attractive target for cancer immunotherapy, and targeting TAMs is an effective cancer immunotherapy strategy. Preclinical and clinical studies have shown that inhibiting TAM recruitment, consumption, and reprogramming can effectively suppress tumor progression and improve the prognosis of cancer patients. Clinically, it is crucial to assess which patients are suitable for macrophage-targeted immunotherapy. Patients with a large number of functionally or phenotypically pro-tumorigenic macrophages in their tumor microenvironment are most suitable for macrophage-targeted immunotherapy. Macrophage subsets are traditionally divided into classically activated macrophages (M1) and alternately activated macrophages (M2), the former having pro-inflammatory and tumor-killing effects, and the latter having anti-inflammatory and pro-tumorigenic functions. However, increasing evidence suggests that this nomenclature is too simplistic because macrophages can express overlapping M1 and M2 genes, failing to accurately label tumor-associated macrophages according to their pro-tumorigenic or anti-tumorigenic functions. Currently, specific biomarkers that can effectively label functional pro-tumorigenic or tumor-suppressive macrophages in the tumor microenvironment are still lacking.

[0012] The purpose of this invention is to overcome the problems existing in the prior art and to provide the application of CREPT-positive tumor-associated macrophages (TAMs) in tumor occurrence and progression, as well as their application in marking tumor occurrence and progression.

[0013] The objective of this invention and the technical problem it solves are achieved by the following technical solutions.

[0014] A first aspect of the present invention is to provide the use of inhibition of CREPT-positive tumor-associated macrophages (TAMs) in the preparation of medicaments that regulate the infiltration of macrophages associated with tumor / inflammatory diseases.

[0015] CREPT (Cell-cycle Regulated and Expression-elevated Protein in Tumor) is a novel gene cloned and named by our team (Gene ID: 58490). The human CREPT gene is located on chromosome 20, has 7 exons, and encodes 326 amino acids. CREPT contains a CID domain (C-terminal domain (CTD)-interacting domain), which is found in many proteins involved in transcription termination and RNA processing and is highly conserved from yeast to humans. The CID domain mediates the interaction with the CTD domain of the largest subunit Rpbl of RNA polymerase II (RNAPII) and plays an important role in the efficient synthesis of mRNA. CREPT is highly expressed in various cancer types, including endometrial cancer, retroperitoneal leiomyosarcoma, non-small cell lung cancer, colorectal cancer, and eight other tumor tissues.

[0016] CREPT promotes tumorigenesis and development primarily through transcriptional upregulation of related gene expression. As transcription nears termination, RNAPII moves to the poly A region. CREPT binds to RNAPII in the promoter and transcription termination regions of the oncogene Cyclin D1, forming a chromatin loop that prevents RNAPII from detaching from the DNA, thus enabling continuous transcription of Cyclin D1. Overexpression of Cyclin D1 accelerates the transition from G1 to S phase in the cell cycle, promoting tumor cell proliferation. This loop formation may further promote gene transcription.

[0017] Tumor-associated macrophages (TAMs) are a type of tumor-associated immune cells that infiltrate or are located in or near tumor tissue and belong to the macrophage lineage.

[0018] CREPT-positive tumor-associated macrophages (TAMs) are tumor-associated macrophages that express CREPT and are detected positively, meaning that CREPT expression can be detected within the cells.

[0019] Tumor: As used herein, the terms “cancer,” “malignant tumor,” “vesicle,” “tumor,” and “carcinoma” are used interchangeably to refer to a disease, symptom, or condition in which cells exhibit or demonstrate relatively abnormal, uncontrolled, and / or autonomous growth, thus exhibiting or demonstrating an abnormally elevated rate of proliferation and / or an abnormal growth phenotype. In some embodiments, for example, as proposed herein, a tumor may include one or more types of cancer. In some embodiments, for example, as proposed herein, a tumor may be or include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, for example, as proposed herein, a tumor may be or include solid tumors. In some embodiments, for example, as proposed herein, a tumor may be or include hematologic malignancies. Generally speaking, examples of different types of cancer known in this art include, for example, colorectal cancer; hematopoietic system cancers including leukemia, lymphoma (Hodgkin and non-Hodgkin), myeloma, and myeloproliferative disorders; sarcoma, melanoma, adenoma, solid tissue cancer, squamous cell carcinoma of the oral cavity, pharynx, laryngeal cavity, and lung cancer; liver cancer; genitourinary system cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer; renal cell carcinoma; bone cancer; pancreatic cancer; skin cancer; melanoma of the skin or eye; endocrine system cancers; thyroid cancer; parathyroid cancer; head and neck cancer; breast cancer; gastrointestinal cancer and nervous system cancers; benign lesions such as papilloma, etc.

[0020] The aforementioned cancers are all within the scope of this invention.

[0021] Solid tumor: As used herein, the term "solid tumor" refers to an abnormal mass of tissue that includes cancer cells. In various embodiments, such as those proposed herein, a solid tumor is or includes an abnormal mass of tissue that does not contain cysts or fluid-filled areas. In some embodiments, such as those proposed herein, a solid tumor may be benign; in some embodiments, a solid tumor may be malignant. Examples of solid tumors include carcinoma, lymphoma, and sarcoma. In some embodiments, such as those proposed herein, a solid tumor may be or includes tumors of the adrenal glands, bile ducts, bladder, bone, brain, breast, cervix, colon, endometrium, esophagus, eye, gallbladder, gastrointestinal tract, kidney, larynx, liver, lung, nasal cavity, nasopharynx, oral cavity, ovary, penis, pituitary gland, prostate, retina, salivary glands, skin, small intestine, stomach, testes, thymus, thyroid gland, uterus, vagina, and / or vulva.

[0022] All of the above-mentioned solid tumors are within the scope of this invention.

[0023] Inflammatory diseases: As used herein, the term "inflammatory disease" refers to a disease caused by a pathological response of the body to various harmful physical, chemical, biological, or other stimuli, primarily as a defense mechanism. In various embodiments, such as those presented herein, inflammatory diseases include polymyositis, diabetic nephropathy, rheumatoid arthritis, and lupus nephritis.

[0024] In some embodiments of the present invention, the tumor includes solid carcinoma. In some embodiments of the present invention, the tumor includes one or more of the following: cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, liver cancer, kidney cancer, glioma, gastric cancer, pancreatic cancer, prostate cancer, melanoma, and endometrial cancer.

[0025] As some embodiments of the present invention, the inflammatory diseases include polymyositis, diabetic nephropathy, rheumatoid arthritis, and lupus nephritis.

[0026] As some embodiments of the present invention, the regulation of tumor / inflammatory disease-associated macrophage infiltration includes reducing serum inflammatory factor levels and reducing the content of M1 macrophages.

[0027] As some embodiments of the present invention, the agents for inhibiting CREPT-positive tumor-associated macrophages (TAMs) include agents that affect CREPT protein expression, agents that knock out CREPT protein, agents that alter CREPT protein, or agents that degrade CREPT protein.

[0028] As some embodiments of the present invention, the formulation affecting CREPT protein expression includes a nucleic acid reagent or a carrier containing a nucleic acid fragment that inhibits CREPT protein expression.

[0029] As some embodiments of the present invention, the formulation for knocking out the CREPT protein includes reagents for homologous recombination and gene editing to eliminate the CREPT gene.

[0030] As some embodiments of the present invention, the formulation for altering the CREPT protein includes reagents that alter the structure of the CREPT protein.

[0031] As some embodiments of the present invention, the formulation for degrading CREPT protein includes reagents via the PROTAC, molecular gel, LYTAC, MoDE, ATAC, Apt-LYTAC, AbTAC, PROTAB, REULR, KineTAC, IFLD, ATTEC, AUTAC, or AUTOTAC pathways.

