WO2023055076A1 - Composition pour inhiber des métastases cancéreuses - Google Patents

Composition pour inhiber des métastases cancéreuses Download PDF

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Publication number
WO2023055076A1
WO2023055076A1 PCT/KR2022/014563 KR2022014563W WO2023055076A1 WO 2023055076 A1 WO2023055076 A1 WO 2023055076A1 KR 2022014563 W KR2022014563 W KR 2022014563W WO 2023055076 A1 WO2023055076 A1 WO 2023055076A1
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cancer
cafs
cells
aposq
wisp
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PCT/KR2022/014563
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English (en)
Korean (ko)
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이지희
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이화여자대학교 산학협력단
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Priority claimed from KR1020220058184A external-priority patent/KR20230046190A/ko
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Publication of WO2023055076A1 publication Critical patent/WO2023055076A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the present invention provides a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured, a method for inhibiting cancer metastasis using the same, and It relates to the use of the pharmaceutical composition.
  • CAFs cancer-associated fibroblasts
  • Lung cancer both small cell and non-small cell cancer
  • Lung cancer is the most common cancer worldwide in both men and women, and is the leading cause of cancer-related death (18.0% of total cancer deaths).
  • About 75% of lung cancer patients are confirmed to have locally advanced or metastatic disease at the time of diagnosis.
  • metastasis is a multistep process including migration and invasion of cancer cells, and becomes a marker of malignant tumors.
  • CAFs Cancer-associated fibroblasts
  • Basal processes such as cancer cell migration and invasion that can promote progression to malignancy and metastatic spread.
  • CAFs physically remodel the matrix in the tumor stroma, allowing cancer cells to invade while still maintaining epithelial properties.
  • the underlying molecular mechanisms of CAFs-mediated regulation in tumor progression are still unclear.
  • Notch signaling in regulating fibroblast activation in the tumor microenvironment (TME) is well established. Activation of Notch signaling is generally tightly regulated by direct interaction with ligand-expressing cells, and problems in the regulation of Notch signaling result in developmental abnormalities or cancer. Interestingly, Notch activity is associated with both oncogenic and tumor-suppressive functions, which depend on the complex microenvironment of cellular responses that Notch induces. Stromal fibroblasts with an activated Notch pathway can attenuate melanoma growth and inhibit tumor angiogenesis, in part through upregulation of Wnt-induced signaling protein-1 (WISP-1). These findings will help elucidate the molecular mechanisms for the Notch1-dependent tumor-regulatory role in CAFs in other cancer types as well.
  • WISP-1 Wnt-induced signaling protein-1
  • TME tumor microenvironment
  • phagocyte-mediated clearance has been reported to suppress antitumor immune responses.
  • tumors can evade immune surveillance by inhibiting recognition for efferocytosis.
  • the anti-inflammatory and anti-inflammatory lipid autacoids specifically inhibit debris-stimulated cancer progression by promoting the clearance of cellular debris through macrophage phagocytosis in multiple tumor types.
  • macrophages exposed to UV-irradiated apoptotic lung cancer cells showed upregulation of exosome phosphatase and tensin homolog (PTEN) and peroxisome proliferator-activated receptor-gamma (PPARr).
  • PTEN exosome phosphatase and tensin homolog
  • PPARr peroxisome proliferator-activated receptor-gamma
  • the present inventors studied how the interaction between CAFs and apoptotic cancer cells regulates the migration and invasion of cancer cells and CAFs. It was confirmed that WISP-1 was induced to inhibit migration and invasion of cancer cells and CAFs themselves. In addition, after intratumoral administration of apoptotic lung cancer cells, inhibition of CAFs activation and cancer cell migration and invasion were confirmed. Furthermore, the present invention was completed by confirming the effect of inhibiting lung metastasis after intratumoral administration of the co-culture of apoptosis lung cancer cells and CAFs.
  • One aspect is to provide a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured.
  • CAFs cancer-associated fibroblasts
  • Another aspect is to provide a pharmaceutical composition for inhibiting cancer metastasis, containing cancer-associated fibroblasts exposed to killed cancer cells.
  • Another aspect is to provide a health functional food for inhibiting cancer metastasis containing a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.
  • Another aspect is to provide a method for preparing a composition for inhibiting cancer metastasis, comprising co-cultivating cancer-related fibroblasts and apoptotic cancer cells.
  • Another aspect is to provide a method for inhibiting cancer metastasis, including the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptosis-killed cancer cells are co-cultured.
  • Another aspect is to provide a method for suppressing cancer metastasis, including treating a subject with Wnt-induced signaling protein-1 (WISP-1).
  • WISP-1 Wnt-induced signaling protein-1
  • Another aspect is to provide a method for preventing or treating cancer, which includes the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptosis cancer cells are co-cultured.
  • Another aspect is to provide a method for preventing or treating cancer, including treating a subject with Wnt-induced signaling protein-1 (WISP-1).
  • WISP-1 Wnt-induced signaling protein-1
  • Another aspect is to provide the use of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured for the manufacture of a drug for inhibiting cancer metastasis.
  • Another aspect is to provide use of WISP-1 (Wnt-induced signaling protein-1) for the manufacture of a drug for suppressing cancer metastasis.
  • WISP-1 Wi-induced signaling protein-1
  • Another aspect is to provide the use of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured for the manufacture of a drug for preventing or treating cancer.
  • WISP-1 Wnt-induced signaling protein-1
  • Another aspect is to provide a use for suppressing cancer metastasis of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.
  • Another aspect is to provide a use for inhibiting cancer metastasis of a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.
  • Another aspect is to provide a use of Wnt-induced signaling protein-1 (WISP-1) to inhibit cancer metastasis.
  • WISP-1 Wnt-induced signaling protein-1
  • Another aspect is to provide a use for preventing or treating cancer of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.
  • Another aspect is to provide a pharmaceutical composition containing a culture medium in which cancer-related fibroblasts and killed cancer cells are co-cultured for use in preventing or treating cancer.
  • Another aspect is to provide a use for cancer prevention or treatment of Wnt-induced signaling protein-1 (WISP-1).
  • WISP-1 Wnt-induced signaling protein-1
  • the present invention provides a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured.
  • CAFs cancer-associated fibroblasts
  • the culture medium in which the CAFs and the apoptotic cancer cells are co-cultured has an effect of inhibiting metastasis of cancer cells.
  • CAFs Cancer-Associated Fibroblasts
  • ⁇ -SMA smooth muscle actin
  • the CAFs are colorectal cancer.
  • its presence has been confirmed in various cancers such as lung cancer, prostate cancer, breast cancer, stomach cancer, cholangiocarcinoma, and basal cell carcinoma.
  • the cancers associated with the CAFs include brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, renal cancer, ureter cancer, and bladder cancer.
  • prostate cancer penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and solid cancer such as skin cancer.
  • the CAFs are related to cancer and may be fibroblasts related to malignant solid tumors.
  • the CAFs are fibroblasts related to sarcomas such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, malignant skin cancer, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, etc. It may be, and may be fibroblasts related to lung cancer, stomach cancer, breast cancer, colon cancer or prostate cancer.
  • the CAFs can have enhanced phagocytic ability by the interaction of Notch1 signaling and BAI1/Rac1 signaling.
  • apoptotic cancer cells may be those in which apoptosis is induced by irradiating cancer cells with light of a specific wavelength.
  • the irradiation of light of the specific wavelength may be ultraviolet (Ultra-violet ray, UV) irradiation.
  • the wavelength may be irradiated for 5 to 30 minutes with a wavelength of 100 to 400 nm.
  • co-culture can be achieved by culturing CAFs and apoptotic cancer cells together.
  • CAFs may be cultured with apoptotic cancer cells in X-VIVO or serum-free DMEM medium for 20 to 30 hours.
  • culture medium means a culture product obtained through co-cultivation of 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.
  • the carcinoma of the killed cancer cells is 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, It may be at least one selected from the group consisting of testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, and leukemia.
  • the carcinoma may be at least one selected from the group consisting of lung cancer, breast cancer, stomach cancer, colon cancer, and prostate cancer
  • the lung cancer may be lung adenocarcinoma or non-small cell lung cancer.
  • the lung adenocarcinoma cells may be the 344SQ cell line
  • the non-small cell lung cancer cells may be the A549 cell line
  • the colon cancer cells may be the HCT116 cell line
  • the breast cancer cells may be the MCF-7 cell line.
  • the culture medium may contain WISP-1 (Wnt-induced signaling protein-1) as an active ingredient.
  • 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. do.
  • the WISP-1 may be generated by Notch1 signaling.
  • Notch1 signaling is cell contact-dependent through Notch receptors and plays an important role in development, regeneration, and maintenance of homeostasis, and the activity of Notch signaling is associated with both oncogenic and tumor-suppressive functions.
  • Notch1-WISP-1 signaling is known to determine the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma invasion and metastasis.
  • the Notch1 signaling may be initiated by Dll1 (Delta-like ligand 1).
  • Dll1 is a type of Notch Delta ligand that plays a role in regulating cell fate decisions during blood formation and is known to be involved in cell-cell communication.
  • the Notch1 signaling may be enhanced by BAI1/Rac1 (Brain angiogenesis inhibitor 1/Ras-related C3 botulinum toxin substrate 1) signaling.
  • BAI1 is a type of aggregation GPCR that acts to inhibit angiogenesis
  • Rac1 is a type of GTPase that plays a role in regulating cell growth, cytoskeleton reorganization, cell cycle, cell-cell aggregation and migration, and the like.
