WO2023182792A1 - Composition for inhibiting cancer growth - Google Patents

Composition for inhibiting cancer growth Download PDF

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Publication number
WO2023182792A1
WO2023182792A1 PCT/KR2023/003762 KR2023003762W WO2023182792A1 WO 2023182792 A1 WO2023182792 A1 WO 2023182792A1 KR 2023003762 W KR2023003762 W KR 2023003762W WO 2023182792 A1 WO2023182792 A1 WO 2023182792A1
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cancer
cells
caf
cafs
pharmaceutical composition
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PCT/KR2023/003762
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French (fr)
Korean (ko)
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이지희
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이화여자대학교 산학협력단
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Priority claimed from KR1020230036937A external-priority patent/KR20230141504A/en
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Publication of WO2023182792A1 publication Critical patent/WO2023182792A1/en

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention provides a pharmaceutical composition for inhibiting cancer growth containing a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and Apoptotic cancer cells, a method for inhibiting cancer growth 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 the leading cause of cancer-related deaths (18.0% of total cancer deaths).
  • Approximately 75% of lung cancer patients are confirmed to have locally advanced or metastatic disease at the time of diagnosis, and metastasis is a multi-step process involving the movement and invasion of cancer cells and serves as a marker for malignant tumor.
  • CAFs Cancer-associated fibroblasts
  • Basal processes such as cancer cell migration and invasion, which can promote malignant tumor progression 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 remain 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 with 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 Notch-induced cellular responses. Stromal fibroblasts with 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 underlying Notch1-dependent tumor-regulatory roles in CAFs, even in other cancer types.
  • WISP-1 Wnt-induced signaling protein-1
  • TEE tumor microenvironment
  • phagocyte-mediated clearance have been reported to suppress anti-tumor immune responses.
  • tumors can also evade immune surveillance by inhibiting recognition for dead cell elimination (efferocytosis).
  • the anti-inflammatory and lytic lipid autacoid specifically inhibits debris-stimulated cancer progression by promoting clearance of cellular debris through macrophage phagocytosis in multiple tumor types.
  • macrophages exposed to UV-irradiated apoptotic lung cancer cells expressed exosomal phosphatase and tensin homolog (PTEN) and peroxisome proliferator-activated receptor-gamma (PPARr).
  • PTEN exosomal phosphatase and tensin homolog
  • PPARr peroxisome proliferator-activated receptor-gamma
  • the present inventors studied how the interaction between CAFs and apoptotic cancer cells regulates cancer cell growth. As a result, exposure of CAFs to apoptotic cancer cells led to Notch1 signaling-dependent WISP-1 production. It was confirmed that it inhibits the proliferation of cancer cells and induces cell death. In addition, inhibition of the RhoA/Rho kinase signaling pathway promoted the efferocytosis of CAFs on apoptotic cancer cells and enhanced Notch1-WISP-1 signaling due to stimulation of apoptotic cancer cells, thereby enhancing the cancer cell growth inhibition effect. . Furthermore, the present invention was completed by confirming that the conditioned medium for co-culture of CAFs and killed cancer cells inhibited tumor growth in mice, and that this cancer growth inhibition effect was WISP-1 dependent.
  • One aspect is to provide a pharmaceutical composition for inhibiting cancer growth, which contains a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs Cancer-Associated Fibroblasts
  • Another aspect is to provide a pharmaceutical composition for inhibiting cancer growth, containing Carcinoma-Associated Fibroblasts (CAFs) exposed to apoptotic cancer cells.
  • CAFs Carcinoma-Associated Fibroblasts
  • Another aspect is to provide a health functional food for inhibiting cancer growth, containing a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs Cancer-Associated Fibroblasts
  • Another aspect is to provide a method for producing a composition for inhibiting cancer growth, comprising co-culturing cancer-associated fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs cancer-associated fibroblasts
  • Another aspect is to provide a method of inhibiting cancer growth, comprising treating a subject with a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs cancer-associated fibroblasts
  • Another aspect is to provide a method of inhibiting cancer growth, comprising treating a subject with WISP-1 (Wnt-induced signaling protein-1).
  • WISP-1 Wi-induced signaling protein-1
  • Another aspect is to provide the use of a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells for the production of a drug for inhibiting cancer growth.
  • CAFs cancer-associated fibroblasts
  • WISP-1 Wi-induced signaling protein-1
  • Another aspect is to provide a use for inhibiting cancer growth in a co-culture of Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs Cancer-Associated Fibroblasts
  • Another aspect provides the use of a pharmaceutical composition for inhibiting cancer growth, comprising a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells. It is done.
  • CAFs Cancer-Associated Fibroblasts
  • Another aspect is to provide a use of WISP-1 (Wnt-induced signaling protein-1) to inhibit cancer growth.
  • WISP-1 Wi-induced signaling protein-1
  • the present invention provides a pharmaceutical composition for inhibiting cancer growth, containing a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs Cancer-Associated Fibroblasts
  • the culture medium co-cultured with the CAFs and killed cancer cells contains WISP-1, which has the effect of inhibiting the growth of cancer cells.
  • CAFs Cancer-Associated Fibroblasts
  • ⁇ -SMA smooth muscle actin
  • the CAFs are colon cancer cells. , its presence has been confirmed in various cancers such as lung cancer, prostate cancer, breast cancer, stomach cancer, bile duct cancer, and basal cell cancer.
  • cancers related to 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, ureteral cancer, and bladder cancer.
  • It may be solid cancer such as prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and skin cancer.
  • the CAFs are cancer-related and may be fibroblasts associated with malignant solid tumors.
  • the CAF may be related to sarcomas such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, malignant skin cancer, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, and osteosarcoma. and may be fibroblasts related to lung cancer, stomach cancer, breast cancer, colon cancer, or prostate cancer.
  • apoptotic cancer cells may be those in which apoptosis is induced by irradiating light of a specific wavelength to cancer cells.
  • Irradiation of light of the specific wavelength may be ultraviolet ray (UV) irradiation.
  • the wavelength may be 100 to 400 nm for 5 to 30 minutes.
  • the UV irradiation may be performed at a wavelength of 150 to 350 nm for 20 minutes or at a wavelength of 200 to 300 nm for 10 to 15 minutes.
  • co-culture can be achieved by culturing CAFs and killed cancer cells together.
  • CAFs can be cultured with killed cancer cells in X-VIVO or serum-free DMEM medium for 20 to 30 hours.
  • culture medium refers to the culture result obtained through co-culture of CAFs and killed cancer cells.
  • the culture medium may be a liquid medium, solid medium, or semi-solid medium.
  • the culture medium may be a conditioned medium.
  • cancer growth is a process of uncontrollable proliferation of tumor cells and is distinguished from metastasis to other tissues.
  • proliferation of the tumor cells may include growth in volume, weight, and diameter, and colony formation.
  • cancer growth inhibition refers to inhibition of proliferation of cancer cells and colonies at the cellular level or inhibition of tumor growth in vivo.
  • 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 one or more 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 one or more 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 signaling. do.
  • the WISP-1 can be produced by Notch1 signaling.
  • Notch1 signaling occurs in a cell contact-dependent manner using the Notch receptor as a mediator, and is known to play an important role in development, regeneration, and homeostasis. Additionally, the activity of Notch signaling is associated with both oncogenic and tumor-suppressive functions.
  • the Notch1 signaling can be enhanced by inhibition of RhoA and Rho kinase.
  • RhoA is one of the Rho-GTPases, which regulates the cytoskeleton and cell division, and increased RhoA activity is known to be associated with cell proliferation.
  • 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 WISP-1, NICD1, and Hes1.
  • the composition can reduce the expression of cell proliferation markers in cancer-related fibroblasts.
  • the cell proliferation marker may be one or more selected from the group consisting of Ki67 and PCNA.
  • the composition can increase apoptosis of cancer cells. Additionally, the composition can increase the expression of pro-apoptotic factors and decrease the expression of anti-apoptotic factors in cancer-related fibroblasts.
  • the pro-apoptotic factor is one or more selected from the group consisting of cleaved caspase 3, cleaved PARP, and Bax
  • the anti-apoptotic factor is Bcl-2, Mcl-1, and Bcl-xL. It may be one or more selected from the group consisting of:
  • the composition may further contain RhoA and Rho kinase inhibitors.
  • the RhoA and Rho kinase inhibitor may be one or more selected from the group consisting of a compound, shRNA, miRNA, siRNA, antibody, and aptamer.
  • the siRNA may have a ribonucleotide unit structure that exists in nature (unmodified), or may be chemically modified, such as synthesized to have one or more chemical modifications.
  • chemical modifications such as enhancing resistance to nuclease, increasing intracellular uptake, improving cell targeting (target specificity), increasing stability, or reducing interferon activity, immune response and sense effect. Effects such as reduced off-target effects and increased RISC loading (increased RNAi activity) can be achieved.
  • the method of chemical modification of the siRNA is not particularly limited, and for example, the sugar structure or base structure of one or more ribonucleotides, or the binding site between the ribonucleic acids may be chemically modified, and may be carried out using methods known in the art.
  • a person skilled in the art can synthesize and modify the siRNA molecules in any desired manner using methods known in the art.
  • the chemical modification may be a nucleobase-modified ribonucleotide, i.e., uridine or cytidine modified at the 5-position (5-(2-amino)propyl uridine, 5-bromouridine, etc.) , adenosine and guanosine modified at position 8 (8-bromo guanosine, etc.), deaza nucleotides (7-deaza-adenosine, etc.), 0- and N-alkylated nucleotides (N6-methyl adenosine, etc.) etc.), it may contain a ribonucleotide containing a non-natural nucleobase instead of a natural nucleobase.
  • the 2'OH group is selected from the group consisting of H, OR, R, halogen (F, Cl, Br or I), SH, SR, NH2, NHR, NR2 and CN. It may be substituted by a substituent (R is C1-C6 alkyl, alkenyl, or alkynyl), and in ribonucleotides, the phosphodiester bond of the backbone is boranophosphate or phosphorothioate. can be replaced.
  • the compound when the RhoA and Rho kinase inhibitor is a compound, the compound may be Y-27632. Additionally, when the RhoA and Rho kinase inhibitors are siRNAs, the siRNAs may be polynucleotides consisting of any one of the nucleotide sequences of SEQ ID NOs: 19 and 20.
  • the pharmaceutical composition according to the present invention may include a “pharmaceutically acceptable carrier.”
  • the pharmaceutically acceptable carrier is commonly used in preparation and includes, but is limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. If necessary, other common additives such as antioxidants and buffers may be added.
  • diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.
  • the formulations can be preferably formulated according to each ingredient using the method disclosed in Remington's literature.
  • the pharmaceutical composition of the present invention is not particularly limited in formulation, but can be formulated as an injection, inhalation agent, topical skin agent, or oral ingestion agent.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, applied to the skin, nasal cavity, or respiratory tract) according to the desired method, and the dosage is determined by the patient's condition, weight, and disease. It varies depending on the degree, drug form, administration route and time, but can be appropriately selected by a person 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 the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine.
  • 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 singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and body weight, and is generally administered at 0.001 to 150 mg, preferably 0.01 to 100 mg, per kg of body weight every day or every other day, or 1 It can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.
  • the present invention provides a pharmaceutical composition for inhibiting cancer growth, which contains Cancer-Associated Fibroblasts (CAFs) exposed to apoptotic cancer cells.
  • CAFs Cancer-Associated Fibroblasts
  • the present invention provides a health functional food for inhibiting cancer growth, containing a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs cancer-associated fibroblasts
  • the health functional food of the present invention may be formulated with one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including one or more of carriers, diluents, excipients, and additives.
  • 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, etc.
  • 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 ingredients selected from the group consisting of pactic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohol, carbonating agents and pulp can be used. .
  • natural carbohydrates examples include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • sweetening agents thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, and alginic acid. and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Additionally, the composition according to the present invention may contain pulp for the production of natural fruit juice and vegetable drinks. These ingredients can be used independently or in combination.
  • the carriers, excipients, diluents and additives include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium.
  • 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 producing a composition for inhibiting cancer growth, comprising the step of co-culturing cancer-associated fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs cancer-associated fibroblasts
  • the culturing step may be culturing in X-VIVO medium for 24 hours to create a serum-starved state.
  • the culture medium can be replaced with X-VIVO or serum-free DMEM medium containing killed cancer cells.
  • the co-culture may be performed for 10 to 30 hours, 15 to 25 hours, or 18 to 24 hours after replacing the medium.
  • the production method may further include secreting WISP-1 through the co-culture.
  • the present invention provides a method of inhibiting cancer growth, comprising treating a subject with a culture medium obtained by co-culturing cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs cancer-Associated Fibroblasts
  • treatment may mean any act of adding or administering the culture medium to an individual according to experimental requirements.
  • “individual” may include mammals.
  • the subject may include humans, monkeys, mice, cows, dogs, horses, pigs, etc.
  • the present invention provides a method of inhibiting cancer growth, comprising treating the subject with WISP-1 (Wnt-induced signaling protein-1).
  • the present invention provides the use of a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells for the production of a drug for inhibiting cancer growth.
  • CAFs cancer-associated fibroblasts
  • the present invention provides the use of WISP-1 (Wnt-induced signaling protein-1) for the production of a drug for inhibiting cancer growth.
  • the present invention provides a use for inhibiting cancer growth in a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
  • CAFs Cancer-Associated Fibroblasts
  • the present invention provides a use for inhibiting cancer growth of a pharmaceutical composition for inhibiting cancer growth, which contains a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells. to provide.
  • a pharmaceutical composition for inhibiting cancer growth which contains a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells. to provide.
  • CAFs cancer-associated fibroblasts
  • the present invention provides a use of WISP-1 (Wnt-induced signaling protein-1) to inhibit cancer growth.
  • WISP-1 is activated through the Notch1 signaling system.
  • the culture medium co-cultured with CAFs and killed cancer cells inhibited the growth of cancer cells at the cellular level, and also that the culture medium inhibited tumor growth in a syngeneic metastatic lung cancer mouse model ( in vivo ). was confirmed to inhibit, and the cancer growth inhibitory effect of the culture medium was confirmed to be WISP-1 dependent.
  • the present invention can be usefully used in a strategy to inhibit the growth of cancer cells, and in particular, the culture medium and conditioned medium of the present invention can be usefully used as a cancer growth inhibitor.
  • Figure 1 is a diagram showing that conditioned medium (CM) containing a culture medium of cancer-associated fibroblasts (CAFs) co-cultured with killed cancer cells inhibits the growth of cancer cells.
  • Figure 1a shows 344SQ, a mouse lung adenocarcinoma cell line, as a control medium, CAF-conditioned medium (CAF CM), 344SQ co-culture conditioned medium with killed CAFs (ApoSQ-CAF CM), and 344SQ co-culture conditioned medium with CAFs killed, respectively.
  • This is a diagram measuring cell survival after treatment with (NecSQ-CAF CM).
  • Figure 1b shows A549, a human non-small cell lung cancer cell line, in control medium, CAF CM, A549 co-culture conditioned with CAFs killed (ApoA-CAF CM), and CAFs necrosed A549 co-culture conditioned medium (NecA-CAF CM), respectively.
  • This is a diagram measuring cell survival after treatment.
  • Figure 1c shows HCT116, a human colon cancer cell line, treated with control, CAF CM, conditioned medium for co-culture of CAFs and killed HCT116 (ApoH-CAF CM), and conditioned medium for co-culture of CAFs and necrotic HCT116 (NecH-CAF CM), respectively. This is a measure of cell survival.
  • Figure 1d shows LoVo, a human colon cancer cell line, treated with control, CAF CM, conditioned medium for co-culture of LoVo killed with CAFs (ApoL-CAF CM), and conditioned medium for co-culture of LoVo killed with CAFs (NecL-CAF CM), respectively. This is a measure of cell survival.
  • Figure 2 is a diagram confirming that the culture medium containing killed cancer cells alone has no effect on inhibiting the growth of cancer cells.
  • Figure 2a is a diagram measuring cell survival by treating 344SQ cells with control group, CAF CM, ApoSQ CM, and NecSQ CM, respectively.
  • Figure 2b is a diagram measuring cell survival by treating A549 cells with control group, CAF CM, ApoA CM, and NecA CM, respectively.
  • Figure 2c is a diagram measuring cell survival by treating HCT116 cells with control group, CAF CM, ApoH CM, and NecH CM, respectively.
  • Figure 2d is a diagram measuring cell survival by treating LoVo cells with control group, CAF CM, ApoL CM, and NecL CM, respectively.
  • Figure 3 is a diagram showing that CAF CM exposed to killed cancer cells inhibits colony formation of lung cancer cells.
  • Figure 3a shows the colony image and the number of colonies measured after treating 344SQ cells with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM for 8 days, respectively.
  • Figure 3b is a diagram showing colony images and colony numbers measured after treating A549 cells with CAF CM, ApoA-CAF CM, and NecA-CAF CM for 9 days, respectively.
  • Figure 4 is a diagram showing that CAF CM exposed to killed cancer cells inhibits the expression of cell proliferation markers in lung cancer cells.
  • Figure 4a is a diagram showing the amount of Ki67 and PCNA mRNA measured by qRT-PCR after treating 344SQ cells with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM for 3 days.
  • Figure 4b is a diagram showing the protein amounts of Ki67 and PCNA measured by immunoblot analysis after treating 344SQ cells with CAF CM and ApoSQ-CAF CM for 3 days.
  • Figure 4c shows the amount of Ki67 and PCNA mRNA measured by qRT-PCR after treating A549 cells with CAF CM, ApoA-CAF CM, and NecA-CAF CM for 3 days.
  • Figure 5 is a diagram confirming through flow cytometry that CAF CM exposed to killed cancer cells promotes cell death.
  • Figure 5a is a diagram showing the degree of cell death measured by flow cytometry using Annexin V-FICT/PI double staining after treating 344SQ cells with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM.
  • Figure 5b is a diagram showing the degree of cell death measured by flow cytometry using Annexin V-FICT/PI double staining after treating A549 cells with CAF CM, ApoA-CAF CM, and NecA-CAF CM.
  • Figure 6 is a diagram confirming through TUNEL analysis that CAF CM exposed to killed cancer cells promotes cell death.
  • Figure 6a is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining after treating 344SQ cells with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM.
  • Figure 6b is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining after treating A549 cells with CAF CM, ApoA-CAF CM, and NecA-CAF CM.
  • Figure 7 is a diagram confirming that CAF CM exposed to apoptotic cancer cells promotes cell death by immunoblot analysis for pro-apoptotic proteins and anti-apoptotic proteins.
  • Figure 7a shows the expression of pro-apoptotic proteins (cleaved caspase 3, cleaved PARP, and Bax) and anti-apoptotic proteins (Bcl-2, Mcl-1) after treating 344SQ cells with CAF CM and ApoSQ-CAF CM. and Bcl-xL) expression by immunoblot analysis.
  • Figure 7b shows the expression of pro-apoptotic proteins (cleaved caspase 3, cleaved PARP, and Bax) and anti-apoptotic proteins (Bcl-2, Mcl-1) after treating A549 cells with CAF CM and ApoA-CAF CM. and Bcl-xL) expression by immunoblot analysis.
  • Figure 8 is a diagram showing that the anti-proliferative effect is reversed when Notch1 is knocked down in CAFs.
  • Figure 8a shows the expression level of Notch1 measured by immunoblot analysis after transfection of CAFs with two types of siRNA (#1 siNotch1 and #2 siNotch2).
  • Figure 8b is a diagram measuring cell survival according to the presence or absence of #1 siNotch1 or #2 siNotch2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 8c shows colony images and colony numbers measured according to the presence or absence of #1 siNotch1 or #2 siNotch2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 9 is a diagram showing that the effect of suppressing cell proliferation marker expression is reversed when Notch1 is knocked down in CAFs.
  • Figure 9a is a diagram showing the amount of Ki67 and PCNA mRNA according to the presence or absence of #1 siNotch1 or #2 siNotch2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 9b is a diagram showing the amount of Ki67 and PCNA proteins according to the presence or absence of #1 siNotch1 or #2 siNotch2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 10 is a diagram showing that the anti-proliferative effect is reversed when the Notch1 signaling pathway is inhibited with a drug in CAFs.
  • Figure 10a is a diagram measuring cell survival with or without LY3039478 (10 ⁇ M), a selective inhibitor of Notch1, after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. .
  • Figure 10b shows colony images and colony numbers measured with or without LY3039478 (10 ⁇ M), a selective inhibitor of Notch1, after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. It is one degree.
  • Figure 11 is a diagram showing that the effect of suppressing cell proliferation marker expression is reversed when the Notch1 signaling pathway is inhibited with a drug.
  • Figure 11a shows the amount of Ki67 and PCNA mRNA with or without LY3039478 (10 ⁇ M), a selective inhibitor of Notch1, after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively.
  • This is a diagram showing .
  • Figure 11b is a diagram showing the amount of Ki67 and PCNA proteins in the presence or absence of LY3039478 (10 ⁇ M), a selective inhibitor of Notch1, after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 12 is a diagram showing that the anti-proliferative effect is reversed when WISP-1 is knocked down in CAFs.
  • Figure 12a shows the expression level of WISP-1 measured by immunoblot analysis after transfection of CAFs with two types of siRNA (#1 siWISP-1 and #2 siWISP-2).
  • Figure 12b is a diagram measuring cell survival according to the presence or absence of #1 siWISP-1 or #2 siWISP-2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 12c shows colony images and colony numbers measured according to the presence or absence of #1 siWISP-1 or #2 siWISP-2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 13 is a diagram showing that the effect of suppressing cell proliferation marker expression is reversed when WISP-1 is knocked down in CAFs.
  • Figure 13a is a diagram showing the amount of Ki67 and PCNA mRNA according to the presence or absence of #1 siWISP-1 or #2 siWISP-2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 13b is a diagram showing the amount of Ki67 and PCNA proteins according to the presence or absence of #1 siWISP-1 or #2 siWISP-2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 14 is a diagram confirming that the anti-proliferative effect is reversed when WISP-1 is neutralized using an anti-WIPS-1 antibody. After treating CAF CM and ApoSQ-CAF CM with 10 ⁇ g/mL of control (isotype IgG) or anti-WISP-1 antibody, cell survival was compared.
  • Figure 15 is a diagram showing that the inhibitory effect on cell proliferation marker expression is reversed when WISP-1 is neutralized using an anti-WIPS-1 antibody.
  • Figure 15a shows the amounts of Ki67 and PCNA mRNA when 344SQ cells were treated with CAF CM and ApoSQ-CAF CM and then further treated with 10 ⁇ g/mL of control (isotype IgG) or anti-WISP-1 antibody. It's a degree.
  • Figure 15b shows the amount of Ki67 and PCNA proteins when 344SQ cells were treated with CAF CM and ApoSQ-CAF CM and then further treated with 10 ⁇ g/mL of control (isotype IgG) or anti-WISP-1 antibody. It's a degree.