[0032] PROTACs, also known as protein degradation chimeras, are heterobifunctional molecules composed of two ligands linked by a linker. One ligand binds to the target protein, while the other targets the E3 ligase. PROTACs can simultaneously bind to both the target protein and the E3 ligase, thus shortening the distance between them, inducing ubiquitination of the target protein, which is then recognized and degraded by the 26S proteasome. Traditional inhibitors employ a "site-driven" mechanism, requiring the drug molecule to bind tightly to the active site of the target protein to inhibit its activity and achieve a pharmacological effect. PROTACs, however, operate on an event-driven mechanism, binding to any site on the target protein without requiring high affinity to potentially induce protein degradation.

[0033] Molecular glue technology is one of the main technologies based on the ubiquitin-proteasome degradation system. In the field of targeted protein degradation, molecular glues are typically monovalent small molecules (molecular weight less than 500 Da). Their specific mechanism of action involves altering the surface of E3 ligases, thereby blocking the binding of E3 ligases to natural substrates, inducing the specific protein to be degraded to bind to the E3 ligase, further promoting the ubiquitination modification of the specific protein, and finally causing it to be degraded by the proteasome. Due to the complexity of their biological characteristics, some disease-related proteins may possess multiple domains or functional sites, making it difficult to form stable bindings with drug molecules. These proteins have not yet been effectively targeted using traditional drugs such as small molecule drugs or antibodies. The development of molecular glue technology offers a possibility for solving this problem.

[0034] Intracellular targeted protein degradation (iTPD) technologies, represented by PROTAC and molecular gels, have developed into an important new mode for small molecule drug development. While iTPD technology is rapidly developing, extracellular targeted protein degradation (eTPD) technology has also made several significant advances.

[0035] LYTAC is a bifunctional molecule with two binding domains. One end is an oligoglycopeptide group that binds to the cell surface transmembrane receptor CI-M6PR (cation-independent mannose-6-phosphate receptor), and the other end is an antibody or small molecule that binds to a target protein. These two binding domains are linked by a chemical linker. The trimer CI-M6PR-LYTAC-target protein complex formed on the plasma membrane is engulfed by the cell membrane, forming a transport vesicle. The vesicle transports the complex to the lysosome, where the target protein is degraded. LYTAC has the potential to degrade membrane proteins and soluble proteins.

[0036] The asialoglycoprotein receptor (ASGPR) is an endocytic cell surface receptor, highly expressed primarily on hepatocytes (up to 500,000 copies per cell), playing a crucial role in the natural process of endogenous protein internalization and degradation within hepatocytes. Multiple target proteins (such as EGFR) can be degraded by conjugating (multiple) targeting ligands of ASGPR, such as tri-GalNac, to target protein-binding antibodies. The bifunctional small-molecule extracellular targeted protein degradation (eTPD) technologies MoDE and ATAC (ASGPR Targeting Chimeras) utilize ASGPR. ATAC uses a high-affinity monodentate ASGPR targeting moiety that is three to four times smaller than tri-GalNAc. Because ASGPR is a liver-specific lysosomal targeted receptor, ATAC technology, compared to LYTAC (CI-M6PR, which is widely expressed in multiple cell types), can degrade extracellular proteins in a cell-type-restricted manner, offering a potential safety advantage.

[0037] Apt-LYTAC is a small aptamer (8-25 kDa) ASGPR adaptor that links tri-GalNac to the 5' end of the aptamer for the soluble growth factor PDGF and the membrane receptor PTK7. It has been demonstrated that PDGF-labeled Apt-LYTAC is degraded in HepG2 cells. PDGF-bound Apt-LYTAC can be delivered to lysosomes.

[0038] Cytokine receptor-targeting chimera (KineTAC) technology is a novel degradative agent that utilizes the decoy circulating receptor CXCR7 to transport cell membrane and extracellular target proteins to lysosomes for degradation. For example, CXCL12 can be internalized after binding to the decoy receptor CXCR7. One end of KineTAC is the chemokine CXCL12, and the other end binds to the target protein. After the complex enters the lysosome, the target protein is degraded. Studies have confirmed that KineTAC technology can degrade membrane proteins such as PD-L1, HER2, and EGFR, as well as soluble proteins VEGF and TNF-α. The KineTAC platform expands the selectivity of eTPD by utilizing a series of circulating receptors with different tissue distributions and levels.

[0039] A novel integrin-facilitated lysosomal degradation (IFLD) strategy based on bifunctional compounds couples a target protein-binding ligand with an integrin-recognizing ligand. The resulting bifunctional compound induces the endocytosis and degradation of extracellular or cell membrane proteins in an integrin- and lysosome-dependent manner. Integrins are cell adhesion receptors expressed on the cell surface and play a crucial role in cell-matrix interactions. Because integrins can bind to ligands containing the Arg-Gly-Asp (RGD) motif and transport them to lysosomes, they represent an attractive degradation system.

[0040] As some embodiments of the present invention, the drug includes a small molecule inhibitor of the CREPT protein.

[0041] Small molecule inhibitors refer to a class of organic compound molecules with a molecular weight of less than 1,000 Daltons that can target proteins, reduce protein activity, or inhibit biochemical reactions. They are highly selective and have cell permeability, and are widely used in signaling pathway research.

[0042] As some embodiments of the present invention, the nucleic acid reagent is selected from siRNA, shRNA, microRNA, piRNA, and ASO.

[0043] Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a double-stranded RNA of 20 to 25 nucleotides in length, with many different uses in biology. siRNA is primarily involved in RNA interference (RNAi) to regulate gene expression in a specific manner.

[0044] Short hairpin RNA (shRNA), cloned into an shRNA expression vector, consists of two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure controlled by the pol III promoter. It is then followed by 5-6 T molecules as a transcription terminator for RNA polymerase III. Cloning the siRNA sequence as a "short hairpin" into a plasmid vector allows for the delivery of "small interfering RNA" (siRNA) in vivo. When introduced into an animal, the hairpin sequence is expressed, forming a "double-stranded RNA" (dsRNA), which is then processed by RNAi channels.

[0045] MicroRNAs (miRNAs) are non-coding RNAs approximately 22 nt in length, widely found in various organisms from viruses to humans. These small RNAs can bind to mRNA and block the expression of protein-coding genes, preventing them from being translated into proteins.

[0046] piRNA (Piwi-interacting RNA) is a class of small RNAs about 30 nt in length isolated from mammalian germ cells. It can specifically bind to PIWI, an analog of Argonaute protein in animal cells. This small RNA can only exert its regulatory role by binding to members of the PIWI protein family. It has the functions of silencing gene transcription, regulating translation and mRNA stability.

[0047] Antisense oligonucleotides (ASOs) are single-stranded oligonucleotide molecules that typically contain 15-25 nucleotides. After entering the cell, they bind to their complementary target mRNA through base pairing under the action of ribonuclease H1, thereby inhibiting the expression of the target gene.

[0048] As some embodiments of the present invention, the vector containing the nucleic acid fragment that inhibits CREPT protein expression includes adenovirus, adeno-associated virus, slow expression virus, and retrovirus.

[0049] As some embodiments of the present invention, the reagents for homologous recombination and gene editing to eliminate the CREPT gene include the CRISPR / Cas9 system.

[0050] A second aspect of the invention provides the use of inhibition of CREPT-positive tumor-associated macrophages (TAMs) in the preparation of formulations that regulate the tumor microenvironment (TME), wherein the regulation of the tumor microenvironment works by reducing the activity of CREPT-positive tumor-associated macrophages (TAMs).

[0051] Malignant tumors constitute a complex ecosystem involving intricate interactions between tumor cells and non-cancer cells, known as the tumor microenvironment (TME). This TME comprises various immune cell types, fibroblasts, endothelial cells, pericytes, and a variety of other tissue-receptor cell types, all known to play crucial roles in the pathogenesis of malignant tumors. The cellular composition and functional state of the TME can vary considerably depending on the organ from which the tumor develops, the inherent characteristics of the cancer cells, the tumor stage, and the patient's specific features.

[0052] As some embodiments of the present invention, the agents for inhibiting CREPT-positive tumor-associated macrophages (TAMs) include agents that affect CREPT protein expression, agents that knock out CREPT protein, agents that alter CREPT protein, or agents that degrade CREPT protein.

