CN119303096A - Composition for treating and/or preventing tumors and its application - Google Patents

Abstract

The invention belongs to the field of medicines, and discloses a composition for treating and/or preventing tumors and application thereof. The composition comprises any one of (1) an ID1 inhibitor and a chemotherapeutic drug, and (2) an ID1 inhibitor and an immune checkpoint inhibitor drug. The chemotherapeutic agent comprises 5-fluorouracil, oxaliplatin, capecitabine or irinotecan. The immune checkpoint inhibitor drug is selected from targeted CTLA-4 antagonistic antibodies. The composition disclosed by the invention is an anti-tumor pharmaceutical composition for inhibiting ID1 activity, and experiments prove that the composition can improve the chemotherapeutic treatment effect or the immunotherapeutic effect of tumor patients, thereby laying a theoretical foundation for formulating a clinical anti-tumor treatment strategy and having wide application prospect.

Description

Composition for treating and/or preventing tumor and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a composition for treating and/or preventing tumors and application thereof.

Background

Tumors are a major public health problem worldwide, with serious threat to human life health. Tumor development is a complex multi-step process involving transformation, proliferation, angiogenesis, immunomodulation, and metastasis, among others. However, the development and progression of tumors is not only induced by the genetic mutations accumulated by the tumor cells themselves, but also by surrounding non-malignant cells.

Tumor-associated macrophages (Tumor associated macrophages, TAM) are macrophages that infiltrate into tumor tissue, the most abundant innate immune cells in tumor tissue. TAMs are highly plastic, often exhibit a pro-neoplastic M2 phenotype, are important inducers of tumorigenesis, metastasis, and are closely related to resistance to chemotherapy, immunotherapy. Multiple studies have found that targeted inhibition of TAM number or activity can significantly improve the resistance of tumor patients to chemotherapeutic agents, or increase the responsiveness of patients to immune checkpoint inhibitor therapies. Therefore, the anti-tumor strategy of targeting TAM shows great clinical application potential, but the tumor promotion mechanism of the TAM is not yet completely elucidated at present, and brand new pharmaceutically acceptable targets of the TAM are urgently needed to be confirmed.

DNA binding inhibitor protein (Inhibitor of DNA binding, ID) is a family of transcription regulatory factor proteins having a Helix-Loop-Helix (Helix-Loop-Helix, HLH) structure. The family has four members ID1-ID4, and is involved in the regulation of various cell functions including development, aging, differentiation, angiogenesis, cell migration and the like, and one of important members ID1 of the family shows the activity of promoting tumors in various tumors. In solid tumors such as colon cancer, pancreatic cancer, ovarian cancer and the like, the expression of the ID1 protein is obviously increased, and the ID1 protein is related to poor prognosis and chemotherapy resistance of patients. The ID1 protein has the function of promoting angiogenesis and is involved in the metastasis and regulation of various tumors. Knocking down the ID1 gene and administering the ID1 inhibitor can obviously inhibit the growth of tumors, and the ID1 is also a new target for tumor treatment and drug discovery.

John E.Dick et al, J CANCER CELL, demonstrated that ID1 and ID3 maintained the self-renewal capacity of colon cancer stem cells by upregulating cyclin-associated protein p21, and that knocking down ID1 and ID3 significantly enhanced the sensitivity of colon cancer cells to the chemotherapeutic agent oxaliplatin. In addition, ID1 can exert a tumorigenic effect in colon cancer by regulating the differentiation balance of myeloid suppressor MDSCs and dendritic cells, but the effect of ID1 in TAM has not been reported yet.

Disclosure of Invention

The invention aims to provide a composition for treating and/or preventing tumors and application thereof, which are used for solving the problems of the prior art, and the composition constructed by combining an ID1 inhibitor and a chemical drug or an immunotherapeutic drug can remarkably inhibit the growth and metastasis of the tumors.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a composition for treating and/or preventing tumors, which comprises a pharmaceutical combination as shown in any one of the following:

(1) ID1 inhibitors and chemotherapeutic agents;

(2) ID1 inhibitors and immune checkpoint inhibitor drugs.

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

Preferably, the small molecule compound comprises ML323.

Preferably, the chemotherapeutic agent comprises 5-fluorouracil, oxaliplatin, capecitabine or irinotecan.

Preferably, the mass fraction of the ML323 and 5-fluorouracil medicinal composition is 3 (1-10).

Preferably, the immune checkpoint inhibitor drug is selected from the group consisting of targeted CTLA-4 antagonistic antibodies, more preferably targeted CTLA-4 antagonistic antibodies comprise ipilimumab.

Preferably, the mass fraction of the ML323 and CTLA-4 antagonistic antibody pharmaceutical composition is 3 (4-8).

The invention also provides the application of the composition in preparing medicines for treating and/or preventing tumors

The prevention is conventional in the art and preferably refers to preventing or reducing the occurrence of tumors after use in the presence of possible tumor factors. The treatment is conventional in the art, preferably refers to reducing the extent of a tumor, or curing a tumor to normalize it, or slowing the progression of a tumor.

Preferably, the tumor comprises a solid tumor with high expression of ID 1.

Preferably, the solid tumors include intestinal cancer and pancreatic cancer.

The invention discloses the following technical effects:

(1) According to the invention, the high expression of the ID1 in the TAM is found for the first time, so that on one hand, the colon cancer dryness is enhanced, and on the other hand, the immune escape is promoted by inhibiting the infiltration of CD8 ⁺ T cells, and the growth of the colon cancer is promoted. It is also proposed for the first time that TAM with high expression of ID1 enhances tumor stem property and inhibits T cell recruitment as a cause of insensitivity to colon cancer chemotherapy or immune checkpoint inhibitor treatment, and that targeting inhibition of tumor cells and ID1 in TAM is a novel strategy for improving the immunotherapeutic effect of colon cancer.

