WO2023055076A1 - Composition for inhibiting cancer metastasis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis, a method for inhibiting cancer metastasis using same, and use of the pharmaceutical composition, the pharmaceutical composition containing a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured. In the present invention, exposing CAFs to apoptotic cancer cells was found to induce Notch1 signaling-dependent WISP-1 generation and thus inhibit the migration and invasion of the cancer cells and CAFs. Therefore, a culture medium, the main component of the culture medium (WISP-1), or CAF cells exposed to the apoptotic cancer cells according to the present invention can be effectively used as a cancer metastasis inhibitor.

Description

Composition for inhibiting cancer metastasis

The present invention provides a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured, a method for inhibiting cancer metastasis using the same, and It relates to the use of the pharmaceutical composition.

Lung cancer (both small cell and non-small cell cancer) is the most common cancer worldwide in both men and women, and is the leading cause of cancer-related death (18.0% of total cancer deaths). About 75% of lung cancer patients are confirmed to have locally advanced or metastatic disease at the time of diagnosis. In this case, metastasis is a multistep process including migration and invasion of cancer cells, and becomes a marker of malignant tumors.

Cancer-associated fibroblasts (CAFs) of various origins are one of the main cell types present in tumor-associated stroma. It is known that paracrine communication between CAFs and cancer cells promotes basal processes such as cancer cell migration and invasion that can promote progression to malignancy and metastatic spread. CAFs physically remodel the matrix in the tumor stroma, allowing cancer cells to invade while still maintaining epithelial properties. However, the underlying molecular mechanisms of CAFs-mediated regulation in tumor progression are still unclear.

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 in the regulation of Notch signaling result in developmental abnormalities or cancer. Interestingly, Notch activity is associated with both oncogenic and tumor-suppressive functions, which depend on the complex microenvironment of cellular responses that Notch induces. Stromal fibroblasts with an activated Notch pathway can attenuate melanoma growth and inhibit tumor angiogenesis, in part through upregulation of Wnt-induced signaling protein-1 (WISP-1). These findings will help elucidate the molecular mechanisms for the Notch1-dependent tumor-regulatory role in CAFs in other cancer types as well.

High levels of apoptosis within the tumor environment and the mechanisms that clear apoptotic cancer cells can greatly influence tumor-specific immunity. In the tumor microenvironment (TME), the immunosuppressive effect of phagocyte-mediated clearance has been reported to suppress antitumor immune responses. Conversely, tumors can evade immune surveillance by inhibiting recognition for efferocytosis.

In addition, the anti-inflammatory and anti-inflammatory lipid autacoids specifically inhibit debris-stimulated cancer progression by promoting the clearance of cellular debris through macrophage phagocytosis in multiple tumor types. Moreover, in previous studies by the present inventors, macrophages exposed to UV-irradiated apoptotic lung cancer cells showed upregulation of exosome phosphatase and tensin homolog (PTEN) and peroxisome proliferator-activated receptor-gamma (PPARr). It has been demonstrated that through the secretion of ligands such as 15-hydroxyeicosatetraenoic acid (HETE), lipoxin A4 and 15d-prostaglandin J2, inhibition of cell polarity disruption, EMT and invasion of cancer cells (patent registration) 10-1804852). However, it has not yet been studied whether CAFs-induced efferocytosis of cancer cells regulates CAFs activation in the TME and blocks cancer progression and metastasis.

Accordingly, the present inventors studied how the interaction between CAFs and apoptotic cancer cells regulates the migration and invasion of cancer cells and CAFs. It was confirmed that WISP-1 was induced to inhibit migration and invasion of cancer cells and CAFs themselves. In addition, after intratumoral administration of apoptotic lung cancer cells, inhibition of CAFs activation and cancer cell migration and invasion were confirmed. Furthermore, the present invention was completed by confirming the effect of inhibiting lung metastasis after intratumoral administration of the co-culture of apoptosis lung cancer cells and CAFs.

One aspect is to provide a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured.

Another aspect is to provide a pharmaceutical composition for inhibiting cancer metastasis, containing cancer-associated fibroblasts exposed to killed cancer cells.

Another aspect is to provide a health functional food for inhibiting cancer metastasis containing a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.

Another aspect is to provide a method for preparing a composition for inhibiting cancer metastasis, comprising co-cultivating cancer-related fibroblasts and apoptotic cancer cells.

Another aspect is to provide a method for inhibiting cancer metastasis, including the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptosis-killed cancer cells are co-cultured.

Another aspect is to provide a method for suppressing cancer metastasis, including treating a subject with Wnt-induced signaling protein-1 (WISP-1).

Another aspect is to provide a method for preventing or treating cancer, which includes the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptosis cancer cells are co-cultured.

Another aspect is to provide a method for preventing or treating cancer, including treating a subject with Wnt-induced signaling protein-1 (WISP-1).

Another aspect is to provide the use of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured for the manufacture of a drug for inhibiting cancer metastasis.

Another aspect is to provide use of WISP-1 (Wnt-induced signaling protein-1) for the manufacture of a drug for suppressing cancer metastasis.

Another aspect is to provide the use of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured for the manufacture of a drug for preventing or treating cancer.

Another aspect is to provide use of Wnt-induced signaling protein-1 (WISP-1) for the manufacture of a drug for preventing or treating cancer.

Another aspect is to provide a use for suppressing cancer metastasis of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.

Another aspect is to provide a use for inhibiting cancer metastasis of a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.

Another aspect is to provide a use of Wnt-induced signaling protein-1 (WISP-1) to inhibit cancer metastasis.

Another aspect is to provide a use for preventing or treating cancer of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.

Another aspect is to provide a pharmaceutical composition containing a culture medium in which cancer-related fibroblasts and killed cancer cells are co-cultured for use in preventing or treating cancer.

Another aspect is to provide a use for cancer prevention or treatment of Wnt-induced signaling protein-1 (WISP-1).

The present invention provides a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-associated fibroblasts (CAFs) and apoptotic cancer cells are co-cultured.

In the present invention, the culture medium in which the CAFs and the apoptotic cancer cells are co-cultured has an effect of inhibiting metastasis of cancer cells.

In the present invention, "Cancer-Associated Fibroblasts (CAFs)" refers to α-SMA (α-smooth muscle actin) positive fibroblasts present inside and/or around cancer lesions, and the CAFs are colorectal cancer. , its presence has been confirmed in various cancers such as lung cancer, prostate cancer, breast cancer, stomach cancer, cholangiocarcinoma, and basal cell carcinoma.

In the present invention, the cancers associated with the CAFs include brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, renal cancer, ureter cancer, and bladder cancer. , prostate cancer, penile cancer, testicular cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, and solid cancer such as skin cancer.

In one embodiment of the present invention, the CAFs are related to cancer and may be fibroblasts related to malignant solid tumors. In one embodiment, the CAFs are fibroblasts related to sarcomas such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, malignant skin cancer, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, etc. It may be, and may be fibroblasts related to lung cancer, stomach cancer, breast cancer, colon cancer or prostate cancer.

In one embodiment of the present invention, the CAFs can have enhanced phagocytic ability by the interaction of Notch1 signaling and BAI1/Rac1 signaling.

In the present invention, “apoptotic cancer cells” may be those in which apoptosis is induced by irradiating cancer cells with light of a specific wavelength. The irradiation of light of the specific wavelength may be ultraviolet (Ultra-violet ray, UV) irradiation. In one embodiment, the wavelength may be irradiated for 5 to 30 minutes with a wavelength of 100 to 400 nm.

In the present invention, "co-culture" can be achieved by culturing CAFs and apoptotic cancer cells together. In one example, CAFs may be cultured with apoptotic cancer cells in X-VIVO or serum-free DMEM medium for 20 to 30 hours.

In the present invention, "culture medium" means a culture product obtained through co-cultivation of CAFs and apoptotic cancer cells. In one embodiment of the present invention, the culture medium may be a liquid medium, a solid medium or a semi-solid medium. In one embodiment, the culture medium may be a conditioned medium.

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 at least one selected from the group consisting of testicular cancer, laryngeal cancer, oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, and leukemia. In one embodiment, the carcinoma may be at least one 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.

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.

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 signals. do.

In one embodiment of the present invention, the WISP-1 may be generated by Notch1 signaling. Notch1 signaling is cell contact-dependent through Notch receptors and plays an important role in development, regeneration, and maintenance of homeostasis, and the activity of Notch signaling is associated with both oncogenic and tumor-suppressive functions. there is. Notch1-WISP-1 signaling is known to determine the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma invasion and metastasis.

In one embodiment of the present invention, the Notch1 signaling may be initiated by Dll1 (Delta-like ligand 1). Dll1 is a type of Notch Delta ligand that plays a role in regulating cell fate decisions during blood formation and is known to be involved in cell-cell communication.

In one embodiment of the present invention, the Notch1 signaling may be enhanced by BAI1/Rac1 (Brain angiogenesis inhibitor 1/Ras-related C3 botulinum toxin substrate 1) signaling. BAI1 is a type of aggregation GPCR that acts to inhibit angiogenesis, and Rac1 is a type of GTPase that plays a role in regulating cell growth, cytoskeleton reorganization, cell cycle, cell-cell aggregation and migration, and the like.

In one embodiment of the present invention, the composition can enhance the phagocytic ability of cancer-related fibroblasts by the interaction of Notch1 signaling and BAI1/Rac1 signaling.

In the present invention, "efferocytosis" means phagocytosis to remove apoptotic cells. The phagocytic ability refers to the ability of CAFs to remove apoptotic cancer cells through phagocytosis.

In one embodiment of the present invention, the "interaction" may mean crosstalk between the Notch1 signaling and the BAI1/Rac1 signaling, and more specifically, may mean positive crosstalk. there is. In the present invention, "crosstalk" means a phenomenon in which one or more components of a signal transduction pathway affect other components. Positive crosstalk refers to a phenomenon in which one signaling reinforces another signaling. Negative crosstalk refers to a phenomenon in which one signaling suppresses another signaling.

In one embodiment of the present invention, the BAI1/Rac1 signaling can promote phagocytosis of apoptotic cancer cells by the cancer-associated fibroblasts.

In one embodiment of the present invention, the composition can enhance WISP-1 production by the interaction of Notch1 signaling and BAI1/Rac1 signaling.

In one embodiment of the present invention, the composition can reduce activation markers in cancer-associated fibroblasts. In one embodiment, the activation marker may be one or more selected from the group consisting of Acta2, Col1α1, Fn, Itg beta 1, Spp1, Pdgfrα, Pdgfr beta, and Mmp1a, 2, 9, and 12.

In one embodiment of the present invention, the composition can reduce growth factors and chemokines in cancer-associated fibroblasts. In one embodiment, the growth factor may be one or more selected from the group consisting of Vegfa, Hgf, Cxcl12 and Cxcl14.

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 Notch1, WISP-1 (Ccn4), Hey1, Hey2, Hes1, and Hes5.

In the present invention, "metastasis" refers to a state in which a malignant tumor has spread to other tissues away from an organ where it has occurred, and can be used as a marker of a malignant tumor. In one embodiment of the present invention, the metastasis may include migration and invasion of cancer cells.

In the present invention, "migration" means that cells move to a specific location in response to a specific external signal, and "invasion" means that cells expand and penetrate other nearby tissues.

The present invention provides a pharmaceutical composition for inhibiting cancer metastasis containing cancer-related fibroblasts exposed to apoptotic cancer cells.

In the present invention, CAFs exposed to the killed cancer cells contain WISP-1, and thus have an effect of suppressing metastasis of cancer cells.

In the present invention, "exposure" means a process of contacting or stimulating apoptosis cancer cells and CAFs to enable cell-to-cell interaction, and is distinguished from a conditioned medium in which apoptosis cancer cells and CAFs are co-cultured.

The pharmaceutical composition according to the present invention may include a "pharmaceutically acceptable carrier". The pharmaceutically acceptable carrier is one commonly used in formulation and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. It is not, and if necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to formulate formulations for injections such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Regarding a suitable pharmaceutically acceptable carrier and formulation, it can be preferably formulated according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in dosage form, but may be formulated as an injection, an inhalant, an external preparation for the skin, or an oral intake.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, applied to the skin, nasal cavity, or respiratory tract) according to the desired method, and the dosage is determined according to the patient's condition and weight, disease Depending on the degree, drug form, administration route and time, it can be appropriately selected by those skilled in the art.

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 a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and activity of the drug of the patient's disease , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg per day or every other day, or 1 It can be administered in 1 to 3 divided doses per day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

The present invention provides a health functional food for inhibiting cancer metastasis, which contains a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.

The health functional food of the present invention may be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations by further including one or more of carriers, diluents, excipients and additives. Examples of foods to which the extract of the present invention can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, and the like.

Additives that may be further included in the present invention include natural carbohydrates, flavors, nutrients, vitamins, minerals (electrolytes), flavors (synthetic flavors, natural flavors, etc.), colorants, fillers (cheese, chocolate, etc.), One or more components selected from the group consisting of pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonating agents and fruit flesh may be used. .

Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the health functional food of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the composition according to the present invention may contain fruit flesh for preparing natural fruit juice and vegetable beverages. These components may be used independently or in combination.

Specific examples of the carrier, excipient, diluent, and additive include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium Silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate And at least one selected from the group consisting of mineral oil is preferably used.

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.

The present invention provides a method for preparing a composition for inhibiting cancer metastasis, comprising co-cultivating cancer-related fibroblasts and apoptotic cancer cells.

In one embodiment of the present invention, the killing of cancer cells may be induced through UV irradiation, and the UV irradiation may be performed for 5 to 30 minutes with a wavelength of 100 to 400 nm. In one embodiment, the UV irradiation may be performed with a wavelength of 150 to 350 nm for 20 minutes or with a wavelength of 200 to 300 nm for 10 to 15 minutes.

In one embodiment of the present invention, the step of culturing may be to culture in X-VIVO medium for 24 hours to make it serum-starved. In one embodiment, the culture medium may be replaced with X-VIVO or serum-free DMEM medium containing killed cancer cells.

Also, in one embodiment, the co-cultivation may be performed for 10 to 30 hours, 15 to 25 hours, or 18 to 24 hours after medium replacement.

