Method for reprogramming type 1 alveolar cells into type 2 alveolar cells using rhosin, dasatinib, or ML141, and composition for inhibiting lung cancer metastasis containing ML141 or dasatinib
Patent Information
- Application Number
- PCT/KR2024/011539
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2023-10-26
- Filing Date
- 2024-08-05
- Publication Date
- 2025-02-13
Smart Images
Figure KR2024011539_13022025_PF_FP_ABST
Abstract
Description
Method for reprogramming type 1 alveolar cells into type 2 alveolar cells using RHOSIN, dasatinib, or ML141 and composition for inhibiting lung cancer metastasis comprising ML 141 or dasatinib
[0001] [Reprogramming Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using Rhosin]
[0002] The present invention relates to a composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, including rhosin as an active ingredient.
[0003] This application claims priority to Korean Patent Application No. 10-2023-0103059, filed on August 7, 2023, the entire disclosure of which is incorporated herein by reference.
[0004]
[0005] [Reprogramming of Type 1 Pneumocytes into Type 2 Pneumocytes Using Dasatinib]
[0006] The present invention relates to a composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, including Dasatinib as an active ingredient.
[0007] This application claims priority to Korean Patent Application No. 10-2023-0123456, filed September 15, 2023, the entire disclosure of which is incorporated herein by reference.
[0008]
[0009] [Reprogramming of Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using ML141]
[0010] The present invention relates to a composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, including ML141 as an active ingredient.
[0011] This application claims priority to Korean Patent Application No. 10-2023-0124261, filed September 18, 2023, the entire disclosure of which is incorporated herein by reference.
[0012]
[0013] [Composition for inhibiting lung cancer metastasis containing ML 141]
[0014] The present invention relates to a composition for inhibiting lung cancer metastasis, etc., comprising ML 141 as an active ingredient.
[0015] This application claims priority to Korean Patent Application No. 10-2023-0144570, filed October 26, 2023, the entire disclosure of which is incorporated herein by reference.
[0016]
[0017] [Composition for inhibiting lung cancer metastasis containing dasatinib]
[0018] The present invention relates to a composition for inhibiting lung cancer metastasis, including Dasatinib as an active ingredient.
[0019] This application claims priority to Korean Patent Application No. 10-2023-0144587, filed October 26, 2023, the entire disclosure of which is incorporated herein by reference.
[0020] [Reprogramming Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using Rhosin]
[0021] Among the various cells that make up the lungs, the cells that perform major functions include adult AT1 (alveolar type 1) cells and stem cells AT2 (alveolar type 2) cells. AT1 cells are cells that make up the alveoli and perform the main function of mediating gas exchange. AT2 cells secrete surfactant to protect and maintain the alveoli, and as precursor cells, they produce AT1 cells to repair damaged epithelial cells.
[0022] AT1 and AT2 cells differ in their internal structure, most notably in F-actin. In AT1 cells, actin forms a structure extending from the inside of the cytoplasm to the outside of the nucleus, but in AT2 cells, actin is not observed in the cytoplasm and does not contact the outside of the nucleus. In addition, with regard to contact with actin, the nucleus of AT1 cells, where actin contacts the outside of the nucleus, has an oval shape, whereas the nucleus of AT2 cells, where actin does not contact the outside of the nucleus, has a relatively close to circular shape.
[0023] Meanwhile, AT2 cells are widely used in research to establish iPSCs (induced pluripotent stem cells) and then differentiate into various cells that make up the respiratory tract. The most common method is to isolate and secure them from surgical tissue. However, the number of AT2 cells that can be isolated and secured from surgical tissue is very limited, and when AT2 cells are cultured in 2D, they have a phenotype similar to that of AT1 cells, making them difficult to culture. Accordingly, it is difficult to secure a sufficient number of AT2 cells compared to their utility.
[0024]
[0025] [Reprogramming of Type 1 Pneumocytes into Type 2 Pneumocytes Using Dasatinib]
[0026] Among the various cells that make up the lungs, the cells that perform major functions include adult AT1 (alveolar type 1) cells and stem cells AT2 (alveolar type 2) cells. AT1 cells are cells that make up the alveoli and perform the main function of mediating gas exchange. AT2 cells secrete surfactant to protect and maintain the alveoli, and as precursor cells, they produce AT1 cells to repair damaged epithelial cells.
