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WO2025249993A1 - Composition pour le traitement de maladies cancéreuses et procédé de criblage associé - Google Patents

Composition pour le traitement de maladies cancéreuses et procédé de criblage associé

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Publication number
WO2025249993A1
WO2025249993A1 PCT/KR2025/095352 KR2025095352W WO2025249993A1 WO 2025249993 A1 WO2025249993 A1 WO 2025249993A1 KR 2025095352 W KR2025095352 W KR 2025095352W WO 2025249993 A1 WO2025249993 A1 WO 2025249993A1
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Prior art keywords
cancer
tam
marker
cells
macrophage
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English (en)
Korean (ko)
Inventor
이지희
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Ewha Womans University
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Ewha Womans University
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Publication of WO2025249993A1 publication Critical patent/WO2025249993A1/fr
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Definitions

  • the present invention relates to a composition for treating cancer and a method for screening the same.
  • Cancer is one of the incurable diseases civilization faces, and massive amounts of capital are being invested globally in the development of treatments. In South Korea, it has been the leading cause of death since 1983, with over 100,000 people diagnosed annually and over 60,000 dying. Carcinogens, including smoking, ultraviolet rays, chemicals, food, and other environmental factors, are known to cause cancer. However, the diverse causes make the development of treatments challenging, and the effectiveness of treatments also varies depending on the site of the disease.
  • anticancer drugs include biological agents such as enzyme preparations or vaccines, purely synthetic drugs, and drugs derived from natural products.
  • anticancer drugs using genes, enzymes, and vaccines are not yet at the practical stage, and anticancer drugs developed through chemotherapy have significant toxicity and have side effects of destroying not only cancer cells but also normal cells because they cannot selectively eliminate only cancer cells.
  • cancer cells have recently developed resistance to these drugs, making them ineffective in cancer treatment. Therefore, there is an urgent need to develop effective anticancer drugs that are less toxic and do not induce resistance in cancer cells for the treatment and prevention of cancer. Accordingly, the development of a simple method for screening new anticancer drugs is also required.
  • the inventors of the present invention completed the present invention by confirming that the expression of tumor-associated macrophage (TAM) markers changed when a culture medium derived from cancer-associated fibroblasts exposed to killed cancer cells was treated.
  • TAM tumor-associated macrophage
  • the present invention provides a pharmaceutical composition for treating cancer, comprising a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured.
  • CAFs cancer-associated fibroblasts
  • the present invention provides a method for screening a cancer treatment agent, comprising the steps of: (a) administering a carcinogenic substance or cancer cells to a non-human experimental animal; (b) administering a test substance to the experimental animal; and (c) confirming the expression levels of tumor suppressive macrophage (M1 TAM) markers and tumor supportive macrophage (M2 TAM) markers in cells of the experimental animal.
  • M1 TAM tumor suppressive macrophage
  • M2 TAM tumor supportive macrophage
  • the description of redundant content will be omitted below.
  • the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.
  • the present invention provides a pharmaceutical composition for treating cancer, comprising a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured.
  • CAFs cancer-associated fibroblasts
  • cancer-associated fibroblast refers to ⁇ -SMA (alpha-smooth muscle actin) positive fibroblasts existing inside and/or around a cancer lesion, and the presence of CAF has been confirmed in various cancers such as colon cancer, lung cancer, prostate cancer, breast cancer, stomach cancer, cholangiocarcinoma, and basal cell carcinoma.
  • CAF cancer-associated fibroblast
  • the CAF may be associated with one or more cancers selected from the group consisting of fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangiosarcoma, malignant skin cancer, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, lung cancer, gastric cancer, breast cancer, colon cancer, and prostate cancer.
  • cancers selected from the group consisting of fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangiosarcoma, malignant skin cancer, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, lung cancer, gastric cancer, breast cancer, colon cancer, and prostate cancer.
  • apoptotic cancer cells may be cancer cells that have been induced to apoptotic by irradiating them with light of a specific wavelength.
  • the irradiation of light of the specific wavelength may be ultraviolet ray (UV) irradiation.
  • the wavelength may be irradiated for 5 to 30 minutes at a wavelength of 100 to 400 nm. More specifically, the UV irradiation may be irradiated for 20 minutes at a wavelength of 150 to 350 nm or for 10 to 15 minutes at a wavelength of 200 to 300 nm.
