New target of ID1 in tumor-associated macrophages as anti-tumor therapeutic drug and application thereof

Abstract

The invention belongs to the field of biological medicine, and discloses a novel target of ID1 protein in tumor-associated macrophages (TAM) as an anti-tumor therapeutic drug and application of the novel target. The invention discloses that the expression of ID1 in TAM is obviously higher than that of para-carcinoma tissue macrophages for the first time, and the ID1 has a tumor promoting effect. Specifically, ID1 highly expressed in TAM can enhance the dryness of tumor cells and promote tumor growth and metastasis by obstructing CD8 + T cell recruitment in tumors. The tumor promoting phenotype of the TAM can be inhibited by reducing the expression of ID1 in the TAM through gene editing. The sensitivity of tumor cells including colon cancer, liver cancer and pancreatic cancer to chemotherapeutic drugs and immune checkpoint inhibitors can be improved by reducing the expression of ID1 in TAM by using a small molecule compound. The invention discloses a new tumor regulation and control promoting signal in a tumor immune microenvironment TAM, and the ID1 protein in the provided TAM can be used as a new tumor treatment target for anti-tumor drug screening. The method lays a theoretical foundation for an anti-tumor research and development strategy, and has a huge potential conversion application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an application of an ID1 gene / protein as a tumor treatment target. Background Art

[0002] Tumors are a major public health problem worldwide and pose a serious threat to human life and health. Tumor development is a complex multi-step process, including transformation, proliferation, angiogenesis, immune regulation, and metastasis. However, the occurrence and development of tumors are not only induced by genetic mutations accumulated in tumor cells themselves, but also affected by surrounding non-malignant cells.

[0003] Tumor associated macrophages (TAMs) are macrophages that infiltrate tumor tissues and are the most abundant innate immune cells in tumor tissues. TAMs are highly plastic and often exhibit a tumor-promoting M2 phenotype. They are important inducing factors for tumor occurrence and metastasis, and are closely related to resistance to chemotherapy and immunotherapy. Many studies have found that targeted inhibition of the number or activity of TAMs can significantly improve the resistance of tumor patients to chemotherapy drugs, or increase the responsiveness of patients to immune checkpoint inhibitor therapy. Therefore, anti-tumor strategies targeting TAMs show great potential for clinical application, but the tumor-promoting mechanism of TAMs has not yet been fully elucidated, and new druggable targets for TAMs are urgently needed to be confirmed.

[0004] Inhibitor of DNA binding (ID) is a family of transcriptional regulator proteins with a helix-loop-helix (HLH) structure. This family has four members, ID1-ID4, which are involved in the regulation of various cell functions, including development, aging, differentiation, angiogenesis, and cell migration. One of its important members, ID1, exhibits tumor-promoting activity in various tumors. John E. Dick's team published an article in the journal Cancer Cell, proving that ID1 and ID3 maintain the self-renewal ability of colon cancer stem cells by upregulating the cell cycle-related protein p21, and knocking down ID1 and ID3 can significantly enhance the sensitivity of colon cancer cells to the chemotherapy drug oxaliplatin. However, the expression of ID1 in TAM, its function in TAM, and whether it has a tumor-promoting effect have not been reported. Therefore, this field still needs to further clarify the relationship between the ID1 protein expressed in TAM and tumor progression, and on this basis, find drugs that can affect the expression of ID1 protein to achieve the purpose of treating the disease.

[0005] In view of this, the present invention experimentally verified for the first time that knocking out ID1 in TAM can significantly reverse the tumor-promoting effect of TAM, and ID1 in TAM can be used as a new target for anti-tumor therapeutic drugs and as a new target for improving the responsiveness of chemotherapy and immunotherapy. So far, there has been no report on the relationship between ID1 in TAM and tumor progression.

[0006] In view of the above problems existing in the prior art, the object of the present invention is to provide ID1 protein in TAM as a new target for anti-tumor therapeutic drugs and its application in the preparation of anti-tumor drugs.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The first aspect of the present invention provides use of an ID1 inhibitor in the preparation of a drug for preventing and / or treating tumors.

[0009] Furthermore, the ID1 inhibitor includes oligonucleotides (shRNA, siRNA) that inhibit ID1 expression and small molecule compounds (ML323, FT-3171, SJB3-019A) that inhibit ID1 expression.

[0010] Furthermore, the ID1 inhibitor reverses the tumor-promoting effect of TAMs by inhibiting ID1 transcription in TAMs or promoting the degradation of ID1 in TAMs.

[0011] In a specific embodiment of the present invention, the inventors of the present invention creatively discovered for the first time through a large number of experimental studies that the expression of ID1 in colon cancer TAMs is significantly higher than that in normal adjacent macrophages, and that high expression of ID1 in TAMs is positively correlated with poor survival prognosis in patients with colon cancer. After using transgenic mice to construct transgenic mice with myeloid cell-specific knockout of ID1, the inventors used a variety of subcutaneous transplant tumor models and tumor metastasis models of colon cancer, liver cancer, etc. to prove that knocking out ID1 in TAMs inhibits tumor progression. Specifically, highly expressed ID1 in TAMs can inhibit the transcription and secretion of CCL4, hindering the proliferation of CD8 + T cell recruitment and enhancement of the stemness characteristics of tumor stem cells by inhibiting SerpinB2 transcription and secretion. The use of ML323, an inhibitor of ID1 deubiquitinase USP1, can significantly inhibit tumor growth and increase tumor effector CD8 + The compound significantly reduced the anti-tumor effect in tumor-bearing mice with ID1 knockout in myeloid cells. These results indicate that ID1 is a new target for anti-tumor drugs and that ID1 inhibitors can be used in the prevention and / or treatment of tumors.

[0012] Furthermore, the ID1 inhibitors described in the present invention refer to any substance that can downregulate the transcription of ID1, protein stability, and reduce the cancer-promoting effect of ID1. These substances can be used in the present invention as substances useful for downregulating ID1, and thus can be used to prevent and / or treat tumor-related diseases. The ID1 inhibitors include but are not limited to: small nucleic acid drugs, small molecule compounds, active polypeptides, etc.