[0027] AT1 and AT2 cells differ in their internal structure, most notably in F-actin. In AT1 cells, actin forms a structure extending from the inside of the cytoplasm to the outside of the nucleus, but in AT2 cells, actin is not observed in the cytoplasm and does not contact the outside of the nucleus. In addition, with regard to contact with actin, the nucleus of AT1 cells, where actin contacts the outside of the nucleus, has an oval shape, whereas the nucleus of AT2 cells, where actin does not contact the outside of the nucleus, has a relatively close to circular shape.
[0028] Meanwhile, AT2 cells are widely used in research to establish iPSCs (induced pluripotent stem cells) and then differentiate into various cells that make up the respiratory tract. The most common method is to isolate and secure them from surgical tissue. However, the number of AT2 cells that can be isolated and secured from surgical tissue is very limited, and when AT2 cells are cultured in 2D, they have a phenotype similar to that of AT1 cells, making them difficult to culture. Accordingly, it is difficult to secure a sufficient number of AT2 cells compared to their utility.
[0029]
[0030] [Reprogramming of Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using ML141]
[0031] Among the various cells that make up the lungs, the cells that perform major functions include adult AT1 (alveolar type 1) cells and stem cells AT2 (alveolar type 2) cells. AT1 cells are cells that make up the alveoli and perform the main function of mediating gas exchange. AT2 cells secrete surfactant to protect and maintain the alveoli, and as precursor cells, they produce AT1 cells to repair damaged epithelial cells.
[0032] AT1 and AT2 cells differ in their internal structure, most notably in F-actin. In AT1 cells, actin forms a structure extending from the inside of the cytoplasm to the outside of the nucleus, but in AT2 cells, actin is not observed in the cytoplasm and does not contact the outside of the nucleus. In addition, with regard to contact with actin, the nucleus of AT1 cells, where actin contacts the outside of the nucleus, has an oval shape, whereas the nucleus of AT2 cells, where actin does not contact the outside of the nucleus, has a relatively close to circular shape.
[0033] Meanwhile, AT2 cells are widely used in research to establish iPSCs (induced pluripotent stem cells) and then differentiate into various cells that make up the respiratory tract. The most common method is to isolate and secure them from surgical tissue. However, the number of AT2 cells that can be isolated and secured from surgical tissue is very limited, and when AT2 cells are cultured in 2D, they have a phenotype similar to that of AT1 cells, making them difficult to culture. Accordingly, it is difficult to secure a sufficient number of AT2 cells compared to their utility.
[0034]
[0035] [Composition for inhibiting lung cancer metastasis containing ML 141]
[0036] Cancer is one of the most common causes of death in modern times. It is a disease that occurs when normal cells change due to genetic mutations caused by various reasons. It refers to a malignant tumor that does not follow the differentiation, proliferation, and growth patterns of normal cells. Among these, lung cancer is the largest cause of cancer-related deaths. Lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), and non-small cell lung cancer accounts for more than 80% of lung cancer. NSCLC has subtypes such as lung adenocarcinoma (LUAD), squamous cell carcinoma, and large cell carcinoma, of which lung adenocarcinoma is known to be the most common type. Furthermore, lung cancer is a cancer that frequently metastasizes compared to other cancers. Even if there is no metastasis at the time of diagnosis, it can metastasize to other organs during treatment. It typically metastasizes to surrounding tissues, such as the other lung, bone, liver, brain, and adrenal gland, before spreading throughout the body. This leads to a very high recurrence rate. Furthermore, metastatic lung cancer is difficult to treat fundamentally through surgery, radiation therapy, and other methods, making its cure rate inevitably low. Therefore, clearly understanding the mechanisms by which lung cancer cells metastasize and effectively suppressing lung cancer metastasis using these mechanisms is expected to significantly improve the survival and cure rates of lung cancer.