  • the composition can enhance the phagocytic ability of cancer-related fibroblasts by the interaction of Notch1 signaling and BAI1/Rac1 signaling.
  • efferocytosis means phagocytosis to remove apoptotic cells.
  • the phagocytic ability refers to the ability of CAFs to remove apoptotic cancer cells through phagocytosis.
  • the "interaction” may mean crosstalk between the Notch1 signaling and the BAI1/Rac1 signaling, and more specifically, may mean positive crosstalk.
  • crosstalk means a phenomenon in which one or more components of a signal transduction pathway affect other components. Positive crosstalk refers to a phenomenon in which one signaling reinforces another signaling. Negative crosstalk refers to a phenomenon in which one signaling suppresses another signaling.
  • the BAI1/Rac1 signaling can promote phagocytosis of apoptotic cancer cells by the cancer-associated fibroblasts.
  • the composition can enhance WISP-1 production by the interaction of Notch1 signaling and BAI1/Rac1 signaling.
  • the composition can reduce activation markers in cancer-associated fibroblasts.
  • the activation marker may be one or more selected from the group consisting of Acta2, Col1 ⁇ 1, Fn, Itg beta 1, Spp1, Pdgfr ⁇ , Pdgfr beta, and Mmp1a, 2, 9, and 12.
  • the composition can reduce growth factors and chemokines in cancer-associated fibroblasts.
  • the growth factor may be one or more selected from the group consisting of Vegfa, Hgf, Cxcl12 and Cxcl14.
  • the composition can increase Notch1-related molecules in cancer-related fibroblasts.
  • the Notch1-related molecule may be one or more selected from the group consisting of Notch1, WISP-1 (Ccn4), Hey1, Hey2, Hes1, and Hes5.
  • metastasis refers to a state in which a malignant tumor has spread to other tissues away from an organ where it has occurred, and can be used as a marker of a malignant tumor.
  • the metastasis may include migration and invasion of cancer cells.
  • migration means that cells move to a specific location in response to a specific external signal
  • invasion means that cells expand and penetrate other nearby tissues.
  • the present invention provides a pharmaceutical composition for inhibiting cancer metastasis containing cancer-related fibroblasts exposed to apoptotic cancer cells.
  • CAFs exposed to the killed cancer cells contain WISP-1, and thus have an effect of suppressing metastasis of cancer cells.
  • exposure means a process of contacting or stimulating apoptosis cancer cells and CAFs to enable cell-to-cell interaction, and is distinguished from a conditioned medium in which apoptosis cancer cells and CAFs are co-cultured.
  • the pharmaceutical composition according to the present invention may include a "pharmaceutically acceptable carrier".
  • the pharmaceutically acceptable carrier is one commonly used in formulation and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. It is not, and if necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants, etc.
  • compositions for injections such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.
  • a suitable pharmaceutically acceptable carrier and formulation it can be preferably formulated according to each component using the method disclosed in Remington's literature.
  • the pharmaceutical composition of the present invention is not particularly limited in dosage form, but may be formulated as an injection, an inhalant, an external preparation for the skin, or an oral intake.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, applied to the skin, nasal cavity, or respiratory tract) according to the desired method, and the dosage is determined according to the patient's condition and weight, disease Depending on the degree, drug form, administration route and time, it can be appropriately selected by those skilled in the art.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and activity of the drug of the patient's disease , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.
  • the composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg per day or every other day, or 1 It can be administered in 1 to 3 divided doses per day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.
  • the present invention provides a health functional food for inhibiting cancer metastasis, which contains a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.
  • the health functional food of the present invention may be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations by further including one or more of carriers, diluents, excipients and additives.
  • carriers diluents, excipients and additives.
  • examples of foods to which the extract of the present invention can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, and the like.
  • Additives that may be further included in the present invention include natural carbohydrates, flavors, nutrients, vitamins, minerals (electrolytes), flavors (synthetic flavors, natural flavors, etc.), colorants, fillers (cheese, chocolate, etc.), One or more components selected from the group consisting of pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonating agents and fruit flesh may be used. .
  • natural carbohydrates examples include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the health functional food of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like.
  • the composition according to the present invention may contain fruit flesh for preparing natural fruit juice and vegetable beverages. These components may be used independently or in combination.
  • carrier examples include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium Silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate And at least one selected from the group consisting of mineral oil is preferably used.
  • the health functional food of the present invention When formulating the health functional food of the present invention, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • the present invention provides a method for preparing a composition for inhibiting cancer metastasis, comprising co-cultivating cancer-related fibroblasts and apoptotic cancer cells.
  • the killing of cancer cells may be induced through UV irradiation, and the UV irradiation may be performed for 5 to 30 minutes with a wavelength of 100 to 400 nm. In one embodiment, the UV irradiation may be performed with a wavelength of 150 to 350 nm for 20 minutes or with a wavelength of 200 to 300 nm for 10 to 15 minutes.
  • the step of culturing may be to culture in X-VIVO medium for 24 hours to make it serum-starved.
  • the culture medium may be replaced with X-VIVO or serum-free DMEM medium containing killed cancer cells.
  • the co-cultivation may be performed for 10 to 30 hours, 15 to 25 hours, or 18 to 24 hours after medium replacement.
  • the present invention provides a method for suppressing cancer metastasis, comprising the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptosis-killed cancer cells are co-cultured.
  • the step of treating the subject with the culture medium is performed by treating the subject with a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured. It could be
  • treatment may mean any act of adding or administering the culture medium to a subject according to experimental requirements.
  • subject means a subject in need of a method for preventing, controlling or treating a disease, and may include a mammal.
  • the mammal may include humans or primates, mice, cows, dogs, horses, pigs, and the like.
  • the present invention provides a method for inhibiting cancer metastasis, comprising treating a subject with Wnt-induced signaling protein-1 (WISP-1).
  • WISP-1 Wnt-induced signaling protein-1
  • the present invention provides a method for preventing or treating cancer, comprising the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.
  • the culture medium in which the cancer-related fibroblasts and apoptosis cancer cells are co-cultured can be treated in the form of a pharmaceutical composition containing the same.
  • prevention may refer to any activity that inhibits or delays the onset of cancer
  • treatment may mean any activity that improves or beneficially changes cancer.
  • the present invention provides a method for preventing or treating cancer, including treating a subject with Wnt-induced signaling protein-1 (WISP-1).
  • WISP-1 Wnt-induced signaling protein-1
  • the present invention provides the use of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured for the manufacture of a drug for inhibiting cancer metastasis.
  • the present invention provides a use of WISP-1 (Wnt-induced signaling protein-1) for the preparation of a drug for suppressing cancer metastasis.
  • WISP-1 Wi-induced signaling protein-1
  • the present invention provides the use of a culture medium in which cancer-related fibroblasts and killed cancer cells are co-cultured for the manufacture of a drug for preventing or treating cancer.
  • the present invention provides a use of WISP-1 (Wnt-induced signaling protein-1) for the manufacture of a drug for preventing or treating cancer.
  • WISP-1 Wi-induced signaling protein-1
  • the present invention provides a use for inhibiting cancer metastasis of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.
  • the present invention provides a use for inhibiting cancer metastasis of a pharmaceutical composition for inhibiting cancer metastasis, which contains a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.
  • the present invention provides a use of WISP-1 (Wnt-induced signaling protein-1) to inhibit cancer metastasis.
  • the present invention provides a use for preventing or treating cancer of a culture medium in which cancer-related fibroblasts and killed cancer cells are co-cultured.
  • the present invention provides a use for preventing or treating cancer of a pharmaceutical composition containing a culture medium in which cancer-related fibroblasts and killed cancer cells are co-cultured.
  • the present invention provides a use of Wnt-induced signaling protein-1 (WISP-1) for preventing or treating cancer.
  • WISP-1 Wnt-induced signaling protein-1
  • CAFs exposed to the culture medium or killed cancer cells of the present invention can be usefully used as cancer metastasis suppressors.
  • FIG. 1 is a diagram showing that a conditioned medium (CM) containing a culture medium of cancer-associated fibroblasts (CAFs) co-cultured with apoptotic cancer cells inhibits migration and invasion of cancer cells.
  • Figure 1a shows a control, CAF conditioned medium (CAF CM), and 344SQ co-culture conditioned medium (ApoSQ-CAF CM) killed with CAFs in the presence or absence of TGF-beta 1 using 344SQ, a lung adenocarcinoma cell line.
  • It is a diagram measuring the migration and invasion of 344SQ cells in the 344SQ co-culture conditioned medium (NecSQ-CAF CM) group with CAFs and necrosis.
  • Figure 1b shows the control, CAF CM, CAFs and apoptosis A549 co-culture conditioned medium (ApoA-CAF CM), CAFs and A549 co-culture in the presence or absence of TGF-beta 1 using A549, a non-small cell lung cancer cell line. It is a diagram measuring migration and invasion of A549 cells in the culture conditioned medium (NecA-CAF CM) group.
  • FIG. 1c is a control, CAF CM, CAFs and apoptosis HCT116 co-culture conditioned medium (ApoH-CAF CM), and CAFs and necrosis HCT116 co-culture in the presence or absence of TGF-beta 1 using HCT116, a colorectal cancer cell line. It is a diagram measuring the migration and invasion of HCT116 cells in the conditioned medium (NecH-CAF CM) group.