  • co-culture may be achieved by co-culturing CAFs with apoptotic cancer cells.
  • CAFs may be cultured with apoptotic cancer cells in X-VIVO or serum-free DMEM medium for 20 to 30 hours.
  • culture medium refers to a culture product obtained through co-culturing CAFs and apoptotic cancer cells.
  • the culture medium may be a liquid medium, a solid medium, or a semi-solid medium.
  • the culture medium may be a conditioned medium (CM).
  • cancer refers to a disease in which abnormally transformed cells proliferate rapidly and uncontrollably due to changes in the genes of cells for various reasons. Cancer cells can spread to various parts of the body through the bloodstream and lymphatic system.
  • the cancer disease may be at least one selected from the group consisting of breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, colon cancer, lung cancer, skin cancer, ovarian cancer, cervical cancer, blood cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, and leukemia, and preferably may be lung cancer.
  • the lung cancer may be lung adenocarcinoma or non-small cell lung cancer.
  • a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells can inhibit tumor growth by increasing the expression level of tumor suppressive macrophage (T1 TAM) markers and decreasing the expression level of tumor supportive macrophage (T2 TAM) markers.
  • CAFs cancer-associated fibroblasts
  • T1 TAM tumor suppressive macrophage
  • T2 TAM tumor supporting macrophage
  • a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured can increase the levels of pro-apoptotic markers including Bax, C-Cas3, and C-PARP, and can decrease the expression levels of anti-apoptotic markers including Mcl-1 and Bcl-xL.
  • CAFs cancer-associated fibroblasts
  • changes in the expression levels of the above markers may be due to the fact that the culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells not only inhibits the survival of M2 macrophages and promotes apoptosis through the WISP-1-STAT1 signaling pathway, but also induces reprogramming from M2 TAMs to TAMs expressing an M1-like phenotype.
  • CAFs cancer-associated fibroblasts
  • WISP-1 is a target protein of the WNT signaling pathway, and WNT signaling plays a role in lung development, regulating both epithelial and mesenchymal development through autocrine and paracrine signals.
  • CD16 is an Fc-gamma receptor, also known as Fc ⁇ RIII, and is found on the surface of NK cells, neutrophils, monocytes, and macrophages.
  • CD32 is an Fc-gamma receptor, also known as Fc ⁇ RII, and corresponds to a surface receptor glycoprotein belonging to the Ig gene family.
  • CD80 is a B7, type I membrane protein belonging to the Ig gene family, and is known to be involved in regulating T cell activation and B cell activity.
  • H2Ab1 corresponds to a variant of a histone protein encoded by the H2AFB1 gene, and activates various functions including peptide antigen binding activity.
  • MHCII refers to the major histocompatibility complex found only in professional antigen-presenting cells such as dendritic cells, macrophages, and B cells.
  • iNOS NOS2
  • Ca 2+ -independent NOS Nitric oxide synthase
  • IFN ⁇ is a dimerized soluble cytokine belonging to the type II interferon group, and is known to have a role in activating macrophages to increase phagocytosis, tumor sterilization, and intracellular apoptosis.
  • IL12p40 is a subunit of the IL-12 cytokine family, functions as a chemoattractant for macrophages, and plays a role in promoting the movement of dendritic cells.
  • TNF ⁇ is a member of the tumor necrosis factor (TNF) family, which is mainly secreted by activated macrophages and is known to play an important role in various immune-mediated inflammatory diseases.
  • IL-1 ⁇ refers to a cytokine protein encoded by IL1B among the interleukin-1 genes, and is involved in various cell activities such as cell proliferation, differentiation, and apoptosis.
  • CD163 is a high-affinity scavenger receptor for the hemoglobin-haptoglobin complex, and is also a marker for cells of the monocyte/macrophage lineage.
  • CD206 is also called mannose receptor and corresponds to C-type lectin that mainly exists on the surface of macrophages, immature dendritic cells, and liver endothelial cells.
  • Arginase 1 is a gene encoding arginase, which is known as a marker of M2a macrophages and myeloid-derived suppressor cells (MDSC), which are major mediators of T cell suppression.
  • TGF ⁇ 1 is a polypeptide member of the transforming growth factor beta family of cytokines, and is a protein that performs various functions including regulation of cell growth, proliferation, differentiation, and apoptosis.