(2) The small molecular compound for inhibiting ID1 can simultaneously reduce the expression of ID1 in tumor-associated macrophages, on one hand, the infiltration and killing effect of CD8 ⁺ T cells are increased by relieving the transcription inhibition effect of ID1 on Ccl4, and immune escape is avoided, and on the other hand, the dryness of colon cancer is reduced by relieving the transcription inhibition effect of ID1 on Serpinb 2.

(3) The anti-tumor pharmaceutical composition based on ID1 inhibition activity provided by the invention has wide application in improving the chemotherapeutic treatment effect or the immune checkpoint inhibitor treatment effect of tumor patients and preparing clinical anti-tumor treatment strategies.

(4) The invention can screen out a proper target patient group based on the anti-tumor pharmaceutical composition for inhibiting the activity of the ID1, namely the patient with solid tumor such as intestinal cancer, pancreatic cancer and the like, and more preferably the patient with the intestinal cancer with high expression of the ID 1.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph of total survival of CRC patients with increased ID1 expression in TAM associated with poor clinical prognosis in colorectal cancer (Colorectal cancer, CRC) patients, representative immunofluorescent-stained images of ID1 and CD68 in tumor tissue and paracancestral normal tissue of CRC patients, (B) statistics of ID1 expression in CD68 ⁺ TAM cells, and (C) CD68 ⁺ TAM intracellular ID1 expression levels;

FIG. 2 is a schematic representation of TAM highly expressing ID1 to promote colon cancer and liver cancer growth, wherein (A) MC38 cells and TAM isolated from Id1 ^Lyz-KO or Id1 ^{f /f} mice are mixed and then secondarily inoculated under the skin of C57BL/6J mice, (B) tumor growth curves of two groups of mice, (C) tumor weights of two groups of mice, (D) tumor injection of culture supernatant of TAM into MC38 subcutaneous transplantation tumor model, (i.t. intratumoral injection, (E) tumor growth curves of two groups of mice, (F) tumor weights of two groups of mice, (G) tumor injection of culture supernatant of TAM into H22 subcutaneous transplantation tumor model, (i.t. intratumoral injection, (H) tumor growth curves of two groups of mice, (I) tumor weights of two groups of mice;

FIG. 3 shows the results of TAM highly expressing ID1 in promoting immune escape of colon cancer and enhancing colon cancer stem property, (A) representative CD8 ⁺ T cell immunofluorescence image and statistical image in tumor tissue of mouse model shown in FIG. 2, and (B) the percentage of interferon-gamma (IFN-gamma) ⁺ and Granzyme B (Granzyme B) ⁺ in CD8 ⁺ T cell isolated from tumor tissue of mouse model shown in FIG. 2, (C) CD45 ^-EpCAM⁺ tumor cells isolated from tumor tissue of Id1 ^Lyz-KO and Id1 ^f/f mouse subcutaneous tumor model, and Cd44 expression level was detected in a flow-type manner;

FIG. 4 is a schematic representation of the transcriptome sequencing of tumor cells isolated from Id1 ^Lyz-KO and Id1 ^f/f mice subcutaneously transplanted tumors by affecting FAK-AKT signaling pathway in tumor cells to maintain colon cancer stem property in TAM with high ID1 expression;

FIG. 5 is a schematic representation of the F4/80 ⁺ macrophages in a model of a subcutaneous engrafting tumor of Id1 ^Lyz-KO mice and Id1 ^f/f mice MC38 and performing transcriptome sequencing by magnetic beads to promote tumor immune escape and maintain colon cancer dryness by inhibiting secretion of CCL4 and SerpinB, respectively, (B) volcanic patterns of the differentially expressed genes of the two groups shown in FIG. 5A, (C) protein abundance of Ccl4 in TAM culture supernatants isolated from Id1 ^Lyz-KO and Id1 ^f/f mice, (D) protein abundance of Serpinb in TAM culture supernatants isolated from Id1 ^Lyz-KO and Id1 ^f/f mice, (E) infection of a model of a subcutaneous engrafting tumor with a BMDM-derived TAM of Id1 ^Lyz-KO or Id1 ^f/f mice with a lentivirus expressing Ccl4-shRNA and Serpinb-shRNA, and collection of cell culture supernatants by intra-injection into the model of a subcutaneous engrafting tumor of the two groups shown in F);

FIG. 6 is an isolated TAM from colon cancer patients, and the effect of ID1 on tumor cell stem mass and CD8 ⁺ T cell migration rate in TAM from knockdown patients was examined by interfering with ID1 expression in TAM using lentiviruses. Schematic of TAM isolation and viral infection in patients, (B) knocking out the effect of ID1 in TAM on HCT116 cell stem property, and (C) knocking out the effect of ID1 in TAM on CD8 ⁺ T cell migration rate.

FIG. 7 is a schematic diagram of research on the influence of an ID1 inhibitor on tumor growth through a mouse colon cancer CT26 subcutaneous tumorigenesis experiment, (A) a schematic diagram of different administration treatments of mouse subcutaneous implantation CT26 cells, (B) different groups of mouse tumor growth curves, (C) immunofluorescence diagrams and statistical diagrams of infiltrated CD8 ⁺ T cells in different groups of mouse tumor tissues, and (D) the expression results of Cd44 in different groups of mouse tumor tissues through flow detection;

FIG. 8 is a graph of a transgenic mouse with ID1 knockdown specifically by wild type C57BL/6J mice and the myeloid lineage, exploring whether ML323 works by reducing ID1 expression in TAM. The method comprises the following steps of (A) establishing a schematic diagram of grouping mice and a subcutaneous transplantation tumor model, (B) establishing a tumor growth curve of each group of mice, and (C) carrying out tumor weight statistics of each group of mice.