  • Figure 16 is a diagram confirming by flow cytometry that Notch1-WISP-1 signaling promotes cell death in CAFs.
  • Figure 16a shows the degree of cell death according to the presence or absence of siNotch1 after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively, using Annexin V-FICT/PI double staining. It is a degree expressed as .
  • Figure 16b shows the degree of cell death according to the presence or absence of siWISP-1 using Annexin V-FICT/PI double staining after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. This is a diagram shown by flow cytometry.
  • Figure 17 is a diagram confirming that recombinant WISP-1 inhibits the proliferation of cancer cells.
  • Figure 17a is a diagram showing cell survival when 344SQ cells or A549 cells were treated with 20, 50, or 100 ng/mL of rWISP-1.
  • Figure 17b is a diagram showing colony images and colony numbers when 344SQ cells or A549 cells were treated with 20, 50, or 100 ng/mL of rWISP-1.
  • Figure 18 is a diagram confirming that recombinant WISP-1 suppresses the expression of cell proliferation markers.
  • Figure 18a is a diagram showing the amount of Ki67 and PCNA mRNA when 344SQ cells or A549 cells were treated with 20, 50, or 100 ng/mL of rWISP-1.
  • Figure 18b is a diagram showing the amount of Ki67 and PCNA proteins when 344SQ cells were treated with 20, 50, or 100 ng/mL of rWISP-1.
  • Figure 19 is a diagram confirming by flow cytometry that recombinant WISP-1 promotes apoptosis of cancer cells.
  • Figure 19a is a diagram showing the degree of cell death by flow cytometry using Annexin V-FICT/PI double staining after treating 344SQ cells with 20, 50, or 100 ng/mL of rWISP-1.
  • Figure 19b is a diagram showing the degree of cell death by flow cytometry using Annexin V-FICT/PI double staining after treating A549 cells with 20, 50, or 100 ng/mL of rWISP-1.
  • Figure 20 is a diagram confirming through TUNEL analysis that recombinant WISP-1 promotes apoptosis of cancer cells.
  • Figure 20a is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining after treating 344SQ cells with 20, 50, or 100 ng/mL of rWISP-1.
  • Figure 20b is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining after treating A549 cells with 20, 50, or 100 ng/mL of rWISP-1.
  • Figure 21 is a diagram confirming that recombinant WISP-1 promotes apoptosis of cancer cells by immunoblot analysis for pro-apoptotic protein and anti-apoptotic protein.
  • Figure 21a shows the expression of pro-apoptotic proteins (cleaved caspase 3, cleaved PARP and Bax) and anti-apoptotic proteins (Bcl-) after treating 344SQ cells with 20, 50 or 100 ng/mL of rWISP-1.
  • This is a diagram showing the expression of Mcl-1 and Bcl-xL) by immunoblot analysis.
  • Figure 21b shows the expression of pro-apoptotic proteins (cleaved caspase 3, cleaved PARP and Bax) and anti-apoptotic proteins (Bcl-) after treating A549 cells with 20, 50 or 100 ng/mL of rWISP-1.
  • This is a diagram showing the expression of Mcl-1 and Bcl-xL) by immunoblot analysis.
  • Figure 22 is a diagram confirming that efferocytosis is promoted when Rho kinase is inhibited in CAFs. After treatment with 30 ⁇ M of Ripasudil, a Rho kinase inhibitor, the phagocytosis of CAFs toward ApoSQ was analyzed by flow cytometry.
  • Figure 23 is a diagram confirming that efferocytosis is promoted when Rho kinase is inhibited in CAFs. Phagocytosis of CAFs was analyzed with and without Ripasudil, and the nucleus and actin cytoskeleton of CAFs were stained with DAPI (blue) and TRITC-conjugated phalloidin (red), respectively.
  • Figure 24 is a diagram confirming that when Rho kinase is inhibited in CAFs, Notch1 signaling protein (NICD1, Hes1, and WISP-1) expression and WISP-1 secretion are promoted.
  • Figure 24a is a diagram showing the expression of Notch1 signaling proteins (NICD1, Hes1, and WISP-1) in CAFs exposed to ApoSQ with or without Ripasudil.
  • Figure 24b is a diagram showing the secretion amount of WISP-1 in CAFs exposed to ApoSQ with or without Ripasudil.
  • Figure 25 is a diagram showing that the anti-proliferative effect is enhanced when RhoA is knocked down in CAFs.
  • Figure 25a shows the expression level of RhoA measured by immunoblot analysis after transfection of CAFs with siRhoA.
  • Figure 25b is a diagram showing cell survival according to the presence or absence of siRhoA after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively.
  • Figure 25c is a diagram showing the colony image and number of colonies according to the presence or absence of siRhoA after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively.
  • Figure 26 is a diagram showing that the effect of suppressing cell proliferation marker expression is enhanced when RhoA is knocked down in CAFs.
  • Figure 26a is a diagram showing the amount of Ki67 and PCNA mRNA according to the presence or absence of siRhoA after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM, CAF CM, or ApoA-CAF CM, respectively.
  • Figure 26b is a diagram showing the amount of Ki67 and PCNA proteins according to the presence or absence of siRhoA after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 27 is a diagram showing that the anti-proliferative effect is enhanced when Rho kinase is inhibited with a drug.
  • Figure 27a shows cell survival measured with or without the Rho kinase inhibitor Y-27632 (30 ⁇ M) after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. am.
  • Figure 27b shows colony images and number of colonies according to the presence or absence of Y-27632 (30 ⁇ M), a Rho kinase inhibitor, after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. It is a measured degree.
  • Figure 28 is a diagram showing that the inhibitory effect on cell proliferation marker expression is strengthened when Rho kinase is inhibited with a drug.
  • Figure 28a shows the mRNA levels of Ki67 and PCNA with or without the Rho kinase inhibitor Y-27632 (30 ⁇ M) after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. It is a degree that represents quantity.
  • Figure 28b is a diagram showing the amount of Ki67 and PCNA proteins with or without the Rho kinase inhibitor Y-27632 (30 ⁇ M) after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
  • Figure 29 is a diagram confirming that cell death is promoted when RhoA is knocked down in CAFs.
  • Figure 29a is a diagram showing the degree of cell death in 344SQ cells or A549 cells according to the presence or absence of siRhoA by flow cytometry using Annexin V-FICT/PI double staining.
  • Figure 29b is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining in 344SQ cells depending on the presence or absence of siRhoA.
  • Figure 30 is a diagram confirming that ApoSQ-CAF CM inhibits the growth of mouse tumors in vivo .
  • Figure 30a is a schematic diagram of the experiment. Syngeneic mice injected with 344SQ were injected with CAF CM, 50% diluted CAF CM, ApoSQ-CAF CM, or 50% diluted ApoSQ-CAF CM, forming a total of 4 groups.
  • Figure 30b is a diagram showing the body weight of four groups of mice.
  • Figure 30c is a diagram showing the weight of tumors in four groups.
  • Figure 30D is a diagram showing photographs of primary tumors in four groups.
  • Figure 30e is a diagram showing the volume of tumors in four groups.
  • Figure 31 is a diagram confirming that ApoSQ-CAF CM inhibits mouse tumor growth through WISP-1 in vivo .
  • Figure 31a is a schematic diagram of the experiment. Syngeneic mice injected with 344SQ were divided into four groups by injecting CAF CM, ApoSQ-CAF CM, ApoSQ-CAF CM and anti-WISP-1 antibody, or ApoSQ-CAF CM and IgG. It was composed.
  • Figure 31b is a diagram showing the body weight of four groups of mice.
  • Figure 31c is a diagram showing the weight of tumors in four groups.
  • Figure 31D is a diagram showing photographs of primary tumors in four groups.
  • Figure 31e is a diagram showing the volume of tumors in four groups.
  • Figure 32 is a diagram confirming that rWISP-1 inhibits mouse tumor growth in vivo .
  • Figure 32a is a schematic diagram of the experiment. rWISP-1 was injected at 12.5 ⁇ g/kg or 25 ⁇ g/kg into syngeneic mice injected with 344SQ, and a total of three groups were formed, including the control group.
  • Figure 32b is a diagram showing the body weight of four groups of mice.
  • Figure 32c is a diagram showing the weight of tumors in four groups.
  • Figure 32D is a diagram showing photographs of primary tumors in four groups.
  • Figure 32e is a diagram showing the volume of tumors in four groups.
  • LY3039478 (HY-12449) was purchased from MedChemExpress (Monmouth Junction, NJ 08852, USA).
  • Ripasudil (K-115) was purchased from Selleck Chemicals (Houston, TX, USA), and Y-27632 (Y0503) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Meanwhile, the list of antibodies used in Western blotting is shown in Table 1 below.
  • 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 cultured in alpha-MEM (Welgene) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (100 U/100 ⁇ g, Welgene), 2 mM L-glutamine (Welgene), and 1 mM sodium pyruvate (Welgene). , Gyeongsan, Korea).
  • alpha-MEM Welgene
  • FBS fetal bovine serum
  • penicillin/streptomycin 100 U/100 ⁇ g
  • Welgene 2 mM L-glutamine
  • Na pyruvate Welgene
  • CAFs were stably transfected with TERT plasmid (pCDH-3xFLAG-TERT, Addgene 51 plasmid #51631) using Lipofactor-EXT (AptaBio, Yongin, Korea).
  • the primary cells used in the experiment were subcultured less than 6 times.
  • Human cancer cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA).
  • 344SQ cells (a lung adenocarcinoma cell line) and various human cancer cell lines [A549 (a non-small cell lung cancer cell line), HCT116 and LoVo (a colon cancer cell line)] were cultured in RPMI 1640 ( HyClone TM , GE Healthcare, Boston, MA).
  • Cancer epithelial cell lines were exposed to ultraviolet irradiation at 254 nm for 15 minutes and then incubated with RPMI-1640 (with 10% FBS) at 37°C and 5% CO 2 for 2 hours. Nuclear morphology was evaluated in Wright-Giemsa-stained samples using light microscopy, and it was confirmed that the irradiated cells were apoptotic. Lytic (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) staining followed by flow cytometric analysis on a FACSCalibur system (BD Bioscience).
  • CAFs were plated at 3 After overnight incubation, cells were serum-depleted with X-VIVO 10 medium (04-380Q, Lonza) for 24 hours prior to stimulation.
  • X-VIVO 10 medium 04-380Q, Lonza
  • the culture medium was replaced with X-VIVO 10 containing killed or necrotic cancer cells (9 After 20 hours, the supernatant was harvested by centrifugation and used as conditioned medium (CM) for stimulation of target cancer epithelial cells ( 5
  • cancer cells 5 Cells were cultured in X-VIVO 10 medium (Lonza, Basel) for 6 hours. Conditioning badges were added to each group. Plates were incubated in an incubator at 37°C and 5% CO 2 for 1-5 days. Afterwards, Cell Counting Kit-8 (CCK-8) solution (Dojindo Molecular Technologies, Rockville, MD) was added to the wells, and the plate was incubated in an incubator at 37°C and 5% CO 2 for 30 minutes. Absorbance was measured using a microplate reader at 450 nm.
  • CCK-8 Cell Counting Kit-8
  • Cancer cells were plated at 500 per well in a 6-well plate (SPL, Pocheon, Korea) along with X-VIVO 10 (control) and conditioned medium. Plates were incubated at 37°C and 5% CO 2 for 8 or 9 days. The medium was changed once every 4 days. The formed colonies were fixed with 95% ethanol at 4°C overnight, stained with 0.1% crystal violet (Sigma-Aldrich), and then diluted with 20% methanol at room temperature overnight. After washing the plate, visible colonies containing more than 50 cells were counted in five sections under a stereomicroscope. Representative colonies were photographed and three independent experiments were performed.
  • annexin V-FITC/propidium iodide (PI) staining kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions.
  • Lung cancer cells were harvested 1 to 3 days after treatment with CM or rWISP-1, resuspended in 150 mL of binding buffer, and stained with 5 ⁇ L FITC-conjugated Annexin V and 5 ⁇ L PI for 15 minutes at room temperature in the dark. did. Afterwards, cells positive for FITC-conjugated Annexin V were detected by flow cytometry (ACEA NovoCyte, San Diego, CA, USA), and data were analyzed using NovoExpress software 1.5.
  • PI idium iodide
  • lung cancer cells were stained using the TUNEL kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Killed cells were observed under a confocal microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany) equipped with a filter set excitation at 488 and 543 nm. Quantification of TUNEL-positive cells was performed in a blinded fashion by manually counting the number of TUNEL-positive cells per 10 randomly selected high-power fields (HPFs) per slide.
  • HPFs high-power fields
  • siRNA targeting CAFs specifically for Notch1 (#1 Bioneer Inc, Daejeon, Korea; #2 Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), WISP-1 (WNT1-inducible-signaling pathway protein 1)
  • two types of siRNA specifically targeting RhoA (#1 Bioneer Inc, Daejeon, Korea; #2 Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA)
  • siRNA specifically targeting RhoA (Bioneer Inc, Daejeon, Korea) or control siRNA (SN-1003 AccuTarget TM Negative Control; Bioneer) using transfection reagent (Lipofectamine RNAi MAX; Invitrogen) at a final concentration of 50 nM according to the manufacturer's instructions. After transfection overnight, cells were cultured in appropriate medium for 24 h and stimulated with ApoSQ or ApoA cells.
  • the siRNA sequences used to target genes are shown in Table 3 below.
  • Conditioned media from CAFs were incubated with 10 ⁇ g/ml of mouse anti-mouse WISP-1 neutralizing antibody (R&D Systems) or 10 ⁇ g/ml of IgG isotype control (R&D Systems) for 2 hours.
  • the neutralizing effect of anti-WISP-1 antibodies was tested using WISP-1 ELISA before use.
  • Phagocytosis of killed cancer cells was evaluated using flow cytometry and immunofluorescence analysis.
  • CAFs were stained with PKH26 (red) before being co-cultured with killed 344SQ cells labeled with PKH67 (green) at a 1:3 ratio for 24 h, and the rate of phagocytosis was assessed by two-color flow cytometry.
  • killed 344SQ cells labeled with PKH67 were co-cultured with CAFs for 24 hours. After washing the CAFs, they were fixed with 3.7% w/v paraformaldehyde and treated with 0.1% Triton X-100 for 15 minutes for permeabilization.
  • F-actin was stained with rhodamine phalloidin (Invitrogen) according to the manufacturer's instructions. Images were acquired with a confocal microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany), and the phagocytosis index was calculated using the equation (number of cell deaths/total 200 CAFs) ⁇ 100.
  • mice were cared for and handled in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals.
  • Lung cancer metastasis research using syngeneic tumor experiments was performed using a known method. Briefly, 344SQ cells (1x10 6 cells in 100 ⁇ l of PBS per mouse) were injected subcutaneously into syngeneic (129/Sv) mice in the right posterior aspect.
  • conditioned medium derived from CAFs 100 ⁇ l per mouse; undiluted or 50% diluted with serum-free medium
  • rWISP-1 12.5 ⁇ g/kg or 25 ⁇ g/kg
  • Mice were monitored daily for tumor growth and sacrificed 6 weeks after injection. An autopsy was then performed to determine the diameter and weight of the subcutaneous tumor mass. All animal experiments and studies were performed with male mice of the same age.
  • the tumor volume was calculated using the formula (L x W x W)/2, where L is the long part of the tumor and W is the short part of the tumor.
  • 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 of 0.05 or less was considered statistically significant, and all data were analyzed using Prism 5 software (GraphPad Software Inc., San Diego, CA, USA).
  • CAFs are known to secrete various factors related to tumor creation, growth, and metastasis
  • in this experiment when CAFs were co-cultured with UV-irradiated killed 344SQ cancer cells, lung cancer cells grew through secretion of bioactive mediators. It was investigated whether this was suppressed.
  • CAFs were isolated from lung tumors of Kras-mutant (Kras LA1 ) mice using the fibroblast-specific marker Thy1 and then treated with the killed 344SQ cell line (ApoSQ) or necrotic 344SQ cell line (NecSQ). At this time, the highly invasive and metastatic lung adenocarcinoma cell line derived from mice co-expressing the Kras LA1 and 53 R172H alleles for 20 hours was used.
  • 344SQ cells were treated with 344SQ co-culture conditioned medium killed with CAFs (ApoSQ-CAF CM) or 344SQ co-culture conditioned medium with CAFs killed (NecSQ-CAF CM) for 1 to 5 days.
  • Cell proliferation analysis was performed using Cell Counting Kit-8 (Dojindo).
  • CM of apoptotic or necrotic cancer cells alone did not affect the growth of cancer cells. could be confirmed ( Figures 2a to 2d).
  • Example 2-1 In order to confirm whether the cancer cell proliferation inhibitory effect of the co-culture conditioned medium of CAFs and killed cancer cells identified in Example 2-1 is due to the promotion of cancer cell death, Annexin V/PI double staining analysis, TUNEL analysis, and Western blotting analysis was performed to evaluate the degree of apoptosis of cancer cells.
  • ApoSQ-CAF CM and ApoA-CAF CM showed an increase in the number of TUNEL-positive cells after 2 days of treatment, but no effect was observed for CAF CM, NecSQ-CM, and NecA-CM ( Figures 6a and 6b).
  • WISP-1 WNT1-inducible-signaling pathway protein 1
  • Notch1-WISP- 1 We investigated whether the signaling system plays an important role in the anti-proliferative and apoptosis-promoting effects of cancer cells.
  • WISP-1 acts in a paracrine manner to exert anti-proliferative and apoptosis-promoting effects
  • 344SQ cells and A549 cells were incubated with mouse recombinant WISP-1 (rWISP-1, 20 to 100 ng). /ml), cell viability, degree of colony formation, expression level of cell proliferation markers, and expression level of cell death markers were analyzed.
  • rWISP-1 suppresses cell survival and colony formation in 344SQ cells and A549 cells in a concentration-dependent manner (FIGS. 17a and 17b), and also reduces mRNA and/or protein expression of Ki67 and PCNA in a concentration-dependent manner. ( Figures 18a and 18b). Additionally, flow cytometry and TUNEL analysis showed that rWISP-1 promoted apoptosis of cancer cells in a concentration-dependent manner ( FIGS. 19A to 20B ).
  • RhoA/Rho kinase signaling pathway enhances Notch1/WISP-1 signaling, anti-proliferative effect, and apoptosis-promoting effect in CAFs.
  • the tumor volume was suppressed by about 54% on the 41st day of injection, but in the case of undiluted ApoSQ-CAF CM, the tumor volume was suppressed by about 86% on the 41st day of injection. It was confirmed that the tumor volume suppression effect of undiluted ApoSQ-CAF CM was superior.
  • anti-WISP-1 neutralizing antibody or IgG isotype was added to the conditioned medium 2 hours before mouse injection (FIG. 31a).
  • rWISP-1 was injected at a concentration of 12.5 ⁇ g/kg or 25 ⁇ g/kg through intratumoral injection three times a week into syngeneic mice two days after injection of 344SQ cells. , the antitumor effect was confirmed (Figure 32a)).

Abstract

The present invention relates to a pharmaceutical composition for inhibiting cancer growth, a method for inhibiting cancer growth using same, and a use of the pharmaceutical composition, wherein the pharmaceutical composition contains a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured. In the present invention, it was confirmed that when CAFs and apoptotic cancer cells are co-cultured, WISP-1 is generated through the Notch1 signaling system to inhibit the growth of cancer cells not only at the cellular level but also in vivo and that when RhoA and Rho kinase are inhibited, the cancer cell growth is further suppressed. Therefore, the culture solution of the present invention, the main component (WISP-1) of the culture solution, CAFs exposed to apoptotic cancer cells, or CAFs simultaneously exposed to apoptotic cancer cells and inhibitors of RhoA and Rho kinase can be advantageously utilized as inhibitors against cancer growth.

Description

암 성장 억제용 조성물Composition for inhibiting cancer growth
본 발명은, 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(Apoptotic cancer cells)를 공동 배양한 배양액을 함유하는 암 성장 억제용 약학적 조성물, 이를 이용한 암 성장 억제 방법 및 상기 약학적 조성물의 용도에 관한 것이다.The present invention provides a pharmaceutical composition for inhibiting cancer growth containing a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and Apoptotic cancer cells, a method for inhibiting cancer growth using the same, and It relates to the use of the pharmaceutical composition.
폐암(소세포암 및 비소세포암 모두)은 남성 및 여성 모두에서 전세 계적으로 가장 흔한 암으로서, 암-관련 사망의 주요 원인이다(총 암 사망의 18.0%). 폐암 환자의 약 75%는 진단시에 국소 진행성 또는 전이성 질환이 존재하는 것으로 확인되며, 이때 전이란 암세포의 이동 및 침윤을 포함하는 다단계 과정으로, 악성 종양의 마커가 된다.Lung cancer (both small cell and non-small cell cancer) is the most common cancer worldwide in both men and women and the leading cause of cancer-related deaths (18.0% of total cancer deaths). Approximately 75% of lung cancer patients are confirmed to have locally advanced or metastatic disease at the time of diagnosis, and metastasis is a multi-step process involving the movement and invasion of cancer cells and serves as a marker for malignant tumor.
다양한 기원을 갖는 암관련 섬유아세포(Cancer-associated fibroblasts, CAFs)는 종양-관련 기질 내에 존재하는 주된 세포 유형 중 하나이다. CAFs와 암 세포 사이의 측분비 교신(paracrine communication)은 악성 종양으로의 진행 및 전이 확산을 촉진할 수 있는 암 세포 이동 및 침윤과 같은 기저 과정을 촉진한다는 것이 알려져 있다. CAFs는 종양 기질에서 매트릭스를 물리적으로 리모델링하여, 암 세포가 여전히 상피 특성을 유지하면서 침윤하도록 한다. 그러나, 종양 진행에 있어서, CAFs-매개된 조절의 근본적인 분자 메카니즘은 여전히 불분명하다.Cancer-associated fibroblasts (CAFs), which have diverse origins, are one of the main cell types present in the tumor-related stroma. It is known that paracrine communication between CAFs and cancer cells promotes basal processes such as cancer cell migration and invasion, which can promote malignant tumor progression and metastatic spread. CAFs physically remodel the matrix in the tumor stroma, allowing cancer cells to invade while still maintaining epithelial properties. However, the underlying molecular mechanisms of CAFs-mediated regulation in tumor progression remain unclear.