[0053] As some embodiments of the present invention, the formulation affecting CREPT protein expression includes a nucleic acid reagent or a carrier containing a nucleic acid fragment that inhibits CREPT protein expression.

[0054] As some embodiments of the present invention, the formulation for knocking out the CREPT protein includes reagents for homologous recombination and gene editing to eliminate the CREPT gene.

[0055] As some embodiments of the present invention, the formulation for altering the CREPT protein includes reagents that alter the structure of the CREPT protein.

[0056] As some embodiments of the present invention, the formulation for degrading CREPT protein includes reagents via the PROTAC, molecular gel, LYTAC, MoDE, ATAC, Apt-LYTAC, AbTAC, PROTAB, REULR, KineTAC, IFLD, ATTEC, AUTAC, or AUTOTAC pathways.

[0057] As some embodiments of the present invention, the drug includes a small molecule inhibitor of the CREPT protein.

[0058] As some embodiments of the present invention, the nucleic acid reagent is selected from siRNA, shRNA, microRNA, piRNA, and ASO.

[0059] As some embodiments of the present invention, the vector containing the nucleic acid fragment that inhibits CREPT protein expression includes adenovirus, adeno-associated virus, slow expression virus, and retrovirus.

[0060] As some embodiments of the present invention, the reagents for homologous recombination and gene editing to eliminate the CREPT gene include the CRISPR / Cas9 system.

[0061] As some embodiments of the present invention, the regulation of the tumor microenvironment includes promoting CD8 + T cell infiltration, weakening of macrophage overall polarization function, reduction of fibroblast proliferation and migration levels, inhibition of tumor-promoting gene expression, and reduction of p65 phosphorylation levels.

[0062] In some implementations, the regulation of the tumor microenvironment includes CD8 + Increased T cell infiltration. In some embodiments, the weakening of macrophage overall polarization includes a reduction in M1 and M2 macrophages. In some embodiments, the regulation of the tumor microenvironment includes a decrease in fibroblast proliferation and migration levels. In some embodiments, the regulation of the tumor microenvironment includes suppression of pro-tumor-related gene expression. As some embodiments of the invention, the regulation of the tumor microenvironment includes a reduction in p65 phosphorylation levels.

[0063] As some embodiments of the present invention, the weakening of the overall polarization function of the macrophage is a reduction in the macrophage polarization towards M1 and M2 types.

[0064] As some embodiments of the present invention, the tumor-promoting genes include SSP1, FN1, and VEGFA.

[0065] Tumor-related genes, also known as oncogenes, are genes that promote tumor growth. Human cells contain proto-oncogenes and tumor suppressor genes. When these two types of genes mutate under certain conditions, cancer can result. The occurrence of tumors is closely related to the activation, mutation, and deletion of these two types of genes. When oncogenes are activated, they lead to the proliferation of tumor cells and the formation of tumors, which are classified as benign or malignant depending on their pathology. Conversely, when tumor suppressor genes mutate or are deleted, they can also cause malignant transformation of cells, leading to the development of tumors.

[0066] As some embodiments of the present invention, the tumor includes solid carcinoma.

[0067] As some embodiments of the present invention, the tumor includes one or more of the following: cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, liver cancer, kidney cancer, glioma, gastric cancer, pancreatic cancer, prostate cancer, melanoma, and endometrial cancer.

[0068] A third aspect of the invention is the use of inhibition of CREPT-positive tumor-associated macrophages (TAMs) in the preparation of medicaments for treating tumors or promoting the function of T lymphocytes.

[0069] As some embodiments of the present invention, the drug reduces the number of tumors, reduces tumor burden, inhibits the proliferation and migration of tumor cells, inhibits tumor growth, and reduces tumor colonization.

[0070] In some embodiments, the drug can treat tumors in situ. In some embodiments, the drug can reduce the number of tumors in situ. In some embodiments, the drug can reduce the tumor burden in situ. In some embodiments, the drug can inhibit the proliferation and migration of tumor cells. In some embodiments, the drug reduces the colonization of metastatic tumors. In some embodiments, the drug inhibits the growth of metastatic tumors.

[0071] As some embodiments of the present invention, the T lymphocyte-promoting effect includes CD8 + Increased infiltration of T cells and INFγ-positive cytotoxic CD8+ T cells.

[0072] In some embodiments, the T-lymphocyte-promoting effect includes CD8 + An increase in the number of T cells. In some embodiments, the T-lymphocyte-promoting effect includes an increase in the number of INFγ-positive cytotoxic CD8+ T cells.

[0073] As some embodiments of the present invention, the tumor includes solid carcinoma.

[0074] As some embodiments of the present invention, the tumors include cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, liver cancer, kidney cancer, glioma, gastric cancer, pancreatic cancer, prostate cancer, melanoma, and endometrial cancer.

[0075] As some embodiments of the present invention, the agents for inhibiting CREPT-positive tumor-associated macrophages (TAMs) include agents that affect CREPT protein expression, agents that knock out CREPT protein, agents that alter CREPT protein, or agents that degrade CREPT protein.

[0076] As some embodiments of the present invention, the formulation affecting CREPT protein expression includes a nucleic acid reagent or a carrier containing a nucleic acid fragment that inhibits CREPT protein expression.

[0077] As some embodiments of the present invention, the formulation for knocking out the CREPT protein includes reagents for homologous recombination and gene editing to eliminate the CREPT gene.

[0078] As some embodiments of the present invention, the formulation for altering the CREPT protein includes reagents that alter the structure of the CREPT protein.

[0079] As some embodiments of the present invention, the formulation for degrading CREPT protein includes reagents via the PROTAC, molecular gel, LYTAC, MoDE, ATAC, Apt-LYTAC, AbTAC, PROTAB, REULR, KineTAC, IFLD, ATTEC, AUTAC, or AUTOTAC pathways.

[0080] As some embodiments of the present invention, the drug includes a small molecule inhibitor of the CREPT protein.

[0081] As some embodiments of the present invention, the nucleic acid reagent is selected from siRNA, shRNA, microRNA, piRNA, and ASO.

[0082] As some embodiments of the present invention, the vector containing the nucleic acid fragment that inhibits CREPT protein expression includes adenovirus, adeno-associated virus, slow expression virus, and retrovirus.

[0083] As some embodiments of the present invention, the reagents for homologous recombination and gene editing to eliminate the CREPT gene include the CRISPR / Cas9 system.

[0084] A fourth aspect of the present invention is to provide a reagent for detecting CREPT-positive tumor-associated macrophages (TAMs) and / or detecting the proportion of CREPT-positive tumor-associated macrophages (TAMs) to the total number of macrophages.

[0085] As some embodiments of the present invention, the reagents include multichromatographic reagents.

[0086] As some embodiments of the present invention, the reagents include reagents for labeling CREPT and reagents for labeling CD68 macrophages.

[0087] A fifth aspect of the invention is to provide the use of the aforementioned reagent or CREPT-positive tumor-associated macrophages (TAMs) in the preparation of a kit for assessing an individual's risk of developing a tumor or for diagnosing whether an individual has a tumor, wherein the proportion of CREPT-positive tumor-associated macrophages (TAMs) to the total number of macrophages is positively correlated with the individual's risk of developing a tumor.

[0088] In some embodiments of the present invention, the tumor includes solid carcinoma. In some embodiments of the present invention, the tumor includes one or more of the following: cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, liver cancer, kidney cancer, glioma, gastric cancer, pancreatic cancer, prostate cancer, melanoma, and endometrial cancer.

[0089] As some embodiments of the present invention, the samples for detecting CREPT-positive tumor-associated macrophages (TAMs) are derived from cancerous tissue or adjacent normal tissue.

[0090] As some embodiments of the present invention, the number of CREPT-positive tumor-associated macrophages (TAMs) accounting for ≥1% of the total number of macrophages indicates that the individual is at risk of developing colorectal cancer or has colorectal cancer; the number of CREPT-positive tumor-associated macrophages (TAMs) accounting for <1% of the total number of macrophages indicates that the individual is not at risk of developing colorectal cancer or has not developed colorectal cancer.