  • Figure 1d is a control, CAF CM, CAFs and MCF-7 co-culture conditioned medium (ApoM-CAF CM), CAFs and necrosis in the presence or absence of TGF-beta 1 using MCF-7, a breast cancer cell line, respectively. It is a diagram measuring the migration and invasion of MCF-7 cells in the MCF-7 co-culture conditioned medium (NecM-CAF CM) group.
  • Figure 2 is a diagram confirming that there is no effect of inhibiting migration and invasion of cancer cells by direct treatment of the killed cancer cell culture medium alone and the killed cancer cells.
  • Figure 2a is a diagram showing the migration and invasion of 344SQ cells in the control, CAF CM, ApoSQ CM and NecSQ CM groups, respectively, in the presence of TGF-beta 1.
  • Figure 2b is a diagram measuring 344SQ cell migration and invasion in the control, ApoSQ, and NecSQ groups, respectively, in the presence or absence of TGF-beta 1.
  • FIG. 3 is a diagram showing that ApoSQ-CAF CM inhibits the signaling pathway activity related to TGF-beta 1 in 344SQ cells using immunoblot analysis.
  • Figure 3a is a diagram showing the results of immunoblot analysis of TGF-beta 1-related signaling pathway expressed after treatment of CAF CM and ApoSQ-CAF CM together with TGF-beta 1 in 344SQ cells.
  • Figure 3b is a diagram showing the total and phosphorylated amounts of Smad2, Smad3, FAK, AKT, SrC, ERK, and P38 expressed after treatment with CAF CM and ApoSQ-CAF CM in 344SQ cells.
  • FIG. 4 is a diagram showing that ApoSQ-CAF CM suppresses MMP2 and MMP12 mRNA expression and protein expression in 344SQ cells using qRT-PCR and immunoblot analysis.
  • A A diagram showing the levels of MMP2 and MMP12 mRNA expression after treatment of CAF CM and ApoSQ-CAF CM with TGF-beta in 344SQ cells.
  • B 344SQ cells treated with CAF CM and ApoSQ-CAF CM, and then immunoblot analysis of MMP2 and MMP12 proteins expressed and a diagram showing the protein amounts.
  • 5 is a diagram showing that only ApoSQ inhibits cell migration and invasion when CAFs are brought into contact with ApoSQ and NecSQ, respectively.
  • Figure 6 shows that when CAFs were exposed to ApoSQ-CAF CM, NecSQ-CAF CM, ApoA-CAF CM, NecA-CAF CM, ApoH-CAF CM and NecH-CAF CM in the presence or absence of TGF-beta 1, respectively, death It is a diagram showing that only the conditioned cancer cells and CAF-conditioned medium inhibit cell migration and invasion.
  • Figure 6a is a diagram showing migration and invasion of CAFs after treatment with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM.
  • Figure 6b is a diagram showing migration and invasion of CAFs after treatment with CAF CM, ApoA-CAF CM, and NecA-CAF CM.
  • Figure 6c is a diagram showing migration and invasion of CAFs after treatment with CAF CM, ApoH-CAF CM, and NecH-CAF CM.
  • Figure 6d is a diagram showing migration and invasion of CAFs after treatment with CAF CM, ApoM-CAF CM, and NecM-CAF CM.
  • FIG. 7 is a diagram confirming that the culture medium of killed cancer cells alone has no effect of inhibiting migration and invasion of CAFs.
  • FIG. 8 is a diagram showing that ApoSQ inhibits the expression of CAF activation markers in CAFs using qRT-PCR, immunoblot analysis, and immunofluorescence staining.
  • Figure 8a is a diagram showing the results of analyzing the mRNA expression level of CAF activation markers when ApoSQ and NecSQ were treated with CAFs, and the results of immunoblot analysis of CAF activation markers and protein amounts when ApoSQ and NecSQ were treated with CAFs.
  • 8b is a diagram showing the fluorescence intensity of ⁇ -SMA when CAFs were treated with ApoSQ and NecSQ.
  • 9 is a diagram showing that ApoSQ inhibits the signaling pathway activity related to TGF-beta 1 in CAFs using immunoblot analysis.
  • 9a is a diagram showing the results of immunoblot analysis of TGF-beta 1-related signaling pathways after CAFs were treated with ApoSQ together with TGF-beta 1;
  • Figure 9b is a diagram showing the total and phosphorylated amounts of signaling pathways, that is, Smad2, Smad3, AKT, FAK, SrC, ERK, and P38, after CAFs were treated with ApoSQ along with TGF-beta 1.
  • FIG. 10 is a diagram showing that ApoSQ inhibits the expression of MMP mRNA and protein in CAFs using qRT-PCR and immunoblot analysis.
  • A A diagram showing the mRNA expression levels of MMP2 and MMP12 after ApoSQ was treated in CAFs.
  • B A diagram showing the results of immunoblot analysis and protein amounts of MMP2 and MMP12 proteins after CAFs were treated with ApoSQ.
  • FIG. 11 is a result of cytokine array analysis in CAF CM, ApoSQ-CAF CM and ApoSQ CM, and the expression of leukemia inhibitory factor (LIF) and WISP-1 among 111 types of cytokines was compared with CAF CM and ApoSQ-CAF CM groups. It is a diagram confirming that the cytokine increased the most in ApoSQ-CAF CM.
  • LIF leukemia inhibitory factor
  • FIG. 12 is a diagram showing that the expression level of WISP-1 decreases when WISP-1 is knocked down.
  • A A diagram showing the results of immunoblot analysis and the level of WISP-1 protein expression measured after transfecting CAFs with WISP-1-specific siRNA.
  • B It is a diagram showing the amount of WISP-1 measured by ELISA in the culture medium after CAFs were transfected with siRNA specific to WISP-1.
  • FIG. 13 is a diagram showing that the effect of inhibiting cell migration and invasion is reversed when CAFs are transfected with WISP-1 siRNA.
  • Figure 13a is a diagram showing migration and invasion of 344SQ cells upon treatment with CAF CM and ApoSQ-CAF CM in the presence or absence of WISP-1 siRNA.
  • Figure 13b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment in the presence or absence of WISP-1 siRNA.
  • FIG. 14 is a diagram showing that the expression level of LIF decreases when LIF is knocked down.
  • A A diagram showing the result of immunoblot analysis and the amount of LIF protein measured after transfection of CAFs with LIF-specific siRNA.
  • B A diagram showing the amount of LIF measured by ELISA in CAF CM and ApoSQ-CAF CM after CAFs were transfected with LIF-specific siRNA.
  • C A diagram showing the amount of WISP-1 measured by ELISA in CAF CM and ApoSQ-CAF CM after CAFs were transfected with LIF-specific siRNA.
  • FIG. 15 is a diagram showing that the effect of inhibiting cell migration and invasion is maintained when CAFs are transfected with LIF siRNA.
  • Figure 15a is a diagram showing migration and invasion of 344SQ cells when treated with CAF CM and ApoSQ-CAF CM in the presence or absence of LIF siRNA.
  • Figure 15b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment in the presence or absence of LIF siRNA.
  • FIG. 16 is a diagram showing that the expression level of WISP-1 increases when WISP-1 is overexpressed.
  • A A diagram showing the results of immunoblot analysis and protein expression level measured after transfection of CAFs with mock vector and WISP-1.
  • B A diagram showing the amount of WISP-1 measured by ELISA in CAF CM and ApoSQ-CAF CM after CAFs were transfected with mock vector and WISP-1.
  • 17 is a diagram showing that cell migration and invasion inhibitory effects are increased when CAFs are transfected with WISP-1 to overexpress WISP-1.
  • 17a is a diagram showing migration and invasion of 344SQ cells when treated with CAF CM and ApoSQ-CAF CM, depending on whether or not WISP-1 was transfected.
  • 17b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment, depending on whether or not WISP-1 was transfected.
  • 18 is a diagram showing that the anti-WISP-1 antibody treatment of ApoSQ-CAF CM reverses the effect of inhibiting migration and invasion of 344SQ cells.
  • FIG. 19 is a diagram showing that migration and invasion of 344SQ cells or CAFs are inhibited in a concentration-dependent manner when recombinant WISP-1 is added.
  • Figure 19a is a diagram showing migration and invasion of 344SQ cells according to rWISP-1 concentration.
  • Figure 19b is a diagram showing migration and invasion of CAFs according to rWISP-1 concentration.
  • FIG. 20 is a diagram showing that rWISP-1 inhibits the activity of the signaling pathway related to TGF-beta 1 and MMP expression in 344SQ cells in a concentration-dependent manner using immunoblot analysis.
  • 20a is a diagram showing the results of immunoblot analysis of TGF-beta 1-related signaling molecules for each concentration of rWISP-1.
  • Figure 20b is a diagram showing the total amount and phosphorylation amount of TGF-beta 1-related signaling pathways, that is, Smad2, Smad3, FAK, AKT, SrC, ERK, and P38, when rWISP-1 was treated by concentration.
  • (A) is a diagram showing the amount of MMP2 and MMP12 mRNA expressed by rWISP-1 concentration.
  • (B) is a diagram showing the results of immunoblot analysis and protein amounts of MMP2 and MMP12 proteins expressed by rWISP-1 concentrations.
  • 21 is a diagram showing that when rWISP-1 is treated with TGF-beta 1, the activity of the signaling pathway related to TGF-beta 1 and the expression of MMP are inhibited in CAFs using immunoblot analysis and qRT-PCR.
  • 21a is a diagram showing the results of immunoblot analysis of the TGF-beta 1-related signaling pathway upon treatment with rWISP-1.