종양미세환경(TME)에서 섬유아세포 활성화를 조절하는 데 있어서 Notch 신호전달(Notch signaling)의 중요성은 잘 확립되어 있다. Notch 신호전달의 활성화는 일반적으로 리간드-발현 세포와의 직접적인 상호작용에 의해 엄격하게 조절되고, Notch 신호전달의 조절에 문제가 생기면 발달 이상이나 암이 생긴다. 흥미롭게도, Notch 활성은 종양원성(oncogenic) 및 종양-억제성 기능 둘 모두와 관련되며, 이는 Notch가 유도하는 세포반응의 복잡한 미세환경에 달려있다. 활성화된 Notch 경로를 갖는 기질 섬유아세포는 부분적으로 WISP-1(Wnt-induced signaling protein-1)의 상향 조절을 통해 흑색종 성장을 약화시키고 종양 혈관신생을 억제할 수 있다. 이러한 발견은 다른 암 유형에 있어서도, CAF에서 Notch1 의존적인 종양-조절 역할에 대한 분자 메커니즘을 밝히는데 도움을 줄 것이다.The importance of 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 with 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 Notch-induced cellular responses. Stromal fibroblasts with 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 underlying Notch1-dependent tumor-regulatory roles in CAFs, even in other cancer types.
종양 환경 내에서 높은 수준의 세포 사멸 및 종양 세포를 사멸시키기 위한 제거 기전은 종양-특이적 면역에 크게 영향을 미칠 수 있다. 종양 미세환경(TME)에서, 식세포 매개 제거의 면역억제 효과는 항종양 면역 반응을 억제하는 것으로 보고된 바 있다. 반대로, 종양은 또한 사멸세포 제거과정(efferocytosis)을 위한 인식을 억제함으로써 면역감시를 회피할 수 있다.High levels of cell death within the tumor environment and clearance mechanisms to kill tumor cells can greatly impact tumor-specific immunity. In the tumor microenvironment (TME), the immunosuppressive effects of phagocyte-mediated clearance have been reported to suppress anti-tumor immune responses. Conversely, tumors can also evade immune surveillance by inhibiting recognition for dead cell elimination (efferocytosis).
또한, 항염증 및 용해 지질 오타코이드(autacoid)는 다중 종양 유형에서 대식세포 탐식작용을 통해 세포 파편의 제거를 촉진시킴으로써 파편-자극된 암 진행을 특이적으로 억제한다. 더욱이, 본 발명자들의 이전 연구에서는, UV-조사된 사멸화(apoptotic) 폐암 세포에 노출된 대식세포가, 엑소좀 포스파타제 및 tensin homolog(PTEN) 및 퍼옥시좀 증식자-활성화 수용체-감마(PPARr) 리간드, 예컨대 15-하이드록시에이코사테트라엔산(HETE), 리폭신 A4 및 15d-프로스타글란딘 J2의 분비를 통해, 세포 극성 파괴, 암세포의 EMT 및 침윤을 억제한다는 것을 입증한 바 있다(특허등록 10-1804852). 또한, 본 발명자들은 이전 연구에서 UV에 조사된 사멸화된 폐암 세포가 CAFs 내에서 Notch1 신호를 통해 WISP-1을 상향 조절함으로써 암세포의 이동 및 침윤을 억제한다는 사실을 밝혀냈다. 그러나, 종양미세환경(TME)에서 사멸화된 암 세포와 CAFs 간 상호작용이 종양 성장을 억제하는지 여부는 아직 연구된 바가 없다.Additionally, the anti-inflammatory and lytic lipid autacoid specifically inhibits debris-stimulated cancer progression by promoting clearance of cellular debris through macrophage phagocytosis in multiple tumor types. Moreover, our previous study showed that macrophages exposed to UV-irradiated apoptotic lung cancer cells expressed exosomal phosphatase and tensin homolog (PTEN) and peroxisome proliferator-activated receptor-gamma (PPARr). It has been demonstrated that it inhibits disruption of cell polarity, EMT and invasion of cancer cells through secretion of ligands such as 15-hydroxyeicosatetraenoic acid (HETE), lipoxin A4 and 15d-prostaglandin J2 (Patent Registration 10) -1804852). Additionally, in a previous study, the present inventors found that UV-irradiated apoptotic lung cancer cells inhibit cancer cell migration and invasion by upregulating WISP-1 through Notch1 signaling within CAFs. However, whether the interaction between killed cancer cells and CAFs in the tumor microenvironment (TME) inhibits tumor growth has not yet been studied.
이에, 본 발명자들은 CAFs와 사멸화(apoptotic) 암 세포의 상호작용 이 암 세포 성장을 어떻게 조절하는지 연구한 결과, 사멸화된 암 세포에 CAFs를 노출시키면 Notch1 신호전달-의존적인 WISP-1 생성을 유도하여, 암 세포의 증식을 억제하고, 세포 사멸을 유도함을 확인하였다. 또한 RhoA/Rho kinase 신호전달 경로의 억제는 CAFs의 사멸암세포 탐식능력(efferocytosis)을 촉진하고 사멸화 암 세포 자극으로 인한 Notch1-WISP-1 신호 전달을 강화하여 암 세포 성장 억제 효과가 증진됨을 확인하였다. 더 나아가 CAFs와 사멸화된 암 세포 공동 배양 조정배지가 생쥐의 종양 성장을 억제하였으며, 이러한 암 성장 억제 효과는 WISP-1 의존적임을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors studied how the interaction between CAFs and apoptotic cancer cells regulates cancer cell growth. As a result, exposure of CAFs to apoptotic cancer cells led to Notch1 signaling-dependent WISP-1 production. It was confirmed that it inhibits the proliferation of cancer cells and induces cell death. In addition, inhibition of the RhoA/Rho kinase signaling pathway promoted the efferocytosis of CAFs on apoptotic cancer cells and enhanced Notch1-WISP-1 signaling due to stimulation of apoptotic cancer cells, thereby enhancing the cancer cell growth inhibition effect. . Furthermore, the present invention was completed by confirming that the conditioned medium for co-culture of CAFs and killed cancer cells inhibited tumor growth in mice, and that this cancer growth inhibition effect was WISP-1 dependent.
일 양상은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 함유하는, 암 성장 억제용 약학적 조성물을 제공하는 것이다.One aspect is to provide a pharmaceutical composition for inhibiting cancer growth, which contains a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
다른 양상은 사멸화된 암 세포(apoptotic cancer cells)에 노출된 암 관련 섬유아세포(Carcinoma-Associated Fibroblasts, CAFs)를 함유하는, 암 성장 억제용 약학적 조성물을 제공하는 것이다.Another aspect is to provide a pharmaceutical composition for inhibiting cancer growth, containing Carcinoma-Associated Fibroblasts (CAFs) exposed to apoptotic cancer cells.
또 다른 양상은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 함유하는, 암 성장 억제용 건강기능식품을 제공하는 것이다.Another aspect is to provide a health functional food for inhibiting cancer growth, containing a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
또 다른 양상은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양하는 단계를 포함하는, 암 성장 억제용 조성물의 제조방법을 제공하는 것이다.Another aspect is to provide a method for producing a composition for inhibiting cancer growth, comprising co-culturing cancer-associated fibroblasts (CAFs) and apoptotic cancer cells.
또 다른 양상은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 개체에 처리하는 단계를 포함하는, 암 성장 억제방법을 제공하는 것이다.Another aspect is to provide a method of inhibiting cancer growth, comprising treating a subject with a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells. .
또 다른 양상은 WISP-1(Wnt-induced signaling protein-1)을 개체에 처리하는 단계를 포함하는, 암 성장 억제방법을 제공하는 것이다.Another aspect is to provide a method of inhibiting cancer growth, comprising treating a subject with WISP-1 (Wnt-induced signaling protein-1).
또 다른 양상은 암 성장 억제용 약제의 제조를 위한 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액의 용도를 제공하는 것이다.Another aspect is to provide the use of a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells for the production of a drug for inhibiting cancer growth.
또 다른 양상은 암 성장 억제용 약제의 제조를 위한 WISP-1(Wnt-induced signaling protein-1)의 용도를 제공하는 것이다.Another aspect provides the use of WISP-1 (Wnt-induced signaling protein-1) for the manufacture of a drug for inhibiting cancer growth.
또 다른 양상은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액의 암 성장 억제 용도를 제공하는 것이다.Another aspect is to provide a use for inhibiting cancer growth in a co-culture of Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
또 다른 양상은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 함유하는, 암 성장 억제용 약학적 조성물의 암 성장 억제 용도를 제공하는 것이다.Another aspect provides the use of a pharmaceutical composition for inhibiting cancer growth, comprising a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells. It is done.
또 다른 양상은 WISP-1(Wnt-induced signaling protein-1)의 암 성장 억제 용도를 제공하는 것이다.Another aspect is to provide a use of WISP-1 (Wnt-induced signaling protein-1) to inhibit cancer growth.
본 발명은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 함유하는, 암 성장 억제용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for inhibiting cancer growth, containing a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
본 발명에서 상기 CAFs와 사멸화된 암 세포를 공동 배양한 배양액은 WISP-1을 함유하고 있어, 암 세포의 성장을 억제하는 효과가 있다.In the present invention, the culture medium co-cultured with the CAFs and killed cancer cells contains WISP-1, which has the effect of inhibiting the growth of cancer cells.
본 발명에서 "암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs)"란 암 병변의 내부 및/또는 주변에 존재하는 α－SMA(α-평활근엑틴) 양성 섬유아세포를 의미하며, 상기 CAFs는 대장암, 폐암, 전립선암, 유방암, 위암, 담관암, 기저세포암 등의 다양한 암에서 그 존재가 확인되고 있다.In the present invention, “Cancer-Associated Fibroblasts (CAFs)” refers to α-SMA (α-smooth muscle actin) positive fibroblasts present within and/or around cancer lesions, and the CAFs are colon cancer cells. , its presence has been confirmed in various cancers such as lung cancer, prostate cancer, breast cancer, stomach cancer, bile duct cancer, and basal cell cancer.
본 발명에서 상기 CAFs와 관련된 암은 뇌종양, 두경부암, 유방암, 폐암, 식도암, 위암, 십이지장암, 충수암, 대장암, 직장암, 간암, 췌장암, 담낭암, 담관암, 항문암, 신암, 수뇨관암, 방광암, 전립선 암, 음경암, 정소암, 자궁암, 난소암, 외음암, 질암, 피부암 등의 고형암일 수 있다.In the present invention, cancers related to 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, ureteral cancer, and bladder cancer. , It may be solid cancer such as prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and skin cancer.
본 발명의 일 실시예에서, 상기 CAFs는 암에 관련된 것으로서 악성고형종양과 관련된 섬유아세포일 수 있다. 일 구체예에서, 상기 CAF는 섬유육종, 악성섬유성조직구종, 지방육종, 횡문 근육종, 평활근육종, 혈관육종, 악성피부암, 림프혈관육종, 활막육종, 연골육종, 골육종 등의 육종에 관련된 것일 수 있으며, 폐암, 위암, 유방암, 대장암 또는 전립선암과 관련된 섬유아세포일 수 있다.In one embodiment of the present invention, the CAFs are cancer-related and may be fibroblasts associated with malignant solid tumors. In one embodiment, the CAF may be related to sarcomas such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, malignant skin cancer, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, and osteosarcoma. and may be fibroblasts related to lung cancer, stomach cancer, breast cancer, colon cancer, or prostate cancer.
본 발명에서 "사멸화된 암 세포(apoptotic cancer cells)"는 암 세포에 특정 파장의 빛을 조사하여 사멸화를 유도한 것일 수 있다. 상기 특정 파장의 빛의 조사는 자외선(Ultra-violet ray, UV) 조사일 수 있다. 일 실시예에서, 상기 파장은 100 내지 400 nm 파장으로 5 내지 30분 동안 조사될 수 있다. 일 구체예에서 상기 UV 조사는 150 내지 350 nm 파장으로 내지 20분 동안 또는 200 내지 300 nm 파장으로 10 내지 15분 동안 수행될 수 있다.In the present invention, “apoptotic cancer cells” may be those in which apoptosis is induced by irradiating light of a specific wavelength to cancer cells. Irradiation of light of the specific wavelength may be ultraviolet ray (UV) irradiation. In one embodiment, the wavelength may be 100 to 400 nm for 5 to 30 minutes. In one embodiment, the UV irradiation may be performed at a wavelength of 150 to 350 nm for 20 minutes or at a wavelength of 200 to 300 nm for 10 to 15 minutes.
본 발명에서 "공동 배양"은 CAFs와 사멸화된 암 세포를 함께 배양하여 이루어질 수 있다. 일 실시예에서, CAFs를 20 내지 30 시간 동안 X-VIVO 또는 무혈청 DMEM 배지에서 사멸화된 암 세포와 함께 배양할 수 있다.In the present invention, “co-culture” can be achieved by culturing CAFs and killed cancer cells together. In one embodiment, CAFs can be cultured with killed cancer cells in X-VIVO or serum-free DMEM medium for 20 to 30 hours.
본 발명에서 "배양액"이란 CAFs와 사멸화된 암세포의 공동 배양을 통해 얻어진 배양 결과물을 의미한다. 본 발명의 일 실시예에서, 상기 배양액은 액체배지, 고체배지 또는 반고체배지일 수 있다. 일 구체예에서, 상기 배양액은 조정배지(Conditioned medium)일 수 있다.In the present invention, “culture medium” refers to the culture result obtained through co-culture of CAFs and killed cancer cells. In one embodiment of the present invention, the culture medium may be a liquid medium, solid medium, or semi-solid medium. In one embodiment, the culture medium may be a conditioned medium.
본 발명에서 "암 성장"이란 통제 불가능한 종양 세포의 증식 과정으로, 타 조직으로의 전이와는 구분된다. 본 발명의 일 실시예에서, 상기 종양 세포의 증식은 부피, 중량 및 직경 생장 및 콜로니 형성을 포함할 수 있다.In the present invention, “cancer growth” is a process of uncontrollable proliferation of tumor cells and is distinguished from metastasis to other tissues. In one embodiment of the present invention, proliferation of the tumor cells may include growth in volume, weight, and diameter, and colony formation.
본 발명에서 "암 성장 억제"란 세포 수준에서 암세포 및 콜로니의 증식 억제 또는 생체 내에서 종양의 성장 억제를 의미한다.In the present invention, “cancer growth inhibition” refers to inhibition of proliferation of cancer cells and colonies at the cellular level or inhibition of tumor growth in vivo.
본 발명의 일 실시예에서, 상기 사멸화된 암 세포의 암종은 유방암, 자궁암, 식도암, 위암, 뇌암, 직장암, 대장암, 폐암, 피부암, 난소암, 자궁경부암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 구강암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종 및 백혈병으로 이루어진 군에서 선택된 하나 이상일 수 있다. 일 구체예에서 상기 암종은 폐암, 유방암, 위암, 대장암 및 전립선암으로 이루어진 군에서 선택된 하나 이상일 수 있으며, 상기 폐암은 폐선암 또는 비소세포폐암일 수 있다.In one embodiment of the present invention, 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 one or more 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. In one embodiment, the carcinoma may be one or more selected from the group consisting of lung cancer, breast cancer, stomach cancer, colon cancer, and prostate cancer, and the lung cancer may be lung adenocarcinoma or non-small cell lung cancer.
본 발명의 일 실시예에서, 상기 폐선암 세포는 344SQ 세포주, 비소세포폐암 세포는 A549 세포주, 대장암 세포는 HCT116 세포주, 유방암 세포는 MCF-7 세포주일 수 있다.In one embodiment of the present invention, 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, and the breast cancer cells may be the MCF-7 cell line.
본 발명의 일 실시예에서, 상기 배양액은 유효성분으로서 WISP-1(Wnt-induced signaling protein-1)을 함유할 수 있다. WISP-1은 WNT 시그널링 경로의 표적 단백질이며, WNT 시그널링은 자가분비(autocrine) 및 주변분비(paracrine) 신호를 통해 상피(epithelial) 및 중간엽(mesenchymal) 발달 모두를 조절하면서, 폐 발달에서 역할을 한다.In one embodiment of the present invention, 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 signaling. do.
본 발명의 일 실시예에서, 상기 WISP-1은 Notch1 시그널링에 의해 생성될 수 있다. Notch1 시그널링은 Notch 수용체를 매개체로 하여 세포접촉의존적으로 이루어지며, 발생, 재생, 항상성 유지 등에 중요한 역할을 하는 것으로 알려져 있다. 또한 Notch 시그널링의 활성은 종양원성(oncogenic) 및 종양-억제성 기능 둘 모두와 연관이 있다.In one embodiment of the present invention, the WISP-1 can be produced by Notch1 signaling. Notch1 signaling occurs in a cell contact-dependent manner using the Notch receptor as a mediator, and is known to play an important role in development, regeneration, and homeostasis. Additionally, the activity of Notch signaling is associated with both oncogenic and tumor-suppressive functions.
본 발명의 일 실시예에서, 상기 Notch1 시그널링은 RhoA 및 Rho kinase의 억제에 의해 강화될 수 있다. RhoA는 Rho-GTPase 중 하나로, 세포 골격 및 세포 분열을 조절하며, RhoA 활성의 증가는 세포 증식과 연관되는 것으로 알려져 있다.In one embodiment of the present invention, the Notch1 signaling can be enhanced by inhibition of RhoA and Rho kinase. RhoA is one of the Rho-GTPases, which regulates the cytoskeleton and cell division, and increased RhoA activity is known to be associated with cell proliferation.
본 발명의 일 실시예에서, 상기 조성물은 암 관련 섬유아세포에서 Notch1 관련 분자를 증가시킬 수 있다. 일 구체예에서, 상기 Notch1 관련 분자는 WISP-1, NICD1 및 Hes1으로 이루어진 군에서 선택되는 하나 이상일 수 있다.In one embodiment of the present invention, the composition can increase Notch1-related molecules in cancer-related fibroblasts. In one embodiment, the Notch1-related molecule may be one or more selected from the group consisting of WISP-1, NICD1, and Hes1.
본 발명의 일 실시예에서, 상기 조성물은 암 관련 섬유아세포에서 세포 증식 마커의 발현을 감소시킬 수 있다. 일 구체예에서, 상기 세포 증식 마커는 Ki67 및 PCNA로 이루어진 군에서 선택되는 하나 이상일 수 있다.In one embodiment of the present invention, the composition can reduce the expression of cell proliferation markers in cancer-related fibroblasts. In one embodiment, the cell proliferation marker may be one or more selected from the group consisting of Ki67 and PCNA.
본 발명의 일 실시예에서, 상기 조성물은 암 세포의 세포 사멸(apoptosis)을 증가시킬 수 있다. 또한, 상기 조성물은 암 관련 섬유아세포에서 세포 사멸 촉진 인자의 발현을 증가시키고, 항-세포 사멸 인자의 발현을 감소시킬 수 있다. 일 구체예에서, 상기 세포 사멸 촉진 인자는 절단된 caspase 3, 절단된 PARP 및 Bax로 이루어진 군에서 선택되는 하나 이상이고, 상기 항-세포 사멸 인자는 Bcl-2, Mcl-1 및 Bcl-xL로 이루어진 군에서 선택되는 하나 이상일 수 있다.In one embodiment of the present invention, the composition can increase apoptosis of cancer cells. Additionally, the composition can increase the expression of pro-apoptotic factors and decrease the expression of anti-apoptotic factors in cancer-related fibroblasts. In one embodiment, the pro-apoptotic factor is one or more selected from the group consisting of cleaved caspase 3, cleaved PARP, and Bax, and the anti-apoptotic factor is Bcl-2, Mcl-1, and Bcl-xL. It may be one or more selected from the group consisting of:
본 발명의 일 실시예에서, 상기 조성물은 RhoA 및 Rho kinase 억제제를 더 함유할 수 있다. 일 구체예에서, 상기 RhoA 및 Rho kinase 억제제는 화합물, shRNA, miRNA, siRNA, 항체 및 앱타머로 이루어진 군에서 선택되는 하나 이상일 수 있다. In one embodiment of the present invention, the composition may further contain RhoA and Rho kinase inhibitors. In one embodiment, the RhoA and Rho kinase inhibitor may be one or more selected from the group consisting of a compound, shRNA, miRNA, siRNA, antibody, and aptamer.
상기 siRNA는 자연에 존재하는(변형되지 않은) 리보뉴클레오티드 단위구조를 가질 수 있으며, 하나 이상의 화학적 변형(modification)을 가지도록 합성되는 등 화학적으로 변형된 것일 수도 있다. 상기 화학적 변형을 통해 뉴클레아제(nuclease)에 대한 저항성 증진, 세포내 흡수(uptake) 증가, 세포 표적화(타겟 특이성) 향상, 안정성 증가, 또는 인터페론 활성 감소, 면역 반응 및 센스(sense) 효과와 같은 타겟 이외 효과(off-target effect) 감소, RISC 로딩(loading) 증가(RNAi 활성 증가) 등의 효과를 얻을 수 있다.The siRNA may have a ribonucleotide unit structure that exists in nature (unmodified), or may be chemically modified, such as synthesized to have one or more chemical modifications. Through the above chemical modifications, such as enhancing resistance to nuclease, increasing intracellular uptake, improving cell targeting (target specificity), increasing stability, or reducing interferon activity, immune response and sense effect. Effects such as reduced off-target effects and increased RISC loading (increased RNAi activity) can be achieved.
상기 siRNA의 화학적 변형 방법은 특별히 제한되지 않으며, 예컨대 하나 이상의 리보뉴클레오티드의 당 구조, 또는 염기 구조, 또는 상기 리보핵산 간의 결합 부위가 화학적으로 변형(modification)된 것일 수 있으며, 당해 기술 분야의 통상의 지식을 가진 당업자라면 당해 기술 분야에 공지된 방법을 이용하여 원하는 방식대로 상기 siRNA 분자를 합성하고 변형시킬 수 있다.The method of chemical modification of the siRNA is not particularly limited, and for example, the sugar structure or base structure of one or more ribonucleotides, or the binding site between the ribonucleic acids may be chemically modified, and may be carried out using methods known in the art. A person skilled in the art can synthesize and modify the siRNA molecules in any desired manner using methods known in the art.
예를 들어, 상기 화학적 변형은 핵염기-변형된 리보뉴클레오티드, 즉, 5번-위치에서 변형된 우리딘 또는 시티딘(5-(2-아미노)프로필 우리딘, 5-브로모 우리딘 등), 8번 위치에서 변형된 아데노신 및 구아노신(8-브로모 구아노신 등), 데아자(deaza) 뉴클레오티드(7-데아자-아데노신 등), 0- 및 N-알킬화된 뉴클레오티드(N6-메틸 아데노신 등)와 같이 천연 핵염기(nucleobase) 대신 비천연 핵염기를 포함하는 리보뉴클레오티드를 포함할 수 있다.For example, the chemical modification may be a nucleobase-modified ribonucleotide, i.e., uridine or cytidine modified at the 5-position (5-(2-amino)propyl uridine, 5-bromouridine, etc.) , adenosine and guanosine modified at position 8 (8-bromo guanosine, etc.), deaza nucleotides (7-deaza-adenosine, etc.), 0- and N-alkylated nucleotides (N6-methyl adenosine, etc.) etc.), it may contain a ribonucleotide containing a non-natural nucleobase instead of a natural nucleobase.