[0013] Furthermore, the tumors described in the present invention include colorectal cancer, pancreatic cancer, liver cancer, gastric cancer, lung cancer, etc., that is, the ID1 inhibitor described in the present invention has a broad spectrum of anti-tumor activity and can be used in the prevention and / or treatment of various tumors.

[0014] The second aspect of the present invention provides a composition for treating and / or preventing tumors, the composition comprising any of the following drug combinations:

[0015] (1) ID1 inhibitors and chemotherapy drugs;

[0016] (2) ID1 inhibitors and immune checkpoint inhibitor drugs.

[0017] Preferably, the ID1 inhibitor is selected from small molecule compounds.

[0018] Preferably, the small molecule compound includes ML323.

[0019] Preferably, the chemotherapy drug includes 5-fluorouracil, oxaliplatin, capecitabine or irinotecan.

[0020] Preferably, the immune checkpoint inhibitor drug is selected from antagonistic antibodies targeting CTLA-4, PD-1 antibodies, and PD-L1 antibodies.

[0021] The present invention also provides the use of the composition in preparing a drug for treating and / or preventing tumors.

[0022] The prevention is conventional prevention in the art, preferably refers to preventing or reducing the occurrence of tumors after use when possible tumor factors exist. The treatment is conventional treatment in the art, preferably refers to reducing the degree of tumors, or curing tumors to normalize them, or slowing down the progression of tumors.

[0023] Preferably, the tumor comprises a solid tumor with high expression of ID1.

[0024] Preferably, the solid tumors include colon cancer, pancreatic cancer, liver cancer, gastric cancer, and lung cancer.

[0025] The present invention discloses the following technical effects:

[0026] (1) The present invention first discovered that ID1 is highly expressed in TAMs, which enhances colon cancer stemness and inhibits CD8 + T cell infiltration promotes immune escape and the growth of colon cancer and liver cancer. At the same time, it is proposed for the first time that TAMs with high expression of ID1 enhance tumor stemness and inhibit T cell recruitment, which is the reason why colon cancer is insensitive to chemotherapy or immune checkpoint inhibitors; targeted inhibition of ID1 in TAMs may be a new strategy to improve the effect of immunotherapy for colon cancer.

[0027] (2) The lentivirus for interfering with ID1 expression prepared by shRNA in the present invention can reduce the expression of ID1 in TAMs of patients with colon cancer, upregulate the transcription and secretion of SerpinB2 and CCL4, inhibit the stemness of tumor cells, and increase the expression of CD8 + Migration ability of T cells.

[0028] (3) The small molecule compounds inhibiting ID1 of the present invention can reduce the expression of ID1 in tumor-associated macrophages, on the one hand, by relieving the transcriptional inhibitory effect of ID1 on Ccl4 and increasing CD8 + T cell infiltration and killing effect can prevent immune escape; on the other hand, it can reduce the stemness of colon cancer by relieving the transcriptional inhibitory effect of ID1 on Serpinb2.

[0029] (4) The anti-tumor drug composition based on inhibiting ID1 activity provided by the present invention has a wide range of applications in improving the therapeutic effect of chemotherapy or immune checkpoint inhibitors on tumor patients, as well as in formulating clinical anti-tumor treatment strategies.

[0030] (5) The present invention can screen out suitable target patient populations for the anti-tumor drug composition based on inhibiting ID1 activity, namely patients with solid tumors such as colorectal cancer, pancreatic cancer, liver cancer, gastric cancer, lung cancer, etc., and more preferably colorectal cancer patients with high ID1 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 Increased expression of ID1 in TAMs is associated with poor clinical prognosis in patients with colorectal cancer (CRC); (A) Representative immunofluorescence staining images of ID1 and CD68 in tumor tissues and adjacent normal tissues of CRC patients; (B) CD68 +Statistical data of ID1 expression in TAMs cells; (C) CD68 + The overall survival rate of CRC patients corresponding to the expression level of ID1 in TAMs cells;

[0033] Figure 2 TAMs with high expression of ID1 promote the growth of colon cancer and liver cancer; (A) MC38 cells and TAMs with high expression of ID1 Lyz-KO or Id1 f / f Schematic diagram of the secondary inoculation of TAMs isolated from mice into the subcutaneous tissue of C57BL / 6J mice after mixing; (B) Tumor growth curves of the two groups of mice; (C) Tumor weights of the two groups of mice; (D) Schematic diagram of the intratumoral injection of TAMs culture supernatant into the MC38 subcutaneous transplant tumor model; it: intratumor injection; (E) Tumor growth curves of the two groups of mice; (F) Tumor weights of the two groups of mice; (G) Schematic diagram of the intratumoral injection of TAMs culture supernatant into the H22 subcutaneous transplant tumor model; it: intratumor injection; (H) Tumor growth curves of the two groups of mice; (I) Tumor weights of the two groups of mice;

[0034] Figure 3 TAMs with high expression of ID1 promote colon cancer immune escape and enhance colon cancer stemness; (A) Figure 2 Representative CD8 in tumor tissue of the mouse model shown in Figure A + T cell immunofluorescence images and statistical graphs; (B) Figure 2 CD8 isolated from tumor tissue of the mouse model shown in Figure A + Interferon-γ (IFN-γ) in T cells + and Granzyme B + Percentage; (C) from Id1 Lyz-KO and Id1 f / f Isolation of CD45 from tumor tissues of subcutaneous tumor models in mice - EpCAM + The expression level of Cd44 was detected by flow cytometry in tumor cells;

[0035] Figure 4 TAMs with high expression of ID1 maintain colon cancer stemness by affecting the FAK-AKT signaling pathway in tumor cells; (A) Lyz-KO and Id1 f / f Schematic diagram of transcriptome sequencing of tumor cells isolated from subcutaneous transplanted tumors in mice; (B) KEGG analysis of Id1 Lyz-KO Bubble diagram of the top 20 signaling pathways that were significantly downregulated in the group;