[0091] A sixth aspect of the invention is to provide the use of the aforementioned reagent or CREPT-positive tumor-associated macrophages (TAMs) in the preparation of a kit for assessing the tumor development stage of an individual with a tumor, wherein the proportion of CREPT-positive tumor-associated macrophages (TAMs) to the total number of macrophages is positively correlated with the tumor development stage of the individual with a tumor.

[0092] In some embodiments of the present invention, the tumor includes solid carcinoma. In some embodiments of the present invention, the tumor includes one or more of the following: cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, liver cancer, kidney cancer, glioma, gastric cancer, pancreatic cancer, prostate cancer, melanoma, and endometrial cancer.

[0093] As some embodiments of the present invention, the samples for detecting CREPT-positive tumor-associated macrophages (TAMs) are derived from cancerous tissue or adjacent normal tissue.

[0094] As some embodiments of the present invention, if the number of CREPT-positive tumor-associated macrophages (TAMs) in an individual suffering from colorectal cancer is ≥10% of the total number of macrophages, it indicates that the individual's tumor is in one of the clinical stages II, III, or IV of colorectal cancer; if the number of CREPT-positive tumor-associated macrophages (TAMs) in an individual suffering from colorectal cancer is <10% of the total number of macrophages, it indicates that the individual's tumor is in the clinical stage I of colorectal cancer.

[0095] By employing the above-described technical solution, the application of the inhibition of CREPT-positive tumor-associated macrophages (TAMs) in regulating tumor / inflammatory diseases has at least the following advantages:

[0096] Through the applications and drugs provided by this invention, the invention can more precisely regulate the infiltration capacity of tumor / inflammatory disease-related macrophages, providing new ideas for controlling tumor proliferation, invasion, and metastasis, judging the stage and progression of tumor patients, and alleviating inflammatory diseases. It also provides research directions for developing a drug to control tumor proliferation, invasion, metastasis, or alleviate inflammatory diseases.

[0097] Through the applications and drugs provided by this invention, the present invention can regulate the tumor microenvironment, thereby promoting CD8. + T cell infiltration, weakened overall macrophage polarization, reduced fibroblast proliferation and migration, inhibition of tumor-related gene expression, and decreased P65 phosphorylation levels all contribute to an environment where the tumor microenvironment is unfavorable for tumor growth.

[0098] Through the applications and drugs provided by this invention, this invention can treat tumors or promote T lymphocytes, reduce the number of in situ tumors, decrease the in situ tumor burden, inhibit the proliferation and migration of tumor cells, inhibit the growth of metastatic tumors, and reduce tumor colonization, while simultaneously promoting CD8. + An increase in the number of T cells and the number of INFγ-positive cytotoxic CD8+ T cells.

[0099] Through the applications and drugs provided by this invention, this invention can also provide biomarkers for an individual's risk of developing tumors and biomarkers for the stage of tumor development in an individual, providing a scientific basis for judging an individual's risk of developing tumors and judging the stage of disease development in an individual who has developed tumors. Attached Figure Description

[0100] Figure 1 The data shows the correlation between CREPT expression levels and macrophage infiltration in various cancer types in the TCGA database.

[0101] Figure 2 The correlation analysis shows the relationship between CREPT expression levels in different cancer types and staining results of CD68-positive macrophages.

[0102] Figure 3 The correlation analysis shows the relationship between CREPT expression levels in different cancer types and multicolor histochemistry (mIHC) results of CD68-positive macrophages.

[0103] Figure 4 The correlation analysis between CREPT expression level and CD68-positive macrophage multicolor histochemistry (mIHC) results in different stages of colorectal cancer is shown.

[0104] Figure 5 Displayed as CREPT flox / flox Mouse construction process;

[0105] Figure 6 The data shows the correlation between macrophage-specific knockout of CREPT and tumor growth, among which... Figure 6 A represents the CREPT myeloid-specific knockout mice obtained by knocking out Lyz-cre+ / - mice in myeloid lineage; Figure 6 B represents subcutaneous implantation of mouse melanoma B16 cells in CREPT knockout mice and control mice; Figure 6 C represents the growth of melanoma B16 cells in CREPT myeloid-specific knockout mice obtained from CREPTflox / flox mice and myeloid-specific knockout Lyz-cre+ / - mice; Figure 6 D represents the growth of CREPT myeloid-specific knockout mouse colon cancer MC38 cells obtained from CREPTflox / flox mice and myeloid-specific knockout Lyz-cre+ / - mice;

[0106] Figure 7 The image shows the physiological state of blood, spleen, and peritoneal macrophages in CREPT myeloid-specific knockout mice 6 hours after intraperitoneal injection of lipopolysaccharide (LPS) to induce systemic acute inflammation.

[0107] Figure 8 The mice were identified as CREPT myeloid-specific knockout mice. CREPT knockout did not affect the growth of the mice or the infiltration of their immune cells, nor did it affect the infiltration level of immune cells in various lymphoid organs.

[0108] Figure 9 The mouse model of CREPT-specific knockout was obtained by subcutaneously injecting B16 and MC38 mouse colorectal cancer cells and melanoma cells. In the resulting mouse models of colorectal cancer and melanoma, tumor growth was significantly inhibited.

[0109] Figure 10 The results show colorectal cancer cells and mouse melanoma cells in mice that underwent tail vein injection of B16 and MC38 in the CREPT myeloid-specific knockout mouse model. The resulting mouse models of colorectal cancer and melanoma showed a significant reduction in tumor colonization in the lungs.

[0110] Figure 11 The image shows the differences in colorectal cancer in situ tumor model of AOM / DSS-induced CREPT-specific knockout mice compared to wild-type mice after CREPT knockout.

[0111] Figure 12 The effect of isolated bone marrow-derived macrophages, along with wild-type mouse tumor cells MC38, co-colonized subcutaneously in mice on tumor growth;

[0112] Figure 13 The image shows the changes in the expression of CREPT, a tumor-promoting gene, in mouse bone marrow-derived macrophages after TCM supernatants from mouse melanoma B16F10 cells and mouse colon cancer cells MC38 were treated with the supernatants.

[0113] Figure 14 The image shows the changes in T cell infiltration levels in mouse tumors after CREPT was knocked out in mouse macrophages.

[0114] Figure 15 The image shows CD8+ levels in tissues of patients with high and low TAM expression in CREPT, as verified by multicolor histochemistry. + Differences in T cell infiltration;

[0115] Figure 16 This indicates the elimination of CD8 in mice. + After T cell administration, there were differences in subcutaneous tumor growth between wild-type mice and CREPT macrophage-specific knockout mice.

[0116] Figure 17 The results show the changes in macrophage infiltration levels in mouse tumor tissue after CREPT knockout, compared with the infiltration levels of other myeloid cells;

[0117] Figure 18 The effect of knocking out CREPT on the polarization of M1 and M2 macrophages is shown.

[0118] Figure 19 The effect of macrophages with CREPT knocked out on the proliferation and migration of tumor cells is shown.

[0119] Figure 20 The results showed that macrophages with CREPT knocked out inhibited the proliferation and migration of fibroblasts.

[0120] Figure 21 The high expression of some genes, including SPP1 and FN1, was observed in TAM patients with high CREPT expression.

[0121] Figure 22 The results showed that after CREPT was knocked out in mice, some genes in macrophages were significantly downregulated;

[0122] Figure 23 The results, as verified by qPCR, show that after knocking out CREPT, some genes were significantly downregulated, while after overexpressing CREPT, these genes were significantly upregulated again.

[0123] Figure 24 CREPT in macrophages is shown to regulate the expression of this tumor-promoting gene by affecting the NF-κB signaling pathway;

[0124] Figure 25 The results showed that after overexpression and knockdown of CREPT, CREPT in macrophages affected the transcriptional activity of p65.

[0125] Figure 26 The results showed that the transcriptional method verified that CREPT can interact with the P65 transcription factor downstream of NF-κB, and the interaction site between the two.