  • IL-10 is an anti-inflammatory cytokine that plays a role in downregulating the expression of Th1 cytokines, MHCII antigens, and co-stimulatory molecules in macrophages.
  • IL-4 is a cytokine that induces differentiation from naive Th0 cells into Th2 cells, and is an important regulator of humoral immunity and acquired immunity.
  • IL-13 is a protein encoded by the IL13 gene, and is a cytokine secreted from Th2 cells, CD4 cells, mast cells, etc.
  • Bax is also known as Bcl-2-like protein 4, which forms a heterodimer with Bcl-2 and functions as an apoptosis activator.
  • C-Cas3 refers to Caspase 3, which is cleaved and activated during cell death, and transmits a cell death signal through enzymatic activity toward downstream targets including PARP and other substrates.
  • C-PARP refers to Poly-ADP-ribose polymerase (PARP) that is cleaved by caspase during caspase-dependent apoptosis.
  • Mcl-1 belongs to the Bcl-2 protein family and plays a role in regulating cell death, cell cycle progression, and mitochondrial homeostasis.
  • Bcl-xL is a member of the Bcl-2 protein family and prevents the release of mitochondrial contents that induce caspase activation and cell death.
  • treatment refers to any act of improving or beneficially changing the symptoms of a cancer disease by administering a composition according to the present invention.
  • composition of the present invention may be formulated and provided in an appropriate form.
  • the composition may be prepared by including one or more pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carriers include, but are not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • the pharmaceutical composition of the present invention may include, but is not limited to, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, and other pharmaceutically acceptable additives.
  • the pharmaceutical composition of the present invention when formulated as a solid oral preparation, it includes tablets, pills, powders, granules, capsules, etc., and such solid preparations may include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., and include, but are not limited to, lubricants such as magnesium stearate and talc.
  • excipient for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc.
  • the pharmaceutical composition of the present invention when formulated as an oral liquid, it includes a suspension, a solution, an emulsion, a syrup, etc., and includes, but is not limited to, a diluent such as water or liquid paraffin, a wetting agent, a sweetener, a fragrance, a preservative, etc.
  • the pharmaceutical composition of the present invention when formulated for parenteral use, it includes a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, and a suppository.
  • Non-aqueous solvents and suspensions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • Bases for suppositories include, but are not limited to, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.
  • composition may be administered in single or multiple doses in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to prevent or treat a disease at a reasonable benefit/risk ratio applicable to medical prevention or treatment, and the effective dosage level may be determined according to factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and excretion rate, the duration of treatment, drugs combined with or used concurrently with the composition of the present invention used, and other factors well known in the medical field.
  • the pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes, for example, but not limited to, oral administration, intrathecal, intra-auricular, intraperitoneal or intravenous, intramuscular, subcutaneous, intrauterine, sublingual, or intracerebrovascular injection.
  • the pharmaceutical composition of the present invention can be formulated into a unit dosage form pharmaceutical preparation suitable for administration into the patient's body according to a conventional method in the pharmaceutical field and administered, and the preparation includes an effective dosage amount through one or more administrations.
  • Preferred dosage forms for this purpose are parenteral administration preparations such as injections and infusions.
  • the administration dosage of the pharmaceutical composition may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.
  • the route and method of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in their method, and any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area.
  • administration refers to introducing a given substance into a human or animal by any suitable method.
  • the therapeutic composition according to the present invention may be administered orally or parenterally via any common route, as long as it can reach the target tissue.
  • the therapeutic composition according to the present invention may be administered by any device capable of transporting the active ingredient to target cells.
  • the present invention provides a method for screening a cancer treatment agent.
  • the screening method for a cancer treatment agent comprises the following steps:
  • the present invention comprises a step of (a) administering a carcinogenic substance or cancer cells to an experimental animal other than a human.
  • any substance known to be a carcinogen can be used to produce an animal model that induces cancer.
  • arsenic, benzene, beryllium, cadmium, hexavalent chromium compounds, ethylene oxide, nickel, radon, or vinyl chloride can be used to induce cancer.
  • any experimental animal commonly used in the art can be treated with arsenic, benzene, beryllium, cadmium, hexavalent chromium compounds, ethylene oxide, nickel, radon, and/or vinyl chloride, and the animal model itself or cells isolated therefrom in which the disease is induced can be used.