[0036] Figure 5TAMs with high expression of ID1 promote tumor immune escape and maintain colon cancer stemness by inhibiting the secretion of CCL4 and SerpinB2; (A) Magnetic bead sorting of Id1 Lyz-KO Mouse and Id1 f / f F4 / 80 in the mouse MC38 subcutaneous transplant tumor model + Schematic diagram of transcriptome sequencing of macrophages; (B) Figure 5 Volcano plot of differentially expressed genes between the two groups shown in A; (C) detection of Id1 Lyz-KO and Id1 f / f The abundance of Ccl4 protein in the culture supernatant of TAMs isolated from mice; (D) Detection of Id1 Lyz-KO and Id1 f / f The protein abundance of Serpinb2 in the culture supernatant of TAMs isolated from mice; (E) Id1 Lyz-KO or Id1 f / f Mouse BMDM-derived TAMs were simultaneously infected with lentivirus expressing Ccl4-shRNA and Serpinb2-shRNA, and the cell culture supernatant was collected and injected intratumorally into the subcutaneous transplanted tumor model; (F) Tumor weights of the two groups shown;

[0037] Figure 6 To isolate TAMs from patients with colon cancer, we used lentivirus to interfere with the expression of ID1 in TAMs and to detect the effects of ID1 knockout on tumor cell stemness and CD8 + Effects of ID1 knockout on TAMs on the migration rate of T cells. (A) Schematic diagram of patient TAMs isolation and virus infection; (B) Effects of ID1 knockout on TAMs on the stemness of HCT116 cells; (C) Effects of ID1 knockout on TAMs on CD8 + Effect of T cell migration rate.

[0038] Figure 7 To study the effect of ID1 inhibitor on tumor growth by subcutaneous tumor formation experiment of mouse colon cancer CT26; (A) Schematic diagram of different drug administration treatments of subcutaneous CT26 cells in mice; (B) Tumor growth curves of mice in different groups; (C) CD8 infiltrating in tumor tissues of mice in different groups + Immunofluorescence images and statistical graphs of T cells; (D) Flow cytometry detection of the expression of Cd44 in tumor tissues of mice in different groups;

[0039] Figure 8 To investigate whether ML323 exerts its effects by reducing the expression of ID1 in TAMs, wild-type C57BL / 6J mice and transgenic mice with myeloid-specific knockout of ID1 were used. (A) Schematic diagram of mouse grouping and construction of subcutaneous transplant tumor model; (B) Tumor growth curves of mice in each group; (C) Statistical data of tumor weight of mice in each group.

[0040] Fig. 9 To study the effect of ID1 inhibitor combined with chemotherapy drug combination on tumor growth through subcutaneous tumor formation experiment of mouse colon cancer cell CT26; (A) Schematic diagram of different drug administration treatments of subcutaneous implantation of CT26 cells in mice; (B) Tumor growth curves of mice in different groups; (C) Tumor weights of mice in different groups;

[0041] Fig.10 To study the effect of ID1 inhibitor combined with immunotherapy drug combination on tumor growth through subcutaneous tumor formation experiment of mouse colon cancer cell CT26; (A) Schematic diagram of different drug administration treatments of subcutaneous implantation of CT26 cells in mice; (B) Tumor growth curves of mice in different groups; (C) Tumor weights of mice in different groups; (D) Flow cytometry detection of CD8 + T cell results; (E) CD8 + IFN-γ and Granzyme B expression in T cells;

[0042] Fig.11 The expression of ID1 in various tumor tissues. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] The main experimental materials and reagents involved in the following embodiments of the present invention are:

[0049] (1) Antibodies

[0050] ID1 polyclonal antibody (Proteintech, cat.#18475-1-AP), CD68 antibody (Abcam, cat.#ab955), CD8a (mouse) (Cell Signaling Technology, cat.#98941S), CD14 antibody (Biolegend, cat.#325606), APC anti-mouse / human CD44 antibody (Biolegend, cat.#103011), PE anti-mouse CD45 recombinant antibody (Biolegend, cat.#157604), APC anti-mouse CD45 antibody (Biolegend, cat.#103111), PE anti-mouse CD326 (Ep -CAM) antibody (Biolegend, cat.#118205), PerCP / Cyanine5.5 anti-mouse CD3ε antibody (Biolegend, cat.#100328), APC / Cyanine7 anti-mouse CD8a antibody (Biolegend, cat.#100714), PE anti-mouse IFN-γ antibody (Biolegend, cat.#505808), PE / Cy7 antibody human / mouse Granzyme B recombinant antibody (Biolegend, cat.#372213), PE anti-mouse F4 / 80 recombinant antibody (Biolegend, cat.#157304), InVivoMAb anti-mouse CTLA-4 (Bio X Cell, cat.#BE0164).

[0051] (2) Reagents

[0052] Matrigel (Corning, 354230), ionomycin (Merck, IO634), GolgiStop (BD Biosciences, 554724), Brefeldin A solution (1000×) (Biolegend, 4200601), Phorbol 12-myristate 13-acetate (PMA) (Merck, P8139), collagenase IV (Merck, C4-BIOC), deoxyribonuclease I (Merck, 10104159001), mounting medium (containing DAPI) (ZSGB-BIO, ZLI-9557), human lymphocyte separation medium (Solarbio, P8900), Recombinant murine m-CSF (Peprotech, 315-02), Recombinant murine IL-4 (Peprotech, 214-14), ML323 (Selleck, S7529, CAS: 1572414-83-5, molecular formula: C 23 H 24 N6, molecular weight: 384.48), 5-fluorouracil (Selleck, S1209, CAS: 51-21-8, molecular formula: C4H3FN2O2, molecular weight: 130.08).