[0126] Figure 27 Both isolated primary mouse macrophages and mouse cell lines confirmed that CREPT can interact with p65.

[0127] Figure 28 This shows the effect of macrophages lacking CREPT on p65 phosphorylation levels;

[0128] Figure 29 This section provides a comprehensive overview of the role of tumor-associated macrophages with high CREPT expression in the tumor microenvironment. Detailed Implementation

[0129] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. 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.

[0130] [Experimental Materials]

[0131] 1. Laboratory Animals and Cells

[0132] Lyz-cre + / - Mice were purchased from the Experimental Animal Center of Tsinghua University. CREPTf / f mice were independently constructed by our research group, and MC38 and B16 cells were preserved by our laboratory.

[0133] 2 antibodies

[0134] The CREPT antibody was purified in-house by our research group, and a patent application was filed on January 13, 2012, with application number 201210009890.3; the CD68 antibody was purchased from Abcam (EPR20545).

[0135] 3 reagents

[0136] The main reagents and their sources are shown in Table 1.

[0137] Table 1. Main Reagents and Their Sources

[0138]

[0139]

[0140] Example

[0141] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0142] Example 1: Immunohistochemical staining showed that CREPT expression was positively correlated with macrophage infiltration.

[0143] The relationship between CREPT expression levels and macrophage infiltration in various cancer types was analyzed using the TCGA database. The results showed (e.g.) Figure 1 As shown in the figure: When CREPT is highly expressed in tissue samples from various tumor patients, the degree of macrophage infiltration is also high, such as in colon cancer, lung cancer, and breast cancer.

[0144] Based on the above results, clinical tissue samples from patients with colorectal cancer, lung cancer, and esophageal cancer were collected in this embodiment (sourced from the General Hospital of the Chinese People's Liberation Army (approved by the hospital's ethics committee). After fixation with paraformaldehyde, paraffin embedding, and preparation of pathological sections, immunohistochemical staining was performed. After dewaxing, hydration, and antigen retrieval, a series of serial sections were stained with mouse-derived CREPT monoclonal antibody and rabbit-derived macrophage CD68 antibody as primary antibodies. The secondary antibody was HRP-labeled, and the sections were stained with DAB staining and hematoxylin staining before mounting.

[0145] Staining results as follows Figure 2 As shown, the staining results indicate that CREPT is significantly highly expressed in these types of tumors, and CD68-positive macrophages are also relatively abundant in patient tissues with high CREPT expression.

[0146] All of the above results indicate that CREPT expression level is positively correlated with macrophage infiltration in the tumor microenvironment.

[0147] Example 2: Analysis of CREPT expression in tumor-associated macrophages

[0148] Since CREPT expression is positively correlated with macrophage invasion, but no studies have yet indicated how CREPT affects macrophage invasion. Current technology shows that CREPT is highly expressed in tumor cells. To investigate whether CREPT is also highly expressed in macrophages, thereby affecting macrophage invasion and function, the following experiments were conducted:

[0149] In this embodiment, CREPT and CD68-positive macrophages were simultaneously labeled on patient tissue sections using multicolor histochemistry (mIHC), and the results are as follows: Figure 3 As shown. Figure 3 The results showed that there is indeed a type of cell that highly expresses CREPT among CD68-positive macrophages. Some CD68-positive macrophages with high CREPT expression (red) (green) were detected in colorectal cancer, esophageal cancer, and lung cancer.

[0150] Example 3: Correlation Study of CREPT-Positive Tumor-Associated Macrophages with Tumor Progression

[0151] The study results indicate a strong correlation between macrophage infiltration and colorectal cancer stage, metastasis, and prognosis. This raises the question of whether these CREPT-positive macrophages influence the development and progression of colorectal cancer.

[0152] In this embodiment, clinical tissue samples (approved by the hospital's ethics committee and sourced from the General Hospital of the Chinese People's Liberation Army) from patients with colorectal cancer at different stages were collected. Multichromatographic histochemistry was used to simultaneously label CREPT and CD68 macrophages. A total of 30 patient samples from different clinical stages of colorectal cancer were collected. The multichromatographic results are as follows: Figure 4 As shown.

[0153] The results showed that in stage I patients, CREPT-positive tumor-associated macrophages were fewer, less than 10% of the total macrophage count. However, as the disease progressed, the number of CREPT-positive macrophages significantly increased in stage II, III, and IV patients, reaching approximately 30% of the total macrophage count. There was no significant difference in the number of CREPT-positive macrophages among stage II, III, and IV patients. This indicates a significant increase in CREPT expression in cells as the tumor progresses from stage I to stage II. However, no significant difference in CREPT expression was observed among stages II, III, and IV. This suggests that CREPT expression is induced early in the transition from stage I to stage II, and after induction, it is sufficient to exert its functional effect. Figure 4 BC).

[0154] The above results indicate that the number of CREPT-positive macrophages increases with disease progression. A CREPT-positive macrophage count of 1% may indicate a higher risk of cancer. A CREPT-positive macrophage count of 10% or higher suggests an increased stage of tumor progression.

[0155] Example 4: Macrophage-specific knockout of CREPT can inhibit tumor growth

[0156] CREPT flox / flox The mouse background is C57BL / 6. A flux (flanked by loxP) sequence is inserted at both ends of the exon of the CREPT sequence to be knocked out (e.g., Figure 5 (As shown).

[0157] CREPT flox / flox Mice and myeloid-specific knockout mice Lyz-cre + / - Mice obtained after mating were tail-cropped and genomic DNA was extracted. PCR identification confirmed the genotype was Lyz-cre. + / - CREPT flox / flox CREPT myeloid-specific knockout mice ( Figure 6 A).

[0158] The primers for genotyping are shown in Table 2.

[0159] Table 2 Primer Sequence List for Genotype Identification

[0160]

[0161]

[0162] PCR reaction system: The PCR reaction used a 2×Taq premixed PCR reaction system (containing dye) purchased from Genstar, as shown in Table 3.

[0163] Table 3. PCR reaction system for rat tail genotype identification

[0164] System components Volume / Dosage PCR mix 10μL Primers (10 μM) 0.5μL c DNA template 1μL <![CDATA[ddH2O]]> 8μL

[0165] The PCR reaction conditions are shown in Table 4: the annealing temperature for CREPT is 54℃, and for Lyz2-Cre it is 60℃.

[0166] Table 4. PCR reaction procedure for rat tail genotype identification

[0167]

[0168] By subcutaneously implanting mouse melanoma B16 cells into CREPT knockout mice and control mice ( Figure 6 B) Monitor tumor growth. Figure 6 The results showed that the tumor growth rate in CREPT knockout mice was significantly reduced compared to the control group. Tumor weight was also significantly lower in CREPT knockout mice. Similar results were obtained by subcutaneously implanting mouse colon cancer cells (MC38 cells). Figure 6 As shown in D.

[0169] These results indicate that macrophage-specific knockout of CREPT can inhibit tumor growth.

[0170] Example 5: CREPT promotes inflammation through macrophages

[0171] By using Lyz2-Cre + / - Mice and CREPT flox / flox After hybridization, a macrophage-specific knockout CREPT mouse strain (Lyz2-Cre) was obtained. + / - CREPT flox / flox (For specific procedures, please refer to Example 4). To investigate the effect of CREPT on inflammation in macrophages, this example induced systemic acute inflammation in mice by intraperitoneal injection of lipopolysaccharide (LPS). Six hours later, blood, spleen, and peritoneal macrophages (e.g., ...) were collected from the mice. Figure 7 As shown in Figure A). ELISA was used to detect the levels of inflammatory factors in mouse serum, and the results showed that the TNFα content in macrophage CREPT knockout mice was significantly decreased (e.g., ...). Figure 7 (As shown in B). The content of CD86-positive M1 macrophages was detected by flow cytometry after isolating mouse spleen cells. Figure 7 As shown in Figure C, the content of inflammation-associated M1 macrophages was significantly reduced in CREPT knockout mice. Simultaneously, the content of inflammation-associated M1 macrophages in mouse peritoneal macrophages was also significantly reduced (e.g., Figure 7 (as shown in D).