또한, 당-변형된 리보뉴클레오티드에 있어서, 2’OH 기는 H, OR, R, 할로젠(F, Cl, Br 또는 I), SH, SR, NH2, NHR, NR2 및 CN으로 이루어진 군으로부터 선택되는 치환기(R은 C1-C6 알킬, 알케닐, 또는 알키닐)에 의하여 치환될 수 있고, 리보뉴클레오티드에 있어서, 백본의 포스포디에스테르 결합은 보라노포스페이트(boranophosphate) 또는 포스포로티오에이트(phosphorothioate)로 치환될 수 있다.Additionally, for sugar-modified ribonucleotides, the 2'OH group is selected from the group consisting of H, OR, R, halogen (F, Cl, Br or I), SH, SR, NH2, NHR, NR2 and CN. It may be substituted by a substituent (R is C1-C6 alkyl, alkenyl, or alkynyl), and in ribonucleotides, the phosphodiester bond of the backbone is boranophosphate or phosphorothioate. can be replaced.
본 발명의 일 실시예에서, 상기 RhoA 및 Rho kinase 억제제가 화합물인 경우, 상기 화합물은 Y-27632일 수 있다. 또한, 상기 RhoA 및 Rho kinase 억제제가 siRNA인 경우, 상기 siRNA는 서열번호 19 및 20의 염기 서열 중 어느 하나의 염기 서열로 이루어진 폴리뉴클레오티드일 수 있다.In one embodiment of the present invention, when the RhoA and Rho kinase inhibitor is a compound, the compound may be Y-27632. Additionally, when the RhoA and Rho kinase inhibitors are siRNAs, the siRNAs may be polynucleotides consisting of any one of the nucleotide sequences of SEQ ID NOs: 19 and 20.
본 발명에 따른 약학적 조성물은 "약학적으로 허용 가능한 담체"를 포함할 수 있다. 상기 약학적으로 허용 가능한 담체는 제제시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 사이클로덱스트린, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 등을 포함하지만 이에 한정되지 않으며, 필요에 따라 항산화제, 완충액 등 다른 통상의 첨가제를 더 포함할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립, 또는 정제로 제제화할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제화에 관해서는 레밍턴의 문헌에 개시되어 있는 방법을 이용하여 각 성분에 따라 바람직하게 제제 화할 수 있다. 본 발명의 약학적 조성물은 제형에 특별한 제한은 없으나 주사제, 흡입제, 피부 외용제, 또는 경구 섭취제 등으로 제제화할 수 있다.The pharmaceutical composition according to the present invention may include a “pharmaceutically acceptable carrier.” The pharmaceutically acceptable carrier is commonly used in preparation and includes, but is limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. If necessary, other common additives such as antioxidants and buffers may be added. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, the formulations can be preferably formulated according to each ingredient using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but can be formulated as an injection, inhalation agent, topical skin agent, or oral ingestion agent.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 피부, 비강, 기도에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, applied to the skin, nasal cavity, or respiratory tract) according to the desired method, and the dosage is determined by the patient's condition, weight, and disease. It varies depending on the degree, drug form, administration route and time, but can be appropriately selected by a person skilled in the art.
본 발명에 따른 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. 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 singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
구체적으로, 본 발명에 따른 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.001 내지 150 mg, 바람직하게는 0.01 내지 100 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and body weight, and is generally administered at 0.001 to 150 mg, preferably 0.01 to 100 mg, per kg of body weight every day or every other day, or 1 It can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.
본 발명은 사멸화된 암 세포(apoptotic cancer cells)에 노출된 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs)를 함유하는, 암 성장 억제용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for inhibiting cancer growth, which contains Cancer-Associated Fibroblasts (CAFs) exposed to apoptotic cancer cells.
본 발명은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 함유하는, 암 성장 억제용 건강기능식품을 제공한다.The present invention provides a health functional food for inhibiting cancer growth, containing a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells.
본 발명의 건강기능식품은 담체, 희석제, 부형제 및 첨가제 중 하나 이상을 더 포함하여 정제, 환제, 산제, 과립제, 분말제, 캡슐제 및 액제 제형으로 이루어진 군에서 선택된 하나로 제형될 수 있다. 본 발명의 추출물을 첨가할 수 있는 식품으로는, 각종 식품류, 분말, 과립, 정제, 캡슐, 시럽제, 음료, 껌, 차, 비타민 복합제, 건강기능성 식품류 등이 있다.The health functional food of the present invention may be formulated with one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further including one or more of carriers, diluents, excipients, and additives. 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, etc.
상기 본 발명에 더 포함될 수 있는 첨가제로는, 천연 탄수화물, 향미제, 영양제, 비타민, 광물(전해질), 풍미제(합성 풍미제, 천연 풍미제 등), 착색제, 충진제(치즈, 초콜렛 등), 팩트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH조절제, 안정화제, 방부제, 산화 방지제, 글리세린, 알콜, 탄산화제 및 과육으로 이루어진 군으로부터 선택된 1종 이상의 성분을 사용할 수 있다. 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 ingredients selected from the group consisting of pactic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohol, carbonating agents and pulp can be used. .
상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상기 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
상기 외에 본 발명의 건강기능식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명에 따른 조성물은 천연 과일 쥬스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, and alginic acid. and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Additionally, the composition according to the present invention may contain pulp for the production of natural fruit juice and vegetable drinks. These ingredients can be used independently or in combination.
상기 담체, 부형제, 희석제 및 첨가제의 구체적인 예로는 이에 한정하는 것은 아니나, 락토즈, 덱스트로즈, 슈크로즈, 솔비톨, 만니톨, 에리스리톨, 전분, 아카시아 고무, 인산칼슘, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 미세결정성 셀룰로즈, 폴리비닐피롤리돈, 셀룰로즈, 폴리비닐피롤리돈, 메틸셀룰로즈, 물, 설탕시럽, 메틸셀룰로즈, 메틸 하이드록시 벤조에이트, 프로필하이드록시 벤조에이트, 활석, 스테아트산 마그네슘 및 미네랄 오일로 이루어진 그룹으로부터 선택된 1종 이상이 사용되는 것이 바람직하다.Specific examples of the carriers, excipients, diluents and additives include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium. Silicates, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate. It is preferred that at least one selected from the group consisting of mineral oil is used.
본 발명의 건강기능식품을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.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.
또한, 본 발명은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양하는 단계를 포함하는, 암 성장 억제용 조성물의 제조방법을 제공한다.Additionally, the present invention provides a method for producing a composition for inhibiting cancer growth, comprising the step of co-culturing cancer-associated fibroblasts (CAFs) and apoptotic cancer cells.
본 발명의 일 실시예에서, 상기 배양하는 단계는 X-VIVO 배지에서 24시간 동안 배양하여 혈청결핍(serum-starved) 상태로 만드는 것 일 수 있다. 일 구체예에서, 배양 배지를 사멸화된 암 세포가 포함된 X-VIVO 또는 무혈청 DMEM 배지로 교체할 수 있다.In one embodiment of the present invention, the culturing step may be culturing in X-VIVO medium for 24 hours to create a serum-starved state. In one embodiment, the culture medium can be replaced with X-VIVO or serum-free DMEM medium containing killed cancer cells.
또한 일 구체예에서, 상기 공동 배양은 배지 교체 후 10 내지 30시간, 15 내지 25시간 또는 18내지 24시간 동안 이루어질 수 있다.Also, in one embodiment, the co-culture may be performed for 10 to 30 hours, 15 to 25 hours, or 18 to 24 hours after replacing the medium.
본 발명의 일 실시예에서, 상기 제조방법은 상기 공동 배양을 통해 WISP-1을 분비시키는 단계를 더 포함할 수 있다.In one embodiment of the present invention, the production method may further include secreting WISP-1 through the co-culture.
본 발명은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 개체에 처리하는 단계를 포함하는, 암 성장 억제방법을 제공한다.The present invention provides a method of inhibiting cancer growth, comprising treating a subject with a culture medium obtained by co-culturing cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
본 발명에서, "처리"란 실험 요건에 따라 상기 배양액을 개체에 첨가 또는 투여하는 모든 행위를 의미할 수 있다.In the present invention, “treatment” may mean any act of adding or administering the culture medium to an individual according to experimental requirements.
본 발명에서, "개체"는 포유동물을 포함할 수 있다. 일 구체예에서, 상기 개체는 인간, 원숭이, 마우스, 소, 개, 말, 돼지 등을 포함할 수 있다.In the present invention, “individual” may include mammals. In one embodiment, the subject may include humans, monkeys, mice, cows, dogs, horses, pigs, etc.
또한, 본 발명은 WISP-1(Wnt-induced signaling protein-1)을 개체에 처리하는 단계를 포함하는, 암 성장 억제방법을 제공한다.Additionally, the present invention provides a method of inhibiting cancer growth, comprising treating the subject with WISP-1 (Wnt-induced signaling protein-1).
본 발명은 암 성장 억제용 약제의 제조를 위한 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액의 용도를 제공한다.The present invention provides the use of a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells for the production of a drug for inhibiting cancer growth.
또한, 본 발명은 암 성장 억제용 약제의 제조를 위한 WISP-1(Wnt-induced signaling protein-1)의 용도를 제공한다.Additionally, the present invention provides the use of WISP-1 (Wnt-induced signaling protein-1) for the production of a drug for inhibiting cancer growth.
본 발명은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액의 암 성장 억제 용도를 제공한다.The present invention provides a use for inhibiting cancer growth in a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
또한, 본 발명은 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 함유하는, 암 성장 억제용 약학적 조성물의 암 성장 억제 용도를 제공한다.In addition, the present invention provides a use for inhibiting cancer growth of a pharmaceutical composition for inhibiting cancer growth, which contains a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells. to provide.
본 발명은 WISP-1(Wnt-induced signaling protein-1)의 암 성장 억제 용도를 제공한다.The present invention provides a use of WISP-1 (Wnt-induced signaling protein-1) to inhibit cancer growth.
본 발명에서는, CAFs와 사멸화된 암 세포의 상호작용이 암 세포 및 CAFs의 성장을 어떻게 조절하는지 연구한 결과, CAFs와 사멸화된 암 세포를 공동 배양할 경우, Notch1 신호체계를 통해 WISP-1이 생성됨으로써, 암 세포의 성장을 억제한다는 것을 확인하였고, RhoA/Rho kinase 신호전달 경로를 억제할 경우 암 세포 성장이 더욱 억제됨을 확인하였다.In the present invention, as a result of studying how the interaction between CAFs and killed cancer cells regulates the growth of cancer cells and CAFs, when CAFs and killed cancer cells are co-cultured, WISP-1 is activated through the Notch1 signaling system. By producing this, it was confirmed that it inhibits the growth of cancer cells, and it was confirmed that cancer cell growth is further inhibited when the RhoA/Rho kinase signaling pathway is inhibited.
또한, CAFs와 사멸화된 암 세포를 공동 배양한 배양액이 세포 수준에서 암 세포의 성장을 억제함을 확인하였을 뿐만 아니라, 동계(syngeneic) 전이성 폐암 마우스 모델(in vivo)에서도 상기 배양액이 종양의 성장을 억제함을 확인하였고, 상기 배양액의 암 성장 억제 효과는 WISP-1 의존적임을 확인하였다.In addition, it was confirmed that the culture medium co-cultured with CAFs and killed cancer cells inhibited the growth of cancer cells at the cellular level, and also that the culture medium inhibited tumor growth in a syngeneic metastatic lung cancer mouse model ( in vivo ). was confirmed to inhibit, and the cancer growth inhibitory effect of the culture medium was confirmed to be WISP-1 dependent.
따라서, 본 발명은 암 세포의 성장 억제 전략에 유용하게 이용될 수 있으며, 특히 본 발명의 배양액 및 조건배지는 암 성장 억제제로서 유용하게 이용될 수 있다. Therefore, the present invention can be usefully used in a strategy to inhibit the growth of cancer cells, and in particular, the culture medium and conditioned medium of the present invention can be usefully used as a cancer growth inhibitor.
도 1은 사멸화된 암 세포와 공동 배양된 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs)의 배양액을 함유하는 조정배지(Conditioned medium, CM)가 암 세포의 성장을 억제함을 나타낸 도이다. 도 1a는 생쥐 폐 선암종(adenocarcinoma) 세포주인 344SQ를 각각 대조군, CAF 조정배지(CAF CM), CAFs와 사멸화된 344SQ 공동 배양 조정배지(ApoSQ-CAF CM), CAFs와 괴사된 344SQ 공동 배양 조정배지(NecSQ-CAF CM)에 처리하여 세포 생존을 측정한 도이다. 도 1b는 인간 비소세포폐암 세포주인 A549를 각각 대조군, CAF CM, CAFs와 사멸화된 A549 공동 배양 조정배지(ApoA-CAF CM), CAFs와 괴사된 A549 공동 배양 조정배지(NecA-CAF CM)에 처리하여 세포 생존을 측정한 도이다. 도 1c는 인간 대장암 세포주인 HCT116를 각각 대조군, CAF CM, CAFs와 사멸화된 HCT116 공동 배양 조정배지(ApoH-CAF CM), CAFs와 괴사된 HCT116 공동 배양 조정배지(NecH-CAF CM)에 처리하여 세포 생존을 측정한 도이다. 도 1d는 인간 대장암 세포주인 LoVo를 각각 대조군, CAF CM, CAFs와 사멸화된 LoVo 공동 배양 조정배지(ApoL-CAF CM), CAFs와 괴사된 LoVo 공동 배양 조정배지(NecL-CAF CM)에 처리하여 세포 생존을 측정한 도이다.Figure 1 is a diagram showing that conditioned medium (CM) containing a culture medium of cancer-associated fibroblasts (CAFs) co-cultured with killed cancer cells inhibits the growth of cancer cells. Figure 1a shows 344SQ, a mouse lung adenocarcinoma cell line, as a control medium, CAF-conditioned medium (CAF CM), 344SQ co-culture conditioned medium with killed CAFs (ApoSQ-CAF CM), and 344SQ co-culture conditioned medium with CAFs killed, respectively. This is a diagram measuring cell survival after treatment with (NecSQ-CAF CM). Figure 1b shows A549, a human non-small cell lung cancer cell line, in control medium, CAF CM, A549 co-culture conditioned with CAFs killed (ApoA-CAF CM), and CAFs necrosed A549 co-culture conditioned medium (NecA-CAF CM), respectively. This is a diagram measuring cell survival after treatment. Figure 1c shows HCT116, a human colon cancer cell line, treated with control, CAF CM, conditioned medium for co-culture of CAFs and killed HCT116 (ApoH-CAF CM), and conditioned medium for co-culture of CAFs and necrotic HCT116 (NecH-CAF CM), respectively. This is a measure of cell survival. Figure 1d shows LoVo, a human colon cancer cell line, treated with control, CAF CM, conditioned medium for co-culture of LoVo killed with CAFs (ApoL-CAF CM), and conditioned medium for co-culture of LoVo killed with CAFs (NecL-CAF CM), respectively. This is a measure of cell survival.
도 2는 사멸화된 암 세포 단독 배양액은 암세포의 성장 억제 효과가 없음을 확인한 도이다. 도 2a는 344SQ 세포를 각각 대조군, CAF CM, ApoSQ CM, NecSQ CM에 처리하여 세포 생존을 측정한 도이다. 도 2b는 A549 세포를 각각 대조군, CAF CM, ApoA CM, NecA CM에 처리하여 세포 생존을 측정한 도이다. 도 2c는 HCT116 세포를 각각 대조군, CAF CM, ApoH CM, NecH CM에 처리하여 세포 생존을 측정한 도이다. 도 2d는 LoVo 세포를 각각 대조군, CAF CM, ApoL CM, NecL CM에 처리하여 세포 생존을 측정한 도이다.Figure 2 is a diagram confirming that the culture medium containing killed cancer cells alone has no effect on inhibiting the growth of cancer cells. Figure 2a is a diagram measuring cell survival by treating 344SQ cells with control group, CAF CM, ApoSQ CM, and NecSQ CM, respectively. Figure 2b is a diagram measuring cell survival by treating A549 cells with control group, CAF CM, ApoA CM, and NecA CM, respectively. Figure 2c is a diagram measuring cell survival by treating HCT116 cells with control group, CAF CM, ApoH CM, and NecH CM, respectively. Figure 2d is a diagram measuring cell survival by treating LoVo cells with control group, CAF CM, ApoL CM, and NecL CM, respectively.
도 3은 사멸화된 암 세포에 노출된 CAF CM이 폐암 세포의 콜로니 형성을 억제함을 나타낸 도이다. 도 3a는 344SQ 세포를 각각 CAF CM, ApoSQ-CAF CM, NecSQ-CAF CM으로 8일간 처리한 후 확인한 콜로니 이미지 및 콜로니 수를 측정한 도이다. 도 3b는 A549 세포를 각각 CAF CM, ApoA-CAF CM, NecA-CAF CM으로 9일간 처리한 후 확인한 콜로니 이미지 및 콜로니 수를 측정한 도이다.Figure 3 is a diagram showing that CAF CM exposed to killed cancer cells inhibits colony formation of lung cancer cells. Figure 3a shows the colony image and the number of colonies measured after treating 344SQ cells with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM for 8 days, respectively. Figure 3b is a diagram showing colony images and colony numbers measured after treating A549 cells with CAF CM, ApoA-CAF CM, and NecA-CAF CM for 9 days, respectively.
도 4는 사멸화된 암 세포에 노출된 CAF CM이 폐암 세포에서 세포 증식 마커 발현을 억제함을 나타낸 도이다. 도 4a는 344SQ 세포를 CAF CM, ApoSQ-CAF CM, NecSQ-CAF CM으로 3일간 처리한 후 qRT-PCR로 측정한 Ki67 및 PCNA의 mRNA의 양을 나타낸 도이다. 도 4b는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 3일간 처리한 후 면역블랏분석으로 측정한 Ki67 및 PCNA의 단백질 양을 나타낸 도이다. 도 4c는 A549 세포를 CAF CM, ApoA-CAF CM, NecA-CAF CM으로 3일간 처리한 후 qRT-PCR로 측정한 Ki67 및 PCNA의 mRNA의 양을 나타낸 도이다.Figure 4 is a diagram showing that CAF CM exposed to killed cancer cells inhibits the expression of cell proliferation markers in lung cancer cells. Figure 4a is a diagram showing the amount of Ki67 and PCNA mRNA measured by qRT-PCR after treating 344SQ cells with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM for 3 days. Figure 4b is a diagram showing the protein amounts of Ki67 and PCNA measured by immunoblot analysis after treating 344SQ cells with CAF CM and ApoSQ-CAF CM for 3 days. Figure 4c shows the amount of Ki67 and PCNA mRNA measured by qRT-PCR after treating A549 cells with CAF CM, ApoA-CAF CM, and NecA-CAF CM for 3 days.
도 5는 사멸화된 암 세포에 노출된 CAF CM이 세포 사멸을 촉진함을 유세포 분석으로 확인한 도이다. 도 5a는 344SQ 세포를 CAF CM, ApoSQ-CAF CM, NecSQ-CAF CM으로 처리한 후, Annexin V-FICT/PI 이중 염색을 이용한 유세포 분석으로 측정한 세포 사멸 정도를 나타낸 도이다. 도 5b는 A549 세포를 CAF CM, ApoA-CAF CM, NecA-CAF CM으로 처리한 후, Annexin V-FICT/PI 이중 염색을 이용한 유세포 분석으로 측정한 세포 사멸 정도를 나타낸 도이다.Figure 5 is a diagram confirming through flow cytometry that CAF CM exposed to killed cancer cells promotes cell death. Figure 5a is a diagram showing the degree of cell death measured by flow cytometry using Annexin V-FICT/PI double staining after treating 344SQ cells with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM. Figure 5b is a diagram showing the degree of cell death measured by flow cytometry using Annexin V-FICT/PI double staining after treating A549 cells with CAF CM, ApoA-CAF CM, and NecA-CAF CM.
도 6은 사멸화된 암 세포에 노출된 CAF CM이 세포 사멸을 촉진함을 TUNEL 분석으로 확인한 도이다. 도 6a는 344SQ 세포를 CAF CM, ApoSQ-CAF CM, NecSQ-CAF CM으로 처리한 후, TUNEL 분석으로 확인한 세포 사멸 이미지 및 DAPI 염색으로 확인한 TUNEL 양성 세포 수를 나타낸 도이다. 도 6b는 A549 세포를 CAF CM, ApoA-CAF CM, NecA-CAF CM으로 처리한 후, TUNEL 분석으로 확인한 세포 사멸 이미지 및 DAPI 염색으로 확인한 TUNEL 양성 세포 수를 나타낸 도이다.Figure 6 is a diagram confirming through TUNEL analysis that CAF CM exposed to killed cancer cells promotes cell death. Figure 6a is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining after treating 344SQ cells with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM. Figure 6b is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining after treating A549 cells with CAF CM, ApoA-CAF CM, and NecA-CAF CM.
도 7은 사멸화된 암 세포에 노출된 CAF CM이 세포 사멸을 촉진함을 세포 사멸 촉진 단백질 및 항-세포 사멸 단백질에 대한 면역블랏분석으로 확인한 도이다. 도 7a는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, 세포 사멸 촉진 단백질(절단된 caspase 3, 절단된 PARP 및 Bax)의 발현 및 항-세포 사멸 단백질(Bcl-2, Mcl-1 및 Bcl-xL)의 발현을 면역블랏분석으로 나타낸 도이다. 도 7b는 A549 세포를 CAF CM, ApoA-CAF CM으로 처리한 후, 세포 사멸 촉진 단백질(절단된 caspase 3, 절단된 PARP 및 Bax)의 발현 및 항-세포 사멸 단백질(Bcl-2, Mcl-1 및 Bcl-xL)의 발현을 면역블랏분석으로 나타낸 도이다.Figure 7 is a diagram confirming that CAF CM exposed to apoptotic cancer cells promotes cell death by immunoblot analysis for pro-apoptotic proteins and anti-apoptotic proteins. Figure 7a shows the expression of pro-apoptotic proteins (cleaved caspase 3, cleaved PARP, and Bax) and anti-apoptotic proteins (Bcl-2, Mcl-1) after treating 344SQ cells with CAF CM and ApoSQ-CAF CM. and Bcl-xL) expression by immunoblot analysis. Figure 7b shows the expression of pro-apoptotic proteins (cleaved caspase 3, cleaved PARP, and Bax) and anti-apoptotic proteins (Bcl-2, Mcl-1) after treating A549 cells with CAF CM and ApoA-CAF CM. and Bcl-xL) expression by immunoblot analysis.