[0053] (3) Test kit

[0054] Four-color multiplex fluorescent immunohistochemistry staining kit (TSA-RM) (PANOVUE Biotechnology Co., LTD, 1100020), cell fixation / permeabilization solution (BD Biosciences, 554714), mouse CC-motif chemokine 4 (C-Cmotifchemokine 4, Ccl4) ELISA detection kit (4ABiotech, MOEB0088), mouse plasminogen activator inhibitor 2 (plasminogen activator inhibitor 2, Serpinb2) ELISA detection kit (4A Biotech, MOEB1945), Anti-PE microbeads (Miltenyi Biotec, 130-048-801), CD326 (EpCAM) microbeads, mouse (Miltenyi Biotec, 130-105-958).

[0055] (4) Cell lines

[0056] Mouse colon cancer cells including MC38, CT26, mouse liver cancer cell line H22, mouse pancreatic cancer cell line Pan02

[0057] (5) ID1 knockdown shRNA lentiviral vector

[0058] The shRNA sequence was: 5′-GAGGAAYYACGTGCTCTGT′, and the lentiviral vector was pHBLV-U6-ZsGreen-Puro, which was commissioned to Hanbio Biotech (Shanghai) Co., Ltd. for construction.

[0059] (6) Experimental animals

[0060] C57BL / 6J mice (male, 6 weeks old), BALB / C mice (male, 6 weeks old), Id1 gene conditional knockout mice (Id1 f / f ) and Lyz-cre mice were purchased from Saiye (Suzhou) Biotechnology Co., Ltd., and the mating of the two can obtain myeloid-specific Id1 knockout transgenic mice (Id1 Lyz-KO ), Id1 Lyz-KO and Id1 f / f The mice were bred by our research group using conventional methods in the art.

[0061] Some of the experiments involved in the following embodiments of the present invention have the following specific steps:

[0062] (1) Construction of subcutaneous transplanted tumor model in mice

[0063] Cell preparation: Trypsinize adherent cells, terminate digestion with serum, blow off the cells with PBS, centrifuge at 1000 rpm for 5 min at room temperature. After counting, dilute the cells to 2×10 with PBS containing 40% low growth factor Matrigel. 6 cells / mL, stored on ice. Animal preparation: Mice were randomly divided into groups, with at least 6 mice in each group, and the hair under the right armpit was shaved. Subcutaneous injection: 100 μL of cell suspension was drawn with a 1 mL syringe, and after the air bubbles were evacuated, the skin under the right armpit of the mouse was lifted and injected. 2×10 CT26 cells, MC38 cells, H22 cells or Pan02 cells were implanted subcutaneously in BALB / C mice. 5 In the therapeutic experiment, the ID1 inhibitor or chemotherapy drug was intraperitoneally injected three days later, and the CTLA-4 antagonist antibody (abbreviated as CTLA-4Ab) was intraperitoneally injected six days after the subcutaneous implantation of CT26 cells or Pan02 cells.

[0064] Tumor size measurement: On the fourth or fifth day after subcutaneous implantation of CT26 cells or Pan02 cells, the length and width of the mouse tumor were measured with a vernier caliper, and the tumor volume was calculated using the following formula: volume = 0.5 × length × width × width. The tumor was measured every other day, and samples were collected on the 20th day to draw growth curves. The weight of the tumors in each group was weighed, and the tumor inhibition rate was calculated using the following calculation method: tumor inhibition rate = (1-tumor volume of the combined drug group / tumor volume of the control group) × 100%

[0065] (2) Establishment of stable cell lines

[0066] Id1 Lyz-KO and Id1 f / f The leg bones of mice were removed and ground with DMEM medium to obtain bone marrow-derived cells. The cell suspension was filtered with a 70 μm cell sieve to obtain a single cell suspension. The suspension was centrifuged at 2000 rpm for 5 min, and the red blood cells were resuspended and lysed with 1 mL of red blood cell lysis buffer. After two minutes, the suspension was centrifuged at 2000 rpm for 5 min to obtain bone marrow-derived macrophages.

[0067] Inoculate Id1 in cell culture dishes Lyz-KO and Id1 f / f Mouse bone marrow-derived macrophages were cultured at 37°C, 5% CO2, and 8 μg / mL of polybrene and lentivirus expressing mouse Ccl4 short hairpin RNA (shRNA) and mouse Serpinb2 shRNA were added. The cells were cultured overnight at 37°C, 5% CO2. After 24 hours, new cell culture medium was replaced, and 20 ng / mL of interleukin 4 (IL-4) and 20 ng / mL of macrophage colony-stimulating factor (m-CSF) were added to stimulate bone marrow-derived macrophages in vitro to obtain the TAMs phenotype, and TAMs with stable knockdown of Ccl4 and Serpinb2 expression were obtained.

[0068] (3) Flow cytometry to detect immune profiles

[0069] The tumor tissue of the tumor-bearing mouse was removed and fresh tumor tissue was cut into 1 mm 3 The small pieces were soaked in digestion buffer (containing collagenase IV (200 U / mL) and deoxyribonuclease I (100 mg / mL)), placed on a shaker, incubated at 110 rpm and 37°C for 45 min, and prepared into a single cell suspension. The T cell content and the expression of its effector molecules were determined. + For T cell ratio, anti-CD45, anti-CD3 and anti-CD8 antibodies were added to the single cell suspension and incubated at room temperature in the dark for 20 min. The cells were then washed three times with MACS buffer (PBS solution containing 0.5% fetal bovine serum and 2 mM EDTA) and resuspended.

[0070] CD8+ T cells as a percentage of total cells in the tumor = CD45 + T cell %×CD3 + / CD45 + T cell % × CD8 + / CD3 + T% cells.

[0071] In order to determine the expression level of effector molecules in effector T cells, T cells were enriched by density gradient centrifugation and cultured in a medium containing PMA (5 ng / mL), ionomycin (500 ng / mL), brefeldinA (1:1000) and Golgi stop (1:1000) at 37°C for 4 h. Anti-CD45, anti-CD3 and anti-CD8 antibodies were added and incubated at room temperature in the dark for 20 min for surface staining. The cells were then washed three times with MACS buffer and resuspended with 1 mL of cell fixation / permeabilization solution (BD Biosciences) and fixed overnight at 4°C in the dark. The next day, the cells were washed with Perm / Wash buffer and incubated with anti-interferon-γ (IFN-γ) + The cells were stained with anti-Granzyme B antibody at room temperature in the dark for 30 min, and the cells were washed three times with MACS buffer. + IFN-γ in T cells + and granzyme B + Percentage (calculation method = IFN-γ + All samples were measured on a BD FACS Verse flow cytometer and analyzed using FCS Express 6 software.