  • the experimental animal model can be, for example, a mouse, hamster, rat, ferret, guinea pig, rabbit, dog, primate, or pig, and more preferably a mouse.
  • any cancer cell for inducing the above cancer may be administered.
  • the cancer cells may be one or more selected from the group consisting of fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangiosarcoma, malignant skin cancer, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, lung cancer, gastric cancer, breast cancer, colon cancer, and prostate cancer.
  • the administration of such cancer cells may be administered in an amount typically known to induce cancer in each experimental animal.
  • the cancer-causing substance or cancer cells may be administered through a route appropriate to their characteristics, for example, through respiration, intramuscular injection, renal injection, intraperitoneal injection, or subcutaneous injection.
  • the substances expressed or activated in vivo in non-cancer and cancer-induced states are different. Therefore, to screen for candidate substances for cancer prevention and treatment, cancer must first be induced.
  • the method for screening a cancer treatment agent according to the present invention comprises the step of (b) administering a test substance to the experimental animal.
  • test material refers to an unknown candidate substance used in screening to determine whether it affects the expression level of a gene or the expression or activity of a protein.
  • the sample includes, but is not limited to, chemicals, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.
  • test substances can be administered via conventional routes of administration, including oral and parenteral administration.
  • oral administration may be used, and parenteral administration may be selected from, but is not limited to, topical application to the skin, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
  • the dosage of the test substance can be adjusted to an appropriate level of dosage according to the type of drug and the drug efficacy, based on the general knowledge of those skilled in the art.
  • the method for screening for cancer comprises the step of (c) confirming the expression level of a tumor suppressive macrophage (M1 TAM) marker and a tumor supporting macrophage (M2 TAM) marker in cells of the experimental animal.
  • M1 TAM tumor suppressive macrophage
  • M2 TAM tumor supporting macrophage
  • the method for screening for cancer according to the present invention may include selecting a test substance that (i) increases the expression level of a tumor suppressive macrophage (M1 TAM) marker and (ii) decreases the expression level of a tumor supportive macrophage (T2 TAM) marker in step (c).
  • M1 TAM tumor suppressive macrophage
  • T2 TAM tumor supportive macrophage
  • tumor-associated macrophages are macrophages that participate in the formation of a tumor microenvironment, and influence tumor angiogenesis, proliferation, metastasis, and immunosuppression by secreting various cytokines, chemokines, and proteolytic enzymes.
  • the tumor-associated macrophages are divided into tumor-suppressive macrophages (M1 TAMs) and tumor-supportive macrophages (M2 TAMs).
  • the above tumor suppressive macrophage (M1 TAM) marker may be any one or more selected from the group consisting of CD16/32, CD80, H2Ab1, MHCII, iNOS (NOS2), IFN ⁇ , IL12p40, TNF ⁇ , and IL-1 ⁇ , but is not limited thereto.
  • the above tumor-supporting macrophage (M2 TAM) marker may be any one or more selected from the group consisting of, but is not limited to, CD163, CD206, Arginase 1 (Arg1), TGF ⁇ 1, IL-10, IL-4, and IL-13.
  • the drug may be considered to have therapeutic efficacy against cancer.
  • the tumor suppressive macrophage (M1 TAM) marker is expressed at a level comparable to that of the positive control, the drug may be considered to have therapeutic efficacy against cancer.
  • the drug may be judged to have therapeutic efficacy against cancer.
  • the tumor-supporting macrophage (M2 TAM) marker is expressed at a level equivalent to that of the positive control, the drug may be judged to have therapeutic efficacy against cancer.
  • a control sample means all sample groups for which the expression levels of tumor suppressive macrophage (M1 TAM) markers and tumor supportive macrophage (M2 TAM) markers can be compared and determined according to test substance treatment, including a normal control group and a therapeutic substance control group known for treating cancer.
  • M1 TAM tumor suppressive macrophage
  • M2 TAM tumor supportive macrophage
  • the expression levels of the above tumor suppressive macrophage (M1 TAM) marker and tumor supporting macrophage (M2 TAM) marker can be measured by any one selected from the group consisting of, but not limited to, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry and Western blotting, and flow cytometry.
  • ELISA enzyme-linked immunosorbent assay
  • qPCR quantitative real-time polymerase chain reaction
  • immunohistochemistry immunohistochemistry and Western blotting
  • flow cytometry any one selected from the group consisting of, but not limited to, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry and Western blotting, and flow cytometry.