[0037]
[0038] [Composition for inhibiting lung cancer metastasis containing dasatinib]
[0039] Cancer is one of the most common causes of death in modern times. It is a disease that occurs when normal cells change due to genetic mutations caused by various reasons. It refers to a malignant tumor that does not follow the differentiation, proliferation, and growth patterns of normal cells. Among these, lung cancer is the largest cause of cancer-related deaths. Lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), and non-small cell lung cancer accounts for more than 80% of lung cancer. NSCLC has subtypes such as lung adenocarcinoma (LUAD), squamous cell carcinoma, and large cell carcinoma, of which lung adenocarcinoma is known to be the most common type. Furthermore, lung cancer is a cancer that frequently metastasizes compared to other cancers. Even if there is no metastasis at the time of diagnosis, it can metastasize to other organs during treatment. It typically metastasizes to surrounding tissues, such as the other lung, bone, liver, brain, and adrenal gland, before spreading throughout the body. This leads to a very high recurrence rate. Furthermore, metastatic lung cancer is difficult to treat fundamentally through surgery, radiation therapy, and other methods, making its cure rate inevitably low. Therefore, clearly understanding the mechanisms by which lung cancer cells metastasize and effectively suppressing lung cancer metastasis using these mechanisms is expected to significantly improve the survival and cure rates of lung cancer.
[0040] [Reprogramming Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using Rhosin]
[0041] The present inventors completed the present invention by confirming that AT1 cells were reprogrammed into AT2 cells as a result of treating airway organoids with Rhosin, a Rho GTPase inhibitor.
[0042] Accordingly, the purpose of the present invention is to provide a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, which contains Rhosin as an active ingredient.
[0043] Another object of the present invention is to provide a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition according to the present invention.
[0044] Another object of the present invention is to provide a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a step of culturing type 1 alveolar cells in a medium composition according to the present invention and inducing them into type 2 alveolar cells.
[0045] Another object of the present invention is to provide a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing the airway organoid in a medium composition according to the present invention to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells.
[0046]
[0047] [Reprogramming of Type 1 Pneumocytes into Type 2 Pneumocytes Using Dasatinib]
[0048] The present inventors completed the present invention by confirming that AT1 cells were reprogrammed into AT2 cells as a result of treating airway organoids with Dasatinib, a tyrosine kinase inhibitor.
[0049] Accordingly, the purpose of the present invention is to provide a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, which contains Dasatinib as an active ingredient.
[0050] Another object of the present invention is to provide a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition according to the present invention.
[0051] Another object of the present invention is to provide a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a step of culturing type 1 alveolar cells in a medium composition according to the present invention and inducing them into type 2 alveolar cells.
[0052] Another object of the present invention is to provide a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing the airway organoid in a medium composition according to the present invention to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells.
[0053]
[0054] [Reprogramming of Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using ML141]
[0055] The present inventors completed the present invention by confirming that AT1 cells were reprogrammed into AT2 cells as a result of treating airway organoids with ML141, a Cdc42 / Rac1 GTPase inhibitor.
[0056] Accordingly, the purpose of the present invention is to provide a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, which contains ML141 as an active ingredient.
[0057] Another object of the present invention is to provide a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition according to the present invention.
[0058] Another object of the present invention is to provide a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a step of culturing type 1 alveolar cells in a medium composition according to the present invention and inducing them into type 2 alveolar cells.
[0059] Another object of the present invention is to provide a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing the airway organoid in a medium composition according to the present invention to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells.
[0060]
[0061] [Composition for inhibiting lung cancer metastasis containing ML 141]
[0062] The present invention was conceived to solve the problems of the prior art as described above, and the inventors of the present invention completed the present invention by confirming that ML 141 reprograms cells in respiratory organ tissue into type 2 alveolar cells (AT2), thereby transforming the shape of the cells into a cubic shape and a shape in which actin is not observed in the cytoplasm, thereby effectively inhibiting the metastasis of cancer cells.
[0063] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for inhibiting metastasis of lung cancer, which contains ML 141 as an active ingredient.
[0064] Another object of the present invention is to provide a food composition for preventing or improving metastasis of lung cancer, comprising ML 141 as an active ingredient.