FIG. 9 is a schematic diagram showing the effect of combination of an ID1 inhibitor and a chemotherapeutic drug composition on tumor growth through subcutaneous tumor formation experiments of mouse colon cancer cells CT26, (A) different drug administration treatments of mice by subcutaneous implantation of CT26 cells, (B) different groups of mouse tumor growth curves, (C) different groups of mouse tumor weights;

FIG. 10 is a schematic diagram of the study of the effect of ID1 inhibitor combined with immunotherapeutic pharmaceutical composition on tumor growth by mouse colon cancer cell CT26 subcutaneous tumorigenesis, (A) different dosing treatments of mouse subcutaneous engrafting CT26 cells, (B) different groups of mouse tumor growth curves, (C) different groups of mouse tumor weights, (D) flow detection of CD8 ⁺ T cell results, (E) IFN-gamma and Granzyme B expression of CD8 ⁺ T cells in different groups of mouse tumor tissues;

FIG. 11 shows the expression of ID1 in various tumor tissues.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The following examples of the invention relate to the main experimental materials and reagents:

(1) Antibodies to

ID1 polyclonal antibody (Proteintech, cat.# 18475-1-AP), CD68 antibody (Abcam, cat.# ab 955), CD8a (mouse) (CELL SIGNALING Technology, cat.# 98941S), CD14 antibody (Biolegend, cat.# 325606), APC-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.# 3228), perCP/Cyanine5.5 anti-mouse CD3 epsilon antibody (Biolegend, cat.# 328), APC/Cyan7 anti-mouse CD8a antibody (Biolegend, cat.# 76), PE anti-mouse CD45 antibody (Biolegend, cat# 37 B#23), PE anti-mouse CD326 (Biolegend, cat# 37 B#23), recombinant PE anti-mouse CD 37 E#23, PE#37 B#23.

(2) Reagent(s)

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), cappings (DAPI-containing) (ZSGB-BIO, ZLI-9557), human lymphocyte isolates (Solarbio,P8900),Recombinant murine m-CSF(Peprotech,315-02),Recombinant murineIL-4(Peprotech,214-14),ML323(Selleck,S7529,CAS：1572414-83-5, molecular formula C ₂₃H₂₄N₆, molecular weight 384.48), 5-fluorouracil (Selleck, S1209, CAS:51-21-8, molecular formula C ₄H₃FN₂O₂, molecular weight 130.08).

(3) Kit for detecting a substance in a sample

Four-color multiplex fluorescence immunohistochemical staining kit (TSA-RM) (PANOVUE Biotechnology Co., LTD, 1100020), cell fixation/permeation solution (BD Biosciences, 554714), mouse C-C-motif chemokine4 (C-C motif chemokine4, ccl 4) ELISA detection kit (4A Biotech,MOEB0088), mouse plasminogen activator inhibitor 2 (plasminogen activator inhibitor 2, serpinb 2) ELISA detection kit (4A Biotech,MOEB1945),Anti-PE microbeads(Miltenyi Biotec,130-048-801),CD326(EpCAM)microbeads,mouse(Miltenyi Biotec,130-105-958).

(4) Cell lines

The colon cancer cells of the mice comprise MC38, CT26, liver cancer cell line H22 of the mice and pancreatic cancer cell line Pan02 of the mice

(5) ID1 knockdown shRNA lentiviral vector

The shRNA sequence is 5'-GAGGAAYYACGTGCTCTGT', the slow virus vector is pHBLV-U6-ZsGreen-Puro, and the construction of China-Hengsheng biotechnology (Shanghai) limited company is entrusted.

(6) Experimental animal

C57BL/6J mice (male, 6 weeks old), BALB/C mice (male, 6 weeks old), id1 conditional knockout mice (Id 1 ^f/f) and Lyz-cre mice were purchased from Sai (Suzhou) Biotechnology Inc., and the two were mated to obtain transgenic mice (Id 1 ^Lyz-KO),Id1^Lyz-KO and Id1 ^f/f mice) with marrow specific knockout Id1, which were bred from the subject group using methods conventional in the art.

The following examples of the invention relate to a part of experiments, and the specific method comprises the following steps:

(1) Mouse subcutaneous transplantation tumor model construction

Cell preparation, in which adherent cells are digested by pancreatin and after termination of the digestion by serum, the cells are blown down with PBS and centrifuged at 1000rpm for 5min at room temperature. After counting, cells were diluted to 2X 10 ⁶ cells/mL in PBS containing 40% low growth factor Matrigel and stored on ice. Animals were prepared by randomly grouping mice, at least 6 mice per group, and shaving the right underarm hair. Subcutaneous injection 100. Mu.L of the cell suspension was aspirated with a 1mL syringe, and after air bubbles were emptied, the right underarm skin injection was initiated. CT26 cells, MC38 cells, H22 cells or Pan02 cells were subcutaneously implanted in BALB/C mice 2X 10 ⁵/mouse. In the therapeutic experiments, the ID1 inhibitor or the chemotherapeutic agent was administered by intraperitoneal injection three days later, and the CTLA-4 antagonistic antibody (abbreviated CTLA-4 Ab) was administered by intraperitoneal injection six days after subcutaneous implantation of CT26 cells or Pan02 cells.