  • the method for screening for cancer according to the present invention may further include the step of (d) confirming (i) an increase in the expression level of a pro-apoptotic marker and (ii) a decrease in the expression level of an anti-apoptotic marker in cells of the experimental animal.
  • the pro-apoptotic marker may be at least one selected from the group consisting of Bax, C-Cas3, and C-PARP, and the anti-apoptotic marker may be at least one selected from the group consisting of Mcl-1 and Bcl-xL.
  • the test substance may be a pharmaceutical composition including a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured.
  • CAFs cancer-associated fibroblasts
  • the present invention comprises the steps of: (a) contacting a test substance with cancer cells;
  • a method for screening a cancer treatment agent comprising the step of selecting a test substance having (i) an increased expression level of the tumor suppressive macrophage (M1 TAM) marker and (ii) a decreased expression level of the tumor supportive macrophage (M2 TAM) marker compared to a control sample;
  • Any cancer cell can be used as the cancer cell, and any type of cancer cell mentioned above can be equally applied to the present screening method.
  • the method for screening a cancer treatment agent of the present invention comprises a step of contacting a test substance with cancer cells.
  • the cancer cells include cells from a patient with cancer, animal cells, or tissues thereof.
  • cells induced with cancer-causing substances can be utilized, and any substance known to be a cancer-causing substance can be utilized.
  • the cancer cells can be induced by treatment with arsenic, benzene, beryllium, cadmium, hexavalent chromium compounds, ethylene oxide, nickel, radon, or vinyl chloride.
  • arsenic, benzene, beryllium, cadmium, hexavalent chromium compounds, ethylene oxide, nickel, radon, or vinyl chloride any laboratory animal commonly used in the art can be treated with arsenic, benzene, beryllium, cadmium, hexavalent chromium compounds, ethylene oxide, nickel, radon, or vinyl chloride, and the resulting animal model, or cells isolated therefrom, can be used.
  • Contact with the above test substance means all contacts in vitro and in vivo , and includes all contacts in which the test substance is treated on cells in vitro or administered to animals, etc. in vivo .
  • the method for screening a cancer treatment agent comprises the steps of: confirming the expression levels of tumor suppressive macrophage (M1 TAM) markers and tumor supportive macrophage (M2 TAM) markers in cancer cells that have come into contact with the test substance; and selecting a test substance in which (i) the expression level of the tumor suppressive macrophage (M1 TAM) marker increases and (ii) the expression level of the tumor supportive macrophage (M2 TAM) marker decreases compared to a control sample.
  • M1 TAM tumor suppressive macrophage
  • M2 TAM tumor supportive macrophage
  • the expression levels of the tumor suppressive macrophage (M1 TAM) marker and the tumor supporting macrophage (M2 TAM) marker can be measured by any one selected from the group consisting of, but not limited to, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry, Western blotting, and flow cytometry.
  • ELISA enzyme-linked immunosorbent assay
  • qPCR quantitative real-time polymerase chain reaction
  • immunohistochemistry Western blotting
  • flow cytometry any one selected from the group consisting of, but not limited to, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry, Western blotting, and flow cytometry.
  • the composition for treating cancer of the present invention can treat cancer by suppressing the survival of M2 macrophages and inducing reprogramming of M2 TAMs into M1 TAMs, thereby inhibiting tumor growth. Furthermore, the screening method of the present invention can be usefully utilized in the development and identification of novel cancer treatment drugs by easily identifying and obtaining substances effective in treating cancer among a large number of therapeutic candidates.
  • Figure 1 is a diagram showing that the total TAM density of a primary tumor is reduced by administration of ApoSQ-CAF CM.
  • Figure 2 is a diagram showing that the M2 TAM fraction of primary tumors is reduced by administration of ApoSQ-CAF CM.
  • Figure 3 is a diagram showing that the M1 TAM fraction of primary tumors increases by administration of ApoSQ-CAF CM.
  • Figure 4 is a diagram confirming that the cell death promotion effect is mediated by WISP-1.
  • Figure 5 is a diagram confirming that reprogramming from M2 TAM to M1 TAM is induced by administration of ApoSQ-CAF CM.