[0172] The above results indicate that CREPT can promote inflammation in macrophages by promoting the secretion of inflammatory factors and regulating the transformation of macrophages into inflammation-associated M1 macrophages.

[0173] Example 6: Conditional knockout of CREPT can inhibit tumor development

[0174] For the CREPT-specific knockout mice constructed in Example 4, knocking out CREPT without any induction had no effect on the growth of the mice or the infiltration of their immune cells. Flow cytometry analysis of the infiltration levels of immune cells in the spleen and lymph nodes of the mice also showed no effect. Figure 8 ).

[0175] Subsequently, colorectal cancer cells and melanoma cells in mice were subcutaneously injected with MC38 and B16. Figure 9 As shown in A), specific knockout of CREPT in macrophages significantly inhibited tumor growth, and this was true in both mouse tumor models. Figure 9 BE).

[0176] Next, we investigated whether CREPT in macrophages could affect tumor metastasis via tail vein injection. Using MC38 (mouse colon cancer) cells labeled with luciferase, we observed a significant reduction in lung colonization after tumor induction and knockout of CREPT in macrophages. Similarly, tail vein injection of B16 (mouse melanoma cells) yielded the same results. Figure 10 AD).

[0177] Example 7: Conditional knockout of CREPT can inhibit the development of in situ tumors.

[0178] This embodiment uses an AOM / DSS-induced experimental colorectal cancer mouse model to discuss the inhibitory effect of CREPT knockout on in situ tumors. It can be seen that during the induction process ( Figure 11 A) The mice showed significant changes in body weight. After CREPT was knocked out, the mice exhibited a relatively stable increase in body weight compared to wild-type mice in the later stages. Figure 11 B).

[0179] After induction, colon cancer tissue was dissected from the mice. It was observed that tumors showed significant growth in wild-type mice, while in mice with CREPT knocked out from macrophages, both the number and size of tumors were significantly reduced. Figure 11 C). HE staining also shows that after macrophage-specific knockout, the colorectal cancer tumors in mice were relatively small, while in wild-type mice, very obvious tumor formation was still observed. Figure 11 D).

[0180] The above discussion is based on myeloid-specific knockout of CREPT. We also isolated bone marrow-derived macrophages from two mouse groups—myeloid-specific knockout mice and a control group—in vitro, and co-colonized them with wild-type mouse tumor cells MC38 subcutaneously in mice to observe their effect on tumor growth. Figure 12 A). It can be seen that knocking out CREPT significantly reduced the tumor growth-promoting effect in macrophages. Compared with wild-type macrophages, CREPT expression in macrophages did indeed promote tumor growth. Figure 12 B).

[0181] Example 8: Tumor cell supernatant can promote the expression of CREPT in macrophages.

[0182] This embodiment first isolates bone marrow-derived macrophages from wild-type B6 mice, and then stimulates the bone marrow-derived macrophages with the supernatant after two days of tumor cell culture. Then, the supernatant (TCM) after two days of culture of mouse melanoma B16 cells and mouse colon cancer cells MC38 are collected separately. Figure 13 A). After treating mouse bone marrow-derived macrophages with TCM from two different sources for 72 hours, qPCR results showed that tumor-promoting genes were significantly upregulated in the induced macrophages. Figure 13 B). The results showed that CREPT expression in primary mouse bone marrow-derived macrophages increased after 2 days of B16-TCM treatment, and significantly increased after 3 days of treatment. Figure 13 C). After treating macrophages with MC38-TCM and B16-TCM for three days, both CREPT mRNA and protein levels increased, with a more significant increase in CREPT expression after B16-TCM treatment. Figure 13 DE). Similarly, stimulation of the mouse macrophage cell line Raw264.7 with tumor cell supernatant yielded the same results. Figure 13 F)

[0183] Example 9: Knockout of CREPT promotes CD8 in mouse tumor tissue + T cell infiltration

[0184] This study investigates the promoting effect of CREPT on tumor growth in macrophages, exploring how it affects the immune microenvironment or how this promoting effect occurs. First, we examined the infiltration level of T cells, the most important cells in the microenvironment, by isolating tumors from mice and analyzing them using flow cytometry. We observed that after CREPT knockout, CD4+ levels in the mouse tumors decreased. + No significant difference was observed in T cell infiltration. Figure 14 A). However, CD8 + The level of T cell infiltration was significantly increased. Figure 14 B). Similarly, the proportion of INFγ-positive cytotoxic CD8+ T cells was also increased. Figure 14 C).

[0185] Next, we need to investigate whether CREPT exerts its effects in macrophages directly by influencing CD8+ T cells. Using multichromosome histochemistry, patients were divided into those with high CREPT TAM expression and those with low TAM expression. We observed that in the tissues of patients with low CREPT TAM expression, CD8+ T cells were significantly lower. + T cell infiltration is abundant, while in patient tissues with high expression of tumor-associated macrophages in CREPT, CD8...+ T cell infiltration was relatively low. Figure 15 This result is consistent with the results of flow cytometry analysis in mice.

[0186] Example 10: The inhibitory effect of CREPT deficiency on tumor growth is not directly dependent on CD8. + T cells

[0187] To investigate whether CREPT affects CD8 in macrophages + T cells were used to influence tumor growth. Subcutaneous tumors formed in CREPT wild-type mice and macrophage-specifically knocked-out CREPT mice. Simultaneously, two groups were divided and intraperitoneally injected with an NKCD8 antibody to clear CD8 from the mice. + T cells were found to be effective in clearing CD8+ cells from mice. + Following T cell intervention, tumor growth differed between wild-type mice and macrophage-specific knockout mice. This suggests that CREPT may not directly affect CD8. + T cells play a role, CD8 + The decrease in T cell infiltration is more likely a result of later stages. Figure 16 AB).

[0188] Next, flow cytometry was used to analyze the level of macrophage infiltration in mouse tissues after CREPT knockout. It can be seen that the proportion of infiltrating macrophages is also reduced in the tumor tissues of mice with macrophage-specific CREPT knockout. Figure 17 A). However, the proportion of infiltration in some other myeloid cells did not change significantly. Figure 17 (BD). Therefore, it can be concluded that in the tumor tissue of mice with knocked-out CREPT, the removal of CREPT also had a certain impact on cell aggregation.

[0189] Further research investigated whether knocking out CREPT also affected other polarization functions. This was verified both in vivo and in vitro using flow cytometry. We found that knocking out CREPT inhibited the activity of M1 and M2 polarization markers such as CD86 and CD206. Figure 18 Therefore, CREPT has a greater impact on the polarization transition of macrophages than on the traditional M1 or M2 type; rather, it weakens the overall polarization function of macrophages.

[0190] Example 11: Knockout of CREPT directly inhibits tumor cell proliferation and migration (the effect of CREPT-positive macrophages on the tumor microenvironment)

[0191] CREPT wild-type and CREPT knockout macrophages were isolated in vitro and co-cultured with tumor cells. Figure 19 A). It can be seen that macrophages specifically knocked out CREPT have an inhibitory effect on the proliferation level of tumor cells. Figure 19 B). Scratch assays also showed that macrophage-specific knockout of CREPT inhibited tumor cell migration. Figure 19 C).

[0192] In preliminary single-cell data, increased fibroblast aggregation (mIHC staining for α-SMA antibody) was observed in the tissues of patients with high CREPT expression of TAM. Figure 20 AB). Further investigation was conducted to determine whether CREPT could affect fibroblast aggregation or proliferation levels within macrophages. Macrophages and fibroblasts from CREPT knockout mice were isolated and co-cultured. The results showed that in the presence of CREPT in macrophages, fibroblast proliferation and migration were significantly promoted compared to mice with specifically knocked-out CREPT. Figure 20 CD).