  • Figure 21b is a diagram showing the total and phosphorylated amounts of TGF-beta 1-related signaling pathways, that is, Smad2, Smad3, FAK, AKT, SrC, ERK and P38 upon rWISP-1 treatment.
  • (A) is a diagram showing the change in the mRNA expression amount of MMP2 and MMP12 when rWISP-1 and TGF-beta 1 are treated together
  • (B) is a diagram showing the change in the mRNA expression amount of rWISP-1 and TGF-beta 1 when treated together with MMP2 and MMP12 It is a diagram showing the results of protein immunoblot analysis and the amount of protein.
  • 22 is a diagram showing that the inhibition of migration and invasion according to the addition of WISP-1 is reversed in 344SQ cells or CAFs when a specific anti-integrin is added.
  • 22a is a diagram showing that the inhibition of migration and invasion according to the addition of WISP-1 is reversed in 344SQ cells when an anti-integrin ⁇ v or beta 3 antibody is added.
  • 22b is a diagram showing that the inhibition of migration and invasion according to the addition of WISP-1 is reversed in CAFs when an anti-integrin ⁇ v or beta 5 antibody is added.
  • 23 is a diagram showing that the activation of the signaling pathway related to TGF-beta 1 and the suppression of MMP expression are reversed in 344SQ cells when a specific anti-integrin is added using immunoblot analysis and qRT-PCR.
  • 23a is a diagram showing the results of immunoblot analysis of TGF-beta 1-related signaling molecules according to the addition of rWISP-1 and anti-integrin ⁇ v or beta 3 antibodies.
  • 23b is a diagram showing the mRNA amounts of MMP2 and MMP12 according to the addition of rWISP-1 and anti-integrin ⁇ v or beta 3 antibodies.
  • 23c is a diagram showing the results of immunoblot analysis of MMP2 and MMP12 proteins according to the addition of WISP-1 and anti-integrin ⁇ v or beta 3 antibodies.
  • FIG. 24 is a diagram showing that when a specific anti-integrin is added using immunoblot analysis and qRT-PCR, the TGF-beta 1-related signaling pathway activity and inhibition of MMP expression are reversed by the addition of WISP-1 in CAFs.
  • 24a is a diagram showing the results of immunoblot analysis of TGF-beta 1 related signaling molecules according to the addition of rWISP-1 and anti-integrin ⁇ v or beta 5 antibodies.
  • 24b is a diagram showing the mRNA amounts of MMP2 and MMP12 according to the addition of rWISP-1 and anti-integrin ⁇ v or beta 5 antibodies.
  • 24c is a diagram showing the results of immunoblot analysis of MMP2 and MMP12 proteins according to the addition of WISP-1 and anti-integrin ⁇ v or beta 5 antibodies.
  • 25 is a diagram showing that the amount of proteins of factors related to the Notch1 signaling pathway expressed in CAFs according to the presence or absence of ApoSQ is increased.
  • A A diagram showing the results of protein immunoblot analysis according to the presence or absence of ApoSQ in CAFs.
  • B A diagram showing the amount of WISP-1 expressed in CAF CM, ApoSQ-CAF CM and NecSQ-CAF CM.
  • 26 is a diagram showing that the mRNA expression levels of Hes1 and WISP-1, which are target genes of the Notch signaling pathway, and the activity of 4 ⁇ CSL luciferase are increased when ApoSQ or NecSQ is treated in CAFs.
  • A A diagram showing the mRNA expression levels of Hes1 and WISP-1 analyzed by qRT-PCR.
  • B A diagram showing the activity of 4 ⁇ CSL luciferase, an overall Notch signal transcriptional effector, upon ApoSQ treatment.
  • FIG. 27 is a diagram showing changes in immunofluorescence staining for NICD1 and WISP-1 according to ApoSQ or NecSQ treatment in CAFs.
  • 28 is a diagram showing that WISP-1 protein expression is suppressed when Notch1 is knocked down through siRNA.
  • 28a is a diagram showing the results of immunoblot analysis measured after CAFs were transfected with Notch1 siRNA.
  • 28B (A) is a diagram showing the immunoblot analysis results and protein amounts for NICD1, Hes1, and WISP-1 measured according to the presence or absence of ApoSQ or NecSQ after CAFs were transfected with Notch1 siRNA, (B) is a diagram showing the ELISA results of WISP-1 in CAF CM and ApoSQ-CAF CM after transfection of CAFs with Notch1 siRNA.
  • FIG. 29 is a diagram showing that when Notch1 is knocked down through siRNA, the anti-migration and anti-invasion effects of ApoSQ-CAF CM and ApoSQ are reversed in 344SQ cells and CAFs, respectively.
  • Figure 29a is a diagram showing migration and invasion of 344SQ cells when treated with CAF CM and ApoSQ-CAF CM in the presence or absence of Notch1 siRNA.
  • Figure 29b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment in the presence or absence of Notch1 siRNA.
  • FIG. 30 is a diagram showing that when Notch1 activity is inhibited through DAPT, the expression of proteins related to the Notch1 signaling pathway is inhibited.
  • A A diagram showing the immunoblot analysis results and protein expression levels for NICD1, Hes1, and WISP-1 measured in the presence or absence of ApoSQ or NecSQ after CAFs were treated with 10 ⁇ M DAPT.
  • B A diagram showing the ELISA results of WISP-1 measured in CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM after CAFs were treated with 10 ⁇ M DAPT.
  • FIG. 31 is a diagram showing that when Notch1 is inhibited through DAPT, the anti-migration and anti-invasion effects of ApoSQ-CAF CM and ApoSQ are reversed in 344SQ cells and CAFs, respectively.
  • Figure 31a is a diagram showing migration and invasion of 344SQ cells upon treatment with CAF CM and ApoSQ-CAF CM in the presence or absence of DAPT.
  • Figure 31b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment in the presence or absence of DAPT.
  • DLL Delta-like ligand
  • JAG Jagged-like
  • JAG2 Jagged-like
  • JAG2 Jagged-like
  • FIG. 33 is a diagram showing that only Dll1 expression is increased in killed cancer cells 344SQ, A549, and HCT116.
  • A A diagram showing the results of immunoblot analysis for Dll1, Dll3, Dll4, JAG1 and JAG2 in apoptotic cancer cells 344SQ, A549 and HCT116.
  • B A diagram showing the amount of Dll1, Dll3, Dll4, JAG1 and JAG2 proteins in the killed cancer cells 344SQ, A549 and HCT116.
  • FIG. 34 is a diagram showing that expression of proteins related to the Notch1 signaling pathway is suppressed in CAFs after ApoSQ treatment when Dll1 is neutralized with an anti-Dll1 antibody.
  • A A diagram showing the immunoblot analysis results and protein amounts for NICD1, Hes1, and WISP-1 measured in the presence or absence of anti-Dll1 antibody in CAFs contacted with ApoSQ.
  • B A diagram showing the ELISA results of WISP-1 measured in the presence or absence of anti-Dll1 antibody in CAFs contacted with ApoSQ.
  • 35 is a diagram showing that expression of proteins related to the Notch1 signaling pathway is suppressed in CAFs when Dll1 is knocked down through siRNA.
  • 35A is a diagram showing the results of immunoblot analysis and the amount of Dll1 protein measured after transfection of 344SQ cells with Dll1 siRNA.
  • FIG. 35b (A) is a diagram showing the immunoblot analysis results and protein amounts for NICD1, Hes1, and WISP-1 measured according to the presence or absence of ApoSQ in CAFs after transfection of 344SQ cells with Dll1 siRNA, (B ) is a diagram showing the ELISA results of WISP-1 measured according to the presence or absence of ApoSQ in CAFs after transfecting 344SQ cells with Dll1 siRNA.
  • FIG. 36 is a diagram showing that, when CAFs in contact with ApoSQ are treated with annexin V, brain-specific angiogenesis inhibitor 1 (BAI1) signals are inhibited, thereby inhibiting ApoSQ phagocytosis, Notch1 activation, and WISP-1 secretion in CAFs.
  • Figure 36a is a diagram showing the percentage of CAFs phagocytosis after exposure to ApoSQ cells in the presence or absence of annexin V by flow cytometry. In FIG.
  • (A) is a diagram showing the immunoblot analysis results for NICD1, Hes1, and WISP-1 in CAFs contacted with ApoSQ in the presence or absence of annexin V
  • (B) is a diagram showing the results of immunoblot analysis in the presence or absence of annexin V.
  • (C) shows the ELISA results of WISP-1 measured in CAFs in contact with ApoSQ in the presence or absence of annexin V. It is also
  • 37 is a diagram showing that when CAFs in contact with ApoSQ were transfected with BAI1 siRNA, phagocytosis of CAFs, Notch1 activation, and WISP-1 secretion were suppressed as BAI1 signal was suppressed.
  • 37a is a diagram showing the results of immunoblot analysis and the amount of BAI1 protein measured after CAFs were transfected with BAI1 siRNA.
  • Figure 37b is a diagram showing the phagocytosis of CAFs measured in % by flow cytometry after CAFs were transfected with BAI1 siRNA and exposed to ApoSQ cells. In FIG.
  • (A) is a diagram showing the immunoblot analysis results for NICD1, Hes1, and WISP-1 measured in the presence or absence of ApoSQ after CAFs were transfected with BAI1 siRNA
  • (B) is a diagram showing CAFs with BAI1 siRNA. After transfection, the amount of NICD1, Hes1, and WISP-1 proteins measured according to the presence or absence of ApoSQ is a diagram. It is a diagram showing the results of ELISA.