The present invention provides a method for suppressing cancer metastasis, comprising the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptosis-killed cancer cells are co-cultured.

In one embodiment of the present invention, the step of treating the subject with the culture medium is performed by treating the subject with a pharmaceutical composition for inhibiting cancer metastasis containing a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured. it could be

In the present invention, "treatment" may mean any act of adding or administering the culture medium to a subject according to experimental requirements.

In the present invention, "subject" means a subject in need of a method for preventing, controlling or treating a disease, and may include a mammal. In one embodiment, the mammal may include humans or primates, mice, cows, dogs, horses, pigs, and the like.

The present invention provides a method for inhibiting cancer metastasis, comprising treating a subject with Wnt-induced signaling protein-1 (WISP-1).

The present invention provides a method for preventing or treating cancer, comprising the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.

In one embodiment of the present invention, the culture medium in which the cancer-related fibroblasts and apoptosis cancer cells are co-cultured can be treated in the form of a pharmaceutical composition containing the same.

The term "prevention" may refer to any activity that inhibits or delays the onset of cancer, and the term "treatment" may mean any activity that improves or beneficially changes cancer.

The present invention provides a method for preventing or treating cancer, including treating a subject with Wnt-induced signaling protein-1 (WISP-1).

The present invention provides the use of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured for the manufacture of a drug for inhibiting cancer metastasis.

The present invention provides a use of WISP-1 (Wnt-induced signaling protein-1) for the preparation of a drug for suppressing cancer metastasis.

The present invention provides the use of a culture medium in which cancer-related fibroblasts and killed cancer cells are co-cultured for the manufacture of a drug for preventing or treating cancer.

The present invention provides a use of WISP-1 (Wnt-induced signaling protein-1) for the manufacture of a drug for preventing or treating cancer.

The present invention provides a use for inhibiting cancer metastasis of a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.

In addition, the present invention provides a use for inhibiting cancer metastasis of a pharmaceutical composition for inhibiting cancer metastasis, which contains a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured.

The present invention provides a use of WISP-1 (Wnt-induced signaling protein-1) to inhibit cancer metastasis.

The present invention provides a use for preventing or treating cancer of a culture medium in which cancer-related fibroblasts and killed cancer cells are co-cultured.

In addition, the present invention provides a use for preventing or treating cancer of a pharmaceutical composition containing a culture medium in which cancer-related fibroblasts and killed cancer cells are co-cultured.

The present invention provides a use of Wnt-induced signaling protein-1 (WISP-1) for preventing or treating cancer.

In the present invention, as a result of studying how the interaction between CAFs and apoptotic cancer cells regulates the migration and invasion of cancer cells and CAFs, a conditioned medium in which apoptotic cancer cells and CAFs are co-cultured (ApoSQ-CAF CM) It was confirmed that this Notch1 signal transduction-dependent WISP-1 production was induced, thereby suppressing the migration and invasion of cancer cells and CAFs themselves.

In addition, using a syngeneic metastatic lung cancer mouse model ( in vivo ), when apoptotic cancer cells are injected, activation of Thy1 ⁺ CAFs isolated from primary tumors is suppressed, and WISP-1 production is increased and Thy1 by Notch1 signal activation ⁺ It was confirmed that WISP-1 was increased in the culture medium and serum of CAFs, and the migration and invasion of isolated CD326 ⁺ tumor cells and Thy1 ⁺ CAFs were also inhibited. When injected with ApoSQ-CAF CM, the number and metastasis rate of nodules metastasized to the lung were suppressed, gene expression related to invasion and metastasis was suppressed in CD326 ⁺ tumor cells, and expression of genes related to cell adhesion and extracellular matrix in Thy1 ⁺ CAFs was confirmed to inhibit.

In addition, it was confirmed that the migration and invasion of isolated CD326 ⁺ tumor cells and Thy1 ⁺ CAFs were also inhibited when ApoSQ-CAF CM was injected.

Therefore, according to the present invention, it can be usefully used for cancer metastasis suppression strategies, and in particular, CAFs exposed to the culture medium or killed cancer cells of the present invention can be usefully used as cancer metastasis suppressors.

1 is a diagram showing that a conditioned medium (CM) containing a culture medium of cancer-associated fibroblasts (CAFs) co-cultured with apoptotic cancer cells inhibits migration and invasion of cancer cells. am. Figure 1a shows a control, CAF conditioned medium (CAF CM), and 344SQ co-culture conditioned medium (ApoSQ-CAF CM) killed with CAFs in the presence or absence of TGF-beta 1 using 344SQ, a lung adenocarcinoma cell line. , It is a diagram measuring the migration and invasion of 344SQ cells in the 344SQ co-culture conditioned medium (NecSQ-CAF CM) group with CAFs and necrosis. Figure 1b shows the control, CAF CM, CAFs and apoptosis A549 co-culture conditioned medium (ApoA-CAF CM), CAFs and A549 co-culture in the presence or absence of TGF-beta 1 using A549, a non-small cell lung cancer cell line. It is a diagram measuring migration and invasion of A549 cells in the culture conditioned medium (NecA-CAF CM) group. 1c is a control, CAF CM, CAFs and apoptosis HCT116 co-culture conditioned medium (ApoH-CAF CM), and CAFs and necrosis HCT116 co-culture in the presence or absence of TGF-beta 1 using HCT116, a colorectal cancer cell line. It is a diagram measuring the migration and invasion of HCT116 cells in the conditioned medium (NecH-CAF CM) group. Figure 1d is a control, CAF CM, CAFs and MCF-7 co-culture conditioned medium (ApoM-CAF CM), CAFs and necrosis in the presence or absence of TGF-beta 1 using MCF-7, a breast cancer cell line, respectively. It is a diagram measuring the migration and invasion of MCF-7 cells in the MCF-7 co-culture conditioned medium (NecM-CAF CM) group.

Figure 2 is a diagram confirming that there is no effect of inhibiting migration and invasion of cancer cells by direct treatment of the killed cancer cell culture medium alone and the killed cancer cells. Figure 2a is a diagram showing the migration and invasion of 344SQ cells in the control, CAF CM, ApoSQ CM and NecSQ CM groups, respectively, in the presence of TGF-beta 1. Figure 2b is a diagram measuring 344SQ cell migration and invasion in the control, ApoSQ, and NecSQ groups, respectively, in the presence or absence of TGF-beta 1.

3 is a diagram showing that ApoSQ-CAF CM inhibits the signaling pathway activity related to TGF-beta 1 in 344SQ cells using immunoblot analysis. Figure 3a is a diagram showing the results of immunoblot analysis of TGF-beta 1-related signaling pathway expressed after treatment of CAF CM and ApoSQ-CAF CM together with TGF-beta 1 in 344SQ cells. Figure 3b is a diagram showing the total and phosphorylated amounts of Smad2, Smad3, FAK, AKT, SrC, ERK, and P38 expressed after treatment with CAF CM and ApoSQ-CAF CM in 344SQ cells.

4 is a diagram showing that ApoSQ-CAF CM suppresses MMP2 and MMP12 mRNA expression and protein expression in 344SQ cells using qRT-PCR and immunoblot analysis. (A) A diagram showing the levels of MMP2 and MMP12 mRNA expression after treatment of CAF CM and ApoSQ-CAF CM with TGF-beta in 344SQ cells. (B) 344SQ cells treated with CAF CM and ApoSQ-CAF CM, and then immunoblot analysis of MMP2 and MMP12 proteins expressed and a diagram showing the protein amounts.

5 is a diagram showing that only ApoSQ inhibits cell migration and invasion when CAFs are brought into contact with ApoSQ and NecSQ, respectively.

Figure 6 shows that when CAFs were exposed to ApoSQ-CAF CM, NecSQ-CAF CM, ApoA-CAF CM, NecA-CAF CM, ApoH-CAF CM and NecH-CAF CM in the presence or absence of TGF-beta 1, respectively, death It is a diagram showing that only the conditioned cancer cells and CAF-conditioned medium inhibit cell migration and invasion. Figure 6a is a diagram showing migration and invasion of CAFs after treatment with CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM. Figure 6b is a diagram showing migration and invasion of CAFs after treatment with CAF CM, ApoA-CAF CM, and NecA-CAF CM. Figure 6c is a diagram showing migration and invasion of CAFs after treatment with CAF CM, ApoH-CAF CM, and NecH-CAF CM. Figure 6d is a diagram showing migration and invasion of CAFs after treatment with CAF CM, ApoM-CAF CM, and NecM-CAF CM.

7 is a diagram confirming that the culture medium of killed cancer cells alone has no effect of inhibiting migration and invasion of CAFs.

8 is a diagram showing that ApoSQ inhibits the expression of CAF activation markers in CAFs using qRT-PCR, immunoblot analysis, and immunofluorescence staining. Figure 8a is a diagram showing the results of analyzing the mRNA expression level of CAF activation markers when ApoSQ and NecSQ were treated with CAFs, and the results of immunoblot analysis of CAF activation markers and protein amounts when ApoSQ and NecSQ were treated with CAFs. 8b is a diagram showing the fluorescence intensity of α-SMA when CAFs were treated with ApoSQ and NecSQ.

9 is a diagram showing that ApoSQ inhibits the signaling pathway activity related to TGF-beta 1 in CAFs using immunoblot analysis. 9a is a diagram showing the results of immunoblot analysis of TGF-beta 1-related signaling pathways after CAFs were treated with ApoSQ together with TGF-beta 1; Figure 9b is a diagram showing the total and phosphorylated amounts of signaling pathways, that is, Smad2, Smad3, AKT, FAK, SrC, ERK, and P38, after CAFs were treated with ApoSQ along with TGF-beta 1.

10 is a diagram showing that ApoSQ inhibits the expression of MMP mRNA and protein in CAFs using qRT-PCR and immunoblot analysis. (A) A diagram showing the mRNA expression levels of MMP2 and MMP12 after ApoSQ was treated in CAFs. (B) A diagram showing the results of immunoblot analysis and protein amounts of MMP2 and MMP12 proteins after CAFs were treated with ApoSQ.

11 is a result of cytokine array analysis in CAF CM, ApoSQ-CAF CM and ApoSQ CM, and the expression of leukemia inhibitory factor (LIF) and WISP-1 among 111 types of cytokines was compared with CAF CM and ApoSQ-CAF CM groups. It is a diagram confirming that the cytokine increased the most in ApoSQ-CAF CM.

12 is a diagram showing that the expression level of WISP-1 decreases when WISP-1 is knocked down. (A) A diagram showing the results of immunoblot analysis and the level of WISP-1 protein expression measured after transfecting CAFs with WISP-1-specific siRNA. (B) It is a diagram showing the amount of WISP-1 measured by ELISA in the culture medium after CAFs were transfected with siRNA specific to WISP-1.

13 is a diagram showing that the effect of inhibiting cell migration and invasion is reversed when CAFs are transfected with WISP-1 siRNA. Figure 13a is a diagram showing migration and invasion of 344SQ cells upon treatment with CAF CM and ApoSQ-CAF CM in the presence or absence of WISP-1 siRNA. Figure 13b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment in the presence or absence of WISP-1 siRNA.

14 is a diagram showing that the expression level of LIF decreases when LIF is knocked down. (A) A diagram showing the result of immunoblot analysis and the amount of LIF protein measured after transfection of CAFs with LIF-specific siRNA. (B) A diagram showing the amount of LIF measured by ELISA in CAF CM and ApoSQ-CAF CM after CAFs were transfected with LIF-specific siRNA. (C) A diagram showing the amount of WISP-1 measured by ELISA in CAF CM and ApoSQ-CAF CM after CAFs were transfected with LIF-specific siRNA.

15 is a diagram showing that the effect of inhibiting cell migration and invasion is maintained when CAFs are transfected with LIF siRNA. Figure 15a is a diagram showing migration and invasion of 344SQ cells when treated with CAF CM and ApoSQ-CAF CM in the presence or absence of LIF siRNA. Figure 15b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment in the presence or absence of LIF siRNA.

16 is a diagram showing that the expression level of WISP-1 increases when WISP-1 is overexpressed. (A) A diagram showing the results of immunoblot analysis and protein expression level measured after transfection of CAFs with mock vector and WISP-1. (B) A diagram showing the amount of WISP-1 measured by ELISA in CAF CM and ApoSQ-CAF CM after CAFs were transfected with mock vector and WISP-1.

17 is a diagram showing that cell migration and invasion inhibitory effects are increased when CAFs are transfected with WISP-1 to overexpress WISP-1. 17a is a diagram showing migration and invasion of 344SQ cells when treated with CAF CM and ApoSQ-CAF CM, depending on whether or not WISP-1 was transfected. 17b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment, depending on whether or not WISP-1 was transfected.

18 is a diagram showing that the anti-WISP-1 antibody treatment of ApoSQ-CAF CM reverses the effect of inhibiting migration and invasion of 344SQ cells.

19 is a diagram showing that migration and invasion of 344SQ cells or CAFs are inhibited in a concentration-dependent manner when recombinant WISP-1 is added. Figure 19a is a diagram showing migration and invasion of 344SQ cells according to rWISP-1 concentration. Figure 19b is a diagram showing migration and invasion of CAFs according to rWISP-1 concentration.

20 is a diagram showing that rWISP-1 inhibits the activity of the signaling pathway related to TGF-beta 1 and MMP expression in 344SQ cells in a concentration-dependent manner using immunoblot analysis. 20a is a diagram showing the results of immunoblot analysis of TGF-beta 1-related signaling molecules for each concentration of rWISP-1. Figure 20b is a diagram showing the total amount and phosphorylation amount of TGF-beta 1-related signaling pathways, that is, Smad2, Smad3, FAK, AKT, SrC, ERK, and P38, when rWISP-1 was treated by concentration. In Figure 20c (A) is a diagram showing the amount of MMP2 and MMP12 mRNA expressed by rWISP-1 concentration. (B) is a diagram showing the results of immunoblot analysis and protein amounts of MMP2 and MMP12 proteins expressed by rWISP-1 concentrations.