도 8은 CAFs에서 Notch1를 넉다운시킬 경우 항증식작용이 역전됨을 나타낸 도이다. 도 8a는 두 종류의 siRNA(#1 siNotch1 및 #2 siNotch2)로 CAFs를 형질감염시킨 뒤 면역블롯분석으로 측정한 Notch1의 발현 정도를 나타낸다. 도 8b는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, #1 siNotch1 또는 #2 siNotch2의 유무에 따른 세포 생존을 측정한 도이다. 도 8c는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, #1 siNotch1 또는 #2 siNotch2의 유무에 따른 콜로니 이미지 및 콜로니 수를 측정한 도이다.Figure 8 is a diagram showing that the anti-proliferative effect is reversed when Notch1 is knocked down in CAFs. Figure 8a shows the expression level of Notch1 measured by immunoblot analysis after transfection of CAFs with two types of siRNA (#1 siNotch1 and #2 siNotch2). Figure 8b is a diagram measuring cell survival according to the presence or absence of #1 siNotch1 or #2 siNotch2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM. Figure 8c shows colony images and colony numbers measured according to the presence or absence of #1 siNotch1 or #2 siNotch2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
도 9는 CAFs에서 Notch1를 넉다운시킬 경우 세포 증식 마커 발현 억제 효과가 역전됨을 나타낸 도이다. 도 9a는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, #1 siNotch1 또는 #2 siNotch2의 유무에 따른 Ki67 및 PCNA의 mRNA의 양을 나타낸 도이다. 도 9b는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, #1 siNotch1 또는 #2 siNotch2의 유무에 따른 Ki67 및 PCNA의 단백질의 양을 나타낸 도이다.Figure 9 is a diagram showing that the effect of suppressing cell proliferation marker expression is reversed when Notch1 is knocked down in CAFs. Figure 9a is a diagram showing the amount of Ki67 and PCNA mRNA according to the presence or absence of #1 siNotch1 or #2 siNotch2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM. Figure 9b is a diagram showing the amount of Ki67 and PCNA proteins according to the presence or absence of #1 siNotch1 or #2 siNotch2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
도 10은 CAFs에서 Notch1 신호 전달 경로를 약물로 억제할 경우 항증식작용이 역전됨을 나타낸 도이다. 도 10a는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, Notch1의 선택적 억제제인 LY3039478(10 μM) 유무에 따른 세포 생존을 측정한 도이다. 도 10b는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, Notch1의 선택적 억제제인 LY3039478(10 μM) 유무에 따른 콜로니 이미지 및 콜로니 수를 측정한 도이다.Figure 10 is a diagram showing that the anti-proliferative effect is reversed when the Notch1 signaling pathway is inhibited with a drug in CAFs. Figure 10a is a diagram measuring cell survival with or without LY3039478 (10 μM), a selective inhibitor of Notch1, after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. . Figure 10b shows colony images and colony numbers measured with or without LY3039478 (10 μM), a selective inhibitor of Notch1, after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. It is one degree.
도 11은 Notch1 신호 전달 경로를 약물로 억제할 경우 세포 증식 마커 발현 억제 효과가 역전됨을 나타낸 도이다. 도 11a는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, Notch1의 선택적 억제제인 LY3039478(10 μM) 유무에 따른 Ki67 및 PCNA의 mRNA의 양을 나타낸 도이다. 도 11b는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, Notch1의 선택적 억제제인 LY3039478(10 μM) 유무에 따른 Ki67 및 PCNA의 단백질의 양을 나타낸 도이다.Figure 11 is a diagram showing that the effect of suppressing cell proliferation marker expression is reversed when the Notch1 signaling pathway is inhibited with a drug. Figure 11a shows the amount of Ki67 and PCNA mRNA with or without LY3039478 (10 μM), a selective inhibitor of Notch1, after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. This is a diagram showing . Figure 11b is a diagram showing the amount of Ki67 and PCNA proteins in the presence or absence of LY3039478 (10 μM), a selective inhibitor of Notch1, after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
도 12는 CAFs에서 WISP-1을 넉다운시킬 경우 항증식작용이 역전됨을 나타낸 도이다. 도 12a는 두 종류의 siRNA(#1 siWISP-1 및 #2 siWISP-2)로 CAFs를 형질감염시킨 뒤 면역블롯분석으로 측정한 WISP-1의 발현 정도를 나타낸다. 도 12b는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, #1 siWISP-1 또는 #2 siWISP-2의 유무에 따른 세포 생존을 측정한 도이다. 도 12c는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, #1 siWISP-1 또는 #2 siWISP-2의 유무에 따른 콜로니 이미지 및 콜로니 수를 측정한 도이다.Figure 12 is a diagram showing that the anti-proliferative effect is reversed when WISP-1 is knocked down in CAFs. Figure 12a shows the expression level of WISP-1 measured by immunoblot analysis after transfection of CAFs with two types of siRNA (#1 siWISP-1 and #2 siWISP-2). Figure 12b is a diagram measuring cell survival according to the presence or absence of #1 siWISP-1 or #2 siWISP-2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM. Figure 12c shows colony images and colony numbers measured according to the presence or absence of #1 siWISP-1 or #2 siWISP-2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
도 13은 CAFs에서 WISP-1을 넉다운시킬 경우 세포 증식 마커 발현 억제 효과가 역전됨을 나타낸 도이다. 도 13a는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, #1 siWISP-1 또는 #2 siWISP-2의 유무에 따른 Ki67 및 PCNA의 mRNA의 양을 나타낸 도이다. 도 13b는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, #1 siWISP-1 또는 #2 siWISP-2의 유무에 따른 Ki67 및 PCNA의 단백질의 양을 나타낸 도이다.Figure 13 is a diagram showing that the effect of suppressing cell proliferation marker expression is reversed when WISP-1 is knocked down in CAFs. Figure 13a is a diagram showing the amount of Ki67 and PCNA mRNA according to the presence or absence of #1 siWISP-1 or #2 siWISP-2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM. Figure 13b is a diagram showing the amount of Ki67 and PCNA proteins according to the presence or absence of #1 siWISP-1 or #2 siWISP-2 after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
도 14는 항-WIPS-1 항체를 통해 WISP-1을 중화할 경우 항증식작용이 역전됨을 확인한 도이다. CAF CM, ApoSQ-CAF CM에 대조군(이소타입 IgG) 또는 항-WISP-1 항체 10 μg/mL를 처리한 후, 세포 생존을 비교하였다.Figure 14 is a diagram confirming that the anti-proliferative effect is reversed when WISP-1 is neutralized using an anti-WIPS-1 antibody. After treating CAF CM and ApoSQ-CAF CM with 10 μg/mL of control (isotype IgG) or anti-WISP-1 antibody, cell survival was compared.
도 15는 항-WIPS-1 항체를 통해 WISP-1을 중화할 경우 세포 증식 마커 발현 억제 효과가 역전됨을 나타낸 도이다. 도 15a는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, 대조군(이소타입 IgG) 또는 항-WISP-1 항체 10 μg/mL를 더 처리하였을 때, Ki67 및 PCNA의 mRNA의 양을 나타낸 도이다. 도 15b는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, 대조군(이소타입 IgG) 또는 항-WISP-1 항체 10 μg/mL를 더 처리하였을 때, Ki67 및 PCNA의 단백질의 양을 나타낸 도이다.Figure 15 is a diagram showing that the inhibitory effect on cell proliferation marker expression is reversed when WISP-1 is neutralized using an anti-WIPS-1 antibody. Figure 15a shows the amounts of Ki67 and PCNA mRNA when 344SQ cells were treated with CAF CM and ApoSQ-CAF CM and then further treated with 10 μg/mL of control (isotype IgG) or anti-WISP-1 antibody. It's a degree. Figure 15b shows the amount of Ki67 and PCNA proteins when 344SQ cells were treated with CAF CM and ApoSQ-CAF CM and then further treated with 10 μg/mL of control (isotype IgG) or anti-WISP-1 antibody. It's a degree.
도 16은 CAFs에서 Notch1-WISP-1 신호가 세포 사멸을 촉진함을 유세포 분석으로 확인한 도이다. 도 16a는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, siNotch1 유무에 따른 세포 사멸 정도를 Annexin V-FICT/PI 이중 염색을 이용한 유세포 분석으로 나타낸 도이다. 도 16b는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, siWISP-1 유무에 따른 세포 사멸 정도를 Annexin V-FICT/PI 이중 염색을 이용한 유세포 분석으로 나타낸 도이다.Figure 16 is a diagram confirming by flow cytometry that Notch1-WISP-1 signaling promotes cell death in CAFs. Figure 16a shows the degree of cell death according to the presence or absence of siNotch1 after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively, using Annexin V-FICT/PI double staining. It is a degree expressed as . Figure 16b shows the degree of cell death according to the presence or absence of siWISP-1 using Annexin V-FICT/PI double staining after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. This is a diagram shown by flow cytometry.
도 17은 재조합 WISP-1이 암 세포의 증식을 억제함을 확인한 도이다. 도 17a는 344SQ 세포 또는 A549 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리하였을 때 세포 생존을 나타낸 도이다. 도 17b는 344SQ 세포 또는 A549 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리하였을 때 콜로니 이미지 및 콜로니 수를 나타낸 도이다.Figure 17 is a diagram confirming that recombinant WISP-1 inhibits the proliferation of cancer cells. Figure 17a is a diagram showing cell survival when 344SQ cells or A549 cells were treated with 20, 50, or 100 ng/mL of rWISP-1. Figure 17b is a diagram showing colony images and colony numbers when 344SQ cells or A549 cells were treated with 20, 50, or 100 ng/mL of rWISP-1.
도 18은 재조합 WISP-1이 세포 증식 마커의 발현을 억제함을 확인한 도이다. 도 18a는 344SQ 세포 또는 A549 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리하였을 때 Ki67 및 PCNA의 mRNA의 양을 나타낸 도이다. 도 18b는 344SQ 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리하였을 때 Ki67 및 PCNA의 단백질의 양을 나타낸 도이다.Figure 18 is a diagram confirming that recombinant WISP-1 suppresses the expression of cell proliferation markers. Figure 18a is a diagram showing the amount of Ki67 and PCNA mRNA when 344SQ cells or A549 cells were treated with 20, 50, or 100 ng/mL of rWISP-1. Figure 18b is a diagram showing the amount of Ki67 and PCNA proteins when 344SQ cells were treated with 20, 50, or 100 ng/mL of rWISP-1.
도 19는 재조합 WISP-1이 암 세포의 세포 사멸을 촉진함을 유세포 분석으로 확인한 도이다. 도 19a는 344SQ 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리한 후, 세포 사멸 정도를 Annexin V-FICT/PI 이중 염색을 이용한 유세포 분석으로 나타낸 도이다. 도 19b는 A549 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리한 후, 세포 사멸 정도를 Annexin V-FICT/PI 이중 염색을 이용한 유세포 분석으로 나타낸 도이다.Figure 19 is a diagram confirming by flow cytometry that recombinant WISP-1 promotes apoptosis of cancer cells. Figure 19a is a diagram showing the degree of cell death by flow cytometry using Annexin V-FICT/PI double staining after treating 344SQ cells with 20, 50, or 100 ng/mL of rWISP-1. Figure 19b is a diagram showing the degree of cell death by flow cytometry using Annexin V-FICT/PI double staining after treating A549 cells with 20, 50, or 100 ng/mL of rWISP-1.
도 20은 재조합 WISP-1이 암 세포의 세포 사멸을 촉진함을 TUNEL 분석으로 확인한 도이다. 도 20a는 344SQ 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리한 후, TUNEL 분석으로 확인한 세포 사멸 이미지 및 DAPI 염색으로 확인한 TUNEL 양성 세포 수를 나타낸 도이다. 도 20b는 A549 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리한 후, TUNEL 분석으로 확인한 세포 사멸 이미지 및 DAPI 염색으로 확인한 TUNEL 양성 세포 수를 나타낸 도이다.Figure 20 is a diagram confirming through TUNEL analysis that recombinant WISP-1 promotes apoptosis of cancer cells. Figure 20a is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining after treating 344SQ cells with 20, 50, or 100 ng/mL of rWISP-1. Figure 20b is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining after treating A549 cells with 20, 50, or 100 ng/mL of rWISP-1.
도 21은 재조합 WISP-1이 암 세포의 세포 사멸을 촉진함을 세포 사멸 촉진 단백질 및 항-세포 사멸 단백질에 대한 면역블랏분석으로 확인한 도이다. 도 21a는 344SQ 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리한 후, 세포 사멸 촉진 단백질(절단된 caspase 3, 절단된 PARP 및 Bax)의 발현 및 항-세포 사멸 단백질(Bcl-2, Mcl-1 및 Bcl-xL)의 발현을 면역블랏분석으로 나타낸 도이다. 도 21b는 A549 세포에 rWISP-1을 20, 50 또는 100 ng/mL로 처리한 후, 세포 사멸 촉진 단백질(절단된 caspase 3, 절단된 PARP 및 Bax)의 발현 및 항-세포 사멸 단백질(Bcl-2, Mcl-1 및 Bcl-xL)의 발현을 면역블랏분석으로 나타낸 도이다.Figure 21 is a diagram confirming that recombinant WISP-1 promotes apoptosis of cancer cells by immunoblot analysis for pro-apoptotic protein and anti-apoptotic protein. Figure 21a shows the expression of pro-apoptotic proteins (cleaved caspase 3, cleaved PARP and Bax) and anti-apoptotic proteins (Bcl-) after treating 344SQ cells with 20, 50 or 100 ng/mL of rWISP-1. 2, This is a diagram showing the expression of Mcl-1 and Bcl-xL) by immunoblot analysis. Figure 21b shows the expression of pro-apoptotic proteins (cleaved caspase 3, cleaved PARP and Bax) and anti-apoptotic proteins (Bcl-) after treating A549 cells with 20, 50 or 100 ng/mL of rWISP-1. 2, This is a diagram showing the expression of Mcl-1 and Bcl-xL) by immunoblot analysis.
도 22는 CAFs에서 Rho kinase를 억제할 경우, 탐식 작용(efferocytosis)이 촉진됨을 확인한 도이다. Rho kinase 억제제인 Ripasudil 30 μM을 처리한 후, CAFs의 ApoSQ에 대한 식세포 작용을 유세포 분석으로 분석하였다.Figure 22 is a diagram confirming that efferocytosis is promoted when Rho kinase is inhibited in CAFs. After treatment with 30 μM of Ripasudil, a Rho kinase inhibitor, the phagocytosis of CAFs toward ApoSQ was analyzed by flow cytometry.
도 23은 CAFs에서 Rho kinase를 억제할 경우, 탐식 작용(efferocytosis)이 촉진됨을 확인한 도이다. Ripasudil 유무에 따른 CAFs의 식세포 작용을 분석하였으며, CAFs의 핵 및 액틴 세포골격은 각각 DAPI(청색) 및 TRITC-conjugated phalloidin(적색)으로 염색하였다.Figure 23 is a diagram confirming that efferocytosis is promoted when Rho kinase is inhibited in CAFs. Phagocytosis of CAFs was analyzed with and without Ripasudil, and the nucleus and actin cytoskeleton of CAFs were stained with DAPI (blue) and TRITC-conjugated phalloidin (red), respectively.
도 24는 CAFs에서 Rho kinase를 억제할 경우, Notch1 신호 단백질(NICD1, Hes1 및 WISP-1) 발현 및 WISP-1 분비가 촉진됨을 확인한 도이다. 도 24a는 ApoSQ에 노출된 CAFs에서 Ripasudil 유무에 따른 Notch1 신호 단백질(NICD1, Hes1 및 WISP-1)의 발현을 나타낸 도이다. 도 24b는 ApoSQ에 노출된 CAFs에서 Ripasudil 유무에 따른 WISP-1의 분비량을 나타낸 도이다.Figure 24 is a diagram confirming that when Rho kinase is inhibited in CAFs, Notch1 signaling protein (NICD1, Hes1, and WISP-1) expression and WISP-1 secretion are promoted. Figure 24a is a diagram showing the expression of Notch1 signaling proteins (NICD1, Hes1, and WISP-1) in CAFs exposed to ApoSQ with or without Ripasudil. Figure 24b is a diagram showing the secretion amount of WISP-1 in CAFs exposed to ApoSQ with or without Ripasudil.
도 25는 CAFs에서 RhoA를 넉다운시킬 경우 항증식작용이 강화됨을 나타낸 도이다. 도 25a는 siRhoA로 CAFs를 형질감염시킨 뒤 면역블롯분석으로 측정한 RhoA의 발현 정도를 나타낸다. 도 25b는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, siRhoA 유무에 따른 세포 생존을 나타낸 도이다. 도 25c는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, siRhoA 유무에 따른 콜로니 이미지 및 콜로니 수를 나타낸 도이다.Figure 25 is a diagram showing that the anti-proliferative effect is enhanced when RhoA is knocked down in CAFs. Figure 25a shows the expression level of RhoA measured by immunoblot analysis after transfection of CAFs with siRhoA. Figure 25b is a diagram showing cell survival according to the presence or absence of siRhoA after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. Figure 25c is a diagram showing the colony image and number of colonies according to the presence or absence of siRhoA after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively.
도 26은 CAFs에서 RhoA를 넉다운시킬 경우 세포 증식 마커 발현 억제 효과가 강화됨을 나타낸 도이다. 도 26a는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, siRhoA 유무에 따른 Ki67 및 PCNA의 mRNA의 양을 나타낸 도이다. 도 26b는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, siRhoA 유무에 따른 Ki67 및 PCNA의 단백질의 양을 나타낸 도이다.Figure 26 is a diagram showing that the effect of suppressing cell proliferation marker expression is enhanced when RhoA is knocked down in CAFs. Figure 26a is a diagram showing the amount of Ki67 and PCNA mRNA according to the presence or absence of siRhoA after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM, CAF CM, or ApoA-CAF CM, respectively. Figure 26b is a diagram showing the amount of Ki67 and PCNA proteins according to the presence or absence of siRhoA after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
도 27은 Rho kinase를 약물로 억제할 경우 항증식작용이 강화됨을 나타낸 도이다. 도 27a는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, Rho kinase 억제제인 Y-27632(30 μM) 유무에 따른 세포 생존을 측정한 도이다. 도 27b는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, Rho kinase 억제제인 Y-27632(30 μM) 유무에 따른 콜로니 이미지 및 콜로니 수를 측정한 도이다.Figure 27 is a diagram showing that the anti-proliferative effect is enhanced when Rho kinase is inhibited with a drug. Figure 27a shows cell survival measured with or without the Rho kinase inhibitor Y-27632 (30 μM) after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. am. Figure 27b shows colony images and number of colonies according to the presence or absence of Y-27632 (30 μM), a Rho kinase inhibitor, after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. It is a measured degree.
도 28은 Rho kinase를 약물로 억제할 경우 세포 증식 마커 발현 억제 효과가 강화됨을 나타낸 도이다. 도 28a는 344SQ 세포 또는 A549 세포를 각각 CAF CM, ApoSQ-CAF CM 또는 CAF CM, ApoA-CAF CM으로 처리한 후, Rho kinase 억제제인 Y-27632(30 μM) 유무에 따른 Ki67 및 PCNA의 mRNA의 양을 나타낸 도이다. 도 28b는 344SQ 세포를 CAF CM, ApoSQ-CAF CM으로 처리한 후, Rho kinase 억제제인 Y-27632(30 μM) 유무에 따른 Ki67 및 PCNA의 단백질의 양을 나타낸 도이다.Figure 28 is a diagram showing that the inhibitory effect on cell proliferation marker expression is strengthened when Rho kinase is inhibited with a drug. Figure 28a shows the mRNA levels of Ki67 and PCNA with or without the Rho kinase inhibitor Y-27632 (30 μM) after treating 344SQ cells or A549 cells with CAF CM, ApoSQ-CAF CM or CAF CM, ApoA-CAF CM, respectively. It is a degree that represents quantity. Figure 28b is a diagram showing the amount of Ki67 and PCNA proteins with or without the Rho kinase inhibitor Y-27632 (30 μM) after treating 344SQ cells with CAF CM and ApoSQ-CAF CM.
도 29는 CAFs에서 RhoA를 넉다운시킬 경우 세포 사멸이 촉진됨을 확인한 도이다. 도 29a는 344SQ 세포 또는 A549 세포에서 siRhoA 유무에 따른 세포 사멸 정도를 Annexin V-FICT/PI 이중 염색을 이용한 유세포 분석으로 나타낸 도이다. 도 29b는 344SQ 세포에서 siRhoA 유무에 따라 TUNEL 분석으로 확인한 세포 사멸 이미지 및 DAPI 염색으로 확인한 TUNEL 양성 세포 수를 나타낸 도이다.Figure 29 is a diagram confirming that cell death is promoted when RhoA is knocked down in CAFs. Figure 29a is a diagram showing the degree of cell death in 344SQ cells or A549 cells according to the presence or absence of siRhoA by flow cytometry using Annexin V-FICT/PI double staining. Figure 29b is a diagram showing the cell death image confirmed by TUNEL analysis and the number of TUNEL-positive cells confirmed by DAPI staining in 344SQ cells depending on the presence or absence of siRhoA.
도 30은 ApoSQ-CAF CM이 in vivo에서 마우스 종양의 성장을 억제함을 확인한 도이다. 도 30a는 실험을 도식화한 것으로, 344SQ를 주사한 동계 마우스에 CAF CM, 50% 희석 CAF CM, ApoSQ-CAF CM 또는 50% 희석 ApoSQ-CAF CM을 주사하여 총 4가지 그룹으로 구성하였다. 도 30b는 4가지 그룹의 마우스 체중을 나타낸 도이다. 도 30c는 4가지 그룹에서의 종양의 중량을 나타낸 도이다. 도 30d는 4가지 그룹에서의 1차 종양의 사진을 나타낸 도이다. 도 30e는 4가지 그룹에서의 종양의 부피를 나타낸 도이다.Figure 30 is a diagram confirming that ApoSQ-CAF CM inhibits the growth of mouse tumors in vivo . Figure 30a is a schematic diagram of the experiment. Syngeneic mice injected with 344SQ were injected with CAF CM, 50% diluted CAF CM, ApoSQ-CAF CM, or 50% diluted ApoSQ-CAF CM, forming a total of 4 groups. Figure 30b is a diagram showing the body weight of four groups of mice. Figure 30c is a diagram showing the weight of tumors in four groups. Figure 30D is a diagram showing photographs of primary tumors in four groups. Figure 30e is a diagram showing the volume of tumors in four groups.