  • FIG. 38 is a diagram showing that when CAFs in contact with ApoSQ are treated with an anti-BAI1 antibody, BAI1 signals are suppressed, and phagocytosis, Notch1 activation, and WISP-1 secretion of CAFs are suppressed.
  • Figure 38a is a diagram showing the percentage of CAFs phagocytosis after exposure of ApoSQ cells in the presence or absence of anti-BAI1 antibody by flow cytometry. In FIG.
  • (A) is a diagram showing the results of immunoblot analysis for NICD1, Hes1, and WISP-1 in CAFs contacted with ApoSQ in the presence or absence of anti-BAI1 antibody
  • (B) is a diagram showing the results of anti-BAI1 antibody
  • (C) is WISP-1 measured in CAFs contacted with ApoSQ in the presence or absence of anti-BAI1 antibody. It is a diagram showing the ELISA result of 1.
  • 39 is a diagram showing that when CAFs contacted with ApoSQ were transfected with BAI1-Flag overexpressing BAI1, phagocytosis of CAFs, Notch1 activation, and WISP-1 secretion increased as BAI1 signal increased.
  • 39a is a diagram showing the results of immunoblot analysis and the amount of BAI1 protein measured after CAFs were transfected with BAI1-Flag.
  • Figure 39b is a diagram showing the phagocytosis of CAFs measured in % by flow cytometry after CAFs were transfected with BAI1-Flag and exposed to ApoSQ cells.
  • (A) is a diagram showing the results of immunoblot analysis for NICD1, Hes1, and WISP-1 measured according to the presence or absence of ApoSQ after CAFs were transfected with BAI1-Flag
  • (B) is a diagram showing CAFs transfected with BAI1-Flag.
  • 40 is a diagram showing that ApoSQ increases the activity of Rac1 in a time-dependent manner in CAFs.
  • 41 is a diagram showing that when CAFs contacted with ApoSQ are treated with NSC23766, a Rac1 inhibitor, phagocytosis of CAFs, Notch1 activation, and WISP-1 secretion are inhibited as Rac1 signaling is inhibited.
  • 41a and 41b are diagrams measuring the phagocytic activity of CAFs in contact with ApoSQ in the presence or absence of 100 ⁇ M NSC23766.
  • 41a is a diagram showing the number of CAFs phagocytosing ApoSQ cells in % by flow cytometry.
  • 41B is a diagram illustrating phagocytic activity as a phagocytic index using a confocal microscope. In FIG.
  • (A) is a diagram showing the immunoblot analysis results for NICD1, Hes1, and WISP-1 in CAFs contacted with ApoSQ in the presence or absence of NSC23766, and (B) is a diagram showing the results of immunoblot analysis for ApoSQ and ApoSQ in the presence or absence of NSC23766.
  • (C) is a diagram showing ELISA results of WISP-1 measured in CAFs contacted with ApoSQ in the presence or absence of NSC23766.
  • FIG. 42 is a diagram showing that when CAFs in contact with ApoSQ are transfected with Notch1 siRNA, the activity of Rac1 and the phagocytosis of CAFs are suppressed according to the inhibition of Notch1 signal.
  • A A diagram showing the activity of Rac1 measured after transfecting CAFs with Notch1 siRNA.
  • B After transfecting CAFs with Notch1 siRNA and exposing ApoSQ cells, the phagocytosis of CAFs was measured in % by flow cytometry.
  • FIG. 43 is a diagram showing that when CAFs contacted with ApoSQ were treated with DAPT, the activity of Rac1 and phagocytosis of CAFs were suppressed as the Notch1 signal was suppressed.
  • A A diagram showing the activity of Rac1 measured in the presence or absence of 20 ⁇ M DAPT.
  • B A diagram showing the phagocytosis of CAFs in contact with ApoSQ in the presence or absence of DAPT through flow cytometry.
  • FIG. 44 is a diagram showing an experimental timeline for injecting 344SQ cells subcutaneously on the flanks of syngeneic mice, subcutaneously administering ApoSQ 2 days later, sacrificing the mice 6 weeks later, and isolating Thy1 CAFs from primary tumor tissue. am.
  • Thy1 + CAFs of the control group CAF activation markers (including Acta2, Col1 ⁇ 1, Fn, Itg beta 1, Spp1, Pdgfr ⁇ , and Pdgfr beta), MMPs (including Mmp1a, 2, 9, and 12), growth factors and chemokines (Vegfa, Hgf, It is a diagram measured by qRT-PCR that the mRNA expression level of Notch1-related molecules (including Notch1, Wisp1 (Ccn4), Hey1, Hey2, and Hes1) is increased, while the mRNA expression level of Cxcl12 and Cxcl14 is decreased.
  • 45A is a diagram showing a heat map made based on qRT-PCR results.
  • 45B is a diagram showing qRT-PCR results for each
  • 46 is a diagram showing the experimental timeline of administering ApoSQ and LY3039478 to mice.
  • mice 47 is a diagram showing that there is no significant difference between each group as a result of measuring mouse weight, tumor weight and tumor volume by administering LY3039478 (8 mg/kg), ApoSQ or ApoSQ and LY3039478 (8 mg/kg) to mice .
  • A A diagram showing the mouse weight, tumor weight and tumor volume in each group.
  • B A diagram showing images of tumors in each group.
  • 51 shows a decrease in the expression of ⁇ -SMA and an increase in the expression of NICD1 and WISP-1 through immunocytochemical analysis in Thy1 + CAFs isolated from primary tumor tissues of mice administered with ApoSQ, but when LY3039478 is additionally administered. It is a diagram showing that the effect is reversed.
  • 51A is a diagram showing the expression level of ⁇ -SMA in each group through immunocytochemical analysis.
  • 51b is a diagram showing the expression levels of NICD1 and WISP-1 in each group through immunocytochemical analysis.
  • FIG. 53 is a diagram showing that cell migration and invasion are inhibited in CD326 + tumor cells and Thy1 + CAFs isolated from primary tumor tissues of mice administered with ApoSQ, but the above effects are reversed when LY3039478 is additionally administered.
  • Figure 53a is a diagram measuring migration and invasion of isolated CD326 + tumor cells.
  • Figure 53b is a diagram showing the migration and invasion of isolated Thy1 + CAFs.
  • FIG. 54 shows that phosphorylation of signaling molecules related to migration and invasion, such as Smad2/3, FAK, ERK, and Akt, and expression of MMP2/12 protein are inhibited in CD326 + tumor cells isolated from primary tumor tissues of mice administered with ApoSQ. , It is a diagram showing that the effect is reversed when LY3039478 is additionally administered.
  • A A diagram showing the results of immunoblot analysis for phosphorylation and MMP2/12 of signaling molecules related to cell migration and invasion in isolated CD326 + tumor cells.
  • (B) It is a diagram measuring the amount of phosphorylation and MMP2/12 of signaling molecules related to cell migration and invasion in isolated CD326 + tumor cells.
  • 55 is a diagram showing the experimental timeline of administering CAF CM, ApoSQ-CAF CM, ApoSQ-CAF CM and anti-WISP-1 antibody, or ApoSQ-CAF CM and IgG to mice after subcutaneous injection of 344SQ cells.
  • 57 is a result of measuring the number of tumor nodules and metastasis rate by administering CAF CM, ApoSQ-CAF CM, ApoSQ-CAF CM and anti-WISP-1 antibody or ApoSQ-CAF CM and IgG to mice, and ApoSQ-CAF CM administration
  • CAF CM ApoSQ-CAF CM
  • ApoSQ-CAF CM ApoSQ-CAF CM
  • anti-WISP-1 antibody ApoSQ-CAF CM
  • A A diagram showing the number and metastasis rate of tumor nodules that metastasized to the lungs in each group.
  • B It is a diagram showing images of tumor nodules that metastasized to the lungs in each group.
  • 58 shows that when ApoSQ-CAF CM was intratumorally administered to mice, compared to the CAF CM experimental group, cell migration and invasion were inhibited in CD326 + tumor cells and Thy1 + CAFs isolated from primary tumor tissue, but anti-WISP -1 It is a diagram showing that the effect is reversed when the antibody is additionally administered.
  • 58a is a diagram showing migration and invasion of CD326 + tumor cells isolated from primary tumor tissue.
  • 58b is a diagram showing the migration and invasion of Thy1 + CAFs isolated from primary tumor tissue.
  • CD326 + tumor cells isolated from primary tumor tissues were associated with migration and invasion, such as Smad2/3, FAK, ERK, and Akt. Phosphorylation of signal molecules and expression of MMP2/12 protein are suppressed, but the effect is reversed when anti-WISP-1 antibody is additionally administered.
  • A A diagram showing the results of immunoblot analysis for molecules involved in cell migration and invasion in CD326 + tumor cells.
  • B A diagram showing the amount of proteins involved in cell migration and invasion in isolated CD326 + tumor cells.
  • 60 shows CAF activity markers (Acta2, Col1 ⁇ 1, Itg beta 1 , Spp1 and Pdgfr ⁇ included ) and MMP (including Mmp1a, 2, and 12) mRNA expression levels are suppressed, but the effect is reversed when an anti-WISP-1 antibody is additionally administered.
  • 61 is a diagram showing an experimental timeline of administration of rWISP-1 at a concentration of 12.5 ⁇ g/kg or 25 ⁇ g/kg after subcutaneous injection of 344SQ cells into mice.