21 is a diagram showing that when rWISP-1 is treated with TGF-beta 1, the activity of the signaling pathway related to TGF-beta 1 and the expression of MMP are inhibited in CAFs using immunoblot analysis and qRT-PCR. 21a is a diagram showing the results of immunoblot analysis of the TGF-beta 1-related signaling pathway upon treatment with rWISP-1. Figure 21b is a diagram showing the total and phosphorylated amounts of TGF-beta 1-related signaling pathways, that is, Smad2, Smad3, FAK, AKT, SrC, ERK and P38 upon rWISP-1 treatment. In Figure 21c, (A) is a diagram showing the change in the mRNA expression amount of MMP2 and MMP12 when rWISP-1 and TGF-beta 1 are treated together, (B) is a diagram showing the change in the mRNA expression amount of rWISP-1 and TGF-beta 1 when treated together with MMP2 and MMP12 It is a diagram showing the results of protein immunoblot analysis and the amount of protein.

22 is a diagram showing that the inhibition of migration and invasion according to the addition of WISP-1 is reversed in 344SQ cells or CAFs when a specific anti-integrin is added. 22a is a diagram showing that the inhibition of migration and invasion according to the addition of WISP-1 is reversed in 344SQ cells when an anti-integrin αv or beta 3 antibody is added. 22b is a diagram showing that the inhibition of migration and invasion according to the addition of WISP-1 is reversed in CAFs when an anti-integrin αv or beta 5 antibody is added.

23 is a diagram showing that the activation of the signaling pathway related to TGF-beta 1 and the suppression of MMP expression are reversed in 344SQ cells when a specific anti-integrin is added using immunoblot analysis and qRT-PCR. 23a is a diagram showing the results of immunoblot analysis of TGF-beta 1-related signaling molecules according to the addition of rWISP-1 and anti-integrin αv or beta 3 antibodies. 23b is a diagram showing the mRNA amounts of MMP2 and MMP12 according to the addition of rWISP-1 and anti-integrin αv or beta 3 antibodies. 23c is a diagram showing the results of immunoblot analysis of MMP2 and MMP12 proteins according to the addition of WISP-1 and anti-integrin αv or beta 3 antibodies.

FIG. 24 is a diagram showing that when a specific anti-integrin is added using immunoblot analysis and qRT-PCR, the TGF-beta 1-related signaling pathway activity and inhibition of MMP expression are reversed by the addition of WISP-1 in CAFs. 24a is a diagram showing the results of immunoblot analysis of TGF-beta 1 related signaling molecules according to the addition of rWISP-1 and anti-integrin αv or beta 5 antibodies. 24b is a diagram showing the mRNA amounts of MMP2 and MMP12 according to the addition of rWISP-1 and anti-integrin αv or beta 5 antibodies. 24c is a diagram showing the results of immunoblot analysis of MMP2 and MMP12 proteins according to the addition of WISP-1 and anti-integrin αv or beta 5 antibodies.

25 is a diagram showing that the amount of proteins of factors related to the Notch1 signaling pathway expressed in CAFs according to the presence or absence of ApoSQ is increased. (A) A diagram showing the results of protein immunoblot analysis according to the presence or absence of ApoSQ in CAFs. (B) A diagram showing the amount of WISP-1 expressed in CAF CM, ApoSQ-CAF CM and NecSQ-CAF CM.

26 is a diagram showing that the mRNA expression levels of Hes1 and WISP-1, which are target genes of the Notch signaling pathway, and the activity of 4×CSL luciferase are increased when ApoSQ or NecSQ is treated in CAFs. (A) A diagram showing the mRNA expression levels of Hes1 and WISP-1 analyzed by qRT-PCR. (B) A diagram showing the activity of 4×CSL luciferase, an overall Notch signal transcriptional effector, upon ApoSQ treatment.

27 is a diagram showing changes in immunofluorescence staining for NICD1 and WISP-1 according to ApoSQ or NecSQ treatment in CAFs.

28 is a diagram showing that WISP-1 protein expression is suppressed when Notch1 is knocked down through siRNA. 28a is a diagram showing the results of immunoblot analysis measured after CAFs were transfected with Notch1 siRNA. 28B, (A) is a diagram showing the immunoblot analysis results and protein amounts for NICD1, Hes1, and WISP-1 measured according to the presence or absence of ApoSQ or NecSQ after CAFs were transfected with Notch1 siRNA, (B) is a diagram showing the ELISA results of WISP-1 in CAF CM and ApoSQ-CAF CM after transfection of CAFs with Notch1 siRNA.

29 is a diagram showing that when Notch1 is knocked down through siRNA, the anti-migration and anti-invasion effects of ApoSQ-CAF CM and ApoSQ are reversed in 344SQ cells and CAFs, respectively. Figure 29a is a diagram showing migration and invasion of 344SQ cells when treated with CAF CM and ApoSQ-CAF CM in the presence or absence of Notch1 siRNA. Figure 29b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment in the presence or absence of Notch1 siRNA.

30 is a diagram showing that when Notch1 activity is inhibited through DAPT, the expression of proteins related to the Notch1 signaling pathway is inhibited. (A) A diagram showing the immunoblot analysis results and protein expression levels for NICD1, Hes1, and WISP-1 measured in the presence or absence of ApoSQ or NecSQ after CAFs were treated with 10 μM DAPT. (B) A diagram showing the ELISA results of WISP-1 measured in CAF CM, ApoSQ-CAF CM, and NecSQ-CAF CM after CAFs were treated with 10 μM DAPT.

31 is a diagram showing that when Notch1 is inhibited through DAPT, the anti-migration and anti-invasion effects of ApoSQ-CAF CM and ApoSQ are reversed in 344SQ cells and CAFs, respectively. Figure 31a is a diagram showing migration and invasion of 344SQ cells upon treatment with CAF CM and ApoSQ-CAF CM in the presence or absence of DAPT. Figure 31b is a diagram showing migration and invasion of CAFs upon ApoSQ treatment in the presence or absence of DAPT.

32 is a result of confirming the expression levels of Delta-like ligand (DLL) 1, Dll3, Dll4, Jagged-like (JAG) 1 and JAG2, which are Notch ligands, in apoptotic cancer cells 344SQ, A549 and HCT116 by flow cytometry, Dll1 It is a diagram showing that expression is increased only in cancer cells that have been killed.

33 is a diagram showing that only Dll1 expression is increased in killed cancer cells 344SQ, A549, and HCT116. (A) A diagram showing the results of immunoblot analysis for Dll1, Dll3, Dll4, JAG1 and JAG2 in apoptotic cancer cells 344SQ, A549 and HCT116. (B) A diagram showing the amount of Dll1, Dll3, Dll4, JAG1 and JAG2 proteins in the killed cancer cells 344SQ, A549 and HCT116.

34 is a diagram showing that expression of proteins related to the Notch1 signaling pathway is suppressed in CAFs after ApoSQ treatment when Dll1 is neutralized with an anti-Dll1 antibody. (A) A diagram showing the immunoblot analysis results and protein amounts for NICD1, Hes1, and WISP-1 measured in the presence or absence of anti-Dll1 antibody in CAFs contacted with ApoSQ. (B) A diagram showing the ELISA results of WISP-1 measured in the presence or absence of anti-Dll1 antibody in CAFs contacted with ApoSQ.

35 is a diagram showing that expression of proteins related to the Notch1 signaling pathway is suppressed in CAFs when Dll1 is knocked down through siRNA. 35A is a diagram showing the results of immunoblot analysis and the amount of Dll1 protein measured after transfection of 344SQ cells with Dll1 siRNA. In Figure 35b (A) is a diagram showing the immunoblot analysis results and protein amounts for NICD1, Hes1, and WISP-1 measured according to the presence or absence of ApoSQ in CAFs after transfection of 344SQ cells with Dll1 siRNA, (B ) is a diagram showing the ELISA results of WISP-1 measured according to the presence or absence of ApoSQ in CAFs after transfecting 344SQ cells with Dll1 siRNA.

36 is a diagram showing that, when CAFs in contact with ApoSQ are treated with annexin V, brain-specific angiogenesis inhibitor 1 (BAI1) signals are inhibited, thereby inhibiting ApoSQ phagocytosis, Notch1 activation, and WISP-1 secretion in CAFs. Figure 36a is a diagram showing the percentage of CAFs phagocytosis after exposure to ApoSQ cells in the presence or absence of annexin V by flow cytometry. In FIG. 36B, (A) is a diagram showing the immunoblot analysis results for NICD1, Hes1, and WISP-1 in CAFs contacted with ApoSQ in the presence or absence of annexin V, and (B) is a diagram showing the results of immunoblot analysis in the presence or absence of annexin V. A diagram measuring the amount of NICD1, Hes1 and WISP-1 proteins in CAFs in contact with ApoSQ, and (C) shows the ELISA results of WISP-1 measured in CAFs in contact with ApoSQ in the presence or absence of annexin V. It is also

37 is a diagram showing that when CAFs in contact with ApoSQ were transfected with BAI1 siRNA, phagocytosis of CAFs, Notch1 activation, and WISP-1 secretion were suppressed as BAI1 signal was suppressed. 37a is a diagram showing the results of immunoblot analysis and the amount of BAI1 protein measured after CAFs were transfected with BAI1 siRNA. Figure 37b is a diagram showing the phagocytosis of CAFs measured in % by flow cytometry after CAFs were transfected with BAI1 siRNA and exposed to ApoSQ cells. In FIG. 37C, (A) is a diagram showing the immunoblot analysis results for NICD1, Hes1, and WISP-1 measured in the presence or absence of ApoSQ after CAFs were transfected with BAI1 siRNA, and (B) is a diagram showing CAFs with BAI1 siRNA. After transfection, the amount of NICD1, Hes1, and WISP-1 proteins measured according to the presence or absence of ApoSQ is a diagram. It is a diagram showing the results of ELISA.

38 is a diagram showing that when CAFs in contact with ApoSQ are treated with an anti-BAI1 antibody, BAI1 signals are suppressed, and phagocytosis, Notch1 activation, and WISP-1 secretion of CAFs are suppressed. Figure 38a is a diagram showing the percentage of CAFs phagocytosis after exposure of ApoSQ cells in the presence or absence of anti-BAI1 antibody by flow cytometry. In FIG. 38B, (A) is a diagram showing the results of immunoblot analysis for NICD1, Hes1, and WISP-1 in CAFs contacted with ApoSQ in the presence or absence of anti-BAI1 antibody, and (B) is a diagram showing the results of anti-BAI1 antibody A diagram showing the amount of NICD1, Hes1 and WISP-1 proteins in CAFs contacted with ApoSQ in the presence or absence, and (C) is WISP-1 measured in CAFs contacted with ApoSQ in the presence or absence of anti-BAI1 antibody. It is a diagram showing the ELISA result of 1.

39 is a diagram showing that when CAFs contacted with ApoSQ were transfected with BAI1-Flag overexpressing BAI1, phagocytosis of CAFs, Notch1 activation, and WISP-1 secretion increased as BAI1 signal increased. 39a is a diagram showing the results of immunoblot analysis and the amount of BAI1 protein measured after CAFs were transfected with BAI1-Flag. Figure 39b is a diagram showing the phagocytosis of CAFs measured in % by flow cytometry after CAFs were transfected with BAI1-Flag and exposed to ApoSQ cells. In FIG. 39C, (A) is a diagram showing the results of immunoblot analysis for NICD1, Hes1, and WISP-1 measured according to the presence or absence of ApoSQ after CAFs were transfected with BAI1-Flag, and (B) is a diagram showing CAFs transfected with BAI1-Flag. A diagram showing the amounts of NICD1, Hes1, and WISP-1 proteins measured according to the presence or absence of ApoSQ after transfection with Flag. It is a diagram showing the ELISA result of -1.

40 is a diagram showing that ApoSQ increases the activity of Rac1 in a time-dependent manner in CAFs.

41 is a diagram showing that when CAFs contacted with ApoSQ are treated with NSC23766, a Rac1 inhibitor, phagocytosis of CAFs, Notch1 activation, and WISP-1 secretion are inhibited as Rac1 signaling is inhibited. 41a and 41b are diagrams measuring the phagocytic activity of CAFs in contact with ApoSQ in the presence or absence of 100 μM NSC23766. 41a is a diagram showing the number of CAFs phagocytosing ApoSQ cells in % by flow cytometry. 41B is a diagram illustrating phagocytic activity as a phagocytic index using a confocal microscope. In FIG. 41C, (A) is a diagram showing the immunoblot analysis results for NICD1, Hes1, and WISP-1 in CAFs contacted with ApoSQ in the presence or absence of NSC23766, and (B) is a diagram showing the results of immunoblot analysis for ApoSQ and ApoSQ in the presence or absence of NSC23766. A diagram showing the amounts of NICD1, Hes1, and WISP-1 proteins in CAFs contacted, and (C) is a diagram showing ELISA results of WISP-1 measured in CAFs contacted with ApoSQ in the presence or absence of NSC23766.

42 is a diagram showing that when CAFs in contact with ApoSQ are transfected with Notch1 siRNA, the activity of Rac1 and the phagocytosis of CAFs are suppressed according to the inhibition of Notch1 signal. (A) A diagram showing the activity of Rac1 measured after transfecting CAFs with Notch1 siRNA. (B) After transfecting CAFs with Notch1 siRNA and exposing ApoSQ cells, the phagocytosis of CAFs was measured in % by flow cytometry.

43 is a diagram showing that when CAFs contacted with ApoSQ were treated with DAPT, the activity of Rac1 and phagocytosis of CAFs were suppressed as the Notch1 signal was suppressed. (A) A diagram showing the activity of Rac1 measured in the presence or absence of 20 μM DAPT. (B) A diagram showing the phagocytosis of CAFs in contact with ApoSQ in the presence or absence of DAPT through flow cytometry.

44 is a diagram showing an experimental timeline for injecting 344SQ cells subcutaneously on the flanks of syngeneic mice, subcutaneously administering ApoSQ 2 days later, sacrificing the mice 6 weeks later, and isolating Thy1 CAFs from primary tumor tissue. am.