  • the upper part is a representative flow cytometry plot for CD11b + TAM, and the lower part shows the TAM ratio (CD163 + /MHCII + TAM ratio).
  • Figure 6 is a diagram showing polarization into M1 and M2 macrophages.
  • FIGS. 1 and (d) are diagrams showing the results of immunoblotting using M2 markers (CD163, CD206, and Arginase1 (Arg1)) and M1 markers (MHCII, iNOS, and IL12p40) on M1 and M2 macrophages (M2) derived from THP-1 cells and BMDM.
  • M2 markers CD163, CD206, and Arginase1 (Arg1)
  • M1 markers MHCII, iNOS, and IL12p40
  • (e) and (f) are diagrams showing the results of immunofluorescence staining for the M1 marker CD86 and the M2 marker CD163.
  • Figure 7 is a diagram confirming in vitro that the apoptosis of M2 macrophages increases by CM administration of CAFs exposed to killed cancer cells.
  • Figure 8 is a diagram confirming in vitro that reprogramming from M2 TAM to M1 TAM is induced by CM administration of CAF exposed to killed cancer cells.
  • (d) and (e) are diagrams showing the results of flow cytometry analysis on CD16 + and CD206 + cells among M2 macrophages derived from THP-1 and BMDM.
  • Mouse rWISP-1 (1680-WS) and human rWISP-1 (1627-WS) were purchased from R&D Systems (Minneapolis, MN, USA).
  • Neutralizing mouse WISP-1 antibody (MAB1680) and IgG (MAB0061) were purchased from R&D Systems (Minneapolis, MN, USA).
  • CAFs were isolated from lung tumors of Kras-mutant (Kras LA1 ) mice by magnetic-activated cell sorting (MACS) using the fibroblast-specific marker Thy1. Isolated CAFs were cultured in ⁇ -MEM medium supplemented with 10% fetal bovine serum, penicillin/streptomycin (100 U/100 ⁇ g), 2 mM L-glutamine, and 1 mM sodium pyruvate. For immortalization, CAFs were stably transfected with the TERT plasmid (pCDH-3xFLAG-TERT; Addgene 51 plasmid #51631) using Lipofector-EXT (AptaBio).
  • Cancer epithelial cell lines were irradiated with 254 nm of UV light for 15 min and then cultured for 2 h at 37°C under 5% CO2 conditions.
  • Light microscopy of Wright-Giemsa-stained samples revealed that most irradiated cells were apoptotic.
  • lysed (necrotic) cancer cells were obtained through multiple freeze-thaw cycles. Apoptosis and necrosis were confirmed by flow cytometric analysis using a FACSCalibur system (BD Biosciences) after staining with annexin V-FITC/propidium iodide (BD Biosciences).
  • CAFs were plated at 3 ⁇ 10 5 cells/ml and incubated overnight at 37°C under 5% CO 2 conditions. After serum-starvation for 24 h, the cells were stimulated with X-VIVO 10 medium (04-380Q). For this, the culture medium was replaced with X-VIVO 10 medium containing killed or necrotic cancer cells (9 ⁇ 10 5 cells/ml). After 20 h, the supernatant was harvested by centrifugation and used as CM for stimulating target cancer epithelial cells (5 ⁇ 10 3 cells/ml). The CM was stored at -80°C for in vivo experiments.
  • CAF CM were incubated with 10 ⁇ g/ml mouse anti-WISP-1 neutralizing antibody (R&D Systems) or 10 ⁇ g/ml IgG isotype control (R&D Systems) for 2 h.
  • the anti-WISP-1 antibody neutralizing effect was tested by WISP-1 ELISA before use.
  • mice were managed and handled in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH).
  • 344SQ cells a lung adenocarcinoma cell line (1 ⁇ 106 cells in 100 ⁇ l of PBS per mouse), were injected subcutaneously into the right posterior flank of syngeneic (129/Sv) mice. 2 days later, CM derived from CAFs (100 ⁇ l per mouse) was injected intratumorally three times a week. In addition, CM with or without neutralizing mouse anti-WISP-1 Ab (10 ⁇ g/ml) or isotype IgG was administered on the same schedule. Tumor growth in the mice was monitored daily, and the mice were sacrificed 6 weeks after injection. Autopsies were then performed to examine the diameter and weight of the subcutaneous tumor masses.