[0193] In summary, CREPT has a certain impact on the tumor microenvironment in tumor-associated macrophages: firstly, it can affect CD8. + T cell infiltration can also affect the aggregation and polarization levels of macrophages, and can promote the proliferation and migration of tumor cells and fibroblasts, thereby promoting tumor development.

[0194] Example 12: CREPT-positive TAM regulates the expression of tumor-related genes

[0195] Further investigation was conducted into changes at the gene level in macrophages with CREPT knockout. First, single-cell sequencing analysis was used to analyze the differences in gene expression levels between patients with high CREPT expression and those with low CREPT expression in tumor-associated macrophages. It was observed that several genes were significantly elevated in patients with high CREPT expression associated with tumor-associated tumors (TAMs), such as SPP1 and FN1, which are clinically considered to be associated with poor prognosis, and metalloproteinases like VEGFA and MMP1, which have been shown to promote tumor development and progression in macrophages. Figure 21 AB).

[0196] Similarly, macrophages were isolated from mouse tumor tissue for RNA-seq analysis. In macrophages with CREPT knocked out, some genes were significantly downregulated. Figure 22 (AB). Consistent with human single-cell sequencing data, the expression of genes such as SPP1 and FN1 was found, and the results of these two experiments and analyses corroborate each other.

[0197] Next, qPCR was used to verify these genes. Indeed, knocking out CREPT significantly downregulated these genes, while overexpressing CREPT significantly restored their expression. Figure 23 AB).

[0198] Example 13: CREPT participates in regulating the maintenance of the macrophage NF-κB signaling pathway

[0199] This embodiment explores how CREPT regulates changes in the expression of certain tumor-promoting genes. Signal pathway enrichment was performed using human single-cell data and mouse RNA-seq data, both of which enriched the NF-κB pathway (…). Figure 24 Therefore, it can be assumed that CREPT may exert its regulatory effect on the expression of this tumor-promoting gene primarily by influencing the NF-κB signaling pathway.

[0200] We first verified this using a luciferase reporter assay. We found that overexpression of CREPT in 293T cells significantly increased the transcriptional activity of NF-κB p65, while knockdown of CREPT significantly downregulated this activity. Therefore, we can conclude that CREPT can indeed affect the transcriptional function of p65. Figure 25 AB).

[0201] To identify the interaction between CREPT and the p65 receptor, an in vitro protein pull-down experiment was designed, with the following steps:

[0202] 1) Transfect the eukaryotic protein to be tested into HEK293T cells. After 24-48 hours of transfection, collect the cells with RIPA lysis buffer and prepare cell lysis buffer.

[0203] 2) Take 30 μl of cell lysis buffer as the input for transfection, add 2x loading buffer, and boil the sample.

[0204] 3) Add purified GST and GST fusion protein to the remaining lysis buffer. Add 30 μl of equilibrated GST Sepharose-4B beads to each sample and incubate overnight at 4°C by rotation.

[0205] 4) Centrifuge at 4℃, 3000rpm for 2min, and discard the supernatant.

[0206] 5) 1 ml of PBST containing 5% Tween 20 resuspended the beads.

[0207] 6) Repeat steps 4-5 four times.

[0208] 7) Centrifuge and discard the supernatant. Add 30-50 μl of loading buffer, mix well, centrifuge briefly, and boil the sample.

[0209] 8) Run the gel, transfer the membrane, incubate the antibody, and detect the protein binding.

[0210] Immunoprecipitation revealed that CREPT interacts with the p65 transcription factor downstream of NF-κB. Overexpression of both CREPT and p65 proteins in 293T cells confirmed this interaction. Figure 26 A). The GST pull-down experiment also found that they interact ( Figure 26 B).

[0211] Since the p65 transcription factor contains different domains, we further investigated at which site p65 can interact with CREPT. We constructed p65 truncated versions of different lengths and ultimately discovered the possible interaction site between CREPT and p65, which is located at amino acid positions 332 to 410 of p65. Figure 26 C).

[0212] CREPT is also divided into the CID domain and the CCT domain. Further investigation revealed which domain of CREPT interacts. Ultimately, it was found that p65 can interact with the CID domain of CREPT. Figure 26 DE).

[0213] We also performed an endogenous immunoprecipitation experiment in macrophages, and found that CREPT could interact with p65 in both mouse cell lines and isolated primary cells. Figure 27 AB).

[0214] The above section demonstrates that CREPT can interact with p65 and regulate downstream transcription levels, but how this transcriptional activity is regulated requires further investigation. Firstly, p65's most important transcriptional function is significantly altered by its phosphorylation level. Our initial goal was to see if knocking out CREPT affected p65 phosphorylation levels. After knocking out CREPT, the results of detecting p65 phosphorylation levels using a phosphorylated p65 antibody showed no effect. Figure 28 A). Over time, it can be seen that CREPT plays an important role in maintaining p65 phosphorylation levels. Figure 28 B).

[0215] Further investigation will reveal whether CREPT can exert its transcriptional promoting effect by binding to the promoter regions of the genes we previously identified.

[0216] We further validated this using CHIP-qPCR experiments. The steps for the chromatin immunoprecipitation (CHIP-qPCR) experiment are as follows:

[0217] 1) Formaldehyde crosslinking: Ensure the culture medium in the petri dish is 10 mL, then directly add 270 μL of 37% formaldehyde to a final concentration of 1%. Immediately mix thoroughly from all sides and incubate on a shaker at room temperature for 10-11 min.

[0218] 2) Termination of cross-linking: Add 1 mL of 1.25 M glycine to the culture medium, mix quickly, and incubate in a shaker at room temperature for 8-10 min.

[0219] 3) Cell harvesting: Discard the culture medium and wash twice with pre-cooled PBS. Scrape off the cells and transfer them to a 1.5 mL centrifuge tube. Centrifuge at 2000 rpm, 4°C, for 2 min. Discard the supernatant. The sample can now be used directly for subsequent experiments or stored long-term at -80°C.

[0220] 4) Cell lysis: Prepare SDS lysis buffer and add protease inhibitor. Resuspend the cells in 250 μL of lysis buffer, mix well, try to avoid generating bubbles, and incubate on ice for 40 min.

[0221] 5) Ultrasound: Use a Minichiller 300 (Diagenode) sonicator with a 10-second sonication cycle followed by a 30-second pause, repeating approximately 10 times. The final DNA fragment should be between 200-1000 bp, with the main band around 500 bp.

[0222] 6) Centrifuge at 14000 rpm and 4℃ for 10 min. Transfer the supernatant to another clean 1.5 mL centrifuge tube.

[0223] 7) Take 10 μL of supernatant and add it to 90 μL of ChIP dilution buffer as input.

[0224] 8) Take 100 μL of supernatant and add 900 μL of ChIP dilution buffer, then add 2 μg of the corresponding antibody. Take another 100 μL of supernatant and add 900 μL of ChIP dilution buffer, then add the corresponding amount of IgG. Incubate overnight at 4°C by rotation.

[0225] 9) Add 30 μL of equilibrated Protein G beads to each sample and incubate at 4°C for 2-4 hours by rotation.

[0226] 10) Centrifuge at 3000 rpm, 4℃ for 1 min. Discard the supernatant, and wash the beads with buffer in the following order, adding 1 mL each time, rotating and washing at 4℃ for 10 min, then centrifuging at 3000 rpm for 2 min. Wash twice with TE buffer, and once with other buffers (low-salt buffer, high-salt buffer, and LiCl buffer, respectively).

[0227] 11) Centrifuge and discard the supernatant. Add 100 μL of freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) and elute by rotation at room temperature for 30 min.

[0228] 12) Centrifuge and collect the supernatant. This is the sample tube and the IgG control tube.

[0229] 13) De-crosslinking: Add 4 μL of 5M NaCl to the input tube, sample tube and IgG control tube respectively, and incubate at 65℃ for 4 h.

[0230] 14) Protein removal: Briefly centrifuge the sample tubes, add 2 μL of 0.5M EDTA, 2 μL of 1M Tris HCl (pH 6.5), and 2 μL of 10 mg / ml proteinase K. Mix well, centrifuge, and incubate at 42°C for 2 hours.