[0065]
[0066] [Composition for inhibiting lung cancer metastasis containing dasatinib]
[0067] The present invention was conceived to solve the problems of the prior art as described above, and the inventors of the present invention completed the present invention by confirming that dasatinib reprograms cells in respiratory tissue into type 2 alveolar cells (AT2), thereby transforming the shape of the cells into a cubic shape and a shape in which actin is not observed in the cytoplasm, thereby effectively inhibiting the metastasis of cancer cells.
[0068] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for inhibiting metastasis of lung cancer, which comprises dasatinib as an active ingredient.
[0069] Another object of the present invention is to provide a food composition for preventing or improving metastasis of lung cancer, comprising dasatinib as an active ingredient.
[0070] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.
[0071] [Reprogramming Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using Rhosin]
[0072] To achieve the above purpose, the present invention provides a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, which contains Rhosin as an active ingredient.
[0073] In addition, the present invention provides a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition according to the present invention.
[0074] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a step of culturing type 1 alveolar cells in a medium composition according to the present invention and inducing them into type 2 alveolar cells.
[0075] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing the airway organoid in a medium composition according to the present invention to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells.
[0076] In one embodiment of the present invention, the rosin may be included at a concentration of 1 to 500 μM relative to the entire composition, but is not limited thereto.
[0077] In another embodiment of the present invention, the type 1 alveolar cells may express the Aquaporin 5 gene, and the type 2 alveolar cells may express the SFTPC gene, but are not limited thereto.
[0078] In another embodiment of the present invention, the culturing may be performed for 1 to 10 days, but is not limited thereto.
[0079] The present invention also provides the use of Rhosin; or a composition thereof; for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells.
[0080]
[0081] [Reprogramming of Type 1 Pneumocytes into Type 2 Pneumocytes Using Dasatinib]
[0082] To achieve the above purpose, the present invention provides a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising Dasatinib as an active ingredient.
[0083] In addition, the present invention provides a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition according to the present invention.
[0084] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a step of culturing type 1 alveolar cells in a medium composition according to the present invention and inducing them into type 2 alveolar cells.
[0085] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing the airway organoid in a medium composition according to the present invention to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells.
[0086] In one embodiment of the present invention, the dasatinib may be included at a concentration of 0.1 to 50 μM relative to the entire composition, but is not limited thereto.
[0087] In another embodiment of the present invention, the type 1 alveolar cells may express the Aquaporin 5 gene, but are not limited thereto.
[0088] In another embodiment of the present invention, the culturing may be performed for 1 to 10 days, but is not limited thereto.
[0089] The present invention also provides the use of dasatinib; or a composition thereof; for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells.
[0090]
[0091] [Reprogramming of Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using ML141]
[0092] To achieve the above purpose, the present invention provides a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising ML141 as an active ingredient.
[0093] In addition, the present invention provides a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition according to the present invention.
[0094] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a step of culturing type 1 alveolar cells in a medium composition according to the present invention and inducing them into type 2 alveolar cells.
[0095] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing the airway organoid in a medium composition according to the present invention to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells.
[0096] In one embodiment of the present invention, the ML141 may be included at a concentration of 1 to 100 μM relative to the entire composition, but is not limited thereto.
[0097] In another embodiment of the present invention, the type 1 alveolar cells may express the Aquaporin 5 gene, but are not limited thereto.
[0098] In another embodiment of the present invention, the culturing may be performed for 1 to 10 days, but is not limited thereto.
[0099] The present invention also provides the use of ML141; or a composition thereof; for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells.
[0100]
[0101] [Composition for inhibiting lung cancer metastasis containing ML 141]
[0102] The present invention provides a pharmaceutical composition for inhibiting metastasis of lung cancer, comprising ML 141 as an active ingredient.
[0103] In addition, the present invention provides a food composition for improving or preventing metastasis of lung cancer, comprising ML 141 as an active ingredient.
[0104] In one specific example of the present invention, the composition may inhibit metastasis of lung cancer by inducing reprogramming into type 2 alveolar cells in respiratory organ tissue, but is not limited thereto.