Tumor size measurement, namely, starting to measure the tumor length and width of a mouse once by using a vernier caliper after the fourth day or the fifth day of subcutaneously planting CT26 cells or Pan02 cells, calculating the tumor volume, wherein the calculation formula is that the volume=0.5×length×width×width is measured once every 1 day, drawing materials on the 20 th day, drawing a growth curve, weighing the weight of each group of tumors, and calculating the tumor inhibition rate, and the calculation method is that the tumor inhibition rate is= (1-combined administration tumor volume/control tumor volume) ×100%

(2) Establishment of stable cell lines

The leg bones of Id1 ^Lyz-KO and Id1 ^f/f mice were removed, bone marrow-derived cells were obtained by grinding the leg bones with DMEM medium, and the cell suspension was filtered with a cell screen of 70 μm to obtain a single cell suspension. Centrifugation at 2000rpm for 5min and re-suspension of lysed erythrocytes with 1mL of erythrocyte lysate, centrifugation at 2000rpm for 5min after two minutes, resulted in bone marrow-derived macrophages.

Cells were inoculated with Id1 ^Lyz-KO and Id1 ^f/f mouse bone marrow-derived macrophages, respectively, cultured at 37℃with 5% CO ₂, added with 8. Mu.g/mL of polybrene and with a mouse Ccl 4-expressing short hairpin RNA (shRNA) lentivirus and a mouse Serpinb shRNA lentivirus, cultured at 37℃with 5% CO ₂ overnight, replaced with new cell culture medium after 24 hours, and added with 20ng/mL of interleukin 4 (IL-4) and 20ng/mL of macrophage colony stimulating factor (m-CSF) to stimulate bone marrow-derived macrophages in vitro to obtain TAM phenotype, resulting in stable knockdown of TAM expressed by Ccl4 and Serpinb.

(3) Flow cytometry detection of immunomaps

Tumor tissue of tumor-bearing mice was removed, fresh tumor tissue was cut into 1mm ³ pieces with a sterile scalpel, immersed in digestion buffer (containing collagenase IV (200U/mL) and deoxyribonuclease I (100 mg/mL)) and placed on a shaking table, incubated at 110rpm at 37℃for 45min, and after preparation of single cell suspensions, the T cell content and expression of effector molecules thereof were determined. To analyze the proportion of CD8 ⁺ T cells therein, anti-CD 45, anti-CD 3 and anti-CD 8 antibodies were added to the single cell suspension and incubated at room temperature for 20min in the absence of light. Cells were then washed 3 times with MACS buffer (PBS solution containing 0.5% fetal bovine serum and 2mm edta) and resuspended.

CD8 ⁺ T cells account for the total cells in tumor%o=cd 45 ⁺ T cells%o×cd3 ⁺/CD45⁺ T cells%o×cd8 ⁺/CD3⁺ T cells.

To determine the expression level of effector molecules in effector T cells, T cells were enriched using density gradient centrifugation, cultured for 4h in medium containing PMA (5 ng/mL), ionomycin (500 ng/mL), brefeldin A (1:1000) and Golgi stop (1:1000) at 37℃and surface stained by adding anti-CD 45, anti-CD 3 and anti-CD 8 antibodies, and incubated at room temperature in the absence of light for 20 min. Cells will then be washed 3 times with MACS buffer and resuspended in 1mL of cell fixation/permeabilization solution (BD Biosciences) and fixed overnight at 4 ℃ protected from light. The following day, cells were washed with Perm/Wash buffer, stained with anti-interferon-gamma (IFN-. Gamma.) ⁺ and anti-Granzyme B (Granzyme B) antibodies for 30min at room temperature in the dark, and washed with MACS buffer three times. The percentage of IFN- γ ⁺ and granzyme B ⁺ in CD8 ⁺ T cells was calculated (calculation method = IFN- γ ⁺/total cell count x 100%). All samples were measured on BDFACSVerse flow cytometer and analyzed with FCS Express 6 software.

(4) Isolation of mouse TAM

Tumor tissue of tumor-bearing mice was removed, fresh tumor tissue was cut into 1mm ³ pieces with a sterile scalpel, immersed in digestion buffer (containing collagenase IV (200U/mL) and deoxyribonuclease I (100 mg/mL), placed on a shaking table, incubated at 110rpm,37℃for 45min, and after preparation of a single cell suspension, TAM was sorted by F4/80 positive sorting beads ((PE-F4/80 antibody, 157304, bioleged) and anti-PE microbeads (130-048-801,Miltenyi Biotec)), and the isolated TAM was cultured in RPMI-1640 medium containing 10% fetal bovine serum.

(5) Isolation of TAM from tumor patients

Tumor tissue of a colon cancer patient was repeatedly washed with Cephalosporium injection, cut into 1mm ³ pieces with a sterile scalpel, immersed in digestion buffer (containing collagenase IV (200U/mL) and deoxyribonuclease I (100. Mu.g/mL), placed on a shaking table, incubated at 110rpm,37℃for 45min, and after preparation of a single cell suspension, TAM was sorted by CD14-PE antibody and anti-PE microbeads (130-048-801,Miltenyi Biotec), isolated TAM was cultured with macrophage medium containing 10% fetal bovine serum, and m-CSF20ng/mL was added.

(6) Transcriptome sequencing

Transcriptome sequencing methods for tumor cells the TAM was isolated from tumor tissue of MC38 subcutaneously transplanted tumor vaccinated in Id1 ^Lyz-KO and Id1 ^{f /f} mice as described above and the transcriptome sequencing was performed as follows. TAM of MC38 subcutaneous transplants in Id1 ^Lyz-KO and Id1 ^f/f were isolated, mixed with MC38 cells at a ratio of 1:3, inoculated subcutaneously in C57BJ/6J mice, and after tumorigenesis, negative sorting (PE anti-mouse CD45 recombinant antibodies (157604, biolegend) and anti-PE magnetic beads (130-048-801,Miltenyi Biotec)) were performed using CD45 magnetic beads, positive sorting (anti-EpCAM magnetic beads (130-105-958, miltenyi-Biotec)) and separation of tumor cells with CD45 ^- and EpCAM ⁺ in tumor tissue for transcriptome sequencing and analysis were performed by Shanghai European biomedical technologies, inc.