  • formalin fixation was performed for 30 min at room temperature, the samples were washed three times for 5 min each with IF-Wash buffer (PBS containing 0.05% NaN 3 , 0.1% BSA, 0.2% Triton X-100, and 0.05% Tween-20) and permeabilized with 0.5% Triton X-100 in PBS (Sigma-Aldrich) for 5 min at room temperature.
  • IF-Wash buffer PBS containing 0.05% NaN 3 , 0.1% BSA, 0.2% Triton X-100, and 0.05% Tween-20
  • permeabilized with 0.5% Triton X-100 in PBS Sigma-Aldrich
  • the target protein was captured with the primary antibody during an 18-hour incubation at 4°C, and the captured protein was visualized with fluorescence-conjugated IgG in a darkroom for 1 hour. After staining, the slides were mounted with VECTASHIELD mounting medium containing DAPI (Vector Laboratories) and imaged using a confocal microscope (LSM5 PASCAL). Information on the antibodies used is shown in Table 1 below.
  • Equal amounts of protein were then dissolved in SDS-PAGE gels (#161-0158, Bio-Rad Laboratories) and transferred to nitrocellulose membranes (10600001, GE Healthcare Life Science) using a wet transfer system (Bio-Rad Laboratories).
  • the membranes were blocked with 5% bovine serum albumin (BSA)-TBST or 5% milk-TBST for 1 h, incubated with labeled primary antibodies overnight, and then incubated with labeled secondary antibodies for 1 h at 37°C.
  • BSA bovine serum albumin
  • the Odyssey image analysis system was used for quantification.
  • THP-1 cells were maintained in RPMI 1640 medium containing 10% FBS at 37°C under humidified conditions containing 5% CO2 .
  • THP-1 cell-derived M1 or M2 macrophages were generated as a macrophage model. Specifically, THP-1 cells were primed with 150 ng/ml PMA for 6 h to produce nonpolarized macrophages. To generate M1 macrophages, nonpolarized macrophages were stimulated with 20 ng/ml IFN ⁇ and 100 ng/ml LPS for 48 h. To generate M2 macrophages, nonpolarized macrophages were further stimulated with 20 ng/ml IL-4 and 20 ng/ml IL-13 for 48 h.
  • BMDMs isolated from the tibia and femur of C57BL/6 mice were cultured with L929 complement DMEM for 7 days and then polarized into M1 and M2 type macrophages.
  • Macrophages (3.5 ⁇ 10 4 ) were plated in 96-well plates containing RPMI-1640 medium and cultured in X-VIVO 10 medium for 6 hours. CM or rWISP-1 was added to each group and incubated for 2–4 days under 5% CO 2 , 37°C conditions. Afterwards, cell counting kit-8 (CCK-8) solution was added to the wells and incubated for 30 minutes. The absorbance was measured at 450 nm using a microplate reader.
  • CM or rWISP-1 was added to each group and incubated for 2–4 days under 5% CO 2 , 37°C conditions.
  • cell counting kit-8 (CCK-8) solution was added to the wells and incubated for 30 minutes. The absorbance was measured at 450 nm using a microplate reader.
  • CAFs or macrophages were transiently transfected with siRNAs specifically targeting WISP1 (Bioneer), STAT1 (Bioneer), or a control siRNA (SN-1003 AccuTarget TM Negative Control) at a final concentration of 50 nM using a transfection reagent (Lipofectamin RNAi MAX; Invitrogen, Carlsbad, CA). After overnight transfection, cells were cultured in appropriate media for 24 h and stimulated with ApoSQ cells.
  • the siRNA sequences used were as follows (gene: sense, antisense):
  • Apoptosis was detected using an annexin V-FITC/propidium iodide (PI) staining kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. After harvesting, macrophages were resuspended in 500 ⁇ l of binding buffer, and 100 ⁇ l of the suspension was stained with 5 ⁇ l of FITC-conjugated annexin V and 5 ⁇ l of PI for 15 min at room temperature in the dark. FITC-conjugated annexin V-positive cells were then detected by flow cytometry (ACEA NovoCyte, San Diego, CA, USA) using 400 ⁇ l of binding buffer, and data were analyzed using NovoExpress software 1.5.