[0105] In another specific embodiment of the present invention, the lung cancer may be, but is not limited to, non-small cell lung cancer.
[0106] In another specific example of the present invention, the composition may additionally include a carrier, excipient, diluent, etc. in addition to the active ingredient.
[0107] In addition, the present invention provides a method for inhibiting metastasis of lung cancer, comprising a step of administering to a subject a pharmaceutically effective amount of a composition comprising ML 141 as an active ingredient.
[0108] In addition, the present invention provides a use of a composition comprising ML 141 as an active ingredient for inhibiting lung cancer metastasis.
[0109] The present invention also provides a use for producing a drug for inhibiting metastasis of lung cancer of ML 141.
[0110]
[0111] [Composition for inhibiting lung cancer metastasis containing dasatinib]
[0112] The present invention provides a pharmaceutical composition for inhibiting metastasis of lung cancer, comprising dasatinib as an active ingredient.
[0113] In addition, the present invention provides a food composition for improving or preventing metastasis of lung cancer, comprising dasatinib as an active ingredient.
[0114] In one specific example of the present invention, the composition may inhibit metastasis of lung cancer by inducing reprogramming into type 2 alveolar cells in respiratory organ tissue, but is not limited thereto.
[0115] In another specific embodiment of the present invention, the lung cancer may be, but is not limited to, non-small cell lung cancer.
[0116] In another specific example of the present invention, the composition may additionally include a carrier, excipient, diluent, etc. in addition to the active ingredient.
[0117] In addition, the present invention provides a method for inhibiting metastasis of lung cancer, comprising a step of administering to a subject a pharmaceutically effective amount of a composition comprising dasatinib as an active ingredient.
[0118] In addition, the present invention provides a use of a composition comprising dasatinib as an active ingredient for inhibiting lung cancer metastasis.
[0119] The present invention also provides a use for producing a drug for inhibiting metastasis of lung cancer using dasatinib.
[0120] [Reprogramming Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using Rhosin]
[0121] According to the present invention, by reprogramming type 1 alveolar cells into type 2 alveolar cells using Rhosin, it is possible to efficiently secure type 2 alveolar cells, which were difficult to secure in sufficient numbers. In addition, rather than culturing single cells, it is expected that cells with physiological activity more similar to actual tissues can be secured by culturing the organoids themselves and reprogramming type 1 alveolar cells into type 2 alveolar cells within the organoids.
[0122]
[0123] [Reprogramming of Type 1 Pneumocytes into Type 2 Pneumocytes Using Dasatinib]
[0124] According to the present invention, type 1 alveolar cells can be reprogrammed into type 2 alveolar cells simply by treatment with Dasatinib without the overexpression process of signal transduction proteins and transcription factors through viruses, pDNA, etc., which are generally required for reprogramming. Therefore, it is expected that type 2 alveolar cells, which were difficult to secure in sufficient numbers, can be efficiently secured. In addition, rather than culturing single cells, by culturing the organoids themselves and reprogramming type 1 alveolar cells into type 2 alveolar cells within the organoids, it is expected that cells with physiological activity more similar to actual tissues can be secured.
[0125]
[0126] [Reprogramming of Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using ML141]
[0127] According to the present invention, type 1 alveolar cells can be reprogrammed into type 2 alveolar cells simply by treatment with ML141 without the overexpression process of signal transduction proteins and transcription factors through viruses, pDNA, etc., which are generally required for reprogramming. Therefore, it is expected that type 2 alveolar cells, which were difficult to secure in sufficient numbers, can be efficiently secured. In addition, rather than culturing single cells, by culturing the organoids themselves and reprogramming type 1 alveolar cells into type 2 alveolar cells within the organoids, it is expected that cells with physiological activity more similar to actual tissues can be secured.