[0072] (4) Isolation of mouse TAMs

[0073] The tumor tissue of the tumor-bearing mouse was removed and fresh tumor tissue was cut into 1 mm 3 The small pieces were soaked in digestion buffer (containing collagenase IV (200U / ml) and deoxyribonuclease I (100mg / mL), placed on a shaker, 110rpm, incubated at 37°C for 45min, and prepared into a single cell suspension. TAMs were sorted using F4 / 80 positive sorting magnetic beads ((PE-F4 / 80 antibody, 157304, Biolegend) and anti-PE microbeads (130-048-801, Miltenyi Biotec)). The separated TAMs were cultured in RPMI-1640 medium containing 10% fetal bovine serum.

[0074] (5) Isolation of TAMs from tumor patients

[0075] The tumor tissue of the colon cancer patient was repeatedly washed with cephalosporin injection and cut into 1 mm pieces with a sterile scalpel. 3 The small pieces were soaked in digestion buffer (containing collagenase IV (200U / ml) and deoxyribonuclease I (100μg / mL), placed on a shaker, 110rpm, 37℃ incubation for 45min, prepared into a single cell suspension, and TAMs were sorted by CD14-PE antibody and anti-PE microbeads (130-048-801, Miltenyi Biotec). The separated TAMs were cultured in macrophage culture medium containing 10% fetal bovine serum, and m-CSF 20ng / ml was added.

[0076] (6) Transcriptome sequencing

[0077] The transcriptome sequencing method for tumor cells is as follows: Lyz-KO and Id1 f / f TAMs were isolated from tumor tissues of MC38 subcutaneous transplanted tumors in mice and transcriptome sequencing was performed as follows. Lyz -KO and Id1 f / f TAMs were isolated from MC38 subcutaneous transplanted tumors in the presence of 1:3 MC38 cells and inoculated into C57BJ / 6J mice subcutaneously. After tumor formation, CD45 magnetic beads were used for negative sorting (PE anti-mouse CD45 recombinant antibody (157604, Biolegend) and anti-PE magnetic beads (130-048-801, Miltenyi Biotec), and EpCAM magnetic beads were used for positive sorting (anti-EpCAM magnetic beads (130-105-958, Miltenyi-Biotec); CD45 in tumor tissue was isolated and obtained. - EpCAM + Transcriptome sequencing and analysis were performed by Shanghai Ouyi Biomedical Technology Co., Ltd.

[0078] The transcriptome sequencing method for TAMs was as follows: As described above, Lyz-KO and Id1 f / f TAMs were isolated from tumor tissues of MC38 subcutaneous transplanted tumors in mice and transcriptome sequencing was performed as follows. Lyz-KO and Id1 f / fAfter the MC38 subcutaneous transplanted tumors were isolated, F4 / 80 magnetic beads were used for sorting (PE anti-F4 / 80 antibody (157304, Biolegend) and anti-PE magnetic beads (130-048-801, Miltenyi Biotec)) to obtain F4 / 80-positive TAMs for transcriptome sequencing. Transcriptome sequencing and analysis were performed by Shanghai Ouyi Biomedical Technology Co., Ltd.

[0079] (7) Lentivirus preparation

[0080] 1*10^6 lenti-HEK-293T cells were inoculated in a 100mm dish and cultured overnight at 37 degrees Celsius with 5% CO2. The next day, the virus packaging plasmid (1.5μg psPAX2 and 500ng pMD2.G) and ID1shRNA plasmid (2μg) were transfected using vigofect transfection reagent. Fresh complete cell culture medium was replaced on the third day. On the fourth day, the cell culture supernatant containing viral particles was collected and the virus was concentrated using genecopia's lentiviral concentrate.

[0081] Example 1 Study on the relationship between ID1 expression in TAMs and patient prognosis by human colon cancer tumor tissue chip detection

[0082] The expression of ID1 in CRC TAMs and its relationship with the survival rate of CRC patients were analyzed by immunofluorescence multicolor staining of colorectal cancer (CRC) patient tumor tissue microarray (HColA180Su17, purchased from Shanghai Xinchao Biotechnology Co., Ltd.). The specific method is as follows:

[0083] The tissue samples fixed in formalin and embedded in paraffin were sliced ​​and subjected to multiple immunohistochemical staining using a four-color kit (TSA-RM) produced by PANOVUE Biotechnology Co., Ltd.

[0084] The results showed that CD68 + TAMs have higher ID1 expression levels than macrophages in adjacent normal tissues (see Figure 1 A and B), CD68 + Higher ID1 expression in TAMs is associated with poor prognosis in CRC patients (see Figure 1 C).

[0085] Example 2 In vivo experiments to study the effects of TAMs with high expression of ID1 on the growth of mouse colon cancer and liver cancer cells

[0086] 2.1 To investigate the effect of TAMs with high expression of ID1 on the growth of mouse colon cancer cells, we used transgenic mice with myeloid knockout of Id1 Lyz-KOand control mice Id1 f / f Mouse colon cancer cells MC38 were inoculated subcutaneously, and TAMs were separated from the subcutaneous tumors of the two groups of mice using F4 / 80 magnetic beads. MC38 and the separated TAMs were mixed at a ratio of 3:1 and inoculated again subcutaneously into C57BL / 6J mice (see Figure 2 A), the length and width of the mouse tumor were measured once every day with a vernier caliper, and the tumor volume was calculated using the formula: volume = 0.5 × length × width × width. The tumor was removed on the 20th day and its weight was measured.