Transcriptome sequencing for TAM was performed by isolating TAM from tumor tissue of MC38 subcutaneously transplanted tumor vaccinated in Id1 ^Lyz-KO and Id1 ^f/f mice as described previously. After isolation of MC38 subcutaneous grafts in Id1 ^Lyz-KO and Id1 ^f/f, F4/80 positive TAM was obtained using F4/80 bead sorting (PE anti-F4/80 antibody (157304, biolegend) and anti-PE beads (130-048-801,Miltenyi Biotec)), for transcriptome sequencing. Transcriptome sequencing and analysis was performed by Shanghai European biomedical technologies Inc.

(7) Lentivirus preparation

1X 10 x 6lenti-HEK-293T cells were seeded in 100mm dishes and incubated overnight at 5% CO2,37 ℃. The next day virus packaging plasmid (1.5. Mu. g psPAX2 and 500ng pMD2. G) and ID1 shRNA plasmid (2. Mu.g) were transfected with vigofect transfection reagent. Fresh complete cell culture medium was changed the third day. The fourth day the cell culture supernatant containing the viral particles was collected and the virus was concentrated using genecopia lentiviral concentrate.

EXAMPLE 1 investigation of the relationship of ID1 expression in TAM to patient prognosis by human colon cancer tumor tissue chip detection

The expression of ID1 in CRC TAM and the survival rate relation with CRC patients are analyzed by immunofluorescence polychromatic staining of tumor tissue chip (HColA 180Su17, purchased from Shanghai Corp Biotechnology Co., ltd.) of colorectal cancer (Colorectal cancer, CRC), and the specific method is as follows:

the formalin-fixed paraffin-embedded tissue samples were sectioned and subjected to multiple immunohistochemical staining using a four-color kit (TSA-RM) manufactured by PANOVUE Biotechnology Inc.

The results indicate that CD68 ⁺ TAM had higher levels of ID1 expression than macrophages in the paracancerous normal tissue (see fig. 1A and B), and that higher ID1 expression in CD68 ⁺ TAM was associated with a poor prognosis for CRC patients (see fig. 1C).

EXAMPLE 2 in vivo experiments to investigate the effect of TAM highly expressing ID1 on the growth of tumor cells of colon cancer and liver cancer in mice

2.1 To investigate the effect of TAM highly expressing ID1 on growth of tumor cells of colon cancer in mice, we inoculated marrow knockout Id1 transgenic mice Id1 ^Lyz-KO and control mice Id1 ^f/f subcutaneously with mouse colon cancer cells MC38, separated TAM from the two groups of mouse subcutaneous tumors by F4/80 magnetic beads, mixed MC38 with the separated TAM at a ratio of 3:1, inoculated again under C57BL/6J mice (see FIG. 2A), measured tumor length and width with vernier calipers once every 1 day, calculated tumor volume, calculated formula: volume=0.5×length×width×width, tumors were taken out on day 20, and weights were measured.

The results showed that the tumor growth rate was significantly slower in the Id1 ^Lyz-KO group (see fig. 2B) and the tumor weight was lower (see fig. 2C). The above results suggest that TAMs with high expression of ID1 promote colon cancer growth.

2.2 To investigate whether TAM highly expressing ID1 affects the growth of colon cancer tumor cells in mice by secreting components, we used TAM in vitro cell cultures sorted out by F4/80 beads as above, collected TAM cell Culture supernatants (Culture media, CM), 200. Mu.L/intratumoral injection into C57BL/6J tumor-bearing mice (see FIG. 2D), plotted tumor growth curves and counted tumor weights.

The results showed that the tumor growth rate was significantly slower in the Id1 ^Lyz-KO group (see fig. 2E) and the tumor weight was lower (see fig. 2F). The above results suggest that TAMs with high expression of ID1 can promote colon cancer growth by secreting components.

2.3 To investigate whether TAM highly expressing ID1 affects the growth of liver cancer tumor cells in mice by secreting components, we used TAM in vitro cell cultures sorted out by F4/80 magnetic beads as above, collected TAM cell Culture supernatants (CM), 200. Mu.L/intratumoral injection into H22 tumor-bearing mice (see FIG. 2G), plotted tumor growth curves and counted tumor weights.

The results showed that the tumor growth rate was significantly slower in the Id1 ^Lyz-KO group (see FIG. 2H) and the tumor weight was lower (see FIG. 2I). The above results suggest that TAMs highly expressing ID1 can promote liver cancer growth by secreting components.

EXAMPLE 3 investigation of the Effect of TAM highly expressing ID1 on the immune escape and tumor stem characteristics of colorectal cancer by immunohistochemical polychromatic staining and flow cytometry experiments

3.1 To investigate the effect of ID1 in TAM on T cells in tumor tissue, we performed immunohistochemical polychromatic staining and flow detection on tumor tissue of C57BJ/6J mice in FIG. 2A.

The results showed that the number of CD8 ⁺ T cells in tumor tissue was increased in Id1 ^Lyz-KO group (see fig. 3A), and the percentage of interferon-gamma (IFN- γ) ⁺ and granzyme B ⁺ was higher in CD8 ⁺ T cells than in control group (see fig. 3B). The above results suggest that ID1 highly expressed in TAM exerts a tumor promoting effect by inhibiting infiltration and killing function of CD8 ⁺ T.