[0128]
[0129] [Composition for inhibiting lung cancer metastasis containing ML 141]
[0130] It was confirmed that the composition comprising ML 141 according to the present invention as an active ingredient reprograms cells in respiratory organ tissue into type 2 alveolar cells, thereby changing the cell shape to a cubic shape and a shape in which actin is not observed in the cytoplasm, thereby inhibiting cancer cell metastasis through changes in the cytoskeleton. Therefore, it is expected that the use of ML 141 can effectively inhibit lung cancer metastasis, thereby lowering the recurrence rate of lung cancer, and thereby increasing the cure rate and survival rate of lung cancer, thereby significantly enhancing the therapeutic effect of lung cancer.
[0131]
[0132] [Composition for inhibiting lung cancer metastasis containing dasatinib]
[0133] The composition comprising dasatinib as an active ingredient according to the present invention was confirmed to inhibit cancer cell metastasis by reprogramming cells in respiratory tissue into type 2 alveolar cells, thereby changing the cell shape to a cubic shape and a form in which actin is not observed within the cytoplasm, i.e., through changes in the cytoskeleton. Therefore, it is expected that the use of dasatinib will effectively inhibit lung cancer metastasis, thereby reducing the recurrence rate of lung cancer, and thereby increasing the cure rate and survival rate of lung cancer, thereby significantly enhancing the therapeutic effect of lung cancer.
[0134] Figure 1 is a schematic diagram illustrating a method for producing patient-derived respiratory organoids.
[0135] Figure 2a is a drawing confirming the expression of AQP5 (Aquaporin 5) to identify AT1 cells in airway organoids cultured for 20 days. The lower image is an enlarged image of the area indicated by the white box in the upper image (D20: Day 20). Scale bar = 50 μm (top), 100 μm (bottom).
[0136] Figure 2b is a drawing showing AT2 cells identified in airway organoids cultured for 20 days using LysoTracker (arrow: airway organoids confirmed to contain AT2 cells using LysoTracker). Scale bar = 200 μm.
[0137] Figure 3a is a drawing showing AT2 cells identified using LysoTracker after treating airway organoids with 100 μM, 500 μM, and 1 mM Rhosin for 7 days.
[0138] Figure 3b is a diagram showing the results of FACS (Fluorescence-Activated Cell Sorting) analysis to determine the proportion of AT2 cells after treating 100 μM Rhosin in the airway organoid for 7 days.
[0139] Figure 4a shows the results of observing airway organoids after treating them with 10 μM, 50 μM, and 100 μM Rhosin for 2 days (195N: airway organoid classification number, AO: Airway organoid, P.6: passage number 6, D2: Day 2).
[0140] Figure 4b is a diagram showing the results of confirming AT2 cells using LysoTracker after treating the airway organoid with 10 μM and 50 μM Rhosin for 2 days.
[0141] Figure 4c is a diagram showing the results of confirming AT2 cells using LysoTracker after treating the airway organoids with 5 μM, 10 μM, and 50 μM Rhosin for 7 days.
[0142] Figure 5 is a diagram showing the results of confirming the expression of SFTPC, a biomarker of AT2 cells, after treating the respiratory organoids with 5 μM, 10 μM, and 50 μM Rhosin for 7 days.
[0143] Figure 6 is a diagram showing that when 5 μM of Rhosin was treated on the airway organoid, AT1 cells, which are the starting cells after reprogramming, were reduced.
[0144] Figure 7 is a schematic diagram illustrating a method for producing patient-derived respiratory organoids.
[0145] Figure 8 is a drawing showing the results of confirming the culture status after treating airway organoids with 1 μM, 5 μM, and 10 μM of dasatinib ((A) after dasatinib treatment, (B) 5 days after dasatinib treatment).
[0146] Figure 9 is a drawing showing AT2 (Alveolar Type 2) cells identified using LysoTracker after treating airway organoids with 1 μM, 5 μM, and 10 μM of dasatinib for 5 days.
[0147] Figure 10 is a drawing showing AT2 (Alveolar Type 2) cells identified using LysoTracker after treating airway organoids with 1 μM, 5 μM, and 10 μM of dasatinib for 5 days.
[0148] Figure 11 is a diagram showing the results of confirming AT1 cells after treating airway organoids with 5 μM dasatinib.
[0149] Figure 12 is a schematic diagram illustrating a method for producing patient-derived respiratory organoids.