도 31은 ApoSQ-CAF CM이 in vivo에서 WISP-1을 통해 마우스 종양의 성장을 억제함을 확인한 도이다. 도 31a는 실험을 도식화한 것으로, 344SQ를 주사한 동계 마우스에 CAF CM, ApoSQ-CAF CM, ApoSQ-CAF CM과 항-WISP-1 항체 또는 ApoSQ-CAF CM과 IgG를 주사하여 총 4가지 그룹으로 구성하였다. 도 31b는 4가지 그룹의 마우스 체중을 나타낸 도이다. 도 31c는 4가지 그룹에서의 종양의 중량을 나타낸 도이다. 도 31d는 4가지 그룹에서의 1차 종양의 사진을 나타낸 도이다. 도 31e는 4가지 그룹에서의 종양의 부피를 나타낸 도이다.Figure 31 is a diagram confirming that ApoSQ-CAF CM inhibits mouse tumor growth through WISP-1 in vivo . Figure 31a is a schematic diagram of the experiment. Syngeneic mice injected with 344SQ were divided into four groups by injecting CAF CM, ApoSQ-CAF CM, ApoSQ-CAF CM and anti-WISP-1 antibody, or ApoSQ-CAF CM and IgG. It was composed. Figure 31b is a diagram showing the body weight of four groups of mice. Figure 31c is a diagram showing the weight of tumors in four groups. Figure 31D is a diagram showing photographs of primary tumors in four groups. Figure 31e is a diagram showing the volume of tumors in four groups.
도 32는 rWISP-1이 in vivo에서 마우스 종양의 성장을 억제함을 확인한 도이다. 도 32a는 실험을 도식화한 것으로, 344SQ를 주사한 동계 마우스에 rWISP-1을 12.5 μg/kg 또는 25 μg/kg으로 주사하여 대조군 포함 총 3가지 그룹으로 구성하였다. 도 32b는 4가지 그룹의 마우스 체중을 나타낸 도이다. 도 32c는 4가지 그룹에서의 종양의 중량을 나타낸 도이다. 도 32d는 4가지 그룹에서의 1차 종양의 사진을 나타낸 도이다. 도 32e는 4가지 그룹에서의 종양의 부피를 나타낸 도이다.Figure 32 is a diagram confirming that rWISP-1 inhibits mouse tumor growth in vivo . Figure 32a is a schematic diagram of the experiment. rWISP-1 was injected at 12.5 μg/kg or 25 μg/kg into syngeneic mice injected with 344SQ, and a total of three groups were formed, including the control group. Figure 32b is a diagram showing the body weight of four groups of mice. Figure 32c is a diagram showing the weight of tumors in four groups. Figure 32D is a diagram showing photographs of primary tumors in four groups. Figure 32e is a diagram showing the volume of tumors in four groups.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실험예 1: 재료 및 방법Experimental Example 1: Materials and Methods
1-1. 시약1-1. reagent
LY3039478(HY-12449)는 MedChemExpress(Monmouth Junction, NJ 08852, USA)사로부터 구입하였다. 마우스 재조합 WISP-1 단백질(1680-WS)은 R&D Systems(Minneapolis, MN, USA)사로부터 구입하였다. Ripasudil(K-115)은 Selleck Chemicals(Houston, TX, USA)에서 구입하였으며, Y-27632(Y0503)는 Sigma-Aldrich(St. Louis, MO, USA)에서 구입하였다. 한편, 웨스턴 블랏팅에 사용된 항체 목록은 하기 표 1과 같다.LY3039478 (HY-12449) was purchased from MedChemExpress (Monmouth Junction, NJ 08852, USA). Mouse recombinant WISP-1 protein (1680-WS) was purchased from R&D Systems (Minneapolis, MN, USA). Ripasudil (K-115) was purchased from Selleck Chemicals (Houston, TX, USA), and Y-27632 (Y0503) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Meanwhile, the list of antibodies used in Western blotting is shown in Table 1 below.
Figure PCTKR2023003762-appb-img-000001
Figure PCTKR2023003762-appb-img-000001
1-2. 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs)의 분리 및 세포 배양1-2. Isolation and cell culture of Cancer-Associated Fibroblasts (CAFs)
Kras-돌연변이체(KrasLA1) 마우스의 폐 종양으로부터, 섬유아세포-특이적 마커인 Thy1로 자기(magnetic)-활성화된 세포 분류를 사용하여, CAFs를 분리하였다. 이어서, CAFs를 10% 소 태아 혈청(FBS), 페니실린/스트렙토마이신(100 U/100μg, Welgene), 2 mM L-글루타민(Welgene), 및 1mM sodium pyruvate(Welgene)가 보충된 알파-MEM(Welgene, Gyeongsan, Korea)에서 배양하였다. 불멸화를 위해, CAFs를 리포펙터-EXT (AptaBio, Yongin, Korea)를 사용하여 TERT 플라스미드 (pCDH-3xFLAG-TERT, Addgene 51 플라스미드 # 51631)로 안정하게 형질감염시켰다. 실험에 사용된 1차 세포는 6회 미만으로 계대배양되었다. 인간 암 세포주는 ATCC(American Type Culture Collection, Manassas, VA)로부터 얻었다. 344SQ 세포(폐선암 세포주) 및 다양한 인간 암 세포주[A549(비소세포폐암 세포주), HCT116 및 LoVo(대장암 세포주)]를 10% FBS 및 페니실린/스트렙토마이신(100U/100μg)이 보충된 RPMI 1640 (HyCloneTM, GE Healthcare, Boston, MA)에서 유지시켰다.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 cultured in alpha-MEM (Welgene) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (100 U/100 μg, Welgene), 2 mM L-glutamine (Welgene), and 1 mM sodium pyruvate (Welgene). , Gyeongsan, Korea). For immortalization, CAFs were stably transfected with TERT plasmid (pCDH-3xFLAG-TERT, Addgene 51 plasmid #51631) using Lipofactor-EXT (AptaBio, Yongin, Korea). The primary cells used in the experiment were subcultured less than 6 times. Human cancer cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA). 344SQ cells (a lung adenocarcinoma cell line) and various human cancer cell lines [A549 (a non-small cell lung cancer cell line), HCT116 and LoVo (a colon cancer cell line)] were cultured in RPMI 1640 ( HyClone ™ , GE Healthcare, Boston, MA).
1-3. 세포 사멸 유도1-3. Induction of cell death
암 상피 세포주를 15분 동안 254 nm에서 자외선 조사에 노출시킨 후, RPMI-1640(10% FBS와 함께)로 37℃ 및 5% CO2에서 2시간 동안 배양하였다. Wright-Giemsa-염색된 샘플에 광 현미경을 사용하여 핵 형태를 평가한 결과, 조사된 세포가 세포사멸되었음을 확인하였다. 수회의 동결-해동 주기에 의해 용해(괴사) 암세포를 얻었다. 세포사멸 및 괴사는 annexin V-FITC/propidium iodide(BD Biosciences, San Jose, CA) 염색에 이어서 FACSCalibur 시스템(BD Bioscience)에서 flow cytometric 분석에 의해 확인되었다.Cancer epithelial cell lines were exposed to ultraviolet irradiation at 254 nm for 15 minutes and then incubated with RPMI-1640 (with 10% FBS) at 37°C and 5% CO 2 for 2 hours. Nuclear morphology was evaluated in Wright-Giemsa-stained samples using light microscopy, and it was confirmed that the irradiated cells were apoptotic. Lytic (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) staining followed by flow cytometric analysis on a FACSCalibur system (BD Bioscience).
1-4. CAFs의 배양 및 CAFs 조정배지의 제조1-4. Culture of CAFs and preparation of CAFs conditioned medium
CAFs를 3 X 105 세포/ml로 플레이팅하고, 37℃ 및 5% CO2에서 적합한 배지로 배양시켰다. 밤새 인큐베이션한 후, 세포 자극 전에 24시간 동안 X-VIVO 10 배지 (04-380Q, Lonza)로 혈청-제거하였다. 자극을 위해, 배양 배지를 사멸화된 또는 괴사된 암 세포(9 X 105 세포/ml)를 함유하는 X-VIVO 10으로 대체하였다. 20시간 후, 상청액을 원심분리에 의해 수확하여, 표적 암 상피 세포(5 X 103 세포/ml)의 자극을 위한 조정배지(CM)로 사용하였다.CAFs were plated at 3 After overnight incubation, cells were serum-depleted with X-VIVO 10 medium (04-380Q, Lonza) for 24 hours prior to stimulation. For stimulation, the culture medium was replaced with X-VIVO 10 containing killed or necrotic cancer cells (9 After 20 hours, the supernatant was harvested by centrifugation and used as conditioned medium (CM) for stimulation of target cancer epithelial cells ( 5
1-5. 세포 생존 분석1-5. Cell survival assay
세포 증식 분석을 위해, 암 세포(5 X 103)를 RPMI-1640 배지와 함께 96-웰 플레이트(SPL, Pocheon, Korea)에 플레이팅하였다. 세포를 X-VIVO 10 배지(Lonza, Basel)에서 6시간 동안 배양시켰다. 조정배지는 각 그룹에 추가되었다. 플레이트를 인큐베이터에서 37℃ 및 5% CO2에서 1-5일간 인큐베이션하였다. 그 후, 세포 계수(計數) 키트-8(CCK-8) 용액(Dojindo Molecular Technologies, Rockville, MD)을 웰에 첨가하고, 플레이트를 인큐베이터에서 37℃ 및 5% CO2에서 30분간 인큐베이션하였다. 흡광도는 450 nm에서 마이크로플레이트 리더를 이용하여 측정하였다.For cell proliferation analysis, cancer cells ( 5 Cells were cultured in X-VIVO 10 medium (Lonza, Basel) for 6 hours. Conditioning badges were added to each group. Plates were incubated in an incubator at 37°C and 5% CO 2 for 1-5 days. Afterwards, Cell Counting Kit-8 (CCK-8) solution (Dojindo Molecular Technologies, Rockville, MD) was added to the wells, and the plate was incubated in an incubator at 37°C and 5% CO 2 for 30 minutes. Absorbance was measured using a microplate reader at 450 nm.
1-6. 콜로니 형성 분석1-6. Colony formation assay
암 세포를 6-웰 플레이트(SPL, Pocheon, Korea)에 X-VIVO 10(대조군)과 조정배지와 함께 웰 당 500개씩 플레이팅하였다. 플레이트를 37℃ 및 5% CO2에서 8일 혹은 9일동안 인큐베이션하였다. 배지는 4일마다 한 번씩 교체하였다. 형성된 콜로니를 95% 에탄올과 함께 4℃에서 밤새 고정시키고 0.1% crystal violet(Sigma-Aldrich)으로 염색한 뒤 20% 메탄올로 상온에서 밤새 희석하였다. 플레이트를 세정한 뒤, 50개 이상의 세포를 포함하는 눈에 보이는 콜로니를 입체현미경으로 5개의 구역에서 계수하였다. 대표적인 콜로니를 촬영하였고 3번의 독립적인 실험을 수행하였다.Cancer cells were plated at 500 per well in a 6-well plate (SPL, Pocheon, Korea) along with X-VIVO 10 (control) and conditioned medium. Plates were incubated at 37°C and 5% CO 2 for 8 or 9 days. The medium was changed once every 4 days. The formed colonies were fixed with 95% ethanol at 4°C overnight, stained with 0.1% crystal violet (Sigma-Aldrich), and then diluted with 20% methanol at room temperature overnight. After washing the plate, visible colonies containing more than 50 cells were counted in five sections under a stereomicroscope. Representative colonies were photographed and three independent experiments were performed.
1-7. 세포 사멸 분석1-7. Cell death assay
세포 사멸은 annexin V-FITC/propidium iodide(PI) 염색 키트(BD Biosciences, San Jose, CA, USA)를 사용하여 제조업체의 지침에 따라 검출하였다. 폐암 세포는 CM 또는 rWISP-1으로 처리한 후, 1 내지 3일 후 수확하였고, 150 mL의 결합 버퍼에 재현탁하였으며, 5 μL FITC-conjugated Annexin V 및 5 μL PI로 암실에서 15분간 실온에서 염색하였다. 이후, FITC-conjugated Annexin V에 양성인 세포를 유세포 측정기(ACEA NovoCyte, San Diego, CA, USA)로 검출하였고, 데이터는 NovoExpress 소프트웨어 1.5를 사용하여 분석하였다.Cell death was detected using annexin V-FITC/propidium iodide (PI) staining kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Lung cancer cells were harvested 1 to 3 days after treatment with CM or rWISP-1, resuspended in 150 mL of binding buffer, and stained with 5 μL FITC-conjugated Annexin V and 5 μL PI for 15 minutes at room temperature in the dark. did. Afterwards, cells positive for FITC-conjugated Annexin V were detected by flow cytometry (ACEA NovoCyte, San Diego, CA, USA), and data were analyzed using NovoExpress software 1.5.
또한, 폐암 세포를 TUNEL 키트(Roche, Basel, Switzerland)를 사용하여 제조업체의 지침에 따라 염색하였다. 사멸된 세포는 488 및 543 nm에서 여기(excitation)되는 필터 세트가 장착된 공초점 현미경(LSM5 PASCAL; Carl Zeiss, Jena, Germany)으로 관찰하였다. TUNEL 양성 세포의 정량화는 맹검 방식(blinded fashion)으로 슬라이드당 무작위로 선택된 10개의 HPF(high-power fields)당 TUNEL 양성 세포의 수를 수동으로 계수하여 수행하였다.Additionally, lung cancer cells were stained using the TUNEL kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Killed cells were observed under a confocal microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany) equipped with a filter set excitation at 488 and 543 nm. Quantification of TUNEL-positive cells was performed in a blinded fashion by manually counting the number of TUNEL-positive cells per 10 randomly selected high-power fields (HPFs) per slide.
1-8. 면역블랏팅 분석1-8. Immunoblotting analysis
전체 세포 추출물을 사용하여 표준 웨스턴 블랏을 수행하였으며, 사용한 항체의 정보는 상기 표 1과 같다. 전체 세포 추출물은 세포 사멸 세포 또는 CM에 노출된 CAFs 또는 암 세포로부터 추출하였다. 세포를 수확하고, 차가운 PBS(phosphate-buffered saline)로 세척한 후, 프로테아제 억제제가 첨가된 방사면역침전(radioimmunoprecipitation) 분석 버퍼[10 mM Tris(pH 7.2), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1.0% Triton X-100 및 5 mM EDTA]로 30분간 얼음 위에서 용해시켰다. 이후, 동일한 양의 단백질을 SDS-PAGE 젤(#161-0158, Bio-Rad Laboratories, Hercules, CA, USA)에 용해시킨 후, wet 이송 시스템(Bio-Rad Laboratories)을 사용하여 니트로셀룰로스 막(10600001, GE Healthcare Life Science, Piscataway, NJ, USA)으로 전송하였다. 막을 5% 소 혈청 알부민(BSA)-TBST 또는 5% 우유-TBST로 1시간 동안 차단한 후, 표지된 1차 항체와 밤새 배양한 다음 표지된 2차 항체와 함께 37℃에서 1시간 동안 배양하였다. 정량화를 위해 Odyssey 이미지 분석 시스템(Licor Biosciences, Lincoln, NE, USA)을 사용하였다.Standard Western blotting was performed using whole cell extracts, and information on the antibodies used is shown in Table 1 above. Whole cell extracts were extracted from apoptotic cells or CAFs or cancer cells exposed to CM. Cells were harvested, washed with cold phosphate-buffered saline (PBS), and incubated in radioimmunoprecipitation assay buffer [10 mM Tris (pH 7.2), 150 mM NaCl, 1% Nonidet P-40 supplemented with protease inhibitors. , 0.5% sodium deoxycholate, 0.1% SDS, 1.0% Triton Afterwards, the same amount of protein was dissolved in an SDS-PAGE gel (#161-0158, Bio-Rad Laboratories, Hercules, CA, USA) and then transferred to a nitrocellulose membrane (10600001) using a wet transfer system (Bio-Rad Laboratories). , GE Healthcare Life Science, Piscataway, NJ, USA). The membrane was blocked with 5% bovine serum albumin (BSA)-TBST or 5% milk-TBST for 1 hour, incubated with labeled primary antibody overnight, and then incubated with labeled secondary antibody for 1 hour at 37°C. . The Odyssey image analysis system (Licor Biosciences, Lincoln, NE, USA) was used for quantification.
1-9. 정량적 실시간 중합효소 연쇄 반응(qRT-PCR)1-9. Quantitative real-time polymerase chain reaction (qRT-PCR)
총 RNA는 암세포로부터 RNAiso Plus reagent(Takara, Tokyo)를 이용해 추출하였고, cDNA는 AccuPower RT PreMix(Bioneer, Daejeon, Korea)를 이용해 제조자의 프로토콜에 따라 합성하였다. qRT-PCR은 StepOnePlus system with TB Green Premix Ex Taq II(Takara)를 통해 수행하였다. mRNA 수준은 Hprt mRNA 수준으로 정규화하였고, 대조군과 비교한 발현의 배수 변화로 나타내었다. 표적 유전자의 증폭에 사용한 프라이머 서열은 하기 표 2와 같다.Total RNA was extracted from cancer cells using RNAiso Plus reagent (Takara, Tokyo), and cDNA was synthesized using AccuPower RT PreMix (Bioneer, Daejeon, Korea) according to the manufacturer's protocol. qRT-PCR was performed using the StepOnePlus system with TB Green Premix Ex Taq II (Takara). mRNA levels were normalized to Hprt mRNA levels and expressed as fold change in expression compared to control. Primer sequences used for amplification of target genes are shown in Table 2 below.
ForwardForward ReverseReverse
HPRTHPRT CCAGTGTCAATTATATCTTCAAC
(서열번호 1)CCAGTGTCAATTATATCTTCAAC
(SEQ ID NO: 1) 		CAGACTGAAGAGCTACTGTAATG
(서열번호 2)CAGACTGAAGAGCTACTGTAATG
(SEQ ID NO: 2)
Ki67Ki67 MouseMouse AATCCAACTCAAGTAAACGGGG
(서열번호 3)AATCCAACTCAAGTAAACGGGG
(SEQ ID NO: 3) 		TTGGCTTGCTTCCATCCTCA
(서열번호 4)TTGGCTTGCTTCCATCCTCA
(SEQ ID NO: 4)
HumanHuman AAGCCCTCCAGCTCCTAGTC
(서열번호 5)AAGCCCTCCAGCTCCTAGTC
(SEQ ID NO: 5) 		GCAGGTTGCCACTCTTTCTC
(서열번호 6)GCAGGTTGCCACTCTTTCTC
(SEQ ID NO: 6)
PCNAPCNA MouseMouse GGCGTGAACCTCACCAGTAT(서열번호 7)GGCGTGAACCTCACCAGTAT (SEQ ID NO: 7) TTCTCCTGGTTTGGTGCTTC
(서열번호 8)TTCTCCTGGTTTGGTGCTTC
(SEQ ID NO: 8)
HumanHuman CCTGCTGGGATATTAGCTCCA(서열번호 9)CCTGCTGGATATTAGCTCCA (SEQ ID NO: 9) CAGCGGTAGGTGTCGAAGC
(서열번호 10)CAGCGGTAGGTGTCGAAGC
(SEQ ID NO: 10)
1-10. 일시적 형질감염1-10. Transient transfection
CAFs를 Notch1을 특이적으로 타겟팅하는 두 종류의 siRNA(#1 Bioneer Inc, Daejeon, Korea; #2 Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), WISP-1(WNT1-inducible-signaling pathway protein 1)을 특이적으로 타겟팅하는 두 종류의 siRNA(#1 Bioneer Inc, Daejeon, Korea; #2 Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), RhoA을 특이적으로 타겟팅하는 siRNA (Bioneer Inc, Daejeon, Korea) 또는 대조군 siRNA(SN-1003 AccuTargetTM Negative Control; Bioneer)와 함께 50 nM의 최종 농도의 형질감염 시약(리포펙타민 RNAi MAX; Invitrogen)을 사용하여 제조자의 지시에 따라 일시적으로 형질감염시켰다. 밤새 형질감염 후, 세포를 적합한 배지에서 24시간 동안 배양하고 ApoSQ 또는 ApoA 세포로 자극하였다. 유전자를 타겟팅하는데 사용된 siRNA 서열은 하기 표 3과 같다.Two types of siRNA targeting CAFs specifically for Notch1 (#1 Bioneer Inc, Daejeon, Korea; #2 Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), WISP-1 (WNT1-inducible-signaling pathway protein 1) ), two types of siRNA specifically targeting RhoA (#1 Bioneer Inc, Daejeon, Korea; #2 Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), and siRNA specifically targeting RhoA (Bioneer Inc, Daejeon, Korea) or control siRNA (SN-1003 AccuTarget TM Negative Control; Bioneer) using transfection reagent (Lipofectamine RNAi MAX; Invitrogen) at a final concentration of 50 nM according to the manufacturer's instructions. After transfection overnight, cells were cultured in appropriate medium for 24 h and stimulated with ApoSQ or ApoA cells. The siRNA sequences used to target genes are shown in Table 3 below.
SenseSense Antisense Antisense
Notch1 #1Notch1#1 5'-CCCUUUGAGUCUUCAUACA-3'
(서열번호 11)5'-CCCUUUGAGUCUUCAUACA-3'
(SEQ ID NO: 11) 		5'-UGUAUGAAGACUCAAAGGG-3'
(서열번호 12)5'-UGUAUGAAGACUCAAAGGG-3'
(SEQ ID NO: 12)
Notch1 #2 Notch1#2 5'-GACGACAAUCAGAACGAG-3'(서열번호 13)5'-GACGACAAUCAGAACGAG-3' (SEQ ID NO: 13) 5'-AUGCGUGAUUGUCGU-3'
(서열번호 14)5'-AUGCGUGAUUGUCGU-3'
(SEQ ID NO: 14)
WISP-1 #1WISP-1 #1 5'-GGAAUCCUAACGAUAUCUU-3'(서열번호 15)5'-GGAAUCCUAACGAUAUCUU-3' (SEQ ID NO: 15) 5'-AAGAUAUCGUUAGGAUUCC-3'
(서열번호 16)5'-AAGAUAUCGUUAGGAUUCC-3'
(SEQ ID NO: 16)
WISP-1 #2WISP-1 #2 5'-GTACCTCAGAACGUGGACUATT-3'(서열번호 17)5'-GTACCTCAGAACGUGGACUATT-3' (SEQ ID NO: 17) 5'-UAUCTCACCUCUGAAGACCTT-3'
(서열번호 18)5'-UAUCTCACCUCUGAAGACCTT-3'
(SEQ ID NO: 18)
RhoARhoA 5'-GUCUCAUGUUAGUUACCUU-3'(서열번호 19)5'-GUCUCAUGUUAGUUACCUU-3' (SEQ ID NO: 19) 5'-AAGGUAAUCAACAUGAGAC-3'
(서열번호 20)5'-AAGGUAAUCAACAUGAGAC-3'
(SEQ ID NO: 20)
1-11. 조정배지에서의 WISP-1 중화1-11. WISP-1 neutralization in conditioned media
CAFs로부터의 조정배지를 마우스의 항-마우스 WISP-1 중화 항체(R&D Systems) 10 μg/ml 또는 IgG 이소타입 대조군(R&D Systems) 10 μg/ml과 함께 2시간 동안 인큐베이션하였다. 항-WISP-1 항체 중화 효과는 사용 전 WISP-1 ELISA를 이용해 검사하였다.Conditioned media from CAFs were incubated with 10 μg/ml of mouse anti-mouse WISP-1 neutralizing antibody (R&D Systems) or 10 μg/ml of IgG isotype control (R&D Systems) for 2 hours. The neutralizing effect of anti-WISP-1 antibodies was tested using WISP-1 ELISA before use.