  • 62 is a diagram showing that when rWISP-1 was intratumorally administered to mice at a concentration of 12.5 ⁇ g/kg or 25 ⁇ g/kg, there was no significant difference in body weight between groups.
  • 63 shows the number of tumor nodules and the metastasis rate measured by administering rWISP-1 to mice at a concentration of 12.5 ⁇ g/kg or 25 ⁇ g/kg. It is a diagram showing a decrease in (A) A diagram showing the number and metastasis rate of tumor nodules that metastasized to the lungs in each group. (B) It is a diagram showing images of tumor nodules that metastasized to the lungs in each group.
  • FIG. 64 is a diagram showing that migration and invasion of CD326 + tumor cells and Thy1 + CAFs are inhibited in a concentration-dependent manner when rWISP-1 is intratumorally administered at a concentration of 12.5 ⁇ g/kg or 25 ⁇ g/kg to mice .
  • Figure 64a is a diagram showing the migration and invasion of CD326 + tumor cells.
  • Figure 64b is a diagram showing the migration and invasion of Thy1 + CAFs.
  • FIG. 65 shows signal molecules related to migration and invasion, such as Smad2/3, FAK, ERK, and Akt, in CD326 + tumor cells when rWISP-1 was intratumorally administered to mice at a concentration of 12.5 ⁇ g/kg or 25 ⁇ g/kg. It is a diagram showing that the phosphorylation of them and the expression of MMP2/12 protein are suppressed.
  • A A diagram showing the results of immunoblot analysis for molecules involved in cell migration and invasion in CD326 + tumor cells.
  • (B) A diagram showing the amount of proteins involved in cell migration and invasion in CD326 + tumor cells.
  • CAF activity markers including Acta2, Col1 ⁇ 1, Itg beta 1, Spp1 and Pdgfr ⁇
  • MMP including Mmp1a, 2, and 12
  • FIG. 68 is a diagram showing that, when ApoSQ-CAF CM was intratumorally administered to mice, gene expression related to cell adhesion and ECM remodeling was down-regulated in Thy1 + CAFs isolated from primary tumor tissues, compared to the CAF CM experimental group.
  • DAPT D5942 was purchased from Sigma-Aldrich (St. Louis, MO, USA), TGF-beta 1 (240-B-010) and mouse rWISP-1 (1680-WS), mouse neutralizing WISP-1 antibody (MAB1680 R&D Systems) and IgG (MAB0061) were purchased from R&D Systems (Minneapolis, MN, USA).
  • LY3039478 HY-12449 was purchased from MedChemExpress (Monmouth Junction, NJ, USA).
  • Antibodies used for Western blot, immunofluorescence staining, flow cytometry and cell sorting are as follows (see Table 1).
  • IHC 1:100 1:400 Goat IgG Alexa 488) Thermo Fisher Scientific A11055 Donkey Not applicable FACS 1:100 Goat IgG (Alexa 647) Thermo Fisher Scientific A21447 Donkey Not applicable FACS 1:100 Sheep IgG (Alexa 594) Thermo Fisher Scientific A11016 Donkey Not applicable IF, IHC 1:400 * Abbreviation: IB-Immunoblot, IHC-Immunohistochemistry, IHC-P-Immunohisrochemistry-paraffin, IF-Immunofluorescence, H-human, M-mouse, R-rat, Rb-rabbit, P-pig, Mi-mink, Hm-hamster , Mk-monkey, Dm-drosophila melanogaster, Z-zebrafish, B-Bovine, Pg-pig, Ce-caenorhabditis elegans, F-frog
  • CAFs Carcinoma-Associated Fibroblasts
  • CAFs were isolated from lung tumors of Kras-mutant (Kras LA1 ) mice using magnetic-activated cell sorting with the fibroblast-specific marker Thy1. CAFs were then supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (100 U/100 ⁇ g, Welgene, Gyeongsan, Korea), 2 mM L-glutamine (Welgene), and 1 mM sodium pyruvate (Welgene). cultured in alpha-MEM (Welgene).
  • FBS fetal bovine serum
  • penicillin/streptomycin 100 U/100 ⁇ g, Welgene, Gyeongsan, Korea
  • Welgene 2 mM L-glutamine
  • Welgene 1 mM sodium pyruvate
  • CAFs were stably transfected with TERT plasmid (pCDH-3xFLAG-TERT, Addgene 51 plasmid # 51631) using Lipofector-EXT (AptaBio, Yongin, Korea). Primary cells used in the experiments were subcultured less than 6 times.
  • Human cancer cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). 344SQ cells (lung adenocarcinoma cell line, University of Texas MD Anderson Cancer Center, USA) and various human cancer cell lines [A549 (non-small cell lung cancer cell line), HCT116 (colorectal cancer cell line) and MCF-7 (breast cancer cell line)] were cultured in 10% FBS. and RPMI 1640 (Welgene) supplemented with penicillin/streptomycin (100 U/100 ⁇ g).
  • Cancer epithelial cell lines were exposed to 254 nm wavelength ultraviolet light for 15 minutes and then incubated with RPMI-1640 (with 10% FBS) at 37° C. and 5% CO 2 for 2 hours. As a result of evaluating nuclear morphology using a light microscope in the Wright-Giemsa-stained samples, it was confirmed that the irradiated cells were apoptotic. Lysed (necrotic) cancer cells were obtained by several freeze-thaw cycles.
  • Apoptosis and necrosis were confirmed by annexin V-FITC/propidium iodide (BD Biosciences, San Jose, CA, USA) staining followed by flow cytometry on a FACSCalibur system (ACEA Novocyte 3000, Agilent, Santa Clara, CA, USA).
  • CAFs were plated at 3 X 10 5 cells/ml and cultured in a suitable medium at 37°C and 5% CO 2 . After overnight incubation, serum was removed with X-VIVO 10 medium (04-380Q, Lonza, Basel, Switzerland) for 24 hours before cell stimulation. For stimulation, the culture medium was replaced with X-VIVO 10 containing killed or necrotic cancer cells (9 X 10 5 cells/ml). After 20 hours, the supernatant was harvested by centrifugation and used as a conditioned medium (CM) for stimulation of target cancer epithelial cells (5 X 10 5 cells/ml) or CAFs (3 X 10 5 cells/ml). For in vivo experiments, the conditioned medium was stored at -80°C until needed.
  • CM conditioned medium
  • CAFs migration and invasion in CAFs were performed 24 h after direct exposure with ApoSQ and stimulation with TGF-beta 1 (10 ng/ml).
  • TGF-beta 1 10 ng/ml
  • CAFs Prior to TGF-beta 1 stimulation, CAFs were directly exposed to apoptotic or necrotic cancer cells and then washed X-VIVO. After fixation in 4% paraformaldehyde, immobile or non-infiltrated cells on the upper surface of the membrane were scraped off with a cotton swab. Cells on the lower surface were stained with 0.1% crystal violet, washed with distilled water, and three random microscope fields were photographed (10X magnification) and counted.
  • a standardized Western blot was performed using whole cell extracts.
  • Whole cell extracts were prepared from killed cells or CAFs or target cancer cells co-cultured with conditioned media. Cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), and washed in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxygenate). cholate, 0.1% SDS, 1.0% Triton X-100, 5 mM EDTA) for 30 min on ice with protease inhibitors.
  • PBS ice-cold phosphate-buffered saline
  • cholate 0.1% SDS, 1.0% Triton X-100, 5 mM EDTA
  • Equal amounts of protein were run on SDS-PAGE gels (#161-0158, Bio-Rad Laboratories, Herc-Res, CA, USA) using a wet transfer system (Bio-Rad Laboratories) and transferred to nitrocellulose membranes (10600001, GE Healthcare Life Science). After blocking with 5% BSA-TBST or 5% milk-TBST for 1 hour, blots were incubated with primary antibody overnight, followed by incubation with secondary antibody for 1 hour at 37°C. Quantification was performed using the Odyssey image analysis system (Licor Biosciences, Lincoln, Iowa).
  • Cytokine arrays were performed using the Proteome Profiler Mouse XL Cytokine Array Kit (#ARY028, R&D Systems, USA) in CAF CM, ApoSQ-CAF CM and ApoSQ CM according to the manufacturer's instructions. Array membranes were incubated with conditioned medium overnight and membrane-bound cytokines were detected using a biotinylated detection antibody and streptavidin-horseradish peroxidase. Pixel density of cytokine spots was analyzed using Image J software (NIH, Bethesda, Maryland, USA; http://rsb). info.nih.gov/ij/).
  • formalin fixation was performed for 30 min at room temperature and IF-Wash buffer (0.05% NaN3, 0.1% BSA, 0.2% Triton X-100 and 0.05% Tween-20 in PBS) was used. .
  • IF-Wash buffer 0.05% NaN3, 0.1% BSA, 0.2% Triton X-100 and 0.05% Tween-20 in PBS
  • samples were washed three times with wash buffer for 5 minutes each and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 5 minutes at room temperature.
  • 5% BSA in PBS with or without mouse IgG blocking reagent was used for immunocytochemistry and immunohistochemistry, respectively.
  • target proteins were captured by each primary antibody for 18 hours at 4°C. Captured proteins were observed for 1 hour in the dark by fluorescence-coupled IgG. After staining, all slides were fixed with Vectashield Mounting medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI (Vector Laboratories, Burlingame, CA, USA) and confocal microscopy (LSM5 PASCAL, Carl Zeiss, Jena, Germany). ) or observed through a fluorescence microscope (Eclipse Ti2-U, Nikon, Tokyo, Japan).