45 shows Thy1 ⁺ CAFs isolated from primary tumor tissue after 6 weeks of subcutaneous administration of ApoSQ 2 days after 344SQ cells were injected subcutaneously into the flanks of syngeneic mice, and mice sacrificed 6 weeks later. Thy1 ⁺ CAFs of the control group , CAF activation markers (including Acta2, Col1α1, Fn, Itg beta 1, Spp1, Pdgfrα, and Pdgfr beta), MMPs (including Mmp1a, 2, 9, and 12), growth factors and chemokines (Vegfa, Hgf, It is a diagram measured by qRT-PCR that the mRNA expression level of Notch1-related molecules (including Notch1, Wisp1 (Ccn4), Hey1, Hey2, and Hes1) is increased, while the mRNA expression level of Cxcl12 and Cxcl14 is decreased. . 45A is a diagram showing a heat map made based on qRT-PCR results. 45B is a diagram showing qRT-PCR results for each mRNA.

46 is a diagram showing the experimental timeline of administering ApoSQ and LY3039478 to mice.

47 is a diagram showing that there is no significant difference between each group as a result of measuring mouse weight, tumor weight and tumor volume by administering LY3039478 (8 mg/kg), ApoSQ or ApoSQ and LY3039478 (8 mg/kg) to mice . (A) A diagram showing the mouse weight, tumor weight and tumor volume in each group. (B) A diagram showing images of tumors in each group.

48 is a result of measuring the number and metastasis rate of tumor nodules metastasized to the lung by administering LY3039478, ApoSQ or ApoSQ and LY3039478 to mice, and as a result, the number and metastasis rate of tumor nodules decreased when ApoSQ was administered, and the effect of additional administration of LY3039478 It is a diagram showing that is reversed. (A) A diagram showing the number of tumor nodules and metastasis rate in each group. (B) It is a diagram showing images of tumor nodules metastasized to the lungs in each group.

49 shows that when ApoSQ was administered, the mRNA expression levels of CAF activity markers, MMPs, growth factors, and chemokines decreased in Thy1 ⁺ CAFs isolated from primary tumor tissue, and the mRNA expression levels of Notch1-related molecules increased, but LY3039478 was additionally administered. It is a diagram showing that the effect is reversed upon administration.

50 shows that in primary tumor tissue through immunofluorescence staining, when ApoSQ was subcutaneously administered, the fluorescence intensity of α-SMA decreased and the fluorescence intensity of NICD1 and WISP-1 increased in Thy-1 ⁺ CAFs, but LY3039478 It is a diagram showing that the effect is reversed when additionally administered.

51 shows a decrease in the expression of α-SMA and an increase in the expression of NICD1 and WISP-1 through immunocytochemical analysis in Thy1 ⁺ CAFs isolated from primary tumor tissues of mice administered with ApoSQ, but when LY3039478 is additionally administered. It is a diagram showing that the effect is reversed. 51A is a diagram showing the expression level of α-SMA in each group through immunocytochemical analysis. 51b is a diagram showing the expression levels of NICD1 and WISP-1 in each group through immunocytochemical analysis.

52 shows that the expression of WISP-1 increases in the culture medium and serum of Thy1 ⁺ CAFs isolated from primary tumor tissues of mice administered with ApoSQ, and the effect is reversed when LY3039478 is additionally administered, but the isolated CD326 ⁺ tumor cells and It is a diagram showing that there is no such effect on CD11b ⁺ tumor-associated macrophages.

53 is a diagram showing that cell migration and invasion are inhibited in CD326 ⁺ tumor cells and Thy1 ⁺ CAFs isolated from primary tumor tissues of mice administered with ApoSQ, but the above effects are reversed when LY3039478 is additionally administered. Figure 53a is a diagram measuring migration and invasion of isolated CD326 ⁺ tumor cells. Figure 53b is a diagram showing the migration and invasion of isolated Thy1 ⁺ CAFs.

54 shows that phosphorylation of signaling molecules related to migration and invasion, such as Smad2/3, FAK, ERK, and Akt, and expression of MMP2/12 protein are inhibited in CD326 ⁺ tumor cells isolated from primary tumor tissues of mice administered with ApoSQ. , It is a diagram showing that the effect is reversed when LY3039478 is additionally administered. (A) A diagram showing the results of immunoblot analysis for phosphorylation and MMP2/12 of signaling molecules related to cell migration and invasion in isolated CD326 ⁺ tumor cells. (B) It is a diagram measuring the amount of phosphorylation and MMP2/12 of signaling molecules related to cell migration and invasion in isolated CD326 ⁺ tumor cells.

55 is a diagram showing the experimental timeline of administering CAF CM, ApoSQ-CAF CM, ApoSQ-CAF CM and anti-WISP-1 antibody, or ApoSQ-CAF CM and IgG to mice after subcutaneous injection of 344SQ cells.

56 shows that when CAF CM, ApoSQ-CAF CM, ApoSQ-CAF CM and anti-WISP-1 antibody or ApoSQ-CAF CM and IgG were intratumorally administered to mice, there was no significant difference in mouse weight between groups. It is also

57 is a result of measuring the number of tumor nodules and metastasis rate by administering CAF CM, ApoSQ-CAF CM, ApoSQ-CAF CM and anti-WISP-1 antibody or ApoSQ-CAF CM and IgG to mice, and ApoSQ-CAF CM administration When compared with the CAF CM experimental group, the number and metastasis rate of tumor nodules metastasized to the lungs decreased, and the effect was reversed when an anti-WISP-1 antibody was additionally administered. (A) A diagram showing the number and metastasis rate of tumor nodules that metastasized to the lungs in each group. (B) It is a diagram showing images of tumor nodules that metastasized to the lungs in each group.

58 shows that when ApoSQ-CAF CM was intratumorally administered to mice, compared to the CAF CM experimental group, cell migration and invasion were inhibited in CD326 ⁺ tumor cells and Thy1 ⁺ CAFs isolated from primary tumor tissue, but anti-WISP -1 It is a diagram showing that the effect is reversed when the antibody is additionally administered. 58a is a diagram showing migration and invasion of CD326 ⁺ tumor cells isolated from primary tumor tissue. 58b is a diagram showing the migration and invasion of Thy1 ⁺ CAFs isolated from primary tumor tissue.

59 shows that when ApoSQ-CAF CM was intratumorally administered to mice, compared to the CAF CM experimental group, CD326 ⁺ tumor cells isolated from primary tumor tissues were associated with migration and invasion, such as Smad2/3, FAK, ERK, and Akt. Phosphorylation of signal molecules and expression of MMP2/12 protein are suppressed, but the effect is reversed when anti-WISP-1 antibody is additionally administered. (A) A diagram showing the results of immunoblot analysis for molecules involved in cell migration and invasion in CD326 ⁺ tumor cells. (B) A diagram showing the amount of proteins involved in cell migration and invasion in isolated CD326 ⁺ tumor cells.

60 shows CAF activity markers (Acta2, Col1α1, Itg beta ¹ , Spp1 and Pdgfrα included ) and MMP (including Mmp1a, 2, and 12) mRNA expression levels are suppressed, but the effect is reversed when an anti-WISP-1 antibody is additionally administered.

61 is a diagram showing an experimental timeline of administration of rWISP-1 at a concentration of 12.5 μg/kg or 25 μg/kg after subcutaneous injection of 344SQ cells into mice.

62 is a diagram showing that when rWISP-1 was intratumorally administered to mice at a concentration of 12.5 μg/kg or 25 μg/kg, there was no significant difference in body weight between groups.

63 shows the number of tumor nodules and the metastasis rate measured by administering rWISP-1 to mice at a concentration of 12.5 μg/kg or 25 μg/kg. It is a diagram showing a decrease in (A) A diagram showing the number and metastasis rate of tumor nodules that metastasized to the lungs in each group. (B) It is a diagram showing images of tumor nodules that metastasized to the lungs in each group.

64 is a diagram showing that migration and invasion of CD326 ⁺ tumor cells and Thy1 ⁺ CAFs are inhibited in a concentration-dependent manner when rWISP-1 is intratumorally administered at a concentration of 12.5 μg/kg or 25 μg/kg to mice . Figure 64a is a diagram showing the migration and invasion of CD326 ⁺ tumor cells. Figure 64b is a diagram showing the migration and invasion of Thy1 ⁺ CAFs.

65 shows signal molecules related to migration and invasion, such as Smad2/3, FAK, ERK, and Akt, in CD326 ⁺ tumor cells when rWISP-1 was intratumorally administered to mice at a concentration of 12.5 μg/kg or 25 μg/kg. It is a diagram showing that the phosphorylation of them and the expression of MMP2/12 protein are suppressed. (A) A diagram showing the results of immunoblot analysis for molecules involved in cell migration and invasion in CD326 ⁺ tumor cells. (B) A diagram showing the amount of proteins involved in cell migration and invasion in CD326 ⁺ tumor cells.

66 shows CAF activity markers (including Acta2, Col1α1, Itg beta 1, Spp1 ^and Pdgfrα) and It is a diagram showing that the mRNA expression level of MMP (including Mmp1a, 2, and 12) is suppressed.

67 shows that when ApoSQ-CAF CM was intratumorally administered to mice, compared to the CAF CM experimental group, the expression of genes related to tumor progression and invasion was downregulated in CD326 ⁺ tumor cells isolated from primary tumor tissues, and tumor progression and It is a diagram showing that gene expression related to invasion inhibition is up-regulated.

68 is a diagram showing that, when ApoSQ-CAF CM was intratumorally administered to mice, gene expression related to cell adhesion and ECM remodeling was down-regulated in Thy1 ⁺ CAFs isolated from primary tumor tissues, compared to the CAF CM experimental group.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Experimental Example 1: Materials and Methods

1-1. reagent

DAPT (D5942) was purchased from Sigma-Aldrich (St. Louis, MO, USA), TGF-beta 1 (240-B-010) and mouse rWISP-1 (1680-WS), mouse neutralizing WISP-1 antibody (MAB1680 R&D Systems) and IgG (MAB0061) were purchased from R&D Systems (Minneapolis, MN, USA). LY3039478 (HY-12449) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

1-2. antibody

Antibodies used for Western blot, immunofluorescence staining, flow cytometry and cell sorting are as follows (see Table 1).

Antigen Vendor Cat. No Source Species cross-reactivity Application Dilution p-Smad2 Cell Signaling 3108 Rabbit monoclonal H, M, R, Mi IB 1:1000 p-Smad3 Cell Signaling 9520 Rabbit monoclonal H, M, R IB 1:1000 p-AKT Cell Signaling 4060 Rabbit monoclonal H, M, R, Hm, Mk, Dm, Z, B IB 1:1000 p-FAK Cell Signaling 3283 Rabbit polyclonal H, M, R, Hm, Pg IB 1:1000 p-Src Cell Signaling 2101 Rabbit polyclonal H, M, R IB 1:1000 p-ERK Cell Signaling 9101 Rabbit polyclonal B, Ce, Dm, Hm, H,
Mi, Mk, M IB 1:1000 p-p38 Santa Cruz sc-17852-R Rabbit polyclonal H, M, R IB 1:1000 Smad2/3 Cell Signaling 3102 Rabbit polyclonal H, M, R, MK IB 1:1000 Akt Cell Signaling 4691 Rabbit monoclonal H, M, R, Mk, Dm IB 1:1000 FAK Cell Signaling 3285 Rabbit polyclonal H, M, R, Hm, B, Pg IB 1:1000 Src Santa Cruz sc-19 Rabbit polyclonal H, M, R IB 1:1000 ERK Santa Cruz sc-93-G Goat polyclonal H, M, R, C, F IB 1:1000 p38 Santa Cruz sc-535-G Goat polyclonal H, M, R IB 1:1000 MMP2 Cell Signaling #87809 Rabbit monoclonal H, M IB 1:1000 MMP12 Santa Cruz sc-390863 Mouse monoclonal H, M, R IB 1:1000 α-SMA Abcam ab7817 Mouse monoclonal H, M, R, Rb, Pg IB, IF, IHC 1:1000
1:200 Col1α GeneTex GRX82721 Rabbit polyclonal H, M, R IB 1:1000 FN Abcam ab2413 Rabbit polyclonal H, M IB 1:1000 LIF Abcam ab113262 Rabbit polyclonal H, M, R IB 1:1000 WISP-1 Abcam ab178547 Rabbit polyclonal H, M, R IB, IF 1:1000 R&D Systems AF1680 Sheep polyclonal M IF, IHC 1:200 NICD1 Cell Signaling #4147 Rabbit monoclonal H, M, R IB, IF, IHC 1:1000
1:200 Abcam ab8925 Rabbit M, H, R IHC-P, IB 1:1000 Notch-1 Santa Cruz sc-373891 Mouse monoclonal H, M, R IHC 1:100 Hes1 Abcam ab71559 Rabbit polyclonal H, M, R IB 1:1000 DLL1 Invitrogen PA5-19106 Goat polyclonal H, M, R IB, FACS
Neutralizing 1:1000 1:100
20 µg/ml DLL3 Invitrogen PA5-26336 Rabbit polyclonal H, M, R IB, FACS 1:1000 1:100 DLL4 Invitrogen PA5-85931 Rabbit polyclonal H, M IB, FACS 1:1000 1:100 Jag1 Cell Signaling 2620 Rabbit monoclonal H, M IB, FACS 1:1000 1:100 Jag2 Santa Cruz sc-515725 Mouse monoclonal H, M, R IB, FACS 1:1000 1:100 beta-Actin Santa Cruz sc-69879 Mouse monoclonal Broad species IB 1:1000 α-Tubulin SIGMA T5168 Mouse monoclonal M,R IB 1:3000 BAI1 R&D Systems AF4969 Sheep polyclonal H, M Neutralizing 8 µg/ml WISP-1 R&D Systems MAB1680 Rat monoclonal M Neutralizing 10 µg/ml IgG R&D Systems MAB0061 Rat monoclonal M Neutralizing 10 µg/ml IgG eBioscience 14-4888 Armenian hamster monoclonal Broad species Neutralizing Integrin αv eBioscience 14-0512 Rat monoclonal H, M Neutralizing Integrin α5 eBioscience 14-0493 Armenian hamster monoclonal M,R Neutralizing 10 μg/ml (CAF) Integrin beta 1 eBioscience 16-0291 Armenian hamster monoclonal M,R Neutralizing 3 µg/ml (344SQ) Integrin beta 3 eBioscience 11-0611 Armenian hamster monoclonal M,R Neutralizing Integrin beta 5 eBioscience 14-0497 Mouse monoclonal Hm, H, M Neutralizing CD31 Abcam ab7388 Rat monoclonal M Cell sorting CD45 Abcam ab25386 Rat monoclonal M Cell sorting CD68 Abcam ab53444 Rat monoclonal M Cell sorting CD326 BD Bioscience 552370 Rat monoclonal M Cell sorting Mouse IgG
(HRP) GeneTex GTX213111 Goat Not applicable IB 1:5000 Rabbit IgG (HRP) GeneTex GTX213110 Goat Not applicable IB 1:5000 Goat IgG
(HRP) Santa Cruz sc-2354 Mouse Not applicable IB 1:5000 Mouse IgG
(Alexa 488) Thermo Fisher
Scientific A11029 Goat Not applicable FACS, IHC 1:100
1:400 Mouse IgG
(Alexa 594) Thermo Fisher
Scientific A11032 Goat Not applicable IHC 1:400 Rabbit IgG
(Alexa 488) Thermo Fisher
Scientific A11034 Goat Not applicable FACS, IF, IHC 1:100
1:400 Rabbit IgG
(Alexa 594) Thermo Fisher
Scientific A11012 Goat Not applicable FACS. IF, IHC 1:100
1:400 Goat IgG
(Alexa 488) Thermo Fisher
Scientific A11055 Donkey Not applicable FACS 1:100 Goat IgG
(Alexa 647) Thermo Fisher
Scientific A21447 Donkey Not applicable FACS 1:100 Sheep IgG
(Alexa 594) Thermo Fisher
Scientific A11016 Donkey Not applicable IF, IHC 1:400 * Abbreviation: IB-Immunoblot, IHC-Immunohistochemistry, IHC-P-Immunohisrochemistry-paraffin, IF-Immunofluorescence, H-human, M-mouse, R-rat, Rb-rabbit, P-pig, Mi-mink, Hm-hamster , Mk-monkey, Dm-drosophila melanogaster, Z-zebrafish, B-Bovine, Pg-pig, Ce-caenorhabditis elegans, F-frog