3.2 To investigate the effect of ID1 in TAM on the stem characteristics of tumor cells, we analyzed the stem marker Cd44 of CD45 ^-EpCAM⁺ tumor cells on the tumor tissue of C57BJ/6J mice in FIG. 2A.

The results indicated that the expression of the stem marker Cd44 was lower in tumor cells of Id1 ^Lyz-KO group than in the control group (see fig. 3C). The results indicate that TAM with high expression of ID1 promotes immune escape of colon cancer and enhances stem cell quality of tumor.

Example 4 study of molecular mechanisms of TAM highly expressing ID1 to maintain colon cancer dryness by transcriptome sequencing

To further explore the molecular mechanism of high expression of ID1 in TAM to maintain tumor stem, we constructed a model of colon cancer cell MC38 subcutaneous transplantation tumor with ID1 ^f/f and ID1 ^Lyz-KO mice, sorted TAM in tumor tissue with F4/80 magnetic beads, mixed with MC38 cells at a ratio of 1:3, inoculated subcutaneously in C57BJ/6J mice, and tumor cells of CD45 ^-EpCAM⁺ in tumor tissue were isolated after tumor formation for transcriptome sequencing (see fig. 4A).

Knocking out Id1 in TAM was found by KEGG analysis to significantly inhibit Focal Adhesion Kinase (FAK) and Phosphatidylinositide-kinase (PI 3K) -Akt signaling pathway (see FIG. 4B), which is critical for maintaining colon cancer dryness. The above results indicate that TAM with high expression of ID1 maintains colon cancer dryness by affecting FAK-Akt signaling in tumor cells.

EXAMPLE 5 TAM highly expressing ID1 promotes tumor immune escape and maintains colon cancer dryness by inhibiting secretion of CCL4 and SerpinB2, respectively

To explore the molecular biological mechanisms of high expression of ID1 in TAMs to promote tumor immune escape and maintain tumor dryness, we constructed a colon cancer cell MC38 subcutaneous engraftment tumor model with ID1 ^f/f and ID1 ^Lyz-ko mice, sorted TAMs in tumor tissue with F4/80 magnetic beads for RNA transcriptome sequencing, and analyzed the two groups of differentially expressed genes (OE Biotech co., ltd.) (see fig. 5A and B). Ccl4 was a tumor suppressor secreted protein that was up-regulated in Id1 ^Lyz-KO group expression and had a T cell infiltration promoting function, and the protein level of Ccl4 in Id1 ^Lyz-KO group cell culture supernatant was significantly increased as confirmed by ELISA kit (4A Biotech,MOEB0088) (see FIG. 5C). The results indicate that TAM with high expression of ID1 inhibits Ccl4 secretion and promotes immune escape of colon cancer. Serpinb2 is a tumor suppressor secreted protein that was up-regulated in Id1 ^Lyz-KO group expression and had an inhibitory FAK-Akt signaling pathway, and the protein level of SerpinB2 was found to be significantly increased in Id1 ^Lyz-KO group TAM cell culture supernatants by ELISA kit (4A Biotech,MOEB1945) (see FIG. 5D). The above results indicate that TAM with high expression of ID1 inhibits SerpinB2 secretion to maintain colon cancer dryness.

To further confirm whether ID1 exerts a tumor promoting function by reducing expression and secretion of Ccl4 and Serpinb2, stable transgenic cell lines were constructed using lentiviruses expressing Ccl4-shRNA and Serpinb-shRNA to obtain TAMs with stable knockdown of Ccl4 and Serpinb2 expression. Intratumorally injecting TAM cell culture supernatants with knockdown of Ccl4 and Serpinb2 expression into MC38 subcutaneously engrafted tumor mice showed that knockdown of Ccl4 and Serpinb2 expression in TAMs reversed the promoting effect of ID1 on colon cancer growth in TAMs (see fig. 5E and F). The results show that TAM with high expression of ID1 promotes immune escape of colon cancer and maintains dryness of colon cancer by inhibiting transcription of Ccl4 and Serpinb, and promotes growth of colon cancer.

Example 6 knockout of ID1 in TAM from colon cancer patients enhances the migratory capacity of CD8 ⁺ T cells and the stem character of HCT116 cells by increasing secretion of CCL4 and SerpinB2

The above experiments were conducted by exploring the biological effect of ID1 in TAM derived from mice, and the inventors further studied the biological effect of ID1 by using TAM derived from tumor patients in order to verify the possibility of using it as an antitumor target in patients. Tumor tissues of colon cancer patients are cut up, digested into single cells by an enzymolysis method, and further subjected to magnetic bead separation to obtain TAM. After adherence, one group of cells was infected with the ID1 shRNA lentivirus prepared by the inventors to investigate the biological effect of knocking down ID1 (see fig. 6A). Elisa assay showed that knocking down ID1 in TAM significantly increased protein levels of CCL4 and SerpinB2 in TAM culture supernatants (see FIG. 6B). In vitro T cell migration experiments showed that knocking down ID1 in TAM significantly enhanced the migration capacity of T cells co-cultured therewith (see fig. 6C). Flow cytometry experiments showed that knocking down ID1 in TAM significantly inhibited the stem fraction of HCT116 cells co-cultured therewith (CD 44 ^high ALDH⁺) (see fig. 6D). The results show that the shRNA is utilized to knock down the ID1 expression in TAM derived from the tissue of a colon cancer patient, so that the transcription and secretion of Ccl4 and Serpinb can be increased, the immune escape of the colon cancer can be improved, the dryness of the colon cancer can be weakened, and the growth of the colon cancer can be inhibited.

To further demonstrate the effect of ID1 on tumor growth, a more specific biological experiment is described below.