  • WISP-1 and LIF in conditioned media and serum were measured using ELISA kits (R&D Systems) according to the manufacturer's instructions.
  • serum cytokine quantification blood was collected from mice via puncture, and serum was isolated by centrifugation at 1600 ⁇ g for 5 minutes at 4° C.
  • CAFs were transfected with WISP-1 (Bioneer Inc, Daejeon, Korea), LIF (Bioneer), Notch1 (Bioneer) or BAI1 ( Dharmacon. CO, USA) was transiently transfected with either specifically targeting siRNA or control siRNA (Bioneer, Dharmacon) at a final concentration of 100 nM. After overnight transfection, cells were cultured in suitable media for 24 hours and stimulated with ApoSQ cells. 344SQ cells were transiently transfected with Dll1-specific siRNA (D-050912-01-0020, Dharmacon) or control siRNA before UV irradiation.
  • the siRNA sequences used to target the genes are as follows (see Table 3).
  • pEBB empty vector or pEBB-BAI-Flag plasmid For BAI1 overexpression, 2.0 ⁇ g of pEBB empty vector or pEBB-BAI-Flag plasmid (Gwangju Institute of Science and Technology, Gwangju, Korea) for 48 h using Lipofectamine 2000 reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. CAFs were transfected. Cell lysates were measured for WISP-1 and BAI1 gene and protein expression to confirm the potency of the plasmids.
  • luciferase assay CAFs were transfected using Lipofectimin LTX Transfection Reagent and PLUS Reagent (Life Technologies, Darmstadt, Germany). Cells were transfected with 800 ng/well 4 ⁇ CSL luciferase plasmid (#41726 Addgene, Wattown, MA) to produce luciferase in response to Notch pathway activation and transfected with 200 ng/well Renilla luciferase plasmid . Luciferase activity was normalized to Renilla activity and values were reported as fold change relative to the PGL2-control vector. All conditions were performed in triplicate for each independent experiment, and luciferase experiments were performed using the Dual-Luciferase Assay System (Promega, Madison, WI, USA).
  • Conditioned medium from CAFs was incubated for 2 hours with 10 ⁇ g/ml mouse anti-mouse WISP-1 neutralizing antibody (R&D Systems) or 10 ⁇ g/ml IgG isotype control (R&D Systems).
  • the neutralizing effect of anti-WISP-1 antibodies was measured by WISP-1 ELISA.
  • UV-irradiated killed cancer cells or live cancer cells were cultured in FACS buffer (0.1% BSA and 0.1% sodium azide in PBS) with anti-Dll1, anti-Dll3, anti-Dll4, anti-Jag1 or anti-Jag2 (1 :100) for 30 minutes at room temperature. Cells were then incubated for 30 minutes at room temperature in FACS buffer with Alexa 488- or 594-labeled secondary antibodies. After incubation, the cells were washed three times with FACS buffer, and the expression level of Notch ligand was analyzed using a flow cytometer (ACEA Novocyte 3000, Agilent, Santa Clara, CA, USA). Data were analyzed using Novoexpress software (Agilent).
  • Phagocytosis analysis of killed cancer cells was performed through the flow cytometry and immunofluorescence method.
  • CAFs were stained with PKH26 (red), and then co-cultured with apoptotic 344SQ cells labeled with PKH67 (green) at a ratio of 1:3 for 24 hours. After washing, the percentage of phagocytosis by CAFs was measured by two-color flow cytometry.
  • killed 344SQ cells labeled with PKH67 were co-cultured with CAFs for 24 hours, washed, and CAFs were fixed with 3.7% w/v paraformaldehyde and 0.1% Triton X-100. Permeabilized for 15 minutes.
  • F-actin was stained with rhodamine phalloidin (Invitrogen) according to the manufacturer's instructions. Images were captured with a confocal microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany), and the phagocytic index was calculated by the formula (killed cells/200 total CAFs) x 100.
  • CAFs were pre-incubated for 2 hours with DAPT (10 or 20 ⁇ M) or transfected with Notch1 or control siRNA for 24 hours before co-culture with ApoSQ at a 1:3 ratio for 24 hours.
  • Rac1 activity was measured using the G-LISA Rac1 activity assay kit (Cytoskeleton, Denver, CO, USA) according to the manufacturer's instructions. That is, cell lysate containing the same amount of protein was added to the Rac1-GTP binding plate and incubated at 4° C. for 30 minutes. The plate was washed with PBS containing 0.05% of Tween 20 and reacted with an antigen presentation buffer. After washing, the plates were reacted with anti-Racl primary and secondary antibodies. The reaction was observed with horseradish peroxidase detection reagent and stopped with stop solution. The plate was measured for absorbance at 490 nm using a spectrophotometer.
  • the selective Notch1 inhibitor LY3039478 (8 mg/kg) was orally administered with 15% sugar gel vehicle three times a week for 6 weeks one day before ApoSQ injection.
  • conditioned media experiments 2 days after injection of 344SQ, conditioned media derived from CAFs (100 ⁇ l per mouse) with or without neutralizing anti-WISP-1 antibody (10 ⁇ g/ml) or isotype IgG was added for 1 week 3 It was administered via intratumor injection. Mice were monitored daily for tumor growth and sacrificed 6 weeks after injection.
  • a necropsy was performed to examine the diameter and weight of the subcutaneous tumor mass, the status of lung metastases (number or incidence of nodules), and histological evaluation of formalin-fixed, paraffin-embedded, immunofluorescence-stained primary tumors.
  • tumor tissue was dissociated using a tumor dissociation kit combined with a MACSTM Dissociator (all Miltenui Biotec Inc., San Diego, CA) according to the manufacturer's instructions. made it Briefly, tumor tissue from each mouse was transferred to a MACS C tube (Miltenui Biotec) containing the enzyme mixture from the kit for enzymatic digestion. After dissociation, each tissue homogenate was filtered through 70- and 40- ⁇ m sterile nylon mesh and red blood cells were lysed with red blood cell lysis buffer.
  • MACSTM Dissociator all Miltenui Biotec Inc., San Diego, CA
  • Thy1 + CAFs were sorted using CD90.2 MicroBeads and a MACS MS column (Miltenyi Biotec) according to the manufacturer's instructions.
  • CD326 + epithelial tumor cells and CD11b + tumor-associated macrophages were isolated using CD326 and CD11b MicroBeads (Miltenyi Biotec), respectively. Isolated cells were cultured in complete medium.
  • Thy1 + cells were cultured in ⁇ -MEM (Welgene) supplemented with 20% FBS and 1% penicillin/streptomycin, and CD326 + cells were cultured in RPMI 1640 (Welgene) supplemented with 10% FBS and 1% penicillin/streptomycin. and CD11b + cells were cultured in DMEM (Welgene) supplemented with 10% FBS and 1% penicillin/streptomycin. Isolated individual cell groups were validated by qRT-PCR, and individual cells were isolated within randomly selected mouse primary tumors within each group of two or three.
  • mouse tumor metastasis RT 2 Profiler TM PCR Array PAMM-028ZA-6, Qiagen, Hilden, Germany
  • mouse extracellular matrix and aggregation molecule RT 2 Profiler TM PCR Array PAMM-013ZA-6, Qiagen
  • RNA isolation, DNase treatment and RNA washing were performed according to the instructions of the manufacturer (Takara Bio). The isolated RNA was reverse transcribed into cDNA using the RT 2 First Strand kit (Qiagen).
  • PCR was performed using RT 2 SYBR Green qPCR Master Mix (Qiagen), QuantStudio 3 Real-Time PCR system and ABI PRISM7900 instrument (Applied Bio-Systems).
  • the expression level data was normalized using the average Ct value of Gapdh (glyceraldehyde 3-phosphate dehydrogenase), a housekeeping gene in the array. Each assay was performed in triplicate.
  • 344SQ cells were treated with CAFs and killed 344SQ co-culture conditioned medium (ApoSQ-CAF CM) or CAFs and necrotic 344SQ co-culture conditioned medium (NecSQ-CAF CM) in the presence or absence of TGF-beta 1 for 48 hours.
  • Transwell migration and invasion assays were performed to analyze cell migration and invasion capabilities against a chemo-attractant gradient.
  • TGF-beta 1 signal when activated, it induces migration and invasion of cancer cells by regulating Smad-dependent and -independent signaling. and suppression of TGF-beta 1 signals such as the p38 MAP kinase signaling system (FIGS. 3a and 3b). In addition, TGF-beta 1 signal induces upregulation of mRNA and protein expression of MMP-2 and MMP-12, which degrade and remodel the extracellular matrix, and it was confirmed that ApoSQ-CAF CM inhibited this (FIG. 4) .
  • the conditioned medium in which CAFs and apoptotic cancer cells were co-cultured not only suppressed the activation of TGF-beta 1 signal-induced migration and invasion-related signaling pathway molecules of cancer cells, but also related to ECM remodeling. It was verified that migration and invasion of cancer cells can be inhibited by inhibiting the expression of mRNA and protein of MMP2/12.
  • the migratory ability of CAFs is related to the activation state of CAFs, ie, activated fibroblasts migrate better than quiescent or resting fibroblasts.
  • CAFs were exposed to ApoSQ or NecSQ for 20 hours, followed by fresh medium in the presence or absence of TGF-beta 1. was replaced for 24 hours.