1-3. Isolation and cell culture of Carcinoma-Associated Fibroblasts (CAFs)

CAFs were isolated from lung tumors of Kras-mutant (Kras ^LA1 ) mice using magnetic-activated cell sorting with the fibroblast-specific marker Thy1. CAFs were then supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (100 U/100 μg, Welgene, Gyeongsan, Korea), 2 mM L-glutamine (Welgene), and 1 mM sodium pyruvate (Welgene). cultured in alpha-MEM (Welgene). For immortalization, CAFs were stably transfected with TERT plasmid (pCDH-3xFLAG-TERT, Addgene 51 plasmid # 51631) using Lipofector-EXT (AptaBio, Yongin, Korea). Primary cells used in the experiments were subcultured less than 6 times. Human cancer cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). 344SQ cells (lung adenocarcinoma cell line, University of Texas MD Anderson Cancer Center, USA) and various human cancer cell lines [A549 (non-small cell lung cancer cell line), HCT116 (colorectal cancer cell line) and MCF-7 (breast cancer cell line)] were cultured in 10% FBS. and RPMI 1640 (Welgene) supplemented with penicillin/streptomycin (100 U/100 μg).

1-4. induction of apoptosis

Cancer epithelial cell lines were exposed to 254 nm wavelength ultraviolet light for 15 minutes and then incubated with RPMI-1640 (with 10% FBS) at 37° C. and 5% CO ₂ for 2 hours. As a result of evaluating nuclear morphology using a light microscope in the Wright-Giemsa-stained samples, it was confirmed that the irradiated cells were apoptotic. Lysed (necrotic) cancer cells were obtained by several freeze-thaw cycles. Apoptosis and necrosis were confirmed by annexin V-FITC/propidium iodide (BD Biosciences, San Jose, CA, USA) staining followed by flow cytometry on a FACSCalibur system (ACEA Novocyte 3000, Agilent, Santa Clara, CA, USA).

1-5. Culture of CAFs and preparation of CAF conditioned medium

CAFs were plated at 3 X 10 ⁵ cells/ml and cultured in a suitable medium at 37°C and 5% CO ₂ . After overnight incubation, serum was removed with X-VIVO 10 medium (04-380Q, Lonza, Basel, Switzerland) for 24 hours before cell stimulation. For stimulation, the culture medium was replaced with X-VIVO 10 containing killed or necrotic cancer cells (9 X 10 ⁵ cells/ml). After 20 hours, the supernatant was harvested by centrifugation and used as a conditioned medium (CM) for stimulation of target cancer epithelial cells (5 X 10 ⁵ cells/ml) or CAFs (3 X 10 ⁵ cells/ml). For in vivo experiments, the conditioned medium was stored at -80°C until needed.

1-6. Migration and invasion assay

Cell migration and invasion were tested using Transwell chambers (Corning Inc, Corning, NY, USA) coated with 30 μg/ml of fibronectin and 300 μg/ml of Matrigel matrix, respectively, according to the manufacturer's instructions. Cancer cells or CAFs pre-cultured in conditioned medium from CAFs in the absence or presence of TGF-beta 1 (10 ng/ml) for 48 or 24 hours (1 X 10 ⁵ cells/well for migration assay, invasion assay 1 X 10 ⁵ cells/well) were plated with serum-free RPMI in the upper chamber, and placed in RPMI 1640 supplemented with 10% FBS in the bottom well at 37°C for 16 hour transfer time or 24 hour infiltration time. In addition, migration and invasion in CAFs were performed 24 h after direct exposure with ApoSQ and stimulation with TGF-beta 1 (10 ng/ml). Prior to TGF-beta 1 stimulation, CAFs were directly exposed to apoptotic or necrotic cancer cells and then washed X-VIVO. After fixation in 4% paraformaldehyde, immobile or non-infiltrated cells on the upper surface of the membrane were scraped off with a cotton swab. Cells on the lower surface were stained with 0.1% crystal violet, washed with distilled water, and three random microscope fields were photographed (10X magnification) and counted.

1-7. Western blotting analysis

A standardized Western blot was performed using whole cell extracts. Whole cell extracts were prepared from killed cells or CAFs or target cancer cells co-cultured with conditioned media. Cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), and washed in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxygenate). cholate, 0.1% SDS, 1.0% Triton X-100, 5 mM EDTA) for 30 min on ice with protease inhibitors. Equal amounts of protein were run on SDS-PAGE gels (#161-0158, Bio-Rad Laboratories, Herc-Res, CA, USA) using a wet transfer system (Bio-Rad Laboratories) and transferred to nitrocellulose membranes (10600001, GE Healthcare Life Science). After blocking with 5% BSA-TBST or 5% milk-TBST for 1 hour, blots were incubated with primary antibody overnight, followed by incubation with secondary antibody for 1 hour at 37°C. Quantification was performed using the Odyssey image analysis system (Licor Biosciences, Lincoln, Nebraska).

1-8. Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the isolated cells using TRIzol reagent (RNAiso plus, Takara Bio Inc., Kusatsu, Japan). After reverse transcription was performed using the ReverTra Ace ^TM qPCR RT Kit and Master Mix (Toyobo, Osaka, Japan), SYBR Green-based qRT-PCR was performed using the QuantStudio ^TM 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was performed using mRNA was extracted from cells grown to 80% confluence on 6-well plates and quantified using a real-time PCR system (StepOnePlus system, Applied Biosystems, Foster City, Calif.). mRNA levels were normalized to Hprt mRNA and reported as fold change in expression relative to control. Primer sequences used for target genes are as follows (see Table 2).

murine genes forward (5' → 3') reverse (5' → 3') Acta2 CCCAGATTATGTTTGAGACCTTC ATCTCCAGAGTCCAGCACAATAC Cav-1 CACACCAAGGAGATTGACCTGG CCTTCCAGATGCCGTCGAAACT Ccn1 TTTACAGTTGGGCTGGAAGC CACCGCTCTGAAAGGGATCT Ccn2 GCTTGGCGATTTTAGGTGTC CAGACTGGAGAAGCAGAGCC Ccn3 GTCACCAACAGGAATCGCCAGT GTAGGTGGATGGCTTTCAGGGA Ccn5 TGTGTGACCAGGCAGTGATGCA CAGGCTGTGCTCCAGTTTGGAC Ccn6 CACCTGTAACGAAGCCGAGATC ACTCACATCCAACTGCCACAAGA Col1a1 CAAGAAGACATCCCTGAAGTC ACAGTCCAGTTCTTCATTGC Cxcl12 TGCATCAGTGACGGTAAACCA CACAGTTTGGAGTGTTGAGGAT Cxcl14 TACCCACACTGCGAGGAGAAGA CGCTTCTCGTTCCAGGCATTGT Dll1 CCGGCTGAAGCTACAGAAAC AGCCCCAATGATGCTAACAG Dll3 CTGGTGTCTTCGAGCTACAAAT TGCTCCGTATAGACCGGGAC Dll4 CCTCTCGAACTTGGACTTGC TGGAAATACAGATGCCCACA Fap GTCACCTGATCGGCAATTTGT CCCCATTCTGAAGGTCGTAGAT Fgf1 CCAAGGAAACGTCCACAGTCAG ACGGCTGAAGACATCCTGTCTC Fn CACGATGCGGGTCACTTG CTGCAACGTCCTCTTCATTCTTC Hes1 CTACCCCAGCCAGTGTCAAC CCTTCGCCTCTTCTCCATGA Hes5 ATCAACAGCAGCATAGAGCAG CGAAGGCTTTGCTGTGTTTCA Hey1 ACATCGTCCCAGGTTTTGGC GCTTCTCAAAGGCACTGGGT Hey2 TCCAGGCTACAGGGGGTAAA CAAGGCCTTCCACTGAGCTT Hgf CCTGACACCACTTGGGAGTA CTTCTCCTTGGCCTTGAATG Hprt CCAGTGTCAATTATATCTTCAAC CAGACTGAAGAGCTACTGTAATG Itg beta 1 CTCCAGAAGGTGGCTTTGATGC GTGAAACCCAGCATCCGTGGAA Jag1 GGAAGTGGAGGAGGATGACA GTCCAGTTCGGGTGTTTTGT Jag2 TCCGAGTACGCTGTGATGAG GGCTTTCTTTGCATTCTTTGC Mmp1a AGGAAGGCGATATTGTGCTCTCC TGGCTGGAAAGTGTGAGCAAGC Mmp2 CAAGGATGGACTCCTGGCACAT TACTCGCCATCAGCGTTCCCAT Mmp3 CTCTGGAACCTGAGACATCACC AGGAGTCCTGAGAGATTTGCGC Mmp9 TGCCCAGCGACCACAACTC CGGACCCGAAGC GGACATT Mmp12 CACACTTCCCAGGAATCAAGCC TTTGGTGACACGACGGAACAGG Mmp14 GTGAGCGTTGTGTGTGGGTA CCCAAGGCA GCAACTTCAG Notch1 GCCGCAAGAGGCTTGAGAT GGAGTCCTGGCATCGTTGG Pdgfrα CTCTTGGAGATAGACTCCGTAGG ACTTCTCTCCTGCGAATGG Pdgfr beta TGGCCTCTGAGGACTAAAGC AACAGAAGACAGCGAGGTGG Spp1 GCTTGGCTTATGGACTGAGGTC CCTTAGACTCACCGCTCTTCATG Tgf beta TGCCGTACAACTCCAGTGAC TGGAGCAACATGTGGAACTC Tnc CCATCAGTACCACGGCTACC CCCTTCATCAGCAGTCCAGG Vegfa GTACCTCCACCATGCCAAGT TCACATCTGCAAGTACGTTCG Wisp-1 (ccn4) CAATAGGAGTGTGTGCACAGGT TACCTGCAGTTGGGTTGGAAG

1-9. cytokine array

Cytokine arrays were performed using the Proteome Profiler Mouse XL Cytokine Array Kit (#ARY028, R&D Systems, USA) in CAF CM, ApoSQ-CAF CM and ApoSQ CM according to the manufacturer's instructions. Array membranes were incubated with conditioned medium overnight and membrane-bound cytokines were detected using a biotinylated detection antibody and streptavidin-horseradish peroxidase. Pixel density of cytokine spots was analyzed using Image J software (NIH, Bethesda, Maryland, USA; http://rsb). info.nih.gov/ij/).

1-10. immunofluorescence

CAFs grown on glass coverslips until confluent were fixed with a 4% paraformaldehyde solution for 8 min at room temperature. For staining of paraffin-embedded tumors, formalin fixation was performed for 30 min at room temperature and IF-Wash buffer (0.05% NaN3, 0.1% BSA, 0.2% Triton X-100 and 0.05% Tween-20 in PBS) was used. . After fixation, samples were washed three times with wash buffer for 5 minutes each and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 5 minutes at room temperature. 5% BSA in PBS with or without mouse IgG blocking reagent was used for immunocytochemistry and immunohistochemistry, respectively. After 1 hour, target proteins were captured by each primary antibody for 18 hours at 4°C. Captured proteins were observed for 1 hour in the dark by fluorescence-coupled IgG. After staining, all slides were fixed with Vectashield Mounting medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI (Vector Laboratories, Burlingame, CA, USA) and confocal microscopy (LSM5 PASCAL, Carl Zeiss, Jena, Germany). ) or observed through a fluorescence microscope (Eclipse Ti2-U, Nikon, Tokyo, Japan).

1-11. ELISA

WISP-1 and LIF in conditioned media and serum were measured using ELISA kits (R&D Systems) according to the manufacturer's instructions. For serum cytokine quantification, blood was collected from mice via puncture, and serum was isolated by centrifugation at 1600×g for 5 minutes at 4° C.