1-12. 식세포 작용(phagocytosis) 분석1-12. Phagocytosis analysis
사멸화된 암 세포의 식세포 작용은 유세포 분석 및 면역형광분석법을 이용하여 평가하였다. CAFs를 PKH67(녹색)로 표지된 사멸화된 344SQ 세포와 1:3 비율로 24시간 동안 공동배양하기 전에, PKH26(적색)으로 염색하였고, 식세포 작용의 비율은 2색 유세포 분석으로 평가하였다. 또한, 면역형광분석을 위해 PKH67로 표지된 사멸화된 344SQ 세포를 CAFs와 24시간 동안 공동배양하였다. CAFs를 세척한 후, 3.7% w/v 파라포름알데히드로 고정하였고, 투과성을 위해 15분 동안 0.1% Triton X-100으로 처리하였다. F-액틴은 로다민 팔로이딘(rhodamine phalloidin; Invitrogen)으로 제조업체에 지침에 따라 염색하였다. 이미지는 공초점 현미경(LSM5 PASCAL; Carl Zeiss, Jena, Germany)으로 획득하였으며, 식세포 작용 지수는 (세포 사멸 수/총 200개의 CAFs) X 100 의 수학식으로 계산하였다.Phagocytosis of killed cancer cells was evaluated using flow cytometry and immunofluorescence analysis. CAFs were stained with PKH26 (red) before being co-cultured with killed 344SQ cells labeled with PKH67 (green) at a 1:3 ratio for 24 h, and the rate of phagocytosis was assessed by two-color flow cytometry. Additionally, for immunofluorescence analysis, killed 344SQ cells labeled with PKH67 were co-cultured with CAFs for 24 hours. After washing the CAFs, they were fixed with 3.7% w/v paraformaldehyde and treated with 0.1% Triton X-100 for 15 minutes for permeabilization. F-actin was stained with rhodamine phalloidin (Invitrogen) according to the manufacturer's instructions. Images were acquired with a confocal microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany), and the phagocytosis index was calculated using the equation (number of cell deaths/total 200 CAFs) × 100.
1-13. 마우스 실험1-13. mouse experiment
Ewha Medical Research Institute의 동물 케어 위원회로부터 하기 실험 프로토콜을 승인받았다(EUM19-0437). 마우스는 연구소 동물의 관리 및 사용을 위한 국립 보건원(National Institute of Health; NIH) 가이드에 따라 관리 및 취급되었다. 동계(syngeneic) 종양 실험을 이용한 폐암 전이 연구를 공지의 방법으로 수행하였다. 요약하면, 344SQ 세포(마우스 당 PBS 100 μl 중 1x106 세포)를 우측 후방 측면에서 동계(syngeneic)(129/Sv) 마우스 내로 피하 주사하였다. The following experimental protocol was approved by the Animal Care Committee of Ewha Medical Research Institute (EUM19-0437). Mice were cared for and handled in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Lung cancer metastasis research using syngeneic tumor experiments was performed using a known method. Briefly, 344SQ cells (1x10 6 cells in 100 μl of PBS per mouse) were injected subcutaneously into syngeneic (129/Sv) mice in the right posterior aspect.
조정배지 실험을 위해, 2일 후에 시작하여, 중화 항-WISP-1 항체 또는 이소타입 IgG의 존재 또는 부재하에 CAFs로부터 유래된 조정 배지(마우스당 100 μl; 무혈청 배지로 희석하지 않거나 50% 희석)를 1주 3회 종양내 주사를 통해 투여하였다. 또한, rWISP-1(12.5 μg/kg 또는 25 μg/kg)을 344SQ 주사 2일 후, 1주 3회 종양내 주사를 통해 투여하였다. 마우스의 종양 성장을 매일 모니터링하고, 주사 6주 후에 희생시켰다. 이후, 부검을 수행하여, 피하 종양 덩어리의 직경 및 중량을 조사하였다. 모든 동물 실험 및 연구는 연령이 동일한 수컷 마우스로 수행하였다.For conditioned medium experiments, starting 2 days later, conditioned medium derived from CAFs (100 μl per mouse; undiluted or 50% diluted with serum-free medium) in the presence or absence of neutralizing anti-WISP-1 antibody or isotype IgG. ) was administered through intratumoral injection three times a week. Additionally, rWISP-1 (12.5 μg/kg or 25 μg/kg) was administered through intratumoral injection three times a week, 2 days after 344SQ injection. Mice were monitored daily for tumor growth and sacrificed 6 weeks after injection. An autopsy was then performed to determine the diameter and weight of the subcutaneous tumor mass. All animal experiments and studies were performed with male mice of the same age.
1-14. 종양 부피1-14. tumor volume
종양의 부피는 (L x W x W)/2의 방식으로 계산하였으며, L은 종양의 긴 부분이고 W는 종양의 짧은 부분을 의미한다.The tumor volume was calculated using the formula (L x W x W)/2, where L is the long part of the tumor and W is the short part of the tumor.
1-15. 통계1-15. statistics
짝(pairwise) 비교는 two-tailed Student's t-테스트를 사용하여 수행하였고, 다중(multiple) 비교는 Kruskal-Wallis 테스트에 이어 Dunn's post hoc 테스트를 사용하여 수행하였다. 0.05 이하의 P 값은 통계적으로 유의한 것으로 간주하였고, 모든 데이터는 Prism 5 소프트웨어(GraphPad Software Inc., San Diego, CA, USA)를 사용하여 분석하였다.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 of 0.05 or less was considered statistically significant, and all data were analyzed using Prism 5 software (GraphPad Software Inc., San Diego, CA, USA).
실험예 2: 실험결과Experimental Example 2: Experimental results
2-1. CAFs와 사멸화된 암 세포의 상호작용이 암 세포 증식을 억제함을 확인2-1. Confirmed that the interaction between CAFs and killed cancer cells inhibits cancer cell proliferation
CAFs는 종양 생성, 성장 및 전이에 관련된 여러 요소를 분비하는 것으로 알려져 있기 때문에, 본 실험에서는 UV-조사되어 사멸화된 344SQ 암 세포와 CAFs를 공동 배양할 때, 생물활성 매개체 분비를 통하여 폐암 세포 성장이 억제되는지 여부를 조사하였다.Since CAFs are known to secrete various factors related to tumor creation, growth, and metastasis, in this experiment, when CAFs were co-cultured with UV-irradiated killed 344SQ cancer cells, lung cancer cells grew through secretion of bioactive mediators. It was investigated whether this was suppressed.
섬유아세포-특이적 마커인 Thy1을 사용하여 Kras-돌연변이체(KrasLA1) 마우스의 폐 종양으로부터 CAFs를 분리한 후, 사멸화된 344SQ 세포주(ApoSQ) 또는 괴사된 344SQ 세포주(NecSQ)로 처리하였으며, 이때 상기 세포주는 20시간 동안 KrasLA1 및 53R172H 대립유전자를 동시발현하는 마우스로부터 유래된 고침윤성 및 전이성 폐 선암종(adenocarcinoma) 세포주를 이용하였다.CAFs were isolated from lung tumors of Kras-mutant (Kras LA1 ) mice using the fibroblast-specific marker Thy1 and then treated with the killed 344SQ cell line (ApoSQ) or necrotic 344SQ cell line (NecSQ). At this time, the highly invasive and metastatic lung adenocarcinoma cell line derived from mice co-expressing the Kras LA1 and 53 R172H alleles for 20 hours was used.
344SQ 세포는 CAFs와 사멸화된 344SQ 공동 배양 조정배지(ApoSQ-CAF CM) 또는 CAFs와 괴사된 344SQ 공동 배양 조정배지(NecSQ-CAF CM)로 1 내지 5일간 처리하였다. 세포 증식 분석은 세포 계수 키트-8(Dojindo)을 사용하여 진행하였다.344SQ cells were treated with 344SQ co-culture conditioned medium killed with CAFs (ApoSQ-CAF CM) or 344SQ co-culture conditioned medium with CAFs killed (NecSQ-CAF CM) for 1 to 5 days. Cell proliferation analysis was performed using Cell Counting Kit-8 (Dojindo).
그 결과, ApoSQ-CAF CM이 3 내지 5일에서 암 세포의 생존을 상당히 억제한 반면, 대조군(CAF CM) 및 NecSQ-CAF CM은 그렇지 않았음을 확인하였다(도 1a). 또한, 비소세포 폐암 세포주 A549 및 대장암 세포주 HCT116 및 LoVo와 같은 다른 유형의 인간 암 세포주에서도 CAFs와 사멸화된 암 세포의 공동 배양 조정배지(ApoA-CAF CM, ApoH-CAF CM 및 ApoL-CAF CM)의 항증식효과를 확인할 수 있었다(도 1b 내지 1d). 또한, 사멸화된 암 세포 또는 괴사된 암 세포 단독의 CM(ApoSQ CM, NecSQ CM, ApoA CM, NecA CM, ApoH CM, NecH CM, ApoL CM 및 NecL CM)은 암 세포의 성장에 영향을 주지 못함을 확인할 수 있었다(도 2a 내지 2d).As a result, it was confirmed that ApoSQ-CAF CM significantly inhibited the survival of cancer cells from 3 to 5 days, while the control group (CAF CM) and NecSQ-CAF CM did not (Figure 1a). In addition, other types of human cancer cell lines, such as non-small cell lung cancer cell line A549 and colon cancer cell lines HCT116 and LoVo, were also cultured in conditioned media for co-culture of CAFs and killed cancer cells (ApoA-CAF CM, ApoH-CAF CM, and ApoL-CAF CM). ) was confirmed to have an anti-proliferative effect (Figures 1b to 1d). Additionally, CM of apoptotic or necrotic cancer cells alone (ApoSQ CM, NecSQ CM, ApoA CM, NecA CM, ApoH CM, NecH CM, ApoL CM, and NecL CM) did not affect the growth of cancer cells. could be confirmed (Figures 2a to 2d).
CAFs 조정배지의 암 세포 성장에 대한 효과를 보다 상세하게 규명하기 위해 콜로니 형성을 조사하였다. 예상된 바와 같이, ApoSQ-CAF CM 및 ApoA-CAF CM은 344SQ 및 A549 폐암 세포의 콜로니 형성을 상당히 억제한 반면, CAF CM, NecSQ-CAF CM 및 NecA-CAF CM은 그렇지 않았음을 확인할 수 있었다(도 3a 및 3b).To further investigate the effect of CAFs-conditioned medium on cancer cell growth, colony formation was examined. As expected, ApoSQ-CAF CM and ApoA-CAF CM significantly inhibited colony formation of 344SQ and A549 lung cancer cells, whereas CAF CM, NecSQ-CAF CM, and NecA-CAF CM did not ( Figures 3a and 3b).
또한, ApoSQ-CAF CM 및 ApoA-CAF CM은 Ki67 및 PCNA와 같은 세포 증식 마커의 mRNA 및/또는 단백질의 발현 수준을 상당히 억제하는 것을 확인할 수 있었다(도 4a 내지 4c). 그러나, CAF CM, NecSQ-CAF CM 및 NecA-CAF CM은 세포 증식 마커에 대한 mRNA 및/또는 단백질의 발현 억제 효과가 없음을 확인하였다.In addition, it was confirmed that ApoSQ-CAF CM and ApoA-CAF CM significantly suppressed the mRNA and/or protein expression levels of cell proliferation markers such as Ki67 and PCNA (Figures 4a to 4c). However, it was confirmed that CAF CM, NecSQ-CAF CM, and NecA-CAF CM had no effect of suppressing the expression of mRNA and/or protein on cell proliferation markers.
상기 실험 결과들을 통해, CAFs와 사멸화된 암 세포의 상호작용이 주변분비(paracrine) 작용을 통해 암 세포의 증식을 억제함을 입증하였다.Through the above experimental results, it was demonstrated that the interaction between CAFs and killed cancer cells inhibits the proliferation of cancer cells through paracrine action.
2-2. CAFs와 사멸화된 암 세포의 상호작용이 암 세포의 세포 사멸을 촉진함을 확인2-2. Confirmed that the interaction between CAFs and killed cancer cells promotes apoptosis of cancer cells
상기 실시예 2-1에서 확인한 CAFs와 사멸화된 암 세포의 공동 배양 조정배지의 암 세포 증식 억제 효과가 암 세포 사멸의 촉진으로 인한 것인지 확인하기 위하여, Annexin V/PI 이중 염색 분석, TUNEL 분석 및 웨스턴 블랏팅 분석을 수행하여 암 세포의 세포 사멸 정도를 평가하였다.In order to confirm whether the cancer cell proliferation inhibitory effect of the co-culture conditioned medium of CAFs and killed cancer cells identified in Example 2-1 is due to the promotion of cancer cell death, Annexin V/PI double staining analysis, TUNEL analysis, and Western blotting analysis was performed to evaluate the degree of apoptosis of cancer cells.
유세포 분석 결과, 폐암 세포를 CAFs와 사멸화된 암 세포의 공동 배양 조정배지(ApoSQ-CAF CM 및 ApoA-CAF CM)로 처리할 경우, 대조군(CAF CM) 또는 CAFs와 괴사된 암 세포의 공동 배양 조정배지(NecSQ-CAF CM 및 NecA-CM)로 처리할 경우보다 1 내지 3일에 폐암 세포의 세포 사멸이 증가하는 것으로 나타났다(도 5a 및 5b).Flow cytometry results showed that when lung cancer cells were treated with conditioned media for co-culture of CAFs and dead cancer cells (ApoSQ-CAF CM and ApoA-CAF CM), the control medium (CAF CM) or co-culture of CAFs and necrotic cancer cells Cell death of lung cancer cells was found to increase on days 1 to 3 compared to treatment with conditioned medium (NecSQ-CAF CM and NecA-CM) (Figures 5a and 5b).
또한, TUNEL 분석 결과, ApoSQ-CAF CM 및 ApoA-CAF CM은 처리 2일 후 TUNEL 양성 세포의 수가 증가하는 것으로 나타났으나, CAF CM, NecSQ-CM 및 NecA-CM의 경우 아무런 효과가 나타나지 않았다(도 6a 및 6b).Additionally, as a result of TUNEL analysis, ApoSQ-CAF CM and ApoA-CAF CM showed an increase in the number of TUNEL-positive cells after 2 days of treatment, but no effect was observed for CAF CM, NecSQ-CM, and NecA-CM ( Figures 6a and 6b).
암 세포의 세포 사멸 정도에 대한 추가적인 분석을 위해, 웨스턴 블랏팅을 수행하여 344SQ 및 A549 세포에서 세포 사멸 관련 단백질(활성화된 caspase 3 및 PARP, 및 Bcl-2 패밀리)의 발현 수준을 측정하였다. 그 결과, 폐암 세포를 ApoSQ-CAF CM 또는 ApoA-CAF CM으로 처리할 경우, CAF CM으로 처리할 경우보다 절단된 caspase 3, 절단된 PARP 및 Bax의 단백질 발현 수준이 증가하나, 항-세포 사멸 단백질인 Bcl-2, Mcl-1 및 Bcl-xL의 발현 수준은 감소함을 확인하였다(도 7a 및 7b).For further analysis of the extent of apoptosis in cancer cells, Western blotting was performed to measure the expression levels of apoptosis-related proteins (activated caspase 3 and PARP, and Bcl-2 family) in 344SQ and A549 cells. As a result, when lung cancer cells were treated with ApoSQ-CAF CM or ApoA-CAF CM, the protein expression levels of cleaved caspase 3, cleaved PARP, and Bax increased compared to when treated with CAF CM, but anti-apoptotic proteins The expression levels of Bcl-2, Mcl-1, and Bcl-xL were confirmed to be decreased (Figures 7a and 7b).
상기 실험 결과들은, CAFs와 사멸화된 암 세포의 공동 배양 조정배지가 세포 사멸을 촉진하여 암 세포의 증식을 억제함을 의미한다.The above experimental results indicate that the co-culture conditioned medium of CAFs and killed cancer cells promotes cell death and inhibits the proliferation of cancer cells.
2-3. NOTCH1/WISP-1 신호의 암 세포에 대한 항증식효과 및 세포 사멸 촉진 효과 확인2-3. Confirmation of anti-proliferative and cell death-promoting effects of NOTCH1/WISP-1 signaling on cancer cells
본 발명자들의 이전 연구 결과로 WISP-1(WNT1-inducible-signaling pathway protein 1)이 Notch1 신호를 통해 사멸화된 암 세포와 공동 배양된 CAFs에서 분비된다는 것이 증명되었기 때문에, 본 실험에서는 Notch1-WISP-1 신호 체계가 암 세포의 항증식효과 및 세포 사멸 촉진 효과에 중요한 역할을 하는지 여부를 조사하였다.Since our previous research results demonstrated that WISP-1 (WNT1-inducible-signaling pathway protein 1) is secreted from CAFs co-cultured with killed cancer cells through Notch1 signaling, in this experiment Notch1-WISP- 1 We investigated whether the signaling system plays an important role in the anti-proliferative and apoptosis-promoting effects of cancer cells.
우선, Notch1에 대한 두 종류의 siRNA를 설계하여 Notch1의 발현을 침묵시킨 결과, ApoSQ-CAF CM의 344SQ 세포 생존 억제 효과 및 콜로니 형성 억제 효과가 역전되었다(도 8a 내지 8c). 또한, CAFs에서 siRNA를 이용하여 Notch1을 넉다운시킬 경우, ApoSQ-CAF CM에 의한 344SQ 세포에서의 Ki67 및 PCNA의 mRNA 및 단백질 발현 감소 효과 역시 역전됨을 확인하였다(도 9a 및 9b).First, two types of siRNA against Notch1 were designed to silence the expression of Notch1, and the inhibitory effects of ApoSQ-CAF CM on 344SQ cell survival and colony formation were reversed (Figures 8a to 8c). In addition, when Notch1 was knocked down in CAFs using siRNA, the effect of reducing the mRNA and protein expression of Ki67 and PCNA in 344SQ cells by ApoSQ-CAF CM was also confirmed to be reversed (Figures 9a and 9b).
또한, CAFs를 ApoSQ 또는 ApoA에 노출시키기 전에, Notch1의 선택적 억제제인 LY3039478(10 μM)로 전처리한 경우에도, ApoSQ-CAF CM 또는 ApoA-CAF CM의 344SQ 세포 또는 A549 세포 생존 억제 효과 및 콜로니 형성 억제 효과가 각각 역전되었고(도 10a 및 10b), 344SQ 세포 또는 A549 세포에서의 Ki67 및 PCNA의 mRNA 및/또는 단백질 발현 감소 효과 역시 역전됨을 확인하였다(도 11a 및 11b).In addition, even when CAFs were pretreated with LY3039478 (10 μM), a selective inhibitor of Notch1, before exposure to ApoSQ or ApoA, the inhibitory effect of ApoSQ-CAF CM or ApoA-CAF CM on 344SQ cell or A549 cell survival and inhibition of colony formation The effect was reversed (FIGS. 10A and 10B), respectively, and the effect of reducing mRNA and/or protein expression of Ki67 and PCNA in 344SQ cells or A549 cells was also confirmed to be reversed (FIGS. 11A and 11B).
Notch1에 대한 상기 실험과 유사하게, WISP-1에 대해서도 두 종류의 siRNA를 설계하여 CAFs에서 WISP-1을 넉다운시킨 결과, ApoSQ-CAF CM의 344SQ 세포 생존 억제 효과 및 콜로니 형성 억제 효과가 역전되었고(도 12a 내지 12c), ApoSQ-CAF CM에 의한 344SQ 세포에서의 Ki67 및 PCNA의 mRNA 및 단백질 발현 감소 효과 역시 역전되었다(도 13a 및 13b).Similar to the above experiment for Notch1, two types of siRNAs were designed for WISP-1 to knock down WISP-1 in CAFs, and the inhibitory effects of ApoSQ-CAF CM on 344SQ cell survival and colony formation were reversed ( 12A to 12C), the effect of reducing the mRNA and protein expression of Ki67 and PCNA in 344SQ cells by ApoSQ-CAF CM was also reversed (FIGS. 13A and 13B).
또한, ApoSQ-CAF CM을 중화 항-WISP-1 항체의 존재 하에 344SQ 세포에 처리할 경우에도, 344SQ 세포 생존 억제 효과가 역전되었고(도 14), Ki67 및 PCNA의 mRNA 및 단백질 발현 감소 효과 역시 역전되었다(도 15a 및 15b).In addition, when ApoSQ-CAF CM was treated with 344SQ cells in the presence of neutralizing anti-WISP-1 antibody, the inhibitory effect on 344SQ cell survival was reversed (Figure 14), and the effect of reducing mRNA and protein expression of Ki67 and PCNA was also reversed. (Figures 15a and 15b).
추가적으로, CAFs와 사멸화된 암 세포의 공동 배양 조정배지의 세포 사멸 촉진 효과에 Nothc1-WISP-1 신호 전달이 필요한지 여부를 확인하기 위해 유세포 분석을 수행하였다. 그 결과, siRNA로 Notch1 및 WISP-1을 넉다운시킬 경우, ApoSQ-CAF CM 또는 ApoA-CAF CM의 344SQ 세포 또는 A549 세포에서의 세포 사멸 촉진 효과가 역전되는 것으로 확인되었다(도 16a 및 16b).Additionally, flow cytometric analysis was performed to determine whether Nothc1-WISP-1 signaling is required for the apoptosis-promoting effect of the co-culture conditioned medium of CAFs and apoptotic cancer cells. As a result, it was confirmed that when Notch1 and WISP-1 were knocked down with siRNA, the apoptosis-promoting effect of ApoSQ-CAF CM or ApoA-CAF CM in 344SQ cells or A549 cells was reversed (Figures 16a and 16b).