[0087] The results showed that Id1 Lyz-KO The tumor growth rate in the group was significantly slowed down (see Figure 2 B), the tumor weight was lower (see Figure 2 C). The above results suggest that TAMs with high expression of ID1 can promote the growth of colon cancer.

[0088] 2.2 In order to explore whether TAMs with high expression of ID1 affect the growth of mouse colon cancer tumor cells through secretory components, we used the same method to culture TAMs separated by F4 / 80 magnetic beads in vitro, collected TAMs cell culture supernatant (Culture medium, CM), and injected 200 μL / mouse into C57BL / 6J tumor-bearing mice intratumorally (see Figure 2 D), draw the tumor growth curve and calculate the tumor weight.

[0089] The results showed that Id1 Lyz-KO The tumor growth rate in the group was significantly slowed down (see Figure 2 E), and lower tumor weight (see Figure 2 F). The above results suggest that TAMs with high expression of ID1 can promote the growth of colon cancer through secretory components.

[0090] 2.3 In order to explore whether TAMs with high expression of ID1 affect the growth of mouse liver cancer tumor cells through secretory components, we used the same method to culture TAMs separated by F4 / 80 magnetic beads in vitro, collected TAMs cell culture supernatant (Culture medium, CM), and injected 200 μL / mouse into H22 tumor-bearing mice intratumorally (see Figure 2 G), draw the tumor growth curve and calculate the tumor weight.

[0091] The results showed that Id1 Lyz-KO The tumor growth rate in the group was significantly slowed down (see Figure 2 H), and the tumor weight was lower (see Figure 2 I). The above results suggest that TAMs with high expression of ID1 can promote the growth of liver cancer through secretory components.

[0092] Example 3: Effects of TAMs with high ID1 expression on immune escape and tumor stemness of colon cancer by immunohistochemical multicolor staining and flow cytometry

[0093] 3.1 To explore the effect of ID1 in TAMs on T cells in tumor tissues, we Figure 2 Tumor tissues of C57BJ / 6J mice in A were subjected to immunohistochemical multicolor staining and flow cytometry.

[0094] The results showed that Id1 Lyz-KO CD8 + The number of T cells increases (see Figure 3 A), CD8 + Interferon-γ (IFN-γ) in T cells + and granzyme B + The percentage was higher than that of the control group (see Figure 3 B) The above results suggest that ID1, which is highly expressed in TAMs, inhibits CD8 + The infiltration and killing functions of T play a tumor-promoting role.

[0095] 3.2 To explore the effect of ID1 in TAMs on the stemness of tumor cells, we Figure 2 Tumor tissues of C57BJ / 6J mice in A, CD45 - EpCAM + The stemness marker Cd44 of tumor cells was analyzed.

[0096] The results showed that Id1 Lyz-KO The expression of stemness marker Cd44 in tumor cells of the group was lower than that of the control group (see Figure 3 C). The above results indicate that TAMs with high expression of ID1 promote the immune escape of colon cancer and enhance the stemness of tumor cells.

[0097] Example 4: Study on the molecular mechanism of TAMs with high expression of ID1 maintaining colon cancer stemness by transcriptome sequencing

[0098] To further explore the molecular mechanism by which highly expressed ID1 in TAMs maintains tumor stemness, we used Id1 f / f and Id1 Lyz-KO The subcutaneous transplanted tumor model of MC38 colon cancer cells was established in mice. TAMs in tumor tissue were sorted out using F4 / 80 magnetic beads, mixed with MC38 cells at a ratio of 1:3, and inoculated subcutaneously in C57BJ / 6J mice. CD45 - EpCAM + Transcriptome sequencing of tumor cells (see Figure 4 A).

[0099] KEGG analysis revealed that knockout of Id1 in TAMs significantly inhibited the focal adhesion kinase (FAK) and phosphatidylinositide 3-kinase (PI3K)-Akt signaling pathways (see Figure 4 B), FAK-Akt signaling pathway is crucial for maintaining colon cancer stemness. The above research results indicate that TAMs with high expression of ID1 maintain colon cancer stemness by affecting the FAK-Akt signaling pathway in tumor cells.

[0100] Example 5 TAMs with high expression of ID1 promote tumor immune escape and maintain colon cancer stemness by inhibiting the secretion of CCL4 and SerpinB2

[0101] To explore the molecular biological mechanism by which high-expression ID1 in TAMs promotes tumor immune escape and maintains tumor stemness, we used Id1 f / f and Id1 Lyz-ko A subcutaneous transplanted tumor model of MC38 colon cancer cells was established in mice. TAMs in tumor tissues were isolated using F4 / 80 magnetic beads for RNA transcriptome sequencing, and differentially expressed genes in the two groups were analyzed (OE Biotech Co., Ltd.) (see Figure 5 A and B). Ccl4 is in Id1 Lyz-KO The up-regulated expression of the tumor suppressor secretory protein in the group and the function of promoting T cell infiltration were confirmed by ELISA kit (4ABiotech, MOEB0088). Lyz-KO The protein level of Ccl4 in the cell culture supernatant of the group was significantly increased (see Figure 5 C) The above results indicate that TAMs with high expression of ID1 inhibit Ccl4 secretion and promote immune escape of colon cancer. Lyz-KO The up-regulated expression of the tumor suppressor secretory protein in the group and the inhibitory FAK-Akt signaling pathway was detected by ELISA kit (4ABiotech, MOEB1945). Lyz-KO The protein level of SerpinB2 in the culture supernatant of TAMs cells in the group A was significantly increased (see Figure 5 D) These results indicate that TAMs with high ID1 expression inhibit SerpinB2 secretion and maintain colon cancer stemness.