[0231] 15) Add 150 μL of water to all sample tubes, then add 250 μL of binding buffer, and extract DNA according to the instructions of the Tiangen Ultra-Thin DNA Extraction Kit.

[0232] The obtained DNA samples were tested to determine the binding status of CREPT to the gene promoter region.

[0233] The buffer formulations used in step 10) above are as follows:

[0234] Low salt buffer: 0.1% SDS, 1.0% Triton X-100, 2.0mM EDTA, 20.0mM Tris-HCl pH 8.1, 150mM NaCl.

[0235] High-salt buffer: 0.1% SDS, 1.0% Triton X-100, 2.0mM EDTA, 20.0mM Tris-HCl, pH 8.1, 500mM NaCl.

[0236] LiCl buffer: 0.25M LiCl, 1.0% Igepal-CA630, 1.0% deoxycholic acid, 1.0mM EDTA, 10.0mM Tris-HCl, pH 8.1.

[0237] TE buffer: 10.0mM Tris-HCl pH 8.0, 1.0mM EDTA pH 8.0.

[0238] The results showed that in the presence of CREPT and p65, the binding of these two genes, VEGF and SPP1, in the promoter region was significantly promoted. Therefore, CREPT can promote transcriptional maintenance by maintaining the phosphorylation level of p65, thereby promoting the expression of downstream genes.

[0239] In the tumor microenvironment, we have observed a type of tumor-associated macrophage that highly expresses CREPT. These macrophages can promote tumor growth by directly promoting the proliferation of tumor cells or by promoting the proliferation and migration of fibroblasts. The ultimate effect is the inhibition of CD8. + T cell infiltration. In these tumor-associated macrophages with high CREPT expression, it can interact with p65, thereby promoting the transcription of downstream genes through the maintenance of p65 phosphorylation levels. Figure 29 ).

[0240] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of inhibition of CREPT-positive tumor-associated macrophages (TAMs) in the preparation of drugs that regulate the infiltration of macrophages associated with tumors / inflammatory diseases.

2. The application according to claim 1, characterized in that, The tumor includes solid carcinoma; The inflammatory diseases mentioned include polymyositis, diabetic nephropathy, rheumatoid arthritis, and lupus nephritis; The regulation of tumor / inflammatory disease-associated macrophage infiltration includes reducing serum inflammatory factor levels and reducing M1 macrophage content; Preferably, the tumor includes one or more of the following: cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, liver cancer, kidney cancer, glioma, gastric cancer, pancreatic cancer, prostate cancer, melanoma, and endometrial cancer.

3. The application of inhibition of CREPT-positive tumor-associated macrophages (TAMs) in the preparation of agents that regulate the tumor microenvironment, characterized in that... The regulation of the tumor microenvironment is achieved by reducing the activity of CREPT-positive tumor-associated macrophages (TAMs). Preferably, the tumor includes solid carcinoma; preferably, the tumor includes one or more of the following: cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, liver cancer, kidney cancer, glioma, gastric cancer, pancreatic cancer, prostate cancer, melanoma, and endometrial cancer.

4. The application of inhibition of CREPT-positive tumor-associated macrophages (TAMs) in the preparation of drugs for treating tumors or promoting T lymphocyte activity; The drug reduces the number of tumors, lowers the tumor burden, inhibits the proliferation and migration of tumor cells, inhibits tumor growth, and reduces tumor colonization. The T-lymphocyte-promoting effect includes CD8 + Increased infiltration of T cells and INFγ-positive cytotoxic CD8+ T cells; Preferably, the tumor includes solid carcinoma; preferably, the tumor includes one or more of the following: cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, liver cancer, kidney cancer, glioma, gastric cancer, pancreatic cancer, prostate cancer, melanoma, and endometrial cancer.

5. The application according to any one of claims 1 to 4, characterized in that, The agents that inhibit CREPT-positive tumor-associated macrophages (TAMs) include agents that affect CREPT protein expression, agents that knock out CREPT protein, agents that alter CREPT protein, or agents that degrade CREPT protein. The formulations that affect CREPT protein expression include nucleic acid reagents or vectors containing nucleic acid fragments that inhibit CREPT protein expression; The formulations for knocking out the CREPT protein include reagents for homologous recombination and gene editing to eliminate the CREPT gene; The formulation that alters the CREPT protein includes reagents that change the structure of the CREPT protein; The formulations that degrade CREPT protein include those using the PROTAC, molecular gel, LYTAC, MoDE, ATAC, Apt-LYTAC, AbTAC, PROTAB, REULR, KineTAC, IFLD, ATTEC, AUTAC, or AUTOTAC pathways. The drug includes a small molecule inhibitor of the CREPT protein; Preferably, the nucleic acid reagent is selected from siRNA, shRNA, microRNA, piRNA, and ASO; Preferably, the vector containing the nucleic acid fragment that inhibits CREPT protein expression includes adenovirus, adeno-associated virus, slow expression virus, and retrovirus; Preferably, the reagents for homologous recombination and gene editing to eliminate the CREPT gene include the CRISPR / Cas9 system.

6. The application according to claim 5, characterized in that, Regulating the tumor microenvironment includes promoting CD8 + T cell infiltration, weakening of macrophage overall polarization function, reduction of fibroblast proliferation and migration levels, inhibition of tumor-promoting gene expression, and reduction of p65 phosphorylation levels; Preferably, the weakening of the overall polarization function of the macrophage is a reduction in the macrophage's polarization towards M1 and M2 types; Preferably, the tumor-promoting genes include SSP1, FN1, and VEGFA.

7. A reagent, characterized in that, The reagent is used to detect CREPT-positive tumor-associated macrophages (TAMs) and / or to detect the proportion of CREPT-positive tumor-associated macrophages (TAMs) to the total number of macrophages; Preferably, the reagent includes a multicolor histochemical reagent; Preferably, the reagents include reagents for labeling CREPT and reagents for labeling CD68 macrophages.

8. The use of the reagent of claim 7 or CREPT-positive tumor-associated macrophages (TAMs) in the preparation of the kit, characterized in that, The kit is used to assess an individual's risk of developing a tumor or to diagnose whether an individual has a tumor. The proportion of CREPT-positive tumor-associated macrophages (TAMs) to the total number of macrophages is positively correlated with the individual's risk of developing a tumor.

9. The use of the reagent of claim 7 or CREPT-positive tumor-associated macrophages (TAMs) in the preparation of the kit, characterized in that, The kit is used to assess the tumor development stage of an individual with a tumor, and the proportion of CREPT-positive tumor-associated macrophages (TAMs) to the total number of macrophages is positively correlated with the tumor development stage of the individual with a tumor.

10. The application according to claim 8 or 9, characterized in that, The tumor includes solid carcinoma; Alternatively, the sample used to detect CREPT-positive tumor-associated macrophages (TAMs) may be derived from cancerous tissue or adjacent tissue. Preferably, the tumor includes one or more of the following: cervical cancer, ovarian cancer, colorectal cancer, lung cancer, breast cancer, esophageal cancer, liver cancer, kidney cancer, glioma, gastric cancer, pancreatic cancer, prostate cancer, melanoma, and endometrial cancer.

11. The application according to claim 8, characterized in that, The presence of CREPT-positive tumor-associated macrophages (TAMs) accounting for ≥1% of the total macrophage count indicates that the individual is at risk of developing colorectal cancer or has colorectal cancer. The number of CREPT-positive tumor-associated macrophages (TAMs) accounting for <1% of the total number of macrophages indicates that the individual is not at risk of developing colorectal cancer or has not developed colorectal cancer.

12. The application according to claim 9, characterized in that, An individual with colorectal cancer whose CREPT-positive tumor-associated macrophages (TAMs) account for ≥10% of the total macrophages is considered to have clinical stage II, III, or IV colorectal cancer. The presence of CREPT-positive tumor-associated macrophages (TAMs) accounting for less than 10% of the total macrophage count in individuals with colorectal cancer indicates that the tumor is in clinical stage I.

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