[0150] Figure 13 is a drawing showing the results of confirming the culture status after treating the respiratory organoid with 5 μM, 10 μM, and 50 μM of ML141 ((A) after ML141 treatment, (B) 5 days after ML141 treatment).
[0151] Figure 14 is a drawing showing AT2 (Alveolar Type 2) cells confirmed using LysoTracker after treating the airway organoid with 5 μM, 10 μM, and 50 μM of ML141 for 5 days.
[0152] Figure 15 is a drawing showing AT2 (Alveolar Type 2) cells confirmed using LysoTracker after treating the airway organoid with 5 μM, 10 μM, and 50 μM of ML141 for 5 days.
[0153] Figure 16 is a drawing showing the results of confirming AT1 cells after treating the airway organoid with 10 μM ML141.
[0154] Figure 17 is a drawing showing the results of treating airway organoids with ML 141 at different concentrations according to one embodiment of the present invention and confirming the reprogramming effect into type 2 alveolar cells using LysoTracker.
[0155] Figure 18 is a drawing showing the result of producing a 3D spheroid of a lung cancer cell line according to one embodiment of the present invention.
[0156] Figure 19 is a drawing showing the results of confirming the lung cancer metastasis inhibition effect of ML 141 according to one embodiment of the present invention.
[0157] Figure 20 is a diagram showing the results of immunofluorescence staining to confirm the cellular characteristics that appear after treating ML 141 in 3D spheroids of lung cancer cell lines according to one embodiment of the present invention. The left column of each group represents an image obtained by merging images stained with DAPI.
[0158] Figure 21 is a drawing showing the results of treating airway organoids with dasatinib at different concentrations according to one embodiment of the present invention and confirming the reprogramming effect into type 2 alveolar cells using LysoTracker.
[0159] Figure 22 is a drawing showing the result of producing a 3D spheroid of a lung cancer cell line according to one embodiment of the present invention.
[0160] Figure 23 is a drawing showing the results of confirming the lung cancer metastasis inhibition effect of dasatinib according to one embodiment of the present invention.
[0161] Figure 24 is a diagram showing the results of immunofluorescence staining to confirm the cellular characteristics of 3D spheroids of lung cancer cell lines treated with dasatinib according to one embodiment of the present invention. The left column of each group represents an image obtained by merging images stained with DAPI.
[0162] [Reprogramming Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using Rhosin]
[0163] The present inventors completed the present invention by confirming that AT1 cells were reprogrammed into AT2 cells as a result of treating airway organoids with Rhosin, a Rho GTPase inhibitor.
[0164] Specifically, according to one embodiment of the present invention, it was confirmed that when airway organoids were produced (cultured) in a medium mixed (added, treated) with Rhosin, type 2 alveolar cells (AT2 cells) increased compared to airway organoids cultured in a medium commonly used for airway organoid production (culture) (see Examples 2 and 4).
[0165] In addition, according to another embodiment of the present invention, since Rhosin can induce cell death, in order to find the optimal conditions for reprogramming AT1 cells into AT2 cells using it, airway organoids were cultured by controlling the concentration and culture period of Rhosin, and it was confirmed that reprogramming into AT2 cells proceeded only by treating with a low concentration of 5 μM or 10 μM for 2 days (see Example 3).
[0166]
[0167] Hereinafter, the present invention will be described in detail.
[0168]
[0169] The present invention provides a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, which comprises Rhosin as an active ingredient. The reprogramming of type 1 alveolar cells (AT1 cells) into type 2 alveolar cells (AT2 cells) may be induced by adding (treating, mixing) Rhosin to airway organoids cultured in a medium, but is not limited thereto, and may also be induced by adding (treating, mixing) Rhosin to type 1 alveolar cells cultured in a medium.
[0170] The term "Rhosin" used herein refers to a RhoA inhibitor that inhibits cancer proliferation and promotes nerve cell regeneration. It is known to specifically target Rho GTPases to prevent the growth of cancer-related cells. Rhosin has been shown to inhibit cancer cell growth in a dose-dependent manner in breast cancer cells, and has also been shown to be effective in treating asthma and diabetes.