[0102] To further confirm whether ID1 promotes tumor growth by reducing the expression and secretion of Ccl4 and Serpinb2, a stable cell line was constructed using lentivirus expressing Ccl4-shRNA and Serpinb2-shRNA to obtain TAMs with stable knockdown of Ccl4 and Serpinb2 expression. The supernatant of TAMs cells with knockdown of Ccl4 and Serpinb2 expression was injected intratumorally into mice with MC38 subcutaneous tumors. The results showed that knockdown of Ccl4 and Serpinb2 expression in TAMs reversed the promotion of ID1 in TAMs on colon cancer growth (see Figure 5 E and F). The above results indicate that TAMs with high expression of ID1 promote colon cancer immune escape and maintain colon cancer stemness by inhibiting Ccl4 and Serpinb2 transcription, thereby promoting colon cancer growth.

[0103] Example 6 Knockout of ID1 in TAMs from colon cancer patients can enhance CD8 by increasing the secretion of CCL4 and SerpinB2 + Migration ability of T cells and stemness characteristics of HCT116 cells

[0104] The above experiments all explored the biological effects of ID1 in mouse-derived TAMs. To verify its possibility as an anti-tumor target in patients, the inventors further used TAMs from tumor patients to study the biological effects of ID1. The tumor tissue of colon cancer patients was minced, digested into single cells by enzymatic hydrolysis, and TAMs were further obtained by magnetic bead sorting. After adhesion, one group of cells was infected with the ID1shRNA lentivirus prepared by the inventors to explore the biological effects of knocking down ID1 (see Figure 6 A). Elisa assay results showed that knocking down ID1 in TAMs could significantly increase the protein levels of CCL4 and SerpinB2 in the TAM culture supernatant (see Figure 6 B). In vitro T cell migration experiments showed that knocking down ID1 in TAMs could significantly enhance the migration ability of T cells co-cultured with them (see Figure 6 C). Flow cytometry experiments showed that knockdown of ID1 in TAMs could significantly inhibit the stemness ratio of HCT116 cells co-cultured with them (CD44 high ALDH + )(See Figure 6 D). The above results indicate that knocking down the expression of ID1 in TAMs derived from colon cancer patient tissues using shRNA can increase the transcription and secretion of Ccl4 and Serpinb2, improve colon cancer immune escape, weaken colon cancer stemness, and inhibit colon cancer growth.

[0105] To further confirm the effect of ID1 on tumor growth, a more specific biological experiment is described below.

[0106] The drugs in the following experimental examples, the dosage and time of administration are as follows: ML323 (3 mg / kg or 5 mg / kg, once a day), CTLA-4 antagonist antibody (abbreviated as CTLA-4Ab) (4 mg / kg, three times on the 6th, 9th and 12th days after tumor inoculation) or 5-fluorouracil (abbreviated as 5-FU) (10 mg / kg, three times on the 8th, 11th and 14th days after tumor inoculation).

[0107] Example 7 Study on the effect of ID1 inhibitor on tumor growth by subcutaneous tumor formation experiment of mouse colon cancer CT26

[0108] The mice were divided into two groups: a control solvent-treated group (normal saline) and an ID1 inhibitor-treated group (ML323, 5 mg / kg, once a day).

[0109] The mouse strain used in the above tumor formation experiment was BALB / c, and the tumor cell line used was the mouse colon cancer cell line CT26.

[0110] Mice transplanted with CT26 subcutaneous tumors were treated with control solvent or ID1 inhibitor alone. The results showed that ID1 inhibitor ML323 could significantly inhibit tumor cell growth (see Figure 7 A and B). The tumor tissue sections of the two groups of mice were subjected to immunohistochemical polychromatic staining (PANOVUE Biotechnology Co., LTD, 1100020) to compare the CD8 + The results showed that the ID1 inhibitor ML323 increased the number of CD8 T cells in tumor tissues. + T cell infiltration (see Figure 7 C). Flow cytometry was used to detect the expression of CD44, a stemness marker in the two groups of tumor cells. The results showed that the ID1 inhibitor ML323 reduced the expression of CD44 in colon cancer cells.

[0111] The above results suggest that ID1 inhibitor ML323 can inhibit tumor growth and increase CD8 + T cells infiltrated and reduced the stemness of colon cancer cells CT26.

[0112] Example 8: Study on whether the anti-tumor effect of ML323 depends on the inhibition of ID1 in TAMs by using transgenic mice with myeloid knockout of Id1

[0113] In control mice (Id1 f / f ) and myeloid Id1 knockout transgenic mice (Id1 Lyz-KO ) was used to construct a MC38 subcutaneous transplant tumor model, and Id1 f / f and Id1 Lyz-KOThe mice were divided into two groups and given solvent control (normal saline) and ML323 (5 mg / kg, once a day) by intraperitoneal injection (see Figure 8 A). The tumor growth rates of the four groups of mice were observed, and the tumor weights were measured after sampling. The results showed that after myeloid knockout of Id1, the tumor inhibition effect of ML323 was significantly weakened, and the tumor inhibition rate dropped from 70% to about 40% (see Figure 8 BD). These results suggest that the anti-tumor effect of ML323 is partly dependent on the inhibition of ID1 in TAMs.

[0114] Example 9 Study on the Effect of ID1 Inhibitor Combined with Chemotherapy Drug Composition on Tumor Growth by Subcutaneous Tumor Formation Experiment of Mouse Colon Cancer CT26

[0115] The mouse experiment was divided into 4 groups: control solvent treatment group (normal saline), ID1 inhibitor single treatment group (ML323), chemotherapy drug single treatment group (5-FU) and ID1 inhibitor and chemotherapy drug combination treatment group (ML323+5-FU).

[0116] The mouse strain used in the above tumor formation experiment was BALB / C, and the tumor cell line used was mouse colon cancer cell line CT26. Three days after subcutaneous implantation of CT26 cells, ID1 inhibitors or chemotherapy drugs were intraperitoneally injected, and samples were collected 17 days after subcutaneous implantation of CT26 cells, tumor growth curves were drawn, and tumor weights were weighed.

[0117] like Fig. 9 As shown, the results showed that ID1 inhibitors reduced the stemness of colon cancer cells CT26, and the combined administration of ID1 inhibitors / chemotherapeutic drugs could better inhibit tumor growth than single administration.