  • this inhibitory effect was not shown when CAFs were treated with NecSQ (FIG. 5).
  • direct exposure of CAFs to ApoSQ or NecSQ in the absence of TGF-beta 1 did not affect basal levels.
  • CAF CM ApoSQ alone medium
  • ApoSQ-CAF CM ApoSQ-CAF CM
  • WISP1-1 knockdown blocked the inhibitory effect of ApoSQ-CAF CM on TGF-beta 1-induced migration and invasion in 344SQ cells (Fig. 13a), and also blocked the direct inhibitory effect of ApoSQ on CAFs migration and invasion (Fig. 13b). In contrast, when LIF was knocked down, there was no effect of inhibiting migration and invasion in 344SQ cells or CAFs (FIGS. 14 to 15b).
  • the present inventors confirmed the effect of increasing ApoSQ-induced WISP-1 secretion by CAFs using WISP-1 overexpressing CAFs.
  • WIPS-1 expression was increased in CAFs transfected with the WISP-1 plasmid, and WISP-1 secretion was increased in CAFs transfected with or without ApoSQ (FIG. 16).
  • the TGF-beta 1-induced migration and invasion of 344SQ cells were further suppressed (Fig. 17a).
  • TGF-beta 1-induced migration and invasion of CAFs were reduced compared to those of control transfected CAFs in the absence of ApoSQ, but were more inhibited than ApoSQ treated control transfected CAFs.
  • the TGF-beta 1-induced migration and invasion inhibitory effects of WISP-1 overexpressed CAFs were reduced compared to control transfected CAFs (FIG. 17b).
  • WISP-1 acts in an autocrine/paracrine manner to induce anti-migration and anti-invasion effects
  • recombinant mouse WISP-1 (rWISP) was used at concentrations of 5 and 10 ng/ml, or 0.75-3 ng/ml. -1) was directly treated with 344SQ or CAFs, respectively, in the presence of TGF-beta 1.
  • treatment with this low dose of rWISP-1 inhibited TGF-beta 1-induced migration and invasion of 344SQ and CAFs in a concentration-dependent manner (FIGS. 19a and 19b).
  • rWISP-1 suppressed the TGF-beta 1-induced Smad or non-Smad signaling system and suppressed the expression of MMP2/MMP12 mRNA and protein in 344SQ cells or CAFs (FIGS. 20a to 21c).
  • WISP-1 is known to bind to integrin, a cell surface receptor, integrins ⁇ v, ⁇ 5, beta 1, beta Experiments were performed using blocking antibodies against 3 or beta 5.
  • WISP-1 inhibits the signaling pathway induced by TGF-beta 1 in an autocrine/paracrine manner through the integrin ⁇ v beta 3 receptor of 344SQ cells and the integrin ⁇ v beta 5 receptor of CAFs, thereby inhibiting cell migration and It was verified that the infiltration was controlled.
  • the Notch1-WISP-1 axis is known to determine the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma invasion and metastasis. Therefore, the present inventors hypothesized that Notch1 signaling-dependent WISP-1 production in CAFs in response to killed lung cancer cells plays a critical role in the anti-migration and anti-invasion effects in cancer cells and CAFs.
  • Notch1 signaling pathway is activated in CAFs in response to ApoSQ.
  • Notch1 signaling proteins including Hes1 and WISP-1, known as sub-targets of the Notch pathway, as well as Notch intracellular domain 1 (NICD1), known to be secreted only when Notch1 signaling is activated, are induced by ApoSQ in CAFs It was confirmed (FIG. 25 (A)).
  • WISP-1 secretion increased in ApoSQ-CAF CM, but not in NecSQ-CAF CM (FIG. 25(B)).
  • Notch signaling is initiated by the interaction of cell-surface receptor Notch with cell-associated ligands, such as Delta-like ligands (DLL) 1,3 and 4 or Jagged-like (JAG) 1 and 2, so Notch-
  • DLL Delta-like ligands
  • JAG Jagged-like
  • BAI1 can contribute to the apoptotic cell removal process of apoptotic cervical cancer cells through primary fibroblasts
  • the thrombospondin type 1 repeats of BAI1 directly recognize phosphatidylserine (PtdSer) and target apoptotic cells
  • PtdSer phosphatidylserine
  • the percentage of PKH26-stained CAFs that phagocytosed PKH67-stained ApoSQ cells was determined using flow cytometry.
  • Annexin V was added, the phagocytosis of ApoSQ by CAFs was reduced (FIG. 36a), and ApoSQ-induced Notch1 activation and WISP-1 secretion were also downregulated (FIG. 36b).
  • BAI1 was knocked down, phagocytosis, Notch1 signaling, and WISP-1 secretion were downregulated in ApoSQ-treated CAFs (FIGS. 37a to 37c), and the same results were obtained when BAI1 was neutralized with an anti-BAI1 antibody. It was confirmed (FIG.
  • BAI1 a protein that binds to ApoSQ, promotes the removal of apoptotic cells in CAFs and regulates the production of WISP-1 in CAFs by being involved in the Notch1 signaling pathway.
  • the present inventors confirmed that a single injection of ApoSQ cells inhibited lung metastasis in syngeneic (129/Sv) immunocompetent mice. After that, the in vivo response of CAFs to ApoSQ injection was investigated. To this end, leukocytes, endothelial cells, and epithelial cells were first removed from mouse primary tumors, and then CAFs were isolated using magnetic-activated cell sorting using the fibroblast-specific marker Thy1 (FIG. 44), followed by qRT-PCR. used to analyze the amount of mRNA of CAF markers.
  • CAF activation markers such as Acta2, Col1 ⁇ 1, Fn, Itg beta 1, Spp1, Pdgfr ⁇ , Pdgfr beta, growth factors including Mmp1a, 2, 9 and 12, and Vegfa, Hgf, Cxcl12 and Cxcl14 after ApoSQ injection /
  • the mRNA expression levels of chemokines were significantly reduced after ApoSQ injection compared to the control group.
  • Notch downlink target genes including Notch1, WISP-1 (Ccn4), Hey1, Hey2, Hes1 and Hes5 were significantly improved compared to the control group, and notch ligands and other CCN gene families (Ccn1, Ccn2, The mRNA expression levels of Ccn5 and Ccn6) were almost unchanged after ApoSQ injection, but Ccn3 mRNA was approximately doubled in Thy1 + CAFs after ApoSQ injection compared to the control group (FIGS. 45a and 45b).
  • LY3039478 a Notch1 selective inhibitor, was orally administered at 8 mg/kg the day before ApoSQ injection, 3 times a week for 6 weeks (FIG. 46). . LY3039478 hardly changed body weight, tumor weight or tumor volume when compared with the control and ApoSQ groups (FIG. 47), but reversed the effect of ApoSQ administration on reducing the number of tumor nodules on the lung surface and the rate of metastasis (FIG. 48).
  • LY3039478 reversed the effect of ApoSQ administration on the decreased mRNA expression level of CAF markers, MMPs, growth factors, and chemokines in isolated Thy1 + CAFs, and also reversed the effect of increasing the mRNA expression level of Notch1 and Notch target genes. (FIG. 49).
  • LY3039478 also reversed the effect of decreasing ⁇ -SMA and increasing NICD1 and WISP-1 expression in Thy1 + CAFs (FIG. 50).
  • FIG. 51a and 51b Western e
  • CAF CM or ApoSQ-CAF CM was intratumorally injected 3 times a week for 6 weeks into syngeneic mice 2 days after 344SQ injection.
  • ApoSQ-CAF CM was pretreated with neutralizing anti-WISP-1 antibody or isotype IgG for 2 hours before injection (FIG. 55).
  • the mRNA expression levels of CAF activity markers namely Acta2, Col1 ⁇ 1, Itg beta 1, Spp1, Pdgfr ⁇ and Mmp1a, Mmp2, and Mmp12, in isolated Thy1 + CAFs were decreased by ApoSQ-CAF CM, but WISP-1 was immunoremoved.
  • the ApoSQ-CAF CM group showed no such inhibitory effect (FIG. 60).
  • rWISP1-1 was injected at a concentration of 12.5 ⁇ g/kg or 25 ⁇ g/kg 344SQ 2 days after injection to syngeneic mice 3 times a week , injected intratumorally for 6 weeks (FIG. 61)
  • CAF activity markers namely Acta2, Col1 ⁇ 1, Itg beta 1, Spp1, Pdgfr ⁇ and Mmp1a, Mmp2, and Mmp12 in Thy1 + CAFs
  • the mRNA expression level of was decreased (FIG. 66).

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Abstract

La présente invention concerne une composition pharmaceutique destinée à inhiber des métastases cancéreuses, un procédé d'inhibition de métastases cancéreuses l'utilisant, et l'utilisation de la composition pharmaceutique, la composition pharmaceutique contenant un milieu de culture dans lequel des fibroblastes associés au cancer (CAF) et des cellules cancéreuses apoptotiques sont mis en co-culture. Dans la présente invention, il a été montré que l'exposition de CAF à des cellules cancéreuses apoptotiques induisait la génération de protéine WISP-1 dépendant de la voie de signalisation Notch1 et inhibait ainsi la migration et l'invasion des cellules cancéreuses et des CAF. Par conséquent, un milieu de culture, le composant principal du milieu de culture (WISP-1) ou des cellules CAF exposées aux cellules cancéreuses apoptotiques selon la présente invention peuvent être efficacement utilisés comme inhibiteur de métastases cancéreuses.
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Citations (2)

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