1-12. Transient transfection and luciferase activity assay

CAFs were transfected with WISP-1 (Bioneer Inc, Daejeon, Korea), LIF (Bioneer), Notch1 (Bioneer) or BAI1 ( Dharmacon. CO, USA) was transiently transfected with either specifically targeting siRNA or control siRNA (Bioneer, Dharmacon) at a final concentration of 100 nM. After overnight transfection, cells were cultured in suitable media for 24 hours and stimulated with ApoSQ cells. 344SQ cells were transiently transfected with Dll1-specific siRNA (D-050912-01-0020, Dharmacon) or control siRNA before UV irradiation. The siRNA sequences used to target the genes are as follows (see Table 3).

sense antisense WISP-1 5'-GGAAUCCUAACGAUAUCUU-3' 5′-AAGAUAUCGUUAGGAUUCC-3′ LIF 5′-CGACCACUCUGACAAAGAA-3′ 5'-UUCUUUGUCAGAGUGGUCG-3' Notch1 5′-CCCUUUGAGUCUUCAUACA-3′ 5′-UGUAUGAAGACUCAAAGGG-3′ Dll1 5′-GCACGGACCUUGAGGACAG-3′ 5'-CUGUCCUCAAGGUCCGUGC-3' BAI1 5'-GUGUCAUCCUCGUACAGU-3' 5'-ACUGUACGAGGAUGACAC-3'

Gene overexpression experiments were performed by plating 0.5 X 10 ⁶ cells/ml of CAFs in a 60-mm dish for 24 hours. WISP-1 overexpression was analyzed using a mouse WISP-1 gene ORF cRNA clone expression plasmid (MG5A0255-CH, Sino Biological, North Wales, PA). CAFs were transfected with 2.5 μg/well of plasmid DNA using Lipofectamine 2000 (#12566014; Thermo Fisher Scientific, Waltham, Mass.) reagent according to the manufacturer's instructions. Cells were ready for further experiments after 24 hours of culture in growth media after transfection. Mock-transfected cells were transfected with the control vector, pcDNA3, in the same way as WISP-1 transfected cells. For BAI1 overexpression, 2.0 μg of pEBB empty vector or pEBB-BAI-Flag plasmid (Gwangju Institute of Science and Technology, Gwangju, Korea) for 48 h using Lipofectamine 2000 reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. CAFs were transfected. Cell lysates were measured for WISP-1 and BAI1 gene and protein expression to confirm the potency of the plasmids.

For the luciferase assay, CAFs were transfected using Lipofectimin LTX Transfection Reagent and PLUS Reagent (Life Technologies, Darmstadt, Germany). Cells were transfected with 800 ng/well 4×CSL luciferase plasmid (#41726 Addgene, Wattown, MA) to produce luciferase in response to Notch pathway activation and transfected with 200 ng/well Renilla luciferase plasmid . Luciferase activity was normalized to Renilla activity and values were reported as fold change relative to the PGL2-control vector. All conditions were performed in triplicate for each independent experiment, and luciferase experiments were performed using the Dual-Luciferase Assay System (Promega, Madison, WI, USA).

1-13. Neutralization of WISP-1 in conditioned media

Conditioned medium from CAFs was incubated for 2 hours with 10 μg/ml mouse anti-mouse WISP-1 neutralizing antibody (R&D Systems) or 10 μg/ml IgG isotype control (R&D Systems). The neutralizing effect of anti-WISP-1 antibodies was measured by WISP-1 ELISA.

1-14. flow cytometry

for Notch ligand staining. UV-irradiated killed cancer cells or live cancer cells were cultured in FACS buffer (0.1% BSA and 0.1% sodium azide in PBS) with anti-Dll1, anti-Dll3, anti-Dll4, anti-Jag1 or anti-Jag2 (1 :100) for 30 minutes at room temperature. Cells were then incubated for 30 minutes at room temperature in FACS buffer with Alexa 488- or 594-labeled secondary antibodies. After incubation, the cells were washed three times with FACS buffer, and the expression level of Notch ligand was analyzed using a flow cytometer (ACEA Novocyte 3000, Agilent, Santa Clara, CA, USA). Data were analyzed using Novoexpress software (Agilent).

1-15. phagocytosis assay

Phagocytosis analysis of killed cancer cells was performed through the flow cytometry and immunofluorescence method. First, CAFs were stained with PKH26 (red), and then co-cultured with apoptotic 344SQ cells labeled with PKH67 (green) at a ratio of 1:3 for 24 hours. After washing, the percentage of phagocytosis by CAFs was measured by two-color flow cytometry. For immunofluorescence, killed 344SQ cells labeled with PKH67 were co-cultured with CAFs for 24 hours, washed, and CAFs were fixed with 3.7% w/v paraformaldehyde and 0.1% Triton X-100. Permeabilized for 15 minutes. F-actin was stained with rhodamine phalloidin (Invitrogen) according to the manufacturer's instructions. Images were captured with a confocal microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany), and the phagocytic index was calculated by the formula (killed cells/200 total CAFs) x 100.

1-16. Rac1 activity assay

CAFs were pre-incubated for 2 hours with DAPT (10 or 20 μM) or transfected with Notch1 or control siRNA for 24 hours before co-culture with ApoSQ at a 1:3 ratio for 24 hours. Rac1 activity was measured using the G-LISA Rac1 activity assay kit (Cytoskeleton, Denver, CO, USA) according to the manufacturer's instructions. That is, cell lysate containing the same amount of protein was added to the Rac1-GTP binding plate and incubated at 4° C. for 30 minutes. The plate was washed with PBS containing 0.05% of Tween 20 and reacted with an antigen presentation buffer. After washing, the plates were reacted with anti-Racl primary and secondary antibodies. The reaction was observed with horseradish peroxidase detection reagent and stopped with stop solution. The plate was measured for absorbance at 490 nm using a spectrophotometer.

1-17. mouse experiment

The following experimental protocol was approved by the Animal Care Committee of Ewha Medical Research Institute (EUM19-0430). Mice were cared for and handled according to the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Lung cancer metastasis studies using syngeneic tumor experiments were performed by a known method. Briefly, 344SQ cells (1×10 ⁶ cells in 100 μl PBS per mouse) were injected subcutaneously into syngeneic (129/Sv) mice in the right posterior flank. Two days after the first injection, a second injection was given with 100 μl of PBS in the presence or absence of 1×10 ⁷ killed 344SQ cells in the same lesion.

For the Notch1 inhibition experiment, the selective Notch1 inhibitor LY3039478 (8 mg/kg) was orally administered with 15% sugar gel vehicle three times a week for 6 weeks one day before ApoSQ injection. For conditioned media experiments, 2 days after injection of 344SQ, conditioned media derived from CAFs (100 μl per mouse) with or without neutralizing anti-WISP-1 antibody (10 μg/ml) or isotype IgG was added for 1 week 3 It was administered via intratumor injection. Mice were monitored daily for tumor growth and sacrificed 6 weeks after injection. A necropsy was performed to examine the diameter and weight of the subcutaneous tumor mass, the status of lung metastases (number or incidence of nodules), and histological evaluation of formalin-fixed, paraffin-embedded, immunofluorescence-stained primary tumors.

1-18. Thy1 from primary tumor ⁺ CAFs, CD326 ⁺ Tumor cells and CD11b ⁺ Isolation of tumor-associated macrophages

To obtain a single cell suspension from the primary tumor of the metastatic mouse model, solid, fresh tumor tissue was dissociated using a tumor dissociation kit combined with a MACSTM Dissociator (all Miltenui Biotec Inc., San Diego, CA) according to the manufacturer's instructions. made it Briefly, tumor tissue from each mouse was transferred to a MACS C tube (Miltenui Biotec) containing the enzyme mixture from the kit for enzymatic digestion. After dissociation, each tissue homogenate was filtered through 70- and 40-μm sterile nylon mesh and red blood cells were lysed with red blood cell lysis buffer. After centrifugation at 300 g for 10 min, the remaining cells were collected with anti-CD45 (Abcam, Cambridge, UK), anti-CD68 (Abcam), anti-CD31 (Abcam) and anti-CD326 (Abcam) to select for non-CAF cells. BD Bioscience, San Diego, CA, USA). Labeled cells were reduced using pre-washed Dynabeads ^™ (Invitrogen) from the total cell suspension and separated from the magnetic beads using a PureProteome ^™ magnetic stand (Millipore, Billerica, MA, USA). In the unlabeled cell supernatant, Thy1 ⁺ CAFs were sorted using CD90.2 MicroBeads and a MACS MS column (Miltenyi Biotec) according to the manufacturer's instructions. In the negatively selected cell supernatant, CD326 ⁺ epithelial tumor cells and CD11b ⁺ tumor-associated macrophages were isolated using CD326 and CD11b MicroBeads (Miltenyi Biotec), respectively. Isolated cells were cultured in complete medium. Thy1 ⁺ cells were cultured in α-MEM (Welgene) supplemented with 20% FBS and 1% penicillin/streptomycin, and CD326 ⁺ cells were cultured in RPMI 1640 (Welgene) supplemented with 10% FBS and 1% penicillin/streptomycin. and CD11b ⁺ cells were cultured in DMEM (Welgene) supplemented with 10% FBS and 1% penicillin/streptomycin. Isolated individual cell groups were validated by qRT-PCR, and individual cells were isolated within randomly selected mouse primary tumors within each group of two or three.

1-19. Reverse transcription-PCR array

For gene expression analysis related to tumor metastasis in CD326 ⁺ tumor cells isolated from primary tumors and gene expression related to extracellular matrix and aggregation molecules in Thy1 ⁺ CAFs isolated from primary tumors, mouse tumor metastasis RT ² Profiler ^TM PCR Array ( PAMM-028ZA-6, Qiagen, Hilden, Germany) and mouse extracellular matrix and aggregation molecule RT ² Profiler ^TM PCR Array (PAMM-013ZA-6, Qiagen) were used. RNA isolation, DNase treatment and RNA washing were performed according to the instructions of the manufacturer (Takara Bio). The isolated RNA was reverse transcribed into cDNA using the RT ² First Strand kit (Qiagen). PCR was performed using RT ² SYBR Green qPCR Master Mix (Qiagen), QuantStudio 3 Real-Time PCR system and ABI PRISM7900 instrument (Applied Bio-Systems). The expression level data was normalized using the average Ct value of Gapdh (glyceraldehyde 3-phosphate dehydrogenase), a housekeeping gene in the array. Each assay was performed in triplicate.

1-20. statistical analysis

Comparison between the two mean values, ±SEM (control group versus experimental group), was performed with two-tailed Student's t -tests, and multiple comparisons were performed with Kruskal-Wallis test and Dunn's post hoc test. A P value of less than 0.05 was considered statistically significant. All data were analyzed using Graph Prism 5 software (GraphPad Software Inc., San Diego, CA, USA).

Experimental Example 2: Experimental results

2-1. Confirmation of cancer cell migration and invasion inhibition of CAFs and apoptosis cancer cell co-culture conditioned medium (ApoSQ-CAF CM)

Since it is known that the paracrine (paracrine) action between CAFs and cancer cells promotes basic processes such as cancer cell migration and invasion, in this experiment, when co-culturing UV-irradiated and killed cancer cells and CAFs, Whether migration and invasion of lung cancer cells are inhibited through the secretion of active mediators was investigated.

After isolating CAFs from lung tumors of Kras-mutant (Kras ^LA1 ) mice using the fibroblast-specific marker Thy1, they were treated with the apoptosis 344SQ cell line (ApoSQ) for 20 hours, at which time the cell line was used as Kras A highly invasive and metastatic lung adenocarcinoma cell line derived from mice co-expressing ^{the LA1} and 53 ^R172H alleles (22) was used.

344SQ cells were treated with CAFs and killed 344SQ co-culture conditioned medium (ApoSQ-CAF CM) or CAFs and necrotic 344SQ co-culture conditioned medium (NecSQ-CAF CM) in the presence or absence of TGF-beta 1 for 48 hours. . Transwell migration and invasion assays were performed to analyze cell migration and invasion capabilities against a chemo-attractant gradient.

As a result, it was confirmed that ApoSQ-CAF CM substantially inhibited the migration and invasion of TGF-beta 1-induced cancer cells, whereas the control or NecSQ-CAF CM did not (FIG. 1a). In addition, other types of human cancer cell lines, such as non-small cell lung cancer cell line A549, colon cancer cell line HCT116, and breast cancer cell line MCF-7, were also co-cultured with CAFs and killed cancer cells in conditioned media (ApoA-CAF CM, ApoH-CAF CM and ApoM-CAF CM) was able to confirm the effect of inhibiting cancer cell migration and invasion (FIGS. 1b to 1d).

Next, we investigated whether migration and invasion of lung cancer cells were affected by CM derived from ApoSQ alone (ApoSQ CM) or direct exposure to ApoSQ. As a result, it was confirmed that direct exposure to ApoSQ CM, NecSQ CM, ApoSQ or NecSQ did not affect the TGF-beta 1-induced migration and invasion of 344SQ cells (FIGS. 2a and 2b). These results suggest that anti-migration and anti-invasion effects require bioactive mediators secreted by CAFs, which are functionally altered by stimulation of apoptotic cancer cells.

On the other hand, when TGF-beta 1 signal is activated, it induces migration and invasion of cancer cells by regulating Smad-dependent and -independent signaling. and suppression of TGF-beta 1 signals such as the p38 MAP kinase signaling system (FIGS. 3a and 3b). In addition, TGF-beta 1 signal induces upregulation of mRNA and protein expression of MMP-2 and MMP-12, which degrade and remodel the extracellular matrix, and it was confirmed that ApoSQ-CAF CM inhibited this (FIG. 4) .

Through the above experiments, the conditioned medium in which CAFs and apoptotic cancer cells were co-cultured not only suppressed the activation of TGF-beta 1 signal-induced migration and invasion-related signaling pathway molecules of cancer cells, but also related to ECM remodeling. It was verified that migration and invasion of cancer cells can be inhibited by inhibiting the expression of mRNA and protein of MMP2/12.

2-2. Confirmation of inhibition of CAF migration and invasion by interaction between CAFs and killed lung cancer cells

The migratory ability of CAFs is related to the activation state of CAFs, ie, activated fibroblasts migrate better than quiescent or resting fibroblasts. To confirm whether direct exposure of CAFs to apoptotic lung cancer cells affects the migration ability of CAFs themselves, CAFs were exposed to ApoSQ or NecSQ for 20 hours, followed by fresh medium in the presence or absence of TGF-beta 1. was replaced for 24 hours. As a result, while the direct interaction between CAFs and ApoSQ inhibited the migration and invasion of CAFs induced by TGF-beta 1, this inhibitory effect was not shown when CAFs were treated with NecSQ (FIG. 5). Moreover, direct exposure of CAFs to ApoSQ or NecSQ in the absence of TGF-beta 1 did not affect basal levels.