The doses and times of administration of ML323 (1 mg/kg or 3mg/kg or 5mg/kg, once a day), CTLA-4 antagonistic antibodies (abbreviated as CTLA-4 Ab) (2 mg/kg or 4mg/kg or 8mg/kg, administered three times at days 6,9,12 after tumor inoculation) or 5-fluorouracil (abbreviated as 5-FU) (1 mg/kg or 10mg/kg or 40mg/kg, administered three times at days 8,11,14 after tumor inoculation) were as follows.

Example 7 study of the Effect of ID1 inhibitors on tumor growth by mouse colon cancer CT26 subcutaneous tumorigenesis experiments

The mice experiments were divided into 2 groups, control solvent treated group (normal saline) and ID1 inhibitor alone treated group (ML 323,5mg/kg, once a day).

The mouse strain adopted in the above-mentioned tumorigenesis experiment is BALB/c, and the tumor cell strain adopted is mouse colon cancer cell strain CT26.

The results of CT26 subcutaneously transplanted tumor mice given either control solvent treatment or ID1 inhibitor alone indicate that ID1 inhibitor ML323 can significantly inhibit tumor cell growth (see fig. 7A and B). Immunohistochemical polychromatic staining (PANOVUE Biotechnology co., LTD, 1100020) was performed on tumor tissue sections of both groups of mice, respectively, and the number of infiltrated CD8 ⁺ T cells in tumor tissue of both groups of mice was compared, which indicated that ID1 inhibitor ML323 increased CD8 ⁺ T cell infiltration in tumor tissue (see fig. 7C). The expression of the stem index CD44 in two groups of tumor cells is detected by flow cytometry, and the result shows that the ID1 inhibitor ML323 reduces the expression of CD44 of colon cancer cells.

The above results suggest that ID1 inhibitor ML323 can inhibit tumor growth and increase CD8 ⁺ T cell infiltration in tumor tissue and reduce the stem nature of colon cancer cells CT 26.

Example 8 transgenic mice with Id1 knockdown by the medullary System explored whether the anti-tumor effect of ML323 was dependent on inhibition of ID1 in TAM

MC38 subcutaneous engrafting tumor models were constructed in control mice (Id 1 ^f/f) and in marrow knockout Id1 transgenic mice (Id 1 ^Lyz-KO), respectively, id1 ^f/f and Id1 ^Lyz-KO mice were each divided into two groups, and solvent control (saline) and ML323 (5 mg/kg, once a day) were administered by intraperitoneal injection, respectively (see FIG. 8A). The tumor growth rate of four groups of mice was observed, and the tumor weight was weighed after sampling. The results showed that after the marrow knockout of Id1, the tumor inhibition by ML323 was significantly reduced, and the tumor inhibition rate was reduced from 70% to about 40% (see FIG. 8B-D). These results suggest that the antitumor effect of ML323 is dependent in part on the inhibition of ID1 in TAM.

Example 9 study of the Effect of ID1 inhibitor in combination with chemotherapeutic pharmaceutical compositions on tumor growth by mouse colon cancer CT26 subcutaneous oncology experiments

The mice experiments were divided into 4 groups, control solvent treated group (physiological saline), ID1 inhibitor-alone treated group (ML 323:1mg/kg,3mg/kg,5 mg/kg), chemotherapeutic drug-alone treated group (5-FU: 1mg/kg,10mg/kg,40 mg/kg), and ID1 inhibitor and chemotherapeutic drug combination treated group (ML 323 (mg/kg): 5-FU (mg/kg): 1:1,1:10,1:40,3:1,3:10,3:40,5:1,5:10, 5:40).

The mouse strain adopted in the above-mentioned tumorigenesis experiment is BALB/C, and the tumor cell strain adopted is mouse colon cancer cell strain CT26. Three days after CT26 cells are subcutaneously planted, ID1 inhibitor is injected into the abdominal cavity, 5-FU is injected into the abdominal cavity after 8 th, 11 th and 14 th days, materials are obtained after CT26 cells are subcutaneously planted for 17 th days, a tumor growth curve is drawn, the weight of a tumor is weighed, and the weight reduction rate of the mouse is calculated. Mice weight loss rate= (1-mice weight on day/mice weight on day x 100%).

As shown in Table 1, considering the tumor suppression rate and the weight reduction rate of mice in combination, the mass fraction ratio of ML323 to 5-FU is preferably 3:10. The dosage of the composition is far less than the common dosage of the antitumor drugs used independently, and the toxic and side effects of the chemotherapeutic drugs on tumor patients and the treatment cost of the patients are reduced.

As shown in FIG. 9, the results show that the ID1 inhibitor reduces the dryness of colon cancer cells CT26, and the combined administration of the ID1 inhibitor and the chemotherapeutic drug can inhibit the tumor growth better than the single administration.

Table 1 use of ID1 inhibitors in combination with chemotherapeutic agents and their effect on tumor inhibition and body weight in tumor-bearing mice

Example 10 study of the Effect of ID1 inhibitor in combination with immunotherapeutic pharmaceutical compositions on tumor growth by mouse colon cancer CT26 subcutaneous tumorigenesis experiments

The mice experiments were divided into 4 groups, control solvent treatment (normal saline), ID1 inhibitor alone (ML 323:1mg/kg,3mg/kg,5 mg/kg), immunotherapeutic drug alone (CTLA-4 Ab:2mg/kg,4mg/kg,8 mg/kg), and ID1 inhibitor and immunotherapeutic drug combination treatment (ML 323 (mg/kg): CTLA-4 Ab (mg/kg): 1:2,1:4,1:8,3:2,3:4,3:8,5:2,5:4, 5:8).