  • PI annexin V-FITC/propidium iodide
  • NovoCyte (Agilent, Santa Clara, CA, USA) was used. All cell suspensions (1 ⁇ 10 6 CD11b + TAMs and M2 macrophages) were placed in 500 ⁇ l buffer (PBS + 2% FBS) and incubated for 60 min with fluorophore-conjugated anti-mouse antibodies at the manufacturer's recommended concentration. All antibodies used are listed in Table 3. Data acquisition was performed on NovoCyte (Agilent, Santa Clara, CA, USA), and NovoExpress Software 1.5 was used for analysis.
  • TNF- ⁇ , IL-1 ⁇ , IL-4, and IL-13 in macrophage cultures were measured using ELISA kits (R&D Systems) according to the manufacturer's instructions.
  • Pairwise comparisons were performed using the two-tailed Student's t-test, and multiple comparisons were performed using the Kruskal-Wallis test followed by Dunn's post hoc test. A P value less than 0.05 was considered statistically significant, and all data were analyzed using Prism 5 software (GraphPad Software Inc., San Diego, CA, USA).
  • CM CM of CAFs
  • ApoSQ-CAF CM CM of CAFs exposed to killed 344SQ cells
  • the CMs were pre-incubated for 2 h with a neutralizing antibody against WISP-1 or an IgG isotype control antibody.
  • Immunofluorescence staining using total TAM markers revealed that administration of ApoSQ-CAF CM significantly reduced total TAM density in both the central and marginal regions of primary tumors compared to administration of CAF CM (Fig. 1).
  • administration of WISP-1 immunodepleted ApoSQ-CAF CM did not change total TAM density, but administration of CM pre-incubated with IgG isotype control antibody showed an effect similar to that of administration of ApoSQ-CAF CM.
  • Example 2 Induction of reprogramming from M2 TAM to M1 TAM by ApoSQ-CM administration.
  • M2 and M1 markers in CD11b + TAMs isolated from primary tumors were analyzed using RT-qPCR arrays, respectively.
  • nine M2-related genes were downregulated more than twofold in the ApoSQ-CAF CM group compared to the CAF CM group (Fig. 5a).
  • seven M1-related genes including Cd32, Ifng, Cd16, Tnf, Nos2, Socs3, and Cd80, were upregulated more than twofold in the ApoSQ-CAF CM group compared to the CAF CM group.
  • qRT-PCR analysis of M2- and M1-specific markers and cytokines was also performed.
  • THP-1 cells and primary mouse bone marrow-derived macrophages were polarized into M1 and M2 macrophages to mimic TAMs (Figs. 6a and 6b).
  • Successful polarization into M1 and M2 macrophages was confirmed by immunoblotting using M2 markers (CD163, CD206, and Arg1) and M1 markers (MHCII, iNOS, and IL12p40) (Figs. 6c and 6d).
  • confocal microscopy analysis also confirmed polarization using CD86 + M1 and CD163 + M2 markers (Figs. 6e and 6f).
  • THP-1-derived M2 macrophages treatment of THP-1-derived M2 macrophages with ApoSQ-CAF CM enhanced the levels of M1 cytokines, including TNF ⁇ and IL-1 ⁇ , whereas suppressed the levels of M2 cytokines, including IL-4 and IL-13 (Fig. 8c).
  • flow cytometry analysis showed that treatment of THP-1-derived M2 macrophages or BMDM-derived M2 macrophages with ApoSQ-CAF CM decreased the surface expression of the CD206 M2 marker and increased the surface expression of the CD16 M1 marker compared to treatment with CAF CM or NecSQ-CAF CM (Figs. 8d and 8e).

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Abstract

La présente invention concerne une composition pour le traitement de maladies cancéreuses et un procédé de criblage associé. La composition pour le traitement de maladies cancéreuses de la présente invention inhibe la survie de macrophages M2 et induit une reprogrammation des TAM M2 en TAM M1 afin d'inhiber la croissance de tumeurs, ce qui permet de traiter des maladies cancéreuses. De plus, le procédé de criblage de la présente invention peut facilement identifier et obtenir des substances efficaces pour traiter des maladies cancéreuses parmi de multiples substances thérapeutiques candidates, et peut donc être utilisé de manière avantageuse pour mettre au point et identifier de nouveaux agents thérapeutiques contre le cancer.
PCT/KR2025/095352 2024-05-29 2025-05-22 Composition pour le traitement de maladies cancéreuses et procédé de criblage associé Pending WO2025249993A1 (fr)

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