In addition, to confirm the anti-migration and anti-invasion effects on CAFs themselves through autocrine secretion, ApoSQ-CAF CM or NecSQ-CAF CM was applied to CAFs monolayers in the presence or absence of TGF-beta 1. added. Similar to the direct effect of ApoSQ, treatment with ApoSQ-CAF CM significantly inhibited TGF-beta 1-induced migration and invasion of CAFs, but NecSQ-CAF CM had little inhibitory effect (Fig. 6a). Similarly, for ApoA-CAF CM, ApoH-CAF CM and ApoM-CAF CM in which CAF CMs were co-cultured with other types of killed cancer cells, such as A549, HCT116 and MCF-7, migration and invasion of CAFs were also observed. inhibited (Figs. 6b to 6d). However, ApoSQ CM and NecSQ CM alone did not have this inhibitory effect (FIG. 7).

Additionally, as it is known that CAFs exhibit a more aggressive phenotype in response to TGF-beta 1 treatment, we investigated whether direct exposure to ApoSQ inhibits CAF activation markers. As a result, the ApoSQ-treated group suppressed TGF-beta 1-induced increases in gene and protein expression of CAF activation markers such as α-smooth muscle actin (SMA), collagen type 1, and fibronectin, whereas the NecSQ-treated group did not. (Fig. 8a). In addition, as a result of immunocytochemical analysis, the expression of TGF-beta 1-induced α-SMA protein in CAFs was decreased by direct exposure to ApoSQ, but not by exposure to NecSQ (Fig. 8b).

Similarly, ApoSQ inhibited the activation of TGF-beta 1-induced Smad or non-Smad signaling in CAFs (FIG. 9a and 9b), and TGF-beta 1-induced MMP-2 and MMP-12 mRNA and It was confirmed that protein expression was also inhibited (FIG. 10).

Through the above experiments, while verifying the effect of inhibiting migration and invasion of CAFs in a conditioned medium in which CAFs and apoptotic cancer cells are co-cultured, when exposing cancer-related fibroblasts to apoptotic cancer cells to cause interaction, In the CAFs, the expression of TGF-beta 1-induced CAF activation markers is suppressed, the activation of cell migration and invasion-related signaling pathways is suppressed, and the expression of MMP2/12 is suppressed at the gene and protein level, resulting in cancer cell migration and It was verified that infiltration can be inhibited.

2-3. Confirmation that WISP-1 is required for inhibition of migration and invasion of cancer cells and CAFs

To confirm that autocrine/paracrine factors secreted from CAFs play an important role in inhibiting the migration and invasion of cancer cells and CAFs, CAF CM, ApoSQ alone medium, or ApoSQ-CAF CM were used to examine up to 111 species in a single sample. A cytokine array analysis capable of measuring cytokines was performed.

As a result, among these cytokines, LIF (leukemia inhibitory factor) and WISP-1 were most highly induced by CAFs in response to ApoSQ, and the increase in these cytokines was not seen in ApoSQ alone CM without CAFs (Fig. 11), we used specific siRNAs to investigate which cytokines are involved in the anti-migration and anti-invasion effects of ApoSQ-CAF CM.

We confirmed knockdown of WISP-1 in CAFs transfected with specific siRNA, and WISP-1 secretion was also reduced in CAFs transfected with or without ApoSQ (FIG. 12). WISP1-1 knockdown blocked the inhibitory effect of ApoSQ-CAF CM on TGF-beta 1-induced migration and invasion in 344SQ cells (Fig. 13a), and also blocked the direct inhibitory effect of ApoSQ on CAFs migration and invasion (Fig. 13b). In contrast, when LIF was knocked down, there was no effect of inhibiting migration and invasion in 344SQ cells or CAFs (FIGS. 14 to 15b).

In addition, the present inventors confirmed the effect of increasing ApoSQ-induced WISP-1 secretion by CAFs using WISP-1 overexpressing CAFs. First, it was confirmed that WIPS-1 expression was increased in CAFs transfected with the WISP-1 plasmid, and WISP-1 secretion was increased in CAFs transfected with or without ApoSQ (FIG. 16). When treated with CAF CM overexpressing WISP-1 in the presence of ApoSQ, compared to control (empty vector) transfected CAFs and ApoSQ co-culture, the TGF-beta 1-induced migration and invasion of 344SQ cells were further suppressed (Fig. 17a). When CAFs overexpressing WISP-1 were treated with ApoSQ, TGF-beta 1-induced migration and invasion of CAFs were reduced compared to those of control transfected CAFs in the absence of ApoSQ, but were more inhibited than ApoSQ treated control transfected CAFs. In the absence of ApoSQ, the TGF-beta 1-induced migration and invasion inhibitory effects of WISP-1 overexpressed CAFs were reduced compared to control transfected CAFs (FIG. 17b). In addition, when treated with ApoSQ-CAF CM after neutralization with anti-WISP-1 antibody, the anti-migration and anti-invasion effects on 344SQ cells were eliminated, whereas these effects were not observed when treated with control IgG (FIG. 18 ).

To demonstrate that WISP-1 acts in an autocrine/paracrine manner to induce anti-migration and anti-invasion effects, recombinant mouse WISP-1 (rWISP) was used at concentrations of 5 and 10 ng/ml, or 0.75-3 ng/ml. -1) was directly treated with 344SQ or CAFs, respectively, in the presence of TGF-beta 1. As expected, treatment with this low dose of rWISP-1 inhibited TGF-beta 1-induced migration and invasion of 344SQ and CAFs in a concentration-dependent manner (FIGS. 19a and 19b). In addition, it was confirmed that rWISP-1 suppressed the TGF-beta 1-induced Smad or non-Smad signaling system and suppressed the expression of MMP2/MMP12 mRNA and protein in 344SQ cells or CAFs (FIGS. 20a to 21c).

The above experimental results indicate that anti-migration and anti-invasion effects are exerted in cancer cells and CAFs through WISP-1, which is specifically highly expressed in ApoSQ-CAF CM, while the WISP-1 in CAFs exhibits ApoSQ It suggests that it is secreted by contacting and interacting with.

Additionally, since WISP-1 is known to bind to integrin, a cell surface receptor, integrins αv, α5, beta 1, beta Experiments were performed using blocking antibodies against 3 or beta 5.

As a result of the experiment, in the case of 344SQ cells, when treated with anti-integrin αv or beta 3 antibodies, the anti-migration and anti-invasion effects of rWISP-1 were reversed compared to the control IgG antibody (FIG. 22a), and in the case of CAFs, the anti-migration and anti-invasion effects were reversed. - It was confirmed that the anti-migration and anti-invasion effects of rWISP-1 were reversed by treatment with integrin αv or beta 5 antibody compared to the control IgG antibody (FIG. 22b). In contrast, treatment of 344SQ cells with anti-integrin α5, beta 1, or beta 5, or treatment of CAFs with anti-integrin α5, beta 1, or beta 3 did not show significant changes in cell migration or invasion. .

In addition, experiments were performed to confirm whether WISP-1 inhibits the signaling pathway induced by TGF-beta 1 through integrin αv and beta 3 in 344SQ cells and through integrin αv and beta 5 in CAFs.

As a result, when 344SQ cells were treated with anti-integrin αv or beta 3 antibodies, the inhibitory effect of rWISP-1 on TGF-beta 1-induced signaling pathway and MMP2/MMP12 mRNA and protein expression was also attenuated (Fig. 23a to 23c), even when CAFs were treated with anti-integrin αv or beta 3 antibody, the inhibitory effect of rWISP-1 on TGF-beta 1-induced signaling pathway and MMP2/MMP12 mRNA and protein expression It was confirmed that was weakened (FIGS. 24a to 24c).

Through the above experiments, WISP-1 inhibits the signaling pathway induced by TGF-beta 1 in an autocrine/paracrine manner through the integrin αv beta 3 receptor of 344SQ cells and the integrin αv beta 5 receptor of CAFs, thereby inhibiting cell migration and It was verified that the infiltration was controlled.

2-4. Confirmation that apoptotic cancer cells induce Notch1 signaling in CAFs resulting in WISP-1 production

The Notch1-WISP-1 axis is known to determine the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma invasion and metastasis. Therefore, the present inventors hypothesized that Notch1 signaling-dependent WISP-1 production in CAFs in response to killed lung cancer cells plays a critical role in the anti-migration and anti-invasion effects in cancer cells and CAFs.

To verify this hypothesis, we first investigated whether the Notch1 signaling pathway is activated in CAFs in response to ApoSQ. As a result of Western blot analysis, Notch1 signaling proteins, including Hes1 and WISP-1, known as sub-targets of the Notch pathway, as well as Notch intracellular domain 1 (NICD1), known to be secreted only when Notch1 signaling is activated, are induced by ApoSQ in CAFs It was confirmed (FIG. 25 (A)). In addition, WISP-1 secretion increased in ApoSQ-CAF CM, but not in NecSQ-CAF CM (FIG. 25(B)).

Moreover, Hes1 and WISP-1 mRNA expression was increased in CAFs, and these increases were maintained 5 and 20 h after ApoSQ treatment, respectively, but had no effect upon NecSQ stimulation, suggesting that Notch-responsiveness, a common transcriptional effector of Notch signaling activity, As a result of the reporter 4x-CSL-Luc activity test, the activation of Notch signaling in CAFs was increased by exposure to ApoSQ up to 20 hours, whereas there was no significant activity when stimulated with NecSQ (FIG. 26). Additionally, as a result of immunocytochemical analysis, it was confirmed that the expression of NICD1 and WISP-1 was increased in CAFs in response to ApoSQ stimulation, and that WISP-1 expression was colocalized in NICD1 stain-positive CAFs located in the nucleus ( Figure 27).

To confirm the hypothesis that ApoSQ-induced Notch1 signaling-dependent WISP-1 secretion eventually mediates the inhibition of migration and invasion of lung cancer cells and CAFs, further experiments were conducted using Notch1 siRNA and the γ-secretase inhibitor DAPT. did As a result, when Notch1 was knocked down in CAFs using specific siRNA, increased NICD1, Hes1, and WISP-1 protein expression was inhibited (Fig. 28a and 28b (A)), and WIPS-1 secretion by ApoSQ treatment was also inhibited. (FIG. 28B (B)), which in turn had the effect of inhibiting the anti-migration and anti-invasion effects in 344SQ (FIG. 29A) and CAFs themselves (FIG. 29B). Even when pre-treatment with 10 μM of DAPT, a Notch1 inhibitor, increased expression of NICD1, Hes1, and WISP-1 in CAFs by ApoSQ treatment was suppressed (FIG. 30(A)), and WISP-1 secretion was also reduced ( Figure 30 (B)). As a result, when CAFs were treated with DAPT, the anti-migration and anti-invasion effects of ApoSQ-CAF CM were significantly inhibited in 344SQ (FIG. 31a). Similarly, the anti-migration and anti-invasion effects of ApoSQ were also completely inhibited by DAPT in CAFs themselves (FIG. 31B).

Through the above experiment, when CAFs and ApoSQ are brought into contact, Notch1 signal is activated and WISP-1 expression is increased, which suppresses cell migration and invasion in 344SQ cells and CAFs, thereby confirming the cancer cell migration of ApoSQ-CAF CM and invasion inhibitory effect and ApoSQ's CAFs migration and invasion inhibitory effect were verified through Noth1-dependent WIPS-1 production.

2-5. Confirmation of increased expression of Dll1 in UV-irradiated apoptotic cancer cells

Most Notch signaling is initiated by the interaction of cell-surface receptor Notch with cell-associated ligands, such as Delta-like ligands (DLL) 1,3 and 4 or Jagged-like (JAG) 1 and 2, so Notch- To highlight the important cell surface interactions required for activation of 1 signaling, we first examined the expression of Notch ligand on the surface of apoptotic cells using flow cytometry.

As a result, it was confirmed that Dll1 was significantly increased in cancer cells including 344SQ, A549 and HCT116 after UV irradiation for 15 minutes, but the expression levels of other ligands were not changed or rather decreased (FIG. 32), and Western blotting The analysis results show that the only expression of Dll1 increased in the killed cancer cell lysate (FIG. 33).

In addition, when Dll1 was neutralized with an anti-Dll1 antibody, the Notch1 signal induced by ApoSQ was reduced, and the secretion of WISP-1 in CAFs was also reduced (FIG. 34). Similarly, before killing through UV irradiation in 344SQ cells, When Dll1 was knocked down using specific siRNA, Notch1 activation and WISP-1 secretion were suppressed in CAFs after ApoSQ treatment (FIGS. 35a and 35b).

The above experimental results indicate that the Notch1 signaling process in CAFs is initiated by interaction with adjacent killed cancer cells expressing Dll1.

2-6. Promote phagocytosis of apoptotic cells by CAFs of BAI1/Rac1 signals and confirm crosstalk with Notch1 signals

Since BAI1 can contribute to the apoptotic cell removal process of apoptotic cervical cancer cells through primary fibroblasts, an experiment was conducted to confirm whether BAI1 signal could affect Notch1 signal in CAFs. Since the thrombospondin type 1 repeats of BAI1 directly recognize phosphatidylserine (PtdSer) and target apoptotic cells, the interaction between BAI1 and ApoSQ in CAFs was reduced by adding annexin V that binds to PtdSer in apoptotic cells. Whether or not Notch1 signaling was suppressed by inhibition was investigated. The percentage of PKH26-stained CAFs that phagocytosed PKH67-stained ApoSQ cells was determined using flow cytometry. When Annexin V was added, the phagocytosis of ApoSQ by CAFs was reduced (FIG. 36a), and ApoSQ-induced Notch1 activation and WISP-1 secretion were also downregulated (FIG. 36b). In addition, when BAI1 was knocked down, phagocytosis, Notch1 signaling, and WISP-1 secretion were downregulated in ApoSQ-treated CAFs (FIGS. 37a to 37c), and the same results were obtained when BAI1 was neutralized with an anti-BAI1 antibody. It was confirmed (FIG. 38a and 38b). Conversely, in the case of BAI1 overexpressing CAFs, the ApoSQ phagocytosis rate was higher than that of mock-transfected cells, and Notch1 signaling and WISP-1 secretion were also increased (FIGS. 39a to 39c).