또한, WISP-1이 주변분비(paracrine) 방식으로 작용하여 항증식효과 및 세포 사멸 촉진 효과를 나타내는지 확인하기 위해, 344SQ 세포 및 A549 세포를 마우스 재조합 WISP-1(rWISP-1, 20 내지 100 ng/ml)으로 처리한 후, 세포 생존력, 콜로니 형성 정도, 세포 증식 마커의 발현 정도 및 세포 사멸 마커의 발현 정도를 분석하였다.In addition, to determine whether WISP-1 acts in a paracrine manner to exert anti-proliferative and apoptosis-promoting effects, 344SQ cells and A549 cells were incubated with mouse recombinant WISP-1 (rWISP-1, 20 to 100 ng). /ml), cell viability, degree of colony formation, expression level of cell proliferation markers, and expression level of cell death markers were analyzed.
그 결과, rWISP-1이 344SQ 세포 및 A549 세포에서 세포 생존 및 콜로니 형성을 농도 의존적으로 억제하고(도 17a 및 17b), Ki67 및 PCNA의 mRNA 및/또는 단백질 발현 또한 농도 의존적으로 감소시킴을 확인하였다(도 18a 및 18b). 또한, 유세포 분석 및 TUNEL 분석 결과, rWISP-1이 농도 의존적으로 암 세포의 세포 사멸을 촉진하는 것으로 나타났다(도 19a 내지 20b). 뿐만 아니라, rWISP-1을 폐암 세포에 처리할 경우, 농도 의존적으로 세포 사멸 촉진 단백질(절단된 caspase 3, 절단된 PARP, 및 Bax)의 발현 수준은 증가하며, 항-세포 사멸 단백질(Bcl-2, Mcl-1 및 Bcl-xL)의 발현 수준은 감소함을 확인하였다(도 21a 및 21b).As a result, it was confirmed that rWISP-1 suppresses cell survival and colony formation in 344SQ cells and A549 cells in a concentration-dependent manner (FIGS. 17a and 17b), and also reduces mRNA and/or protein expression of Ki67 and PCNA in a concentration-dependent manner. (Figures 18a and 18b). Additionally, flow cytometry and TUNEL analysis showed that rWISP-1 promoted apoptosis of cancer cells in a concentration-dependent manner ( FIGS. 19A to 20B ). In addition, when rWISP-1 is treated with lung cancer cells, the expression levels of pro-apoptotic proteins (cleaved caspase 3, cleaved PARP, and Bax) increase in a concentration-dependent manner, and the expression levels of anti-apoptotic proteins (Bcl-2) increase. , Mcl-1 and Bcl-xL) expression levels were confirmed to be decreased (Figures 21a and 21b).
상기 실험들을 통해, CAFs와 사멸화된 암 세포의 공동 배양 조정배지의 항증식효과 및 세포 사멸 촉진 효과가 CAFs에서 분비되는 Notch1-의존성-WISP-1에 의해 매개되는 것임을 입증하였다.Through the above experiments, it was demonstrated that the anti-proliferative effect and apoptosis-promoting effect of the co-culture conditioned medium of CAFs and dead cancer cells were mediated by Notch1-dependent-WISP-1 secreted by CAFs.
2-4. CAFs에서 RhoA/Rho kinase 신호 경로의 억제가 Notch1/WISP-1 신호, 항증식효과 및 세포 사멸 촉진 효과를 강화시킴을 확인2-4. Confirmed that inhibition of RhoA/Rho kinase signaling pathway enhances Notch1/WISP-1 signaling, anti-proliferative effect, and apoptosis-promoting effect in CAFs.
RhoA/Rho kinase 신호 전달을 억제할 경우, CAFs의 탐식 작용(efferocytosis), Notch1 신호 전달 및 WISP-1 분비에 미치는 영향을 분석하기 위해 하기 실험을 수행하였다.The following experiment was performed to analyze the effects of inhibiting RhoA/Rho kinase signaling on efferocytosis, Notch1 signaling, and WISP-1 secretion of CAFs.
우선, 정량적 유세포 분석으로, 공동 배양 24시간 후 PKH67로 염색된 ApoSQ를 삼킨 PKH26-염색 CAFs의 비율을 측정하였다. 그 결과, Rho kinase 억제제인 ripasudil 30 μM을 처리할 경우, CAFs의 ApoSQ에 대한 식세포 작용이 향상됨을 확인하였다(도 22).First, by quantitative flow cytometry, we measured the proportion of PKH26-stained CAFs that engulfed PKH67-stained ApoSQ after 24 hours of co-culture. As a result, it was confirmed that when treated with 30 μM of ripasudil, a Rho kinase inhibitor, the phagocytosis of CAFs toward ApoSQ was improved (Figure 22).
유세포 분석 결과와 유사하게, 공초점 현미경 분석 결과 ripasudil의 처리에 따라 CAFs의 식세포 작용이 향상됨을 확인하였다(도 23). 또한, ripasudil 처리에 따라 CAFs에서 ApoSQ에 의해 유도되는 Notch1 신호 단백질(NICD1, Hes1 및 WISP-1) 발현 및 WISP-1의 분비 또한 유의하게 증가하는 것으로 나타났다(도 24a 및 24b).Similar to the flow cytometry results, confocal microscopy analysis confirmed that the phagocytosis of CAFs was improved by treatment with ripasudil (FIG. 23). In addition, the expression of Notch1 signaling proteins (NICD1, Hes1, and WISP-1) and secretion of WISP-1 induced by ApoSQ in CAFs were significantly increased following ripasudil treatment (Figures 24a and 24b).
또한, CAFs를 ApoSQ 또는 ApoA에 노출시키기 전에, RhoA siRNA로 형질감염시켜 RhoA/Rho kinase 신호 전달을 억제한 후, 세포 생존력, 콜로니 형성 정도 및 세포 증식 마커의 발현 정도를 측정하였다. 그 결과, RhoA siRNA로 전처리할 경우, 세포 생존 및 콜로니 형성이 더욱 억제되었으며(도 25a 내지 25c), Ki67 및 PCNA의 mRNA 및/또는 단백질 발현 또한 더욱 억제되었다(도 26a 및 26b).In addition, before exposing CAFs to ApoSQ or ApoA, they were transfected with RhoA siRNA to inhibit RhoA/Rho kinase signaling, and then cell viability, colony formation, and expression of cell proliferation markers were measured. As a result, when pretreated with RhoA siRNA, cell survival and colony formation were further inhibited (Figures 25a to 25c), and mRNA and/or protein expression of Ki67 and PCNA were also further inhibited (Figures 26a and 26b).
이와 유사하게, CAFs를 ApoSQ 또는 ApoA에 노출시키기 전에, Rho kinase 억제제인 Y-27632 30 μM을 처리할 경우에도, 세포 생존 및 콜로니 형성이 더욱 억제되며(도 27a 및 27b), Ki67 및 PCNA의 mRNA 및/또는 단백질 발현 또한 더욱 억제됨을 확인하였다(도 28a 및 28b).Similarly, when CAFs were treated with 30 μM of the Rho kinase inhibitor Y-27632 before exposure to ApoSQ or ApoA, cell survival and colony formation were further inhibited (Figures 27a and 27b), and the mRNA of Ki67 and PCNA And/or protein expression was also confirmed to be further suppressed (Figures 28a and 28b).
반면에, 유세포 분석 및 TUNEL 분석 결과, RhoA를 넉다운시킨 CAFs를 ApoSQ 또는 ApoA와 공동 배양할 경우, 344SQ 세포 및 A549 세포에서의 세포 사멸 효과는 더욱 향상되는 것으로 나타났다(도 29a 및 29b).On the other hand, flow cytometry and TUNEL analysis results showed that when CAFs with RhoA knocked down were co-cultured with ApoSQ or ApoA, the cell death effect in 344SQ cells and A549 cells was further improved (FIGS. 29a and 29b).
상기 실험들을 통해, RhoA/Rho kinase 신호 전달을 억제할 경우, CAFs에 의한 암 세포 사멸이 향상되고, Notch1-WISP-1 신호 전달 경로가 강화되어, CAFs와 사멸화된 암 세포의 공동 배양 조정배지의 항증식효과 및 세포 사멸 촉진 효과가 더욱 강화됨을 증명하였다.Through the above experiments, when RhoA/Rho kinase signaling was inhibited, cancer cell death by CAFs was improved and the Notch1-WISP-1 signaling pathway was strengthened, co-culture of CAFs and killed cancer cells in conditioned medium. It was proven that the antiproliferative effect and cell death promoting effect were further strengthened.
2-5. 2-5. in vivoin vivo 에서 ApoSQ-CAF CM의 종양 성장 억제 효과 확인Confirmed the tumor growth inhibition effect of ApoSQ-CAF CM
본 발명자들의 이전 연구에서 동계(syngeneic)(129/Sv) 면역적격 마우스에서 ApoSQ 세포의 단일 주입이 폐 전이를 억제함을 확인하였는 바, 본 실험에서는 이 동계 마우스를 통해 ApoSQ-CAF CM의 종양 성장에 대한 생체 내 효과를 조사하였다. 344SQ 세포 주사 이틀 후, 동계 마우스에 1주 3회 종양내 주사를 통하여 희석하지 않은 CAF CM, 50% 희석 CAF CM, 희석하지 않은 ApoSQ-CAF CM 또는 50% 희석 ApoSQ-CAF CM을 주사하였다(도 30a). In our previous study, we confirmed that a single injection of ApoSQ cells inhibited lung metastasis in syngeneic (129/Sv) immunocompetent mice, and in this experiment, tumor growth of ApoSQ-CAF CM was performed in these syngeneic mice. The in vivo effect was investigated. Two days after injection of 344SQ cells, syngeneic mice were injected with undiluted CAF CM, 50% diluted CAF CM, undiluted ApoSQ-CAF CM, or 50% diluted ApoSQ-CAF CM via intratumoral injection three times a week (Figure 30a).
그 결과, 마우스 그룹 간 체중의 차이는 크지 않았으나(도 30b), ApoSQ-CAF CM 주사 그룹의 경우 대조군(CAF CM 처리 그룹)에 비하여 1차 종양의 중량이 상당히 감소하였고, 종양의 성장 또한 상당히 억제된 것으로 나타났다(도 30c 및 30d). 이와 유사하게, ApoSQ-CAF CM 주사 그룹의 경우 대조군에 비해 종양의 부피가 상당히 감소하였다(도 30e). 특히, 50% 희석 ApoSQ-CAF CM의 경우, 주사 41일차에 있어서 종양 부피가 약 54% 억제되었으나, 희석하지 않은 ApoSQ-CAF CM의 경우 주사 41일차에 종양 부피가 약 86% 억제되는 것으로 나타나, 희석하지 않은 ApoSQ-CAF CM의 종양 부피 억제 효과가 보다 우수함을 확인하였다.As a result, the difference in body weight between mouse groups was not significant (Figure 30b), but in the ApoSQ-CAF CM injection group, the weight of the primary tumor was significantly reduced compared to the control group (CAF CM treatment group), and tumor growth was also significantly inhibited. It was found that (Figures 30c and 30d). Similarly, in the ApoSQ-CAF CM injection group, the tumor volume was significantly reduced compared to the control group (Figure 30e). In particular, in the case of 50% diluted ApoSQ-CAF CM, the tumor volume was suppressed by about 54% on the 41st day of injection, but in the case of undiluted ApoSQ-CAF CM, the tumor volume was suppressed by about 86% on the 41st day of injection. It was confirmed that the tumor volume suppression effect of undiluted ApoSQ-CAF CM was superior.
이후, 생체 내 ApoSQ-CAF CM에서 WISP-1이 가지는 항종양 효과를 확인하기 위해, 조정배지에 항-WISP-1 중화 항체 또는 IgG 이소타입을 마우스 주사 2시간 전 첨가하였다(도 31a).Then, to confirm the antitumor effect of WISP-1 on ApoSQ-CAF CM in vivo, anti-WISP-1 neutralizing antibody or IgG isotype was added to the conditioned medium 2 hours before mouse injection (FIG. 31a).
그 결과, 항-WISP-1 중화 항체 또는 IgG 이소타입의 처리에 따라 마우스 체중에는 유의한 변화가 나타나지 않았다(도 31b). 한편, 항-WISP-1 중화 항체가 투여되어 WIPS-1이 면역제거된 ApoSQ-CAF CM의 경우 종양의 중량 및 부피가 변하지 않았고 종양의 성장 또한 억제되지 않았으나, 대조군인 IgG 이소타입을 첨가한 그룹에서는 ApoSQ-CAF CM의 항종양 효과가 남아있음을 확인할 수 있었다(도 31c 내지 31e).As a result, there was no significant change in mouse body weight depending on treatment with anti-WISP-1 neutralizing antibody or IgG isotype (FIG. 31b). Meanwhile, in the case of ApoSQ-CAF CM in which WIPS-1 was immunized by administration of anti-WISP-1 neutralizing antibody, the weight and volume of the tumor did not change and tumor growth was not inhibited, but in the group to which the control group, IgG isotype, was added. It was confirmed that the antitumor effect of ApoSQ-CAF CM remained (Figures 31c to 31e).
WISP-1의 항종양 효과를 추가적으로 검증하기 위해, 344SQ 세포 주사 이틀 후, 동계 마우스에 1주 3회 종양내 주사를 통하여 rWISP-1을 12.5 μg/kg 또는 25 μg/kg의 농도로 주입한 후, 항종양 효과를 확인하였다(도 32a)).To further verify the anti-tumor effect of WISP-1, rWISP-1 was injected at a concentration of 12.5 μg/kg or 25 μg/kg through intratumoral injection three times a week into syngeneic mice two days after injection of 344SQ cells. , the antitumor effect was confirmed (Figure 32a)).
그 결과, ApoSQ-CAF CM의 항종양 효과와 유사하게, rWISP-1을 처리할 경우, 대조군과 마우스 체중에서는 유의한 차이가 나타나지 않았으나(도 32b), 1차 종양의 중량이 유의하게 감소하며, 종양의 성장 또한 상당히 억제되었고(도 32c 및 도 32d), 종양의 부피 또한 유의하게 감소됨을 확인하였다(도 32e).As a result, similar to the anti-tumor effect of ApoSQ-CAF CM, when treated with rWISP-1, there was no significant difference in body weight between the control group and the mouse (Figure 32b), but the weight of the primary tumor was significantly reduced; Tumor growth was also significantly suppressed (Figures 32c and 32d), and tumor volume was also significantly reduced (Figure 32e).
상기 실험들을 통해, ApoSQ-CAF CM이 in vivo에서도 항종양 효과를 나타내며, ApoSQ-CAF CM이 WISP-1을 통해 항종양 효과를 나타낼 수 있음을 재차 검증하였다. 따라서, 본 발명의 CAFs 및 사멸화된 암 세포의 공동 배양 조정배지는 암의 성장을 억제하는 데에 유용하게 활용될 수 있을 것으로 기대된다.Through the above experiments, it was again verified that ApoSQ-CAF CM exhibits an anti-tumor effect in vivo and that ApoSQ-CAF CM can exhibit an anti-tumor effect through WISP-1. Therefore, it is expected that the co-culture conditioned medium of CAFs and killed cancer cells of the present invention can be usefully used to inhibit cancer growth.

Claims (26)

  1. 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 함유하는, 암 성장 억제용 약학적 조성물.A pharmaceutical composition for inhibiting cancer growth, comprising a culture medium co-cultured with Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
  2. 청구항 1에 있어서, 상기 암 관련 섬유아세포는 섬유육종, 악성섬유성조직구종, 지방육종, 횡문 근육종, 평활근육종, 혈관육종, 악성피부암, 림프혈관육종, 활막육종, 연골육종, 골육종, 폐암, 위암, 유방암, 대장암 및 전립선암으로 이루어진 군에서 선택되는 하나 이상의 암과 관련된 섬유아세포인 것인, 약학적 조성물.The method of claim 1, wherein the cancer-related fibroblasts include fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, malignant skin cancer, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, lung cancer, and stomach cancer. , a pharmaceutical composition that is fibroblasts associated with one or more cancers selected from the group consisting of breast cancer, colon cancer, and prostate cancer.
  3. 청구항 1에 있어서, 상기 사멸화된 암 세포는 자외선(Ultra-violet ray, UV) 조사에 의해 사멸된 것인, 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the killed cancer cells are killed by ultraviolet ray (UV) irradiation.
  4. 청구항 1에 있어서, 상기 사멸화된 암 세포의 암종은 유방암, 자궁암, 식도암, 위암, 뇌암, 직장암, 대장암, 폐암, 피부암, 난소암, 자궁경부암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 구강암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종 및 백혈병으로 이루어진 군에서 선택되는 하나 이상인 것인, 약학적 조성물.The method according to claim 1, wherein 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, testicular cancer, and laryngeal cancer. , a pharmaceutical composition that is one or more selected from the group consisting of oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, and leukemia.
  5. 청구항 4에 있어서, 상기 암종은 폐암, 유방암, 위암, 대장암 및 전립선암으로 이루어진 군에서 선택되는 하나 이상인 것인, 약학적 조성물.The pharmaceutical composition according to claim 4, wherein the carcinoma is at least one selected from the group consisting of lung cancer, breast cancer, stomach cancer, colon cancer, and prostate cancer.
  6. 청구항 1에 있어서, 상기 배양액은 WISP-1(Wnt-induced signaling protein-1)을 함유하는 것인, 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the culture medium contains WISP-1 (Wnt-induced signaling protein-1).
  7. 청구항 6에 있어서, 상기 WISP-1은 Notch1 시그널링에 의해 생성되는 것인, 약학적 조성물.The pharmaceutical composition of claim 6, wherein the WISP-1 is produced by Notch1 signaling.
  8. 청구항 7에 있어서, 상기 Notch1 시그널링은 RhoA 및 Rho kinase의 억제에 의해 강화되는 것인, 약학적 조성물.The pharmaceutical composition according to claim 7, wherein the Notch1 signaling is enhanced by inhibition of RhoA and Rho kinase.
  9. 청구항 1에 있어서, 상기 조성물은 암 관련 섬유아세포에서 Notch1 관련 분자를 증가시키는 것인, 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the composition increases Notch1-related molecules in cancer-related fibroblasts.
  10. 청구항 9에 있어서, 상기 Notch1 관련 분자는 WISP-1, NICD1 및 Hes1으로 이루어진 군에서 선택되는 하나 이상인 것인, 약학적 조성물.The pharmaceutical composition according to claim 9, wherein the Notch1-related molecule is at least one selected from the group consisting of WISP-1, NICD1, and Hes1.
  11. 청구항 1에 있어서, 상기 조성물은 암 관련 섬유아세포에서 세포 증식 마커의 발현을 감소시키는 것인, 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the composition reduces the expression of cell proliferation markers in cancer-related fibroblasts.
  12. 청구항 11에 있어서, 상기 세포 증식 마커는 Ki67 및 PCNA로 이루어진 군에서 선택되는 하나 이상인 것인, 약학적 조성물.The pharmaceutical composition according to claim 11, wherein the cell proliferation marker is at least one selected from the group consisting of Ki67 and PCNA.
  13. 청구항 1에 있어서, 상기 조성물은 암 세포의 세포 사멸(apoptosis)을 증가시키는 것인, 약학적 조성물.The pharmaceutical composition of claim 1, wherein the composition increases apoptosis of cancer cells.
  14. 청구항 1에 있어서, 상기 조성물은 암 관련 섬유아세포에서 세포 사멸 촉진 인자의 발현을 증가시키고, 항-세포 사멸 인자의 발현을 감소시키는 것인, 약학적 조성물.The pharmaceutical composition of claim 1, wherein the composition increases the expression of pro-apoptotic factors and reduces the expression of anti-apoptotic factors in cancer-related fibroblasts.
  15. 청구항 14에 있어서, 상기 세포 사멸 촉진 인자는 절단된 caspase 3, 절단된 PARP 및 Bax로 이루어진 군에서 선택되는 하나 이상이고, 상기 항-세포 사멸 인자는 Bcl-2, Mcl-1 및 Bcl-xL로 이루어진 군에서 선택되는 하나 이상인 것인, 약학적 조성물.The method of claim 14, wherein the cell death promoting factor is at least one selected from the group consisting of cleaved caspase 3, cleaved PARP, and Bax, and the anti-cell death factor is Bcl-2, Mcl-1, and Bcl-xL. A pharmaceutical composition comprising at least one selected from the group consisting of:
  16. 청구항 1에 있어서, 상기 조성물은 RhoA 및 Rho kinase 억제제를 더 함유하는 것인, 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the composition further contains RhoA and Rho kinase inhibitors.
  17. 청구항 1에 있어서, 상기 RhoA 및 Rho kinase 억제제는 화합물, shRNA, miRNA, siRNA, 항체 및 앱타머로 이루어진 군에서 선택되는 하나 이상인 것인, 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the RhoA and Rho kinase inhibitor is one or more selected from the group consisting of compounds, shRNA, miRNA, siRNA, antibodies, and aptamers.
  18. 청구항 17에 있어서, 상기 화합물은 Y-27632인, 약학적 조성물.The pharmaceutical composition of claim 17, wherein the compound is Y-27632.
  19. 청구항 17에 있어서, 상기 siRNA는 서열번호 19 및 20의 염기 서열 중 어느 하나의 염기 서열로 이루어진 폴리뉴클레오티드인, 약학적 조성물.The pharmaceutical composition according to claim 17, wherein the siRNA is a polynucleotide consisting of any one of the base sequences of SEQ ID NOs: 19 and 20.
  20. 사멸화된 암 세포(apoptotic cancer cells)에 노출된 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs)를 함유하는, 암 성장 억제용 약학적 조성물.A pharmaceutical composition for inhibiting cancer growth, comprising Cancer-Associated Fibroblasts (CAFs) exposed to apoptotic cancer cells.
  21. 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 함유하는, 암 성장 억제용 건강기능식품.A health functional food for inhibiting cancer growth, containing a culture medium co-cultured with cancer-associated fibroblasts (CAFs) and apoptotic cancer cells.
  22. 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양하는 단계를 포함하는, 암 성장 억제용 조성물의 제조방법.A method for producing a composition for inhibiting cancer growth, comprising the step of co-culturing cancer-associated fibroblasts (CAFs) and apoptotic cancer cells.
  23. 청구항 22에 있어서, 상기 공동 배양을 통해 WISP-1을 분비시키는 단계를 더 포함하는, 암 성장 억제용 조성물의 제조방법.The method of claim 22, further comprising secreting WISP-1 through the co-culture.
  24. 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액을 개체에 처리하는 단계를 포함하는, 암 성장 억제방법.A method of inhibiting cancer growth, comprising treating a subject with a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured.
  25. 암 관련 섬유아세포(Cancer-Associated Fibroblasts, CAFs) 및 사멸화된 암 세포(apoptotic cancer cells)를 공동 배양한 배양액의 암 성장 억제 용도.For use in inhibiting cancer growth in co-cultures of Cancer-Associated Fibroblasts (CAFs) and apoptotic cancer cells.
  26. WISP-1(Wnt-induced signaling protein-1)의 암 성장 억제 용도.Use of WISP-1 (Wnt-induced signaling protein-1) to inhibit cancer growth.
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