The mouse strain adopted in the above-mentioned tumorigenesis experiment is BALB/C, and the tumor cell strain adopted is mouse colon cancer cell strain CT26. Intraperitoneal injection of ID1 inhibitor 3 days after subcutaneous implantation of CT26 cells, intraperitoneal injection of CTLA-4Ab 6 days after subcutaneous implantation of CT26 cells, sampling after subcutaneous implantation of CT26 cells for 20 days, drawing tumor growth curve, weighing tumor weight, calculating weight reduction rate of mice, and detecting infiltration and killing activity of CD8 ⁺ T cells in each group of tumors by flow cytometry. Mice weight loss rate= (1-mice weight on day/mice weight on day x 100%).

As shown in Table 2, considering the tumor suppression rate and the weight reduction rate of mice, the mass ratio of ML323 to CTLA-4 Ab is preferably 3:4. The dosage of the composition is far less than the common dosage of the antitumor drugs used independently, and the toxic and side effects of the chemotherapeutic drugs on tumor patients and the treatment cost of the patients are reduced.

As shown in figure 10, the result shows that the combination of the ID1 inhibitor and the immune treatment drug increases the infiltration and killing activity of CD8 ⁺ T cells, and the combination of the ID1 inhibitor and the immune checkpoint inhibitor drug can inhibit the tumor growth better than the combination of the ID1 inhibitor and the immune checkpoint inhibitor drug alone.

Table 2 effects of doses of checkpoint inhibitor pharmaceutical compositions on tumor inhibition and body weight of tumor-bearing mice

Example 11 on-line analysis platform by Gepia demonstrated that ID1 expression was higher in pancreatic cancer tumor tissue than in paracancerous tissue

To further understand the expression of ID1 in tumor tissues other than colorectal cancer, we analyzed the cancer Genome map database (THE CANCER Genome Atlas, TCGA) by Gepia on-line analysis platform (http:// gepia2.Cancer-pku. Cn/# index), which showed that ID1 also exhibited significantly higher expression levels in tumor tissues than in paracancerous tissues in pancreatic cancer (PANCREATIC ADENOCARCINOMA, PAAD) patients, consistent with the expression pattern in colorectal cancer (see fig. 11).

EXAMPLE 12 investigation of the Effect of ID1 inhibitor and chemotherapeutic pharmaceutical composition or ID1 inhibitor and immunotherapeutic pharmaceutical composition on tumor growth by mouse pancreatic cancer cell Pan02 subcutaneous tumorigenesis experiment

This example further evaluates the anti-tumor effect of ID1 inhibitors and their combination therapies in pancreatic cancer.

The mice experiments were divided into 6 groups, control solvent treatment (normal saline), ID1 inhibitor alone (ML 323:3 mg/kg), chemotherapeutic drug alone (5-FU: 10 mg/kg), ID1 inhibitor and chemotherapeutic drug combination (ML 323:5-FU:3: 10), immunotherapeutic drug alone (CTLA-4 Ab:4 mg/kg), and ID1 inhibitor and immunotherapeutic drug combination (ML 323 (mg/kg): CTLA-4 Ab (mg/kg): 3: 4).

The mouse strain adopted in the tumor forming experiment is C57BL/6J, the adopted tumor cell strain is a mouse pancreatic cancer cell strain Pan02, and the construction method of the mouse subcutaneous transplantation tumor model is the same as that described above. The method comprises the steps of subcutaneously planting Pan02 cells 2×10 ⁵/patient, carrying out intraperitoneal injection of an ID1 inhibitor or a chemotherapeutic drug after 3 days, carrying out intraperitoneal injection of CTLA-4 Ab after 6 days of subcutaneously planting Pan02 cells, measuring the tumor volume after 4 days of subcutaneously planting Pan02 cells, obtaining materials after 20 days of subcutaneously planting Pan02 cells, weighing the weight of each group of tumors, and calculating the tumor inhibition rate, wherein the calculation method is that the tumor inhibition rate= (1-combined administration of the tumor volume of the group/the tumor volume of the control group) ×100%.

TABLE 3 Effect of combination treatment of tumor volume and tumor inhibition in tumor-bearing mice

Medicament	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%

As shown in Table 3, the results indicate that the ID1 inhibitor reduces the growth of pancreatic cancer cells Pan02, and that the combination of ID1 inhibitor/chemotherapeutic agent and the combination of ID1 inhibitor/immunotherapeutic agent inhibit tumor growth better than the combination alone.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (8)

1. A composition for treating and/or preventing tumors, characterized in that the composition comprises any of the following drug combinations: (1) ID1 inhibitors and chemotherapy drugs; (2) ID1 inhibitors and immune checkpoint inhibitor drugs. 2. The composition according to claim 1, characterized in that the substance that inhibits the expression of ID1 in TAM includes a compound that inhibits the transcription, translation or protein stability of ID1, including but not limited to small nucleic acids such as shRNA that inhibit ID1 transcription, small molecule compounds that inhibit the stability of ID1 protein, and pharmaceutically acceptable carriers or excipients. 3. The composition according to claim 2, characterized in that the small molecule compound includes but is not limited to ML323. 4. The composition according to claim 1, characterized in that the chemotherapy drugs include but are not limited to 5-fluorouracil, oxaliplatin, capecitabine or irinotecan. 5. The composition according to claim 1, characterized in that the immune checkpoint inhibitor drugs include but are not limited to CTLA-4 antibodies, PD-1 antibodies, and PD-L1 antibodies. 6. Use of the composition according to any one of claims 1 to 5 in the preparation of a drug for treating and/or preventing tumors. 7. The use according to claim 6, characterized in that the tumor comprises a solid tumor with high expression of ID1. 8. The use according to claim 7, characterized in that the tumor includes but is not limited to intestinal cancer, liver cancer, gastric cancer, lung cancer, pancreatic cancer, etc.