The above experimental results indicate that BAI1, a protein that binds to ApoSQ, promotes the removal of apoptotic cells in CAFs and regulates the production of WISP-1 in CAFs by being involved in the Notch1 signaling pathway.

Additionally, in order to analyze the relationship between the apoptotic cell elimination process of CAFs and the Notch1 signal in detail, the Rac1 inhibitor NSC23766, which down-regulates the phagocytosis of apoptotic cells, was used. ApoSQ increased Rac1 activity in CAFs in a time-dependent manner, and Rac1 activity was highest after 24 hours (FIG. 40). Similar to the results of flow cytometry, it was confirmed by confocal microscopy that phagocytosis of CAFs was reduced when treated with 100 μM of NSC23766. In addition, it was confirmed that Notch1 signal and WISP-1 secretion were decreased when treated with NSC23766 (FIGS. 41a to 41c).

In addition, when Notch1 was knocked down, Rac1 activity and ApoSQ scavenging action of CAFs were reduced (FIG. 42), and Rac1 activity and ApoSQ scavenging action of CAFs were also reduced when 20 mM DAPT was treated (FIG. 43).

The above experimental results suggest that the Notch1 signal and the BAI1/Rac1 signal pathway can exhibit positive crosstalk when apoptotic cancer cells are stimulated.

2-7. in vivo confirmed that WISP-1 production was promoted by Notch1 signaling during ApoSQ treatment, and CAF activation and lung metastasis were suppressed.

In a previous study, the present inventors confirmed that a single injection of ApoSQ cells inhibited lung metastasis in syngeneic (129/Sv) immunocompetent mice. After that, the in vivo response of CAFs to ApoSQ injection was investigated. To this end, leukocytes, endothelial cells, and epithelial cells were first removed from mouse primary tumors, and then CAFs were isolated using magnetic-activated cell sorting using the fibroblast-specific marker Thy1 (FIG. 44), followed by qRT-PCR. used to analyze the amount of mRNA of CAF markers.

As a result, typical CAF activation markers such as Acta2, Col1α1, Fn, Itg beta 1, Spp1, Pdgfrα, Pdgfr beta, growth factors including Mmp1a, 2, 9 and 12, and Vegfa, Hgf, Cxcl12 and Cxcl14 after ApoSQ injection / The mRNA expression levels of chemokines were significantly reduced after ApoSQ injection compared to the control group. However, mRNA expression levels of Notch downlink target genes including Notch1, WISP-1 (Ccn4), Hey1, Hey2, Hes1 and Hes5 were significantly improved compared to the control group, and notch ligands and other CCN gene families (Ccn1, Ccn2, The mRNA expression levels of Ccn5 and Ccn6) were almost unchanged after ApoSQ injection, but Ccn3 mRNA was approximately doubled in Thy1 ⁺ CAFs after ApoSQ injection compared to the control group (FIGS. 45a and 45b).

To confirm the inhibitory effect of Notch1-WISP-1 signaling on CAF activation and tumor development in vivo, LY3039478, a Notch1 selective inhibitor, was orally administered at 8 mg/kg the day before ApoSQ injection, 3 times a week for 6 weeks (FIG. 46). . LY3039478 hardly changed body weight, tumor weight or tumor volume when compared with the control and ApoSQ groups (FIG. 47), but reversed the effect of ApoSQ administration on reducing the number of tumor nodules on the lung surface and the rate of metastasis (FIG. 48).

In addition, LY3039478 reversed the effect of ApoSQ administration on the decreased mRNA expression level of CAF markers, MMPs, growth factors, and chemokines in isolated Thy1 ⁺ CAFs, and also reversed the effect of increasing the mRNA expression level of Notch1 and Notch target genes. (FIG. 49). As a result of immunohistochemical analysis of primary tumor tissue, LY3039478 also reversed the effect of decreasing α-SMA and increasing NICD1 and WISP-1 expression in Thy1 ⁺ CAFs (FIG. 50). In addition, as a result of immunocytochemical analysis of Thy1 ⁺ CAFs isolated from the primary tumor, it was confirmed that the protein expression pattern was reversed (FIGS. 51a and 51b).

On the other hand, the increased WISP-1 protein in the culture medium and serum of Thy1 ⁺ CAFs isolated from the primary tumor tissue of the ApoSQ-administered mouse group was decreased when LY3039478 was treated, but the primary tumor tissue of the ApoSQ-administered group and the ApoSQ + LY3039478-administered group In the culture media of CD326 ⁺ tumor cells and CD11b ⁺ tumor-associated macrophages isolated from WISP-1, no change in protein level was observed compared to the control group (FIG. 52).

In addition, injection of ApoSQ inhibited the migration and invasion of isolated CD326 ⁺ tumor cells and Thy1 ⁺ CAFs, and these effects were reversed when co-administered with LY3039478 (FIGS. 53A and 53B). Similarly, activation of signaling molecules related to migration and invasion, such as Smad2/3, FAK, ERK, and Akt, and protein expression, such as MMP2 and MMP12, were suppressed in isolated CD326 ⁺ tumor cells upon injection of ApoSQ, but LY3039478 Administration reversed this inhibitory effect (FIG. 54).

Through the above experiment, it was verified that when ApoSQ was administered in vivo , Notch1 signal was activated in CAFs, WISP-1 production was promoted, and the expression level of CAF activation markers was reduced, thereby inhibiting migration and invasion of cancer cells and CAFs.

2-8. ApoSQ-CAF CM in vivo confirmed to inhibit tumor progression and lung metastasis in

To investigate the in vivo effect of ApoSQ-CAF CM on tumor progression and lung metastasis, CAF CM or ApoSQ-CAF CM was intratumorally injected 3 times a week for 6 weeks into syngeneic mice 2 days after 344SQ injection. In addition, to confirm the role of WISP-1 in CM in vivo, ApoSQ-CAF CM was pretreated with neutralizing anti-WISP-1 antibody or isotype IgG for 2 hours before injection (FIG. 55).

As a result, there was no significant difference in body weight between groups (FIG. 56). In addition, similar to the effect of single injection ApoSQ, the group injected with ApoSQ-CAF CM decreased the number of tumor nodules and metastasis rate on the lung surface 6 weeks after 344SQ injection compared to the control group, and the same pattern was observed in the isotype IgG administered group. However, in the ApoSQ-CAF CM group in which WISP-1 was immunosuppressed, it was confirmed that there was no change in the number of tumor nodules and the rate of metastasis (FIG. 57).

Similar to the results of in vitro experiments, injection of ApoSQ-CAF CM significantly reduced the migration and invasion of isolated CD326 ⁺ tumor cells and Thy1 ⁺ CAFs, whereas in the WISP-1 immunodepleted ApoSQ-CAF CM group, these No inhibitory effect was seen (FIGS. 58A and 58B). In addition, the activation of signaling pathways involved in migration and invasion, namely Smad2/3, FAK, ERK, and Akt, and the expression levels of MMP2 and MMP12 in isolated CD326 ⁺ tumor cells were decreased by ApoSQ-CAF CM, but WISP- The ApoSQ-CAF CM group in which 1 was immunodepleted did not show this inhibitory effect (FIG. 59). In addition, the mRNA expression levels of CAF activity markers, namely Acta2, Col1α1, Itg beta 1, Spp1, Pdgfrα and Mmp1a, Mmp2, and Mmp12, in isolated Thy1 ⁺ CAFs were decreased by ApoSQ-CAF CM, but WISP-1 was immunoremoved. The ApoSQ-CAF CM group showed no such inhibitory effect (FIG. 60).

In addition, to confirm the in vivo effect of WISP-1 on anti-tumor progression and anti-metastasis, rWISP1-1 was injected at a concentration of 12.5 μg/kg or 25 μg/kg 344SQ 2 days after injection to syngeneic mice 3 times a week , injected intratumorally for 6 weeks (FIG. 61)

As a result, similar to the case of ApoSQ-CAF CM injection, there was no significant difference in body weight between the groups (FIG. 62), and the number of tumor nodules on the lung surface was reduced in the group injected with rWISP-1 compared to the control group. The transition rate decreased in a concentration dependent manner (FIG. 63). In addition, rWISP-1 inhibited the migration and invasion of CD326 ⁺ tumor cells and Thy1 ⁺ CAFs in a concentration-dependent manner (Fig. 64a and 64b), and the signaling pathway involved in migration and invasion in CD326 ⁺ tumor cells, that is, Smad2/3 , FAK, ERK, Akt activation and MMP2 and MMP12 expression levels were reduced (FIG. 65), and CAF activity markers, namely Acta2, Col1α1, Itg beta 1, Spp1, Pdgfrα and Mmp1a, Mmp2, and Mmp12 in Thy1 ⁺ CAFs The mRNA expression level of was decreased (FIG. 66).

Through the above experiments, it was demonstrated that the tumor suppression effect of ApoSQ-CAF CM was mediated by WISP-1, and it was confirmed that rWISP-1 could exhibit the same effect as that of ApoSQ-CAF CM in vivo .

Additionally, to analyze the in vivo anti-metastatic effect of ApoSQ-CAF CM at the gene level, we simultaneously analyzed the expression of 84 genes related to invasion and metastasis using the mouse tumor metastasis RT ² Profiler PCR array in isolated CD326 ⁺ tumor cells. did As a result, compared to the CAF CM group, the expression of 15 genes, including MMP2, Cdh6, Smad2, Kras, and Igf1, related to tumor progression and invasion, were down-regulated more than 2-fold when injected with ApoSQ-CAF CM, and tumor progression and The expression of genes related to inhibition of invasion (8 types including Cdkn2a, Ror beta, Ctnna1, etc.) was up-regulated more than twice (FIG. 67).

In addition, to verify the mechanism by which ApoSQ-CAF CM reduces CAF invasion, 84 genes related to cell adhesion and ECM remodeling in isolated Thy1 ⁺ CAFs were analyzed by qRT-PCR array. As a result, compared to the CAF CM group, in the case of the ApoSQ-CAF CM group, the expression of 8 cell aggregation-related genes, such as Cdh2, Selp, Itg beta 1, Cdh3, Itga3, Ncam1, Vacm1, and Ncam2, was downregulated more than 2-fold. and the expression of ECM remodeling-related genes, that is, ECM components (4 genes) and MMPs (8 genes), was also down-regulated more than 2-fold (FIG. 68).

Through the above experiments, it was verified that the in vivo administration of ApoSQ-CAF CM can inhibit the migration and invasion of cancer cells and CAFs by reducing the expression of genes related to cancer cell invasion and metastasis and CAFs invasion.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (26)

Cancer-associated fibroblasts (CAFs) and apoptotic cancer cells (apoptotic cancer cells) containing the co-cultivated culture medium, a pharmaceutical composition for inhibiting cancer metastasis. The method according to claim 1, wherein the cancer-related fibroblasts are fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, malignant skin cancer, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, lung cancer, gastric cancer , Breast cancer, colorectal cancer and prostate cancer will be one or more cancer-related fibroblasts selected from the group consisting of, a pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the killed cancer cells are killed by ultraviolet (Ultra-violet ray, UV) irradiation. 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, hematological cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer , Oral cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, at least one selected from the group consisting of lymphoma and leukemia, a pharmaceutical composition. 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. The pharmaceutical composition according to claim 1, wherein the culture medium contains WISP-1 (Wnt-induced signaling protein-1). The pharmaceutical composition according to claim 6, wherein the WISP-1 is produced by Notch1 signaling. The pharmaceutical composition according to claim 7, wherein the Notch1 signaling is initiated by Dll1 (Delta-like ligand 1). The pharmaceutical composition of claim 7, wherein the Notch1 signaling is enhanced by BAI1/Rac1 (Brain angiogenesis inhibitor 1/Ras-related C3 botulinum toxin substrate 1) signaling. The pharmaceutical composition according to claim 9, wherein the composition enhances the phagocytic ability of cancer-related fibroblasts by the interaction of Notch1 signaling and BAI1/Rac1 signaling. The pharmaceutical composition according to claim 9, wherein the composition enhances WISP-1 production by the interaction of Notch1 signaling and BAI1/Rac1 signaling. The pharmaceutical composition according to claim 1, wherein the composition reduces activation markers in cancer-related fibroblasts. The pharmaceutical composition according to claim 12, wherein the activation marker is one or more selected from the group consisting of Acta2, Col1α1, Fn, Itg beta 1, Spp1, Pdgfrα, Pdgfr beta and Mmp1a, 2, 9 and 12. The pharmaceutical composition according to claim 1, characterized in that the composition reduces growth factors and chemokines in cancer-associated fibroblasts. The pharmaceutical composition according to claim 14, wherein the growth factor is at least one selected from the group consisting of Vegfa, Hgf, Cxcl12 and Cxcl14. The pharmaceutical composition according to claim 1, wherein the composition increases Notch1-related molecules in cancer-related fibroblasts. The pharmaceutical composition according to claim 16, wherein the Notch1-related molecule is one or more selected from the group consisting of WISP-1 (Ccn4), Hey1, Hey2, Hes1 and Hes5. The pharmaceutical composition according to claim 1, wherein the cancer metastasis includes migration and invasion of cancer cells. A pharmaceutical composition for inhibiting cancer metastasis, containing cancer-related fibroblasts exposed to apoptotic cancer cells. A health functional food for inhibiting cancer metastasis, containing a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured. A method for preparing a composition for inhibiting cancer metastasis, comprising co-cultivating cancer-related fibroblasts and apoptotic cancer cells. The method of claim 21, further comprising secreting WISP-1 through the co-culture. A method for suppressing cancer metastasis, comprising the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured. A method for preventing or treating cancer, comprising the step of treating a subject with a culture medium in which cancer-related fibroblasts and apoptotic cancer cells are co-cultured. Use of a culture solution in which cancer-related fibroblasts and killed cancer cells are co-cultured for prevention or treatment of cancer. Use of WISP-1 (Wnt-induced signaling protein-1) for cancer prevention or treatment.

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