[0118] Example 10 Study on the effect of ID1 inhibitor combined with immunotherapy drug composition on tumor growth through subcutaneous tumor formation experiment of mouse colon cancer CT26

[0119] The mouse experiment was divided into 4 groups: control solvent treatment group (normal saline), ID1 inhibitor single treatment group (ML323), immunotherapy drug single treatment group (CTLA-4Ab) and ID1 inhibitor and immunotherapy drug combined treatment group (ML323+CTLA-4Ab).

[0120] The mouse strain used in the above tumor formation experiment was BALB / C, and the tumor cell line used was mouse colon cancer cell line CT26. ID1 inhibitor was intraperitoneally injected 3 days after subcutaneous implantation of CT26 cells; CTLA-4Ab was intraperitoneally injected 6 days after subcutaneous implantation of CT26 cells. Samples were collected 20 days after subcutaneous implantation of CT26 cells, tumor growth curves were drawn, tumor weights were weighed, and CD8 in each group of tumors was detected by flow cytometry. + T cell infiltration and cytotoxic activity.

[0121] like Fig.10 The results showed that the combination of ID1 inhibitors / immunotherapeutic drugs increased CD8 + T cell infiltration and killing activity, combined administration of ID1 inhibitors / immune checkpoint inhibitors can better inhibit tumor growth than single administration.

[0122] Example 11 Gepia online analysis platform proves that the expression of ID1 in pancreatic cancer tumor tissue is higher than that in adjacent tissues

[0123] To further understand the expression of ID1 in tumor tissues other than colorectal cancer, we analyzed the Cancer Genome Atlas (TCGA) database using the Gepia online analysis platform (http: / / gepia2.cancer-pku.cn / #index). The results showed that, consistent with the expression pattern in colorectal cancer, the expression level of ID1 in tumor tissues of pancreatic adenocarcinoma (PAAD) patients was significantly higher than that in adjacent tissues (see Fig.11 ).

[0124] Example 12 Studying the Effects of ID1 Inhibitor and Chemotherapeutic Drug Combination or ID1 Inhibitor and Immunotherapy Drug Combination on Tumor Growth by Subcutaneous Tumor Formation Experiment of Pan02 Mouse Pancreatic Cancer Cells

[0125] This example further evaluated the anti-tumor effects of ID1 inhibitors and their combination therapy in pancreatic cancer.

[0126] The mouse experiment was divided into 6 groups: control solvent treatment group (normal saline), ID1 inhibitor single treatment group (ML323), chemotherapy drug single treatment group (5-FU), ID1 inhibitor and chemotherapy drug combination treatment group (ML323+5-FU), immunotherapy drug single treatment group (CTLA-4Ab) and ID1 inhibitor and immunotherapy drug combination treatment group (ML323+CTLA-4Ab).

[0127] The mouse strain used in the above tumor formation experiment was C57BL / 6J, the tumor cell line used was the mouse pancreatic cancer cell line Pan02, and the method for constructing the mouse subcutaneous transplant tumor model was the same as above. 2×10 Pan02 cells were implanted subcutaneously 5 / mouse, and 3 days later, ID1 inhibitor or chemotherapy drugs were intraperitoneally injected. 6 days after subcutaneous implantation of Pan02 cells, CTLA-4Ab was intraperitoneally injected. The tumor volume was measured starting from the 4th day after subcutaneous implantation of Pan02 cells, using the same method as above. Samples were collected 20 days after subcutaneous implantation of Pan02 cells, the weight of the tumor in each group was weighed, and the tumor inhibition rate was calculated as follows: tumor inhibition rate = (1-tumor volume of the combined drug group / tumor volume of the control group) × 100%.

[0128] Table 1 Effects of combined drug combination therapy on tumor volume and tumor inhibition rate in tumor-bearing mice

[0129] drug <![CDATA[Tumor volume (mm 3 )]]> Tumor inhibition rate Normal saline (control) 1093.21±133.76 0％ ML323 765.14±144.21 30.09％ 5-FU 725.38±150.74 33.64％ CTLA-4Ab 428.92±74.91 60.76％ ML323+5-FU 237.13±100.48 78.30％ ML323+CTLA-4Ab 102.04±50.18 90.66％

[0130] As shown in Table 1, the results showed that: ID1 inhibitor reduced the growth of pancreatic cancer cell Pan02, and the combination of ID1 inhibitor / chemotherapeutic drug and ID1 inhibitor / immunotherapeutic drug could better inhibit tumor growth than single administration.

[0131] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of ID1 inhibitors in macrophages in the preparation of anti-tumor therapeutic drugs.

2. The use according to claim 1, characterized in that The ID1 inhibitor in macrophages includes compounds that inhibit ID1 transcription, translation or protein stability, and small nucleic acids that inhibit ID1 transcription.

3. The use according to claim 2, characterized in that The small nucleic acids inhibiting ID1 transcription include shRNA and siRNA, and the small molecule compounds inhibiting ID1 protein stability include ML323, FT-3171, and SJB3-019A.

4. The use according to claim 2, characterized in that The ID1 inhibitor in macrophages inhibits the tumor-promoting phenotype of TAM, promotes the recruitment of effector T cells in the tumor immune microenvironment and inhibits the stemness characteristics of tumor cells, ultimately inhibiting the growth and metastasis characteristics of the tumor.

5. The use according to claim 2, characterized in that ID1 inhibitors in macrophages exert anti-tumor effects by increasing the activity of chemotherapy drugs and immune checkpoint inhibitors; the chemotherapy drugs include 5-fluorouracil, oxaliplatin, and irinotecan, and the immune checkpoint inhibitors include CTLA-4 antibodies, PD-1 antibodies, and PD-L1 antibodies.

6. Application of ID1 in macrophages as a target in screening anti-tumor drugs.

7. Application of ID1 in macrophages in screening ID1 inhibitors that can enhance the effects of chemotherapy and immunotherapy.

8. The use according to any one of claims 1 to 7, characterized in that The tumors include colon cancer, liver cancer, stomach cancer, lung cancer, pancreatic cancer and the like.

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