[0171] In the present invention, the rosin may be included at a concentration of 1 to 500 μM, 5 to 500 μM, 10 to 500 μM, 50 to 500 μM, 100 to 500 μM, 1 to 100 μM, 5 to 100 μM, 10 to 100 μM, 50 to 100 μM, 1 to 50 μM, 5 to 50 μM, 10 to 50 μM, 1 to 10 μM, 5 to 10 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM relative to the entire composition, and according to one embodiment of the present invention, the rosin may be included at a concentration of 5 μM or 10 μM relative to the entire composition, Without being limited thereto, it may be included in an appropriate range of concentrations capable of reprogramming AT1 cells into AT2 cells without inducing cell death relative to the overall composition.
[0172] According to one embodiment of the present invention, the rosin is characterized by reprogramming type 1 alveolar cells into type 2 alveolar cells.
[0173] As used herein, the term "reprogramming" refers to a method of converting a specific cell into a desired cell by controlling the global gene expression pattern of the specific cell. Specifically, reprogramming in the present invention refers to a method of converting a specific cell into a cell with completely different characteristics by artificially manipulating the specific cell, and for the purpose of the present invention, the reprogramming may be performed by treating type 1 alveolar cells or airway organoids containing the same with rosin. For example, reprogramming may include, but is not limited to, dedifferentiation of cells, direct reprogramming (direct conversion), or direct transdifferentiation.
[0174] The term “trans-differentiation” in this specification, also called lineage reprogramming, refers to the transformation of mature somatic cells into other mature somatic cells without passing through an intermediate pluripotent or progenitor cell stage (Graf, T.; Enver, T. (2009). "Forcing cells to change lineages". Nature 462 (7273): 587-594).
[0175] That is, transdifferentiation (direct cross-differentiation) is the conversion of one type of mature cell into another type of mature cell, and the conversion can be confirmed by detecting the morphology of each cell or the expression of specific genes. In the present invention, the differentiation into type 2 alveolar cells can be confirmed through the expression of SFTPC (Surfactant Protein C), a marker specifically expressed in type 2 alveolar cells.
[0176] In this specification, the term “direct reprogramming” is differentiated from the technology of producing induced pluripotent stem cells with pluripotency through a reprogramming process, and is a technology that directly induces conversion into a desired target cell through reprogramming culture. Conventional somatic cell nuclear transfer has the disadvantage of requiring the use of oocytes, which makes it less applicable than other cell reprogramming technologies. In addition, since induced pluripotent stem cell reprogramming technology inherently passes through pluripotent stem cells, there is the disadvantage that the presence of undifferentiated cells and the assurance of safety must be verified. However, the present invention is expected to provide an alternative that can overcome the problems of the above technology, such as production time, cost, efficiency, and safety, by directly producing type 2 alveolar cells, which are target cells, from type 1 alveolar cells through direct reprogramming. For the purposes of the present invention, direct reprogramming may be used interchangeably with direct dedifferentiation, direct differentiation, direct conversion, direct transdifferentiation, and transdifferentiation. In the present invention, direct reprogramming may mean direct dedifferentiation or cross-differentiation into type 2 alveolar cells.
[0177] As used herein, the term "medium" refers to a mixture of nutrients that enable the growth, survival, expansion, or differentiation of cells or organoids in vitro, and includes all suitable conventional media used in the art. The type of medium and culture conditions can be selected based on the type of cell or organoid within the skill of the art.
[0178] In the present invention, the medium composition may additionally include a basal medium for cell or organoid culture containing a carbon source, a nitrogen source, and trace elements, and the basal medium may include, for example, DMEM (Dulbecco's Modified Eagle Medium), Ham's F12, DMEM / F12, and Advanced DMEM / F12. According to one embodiment of the present invention, the basic medium may be Advanced DMEM / F12, and preferably, it may be a medium for culturing airway organoids containing a Wnt agonist (R-Spondin-1), a mitogenic growth factor (FGF-7 and FGF-10), a TGF-β inhibitor (Noggin and A83-01), a ROCK inhibitor (Y-27632), a p38 MAPK inhibitor (SB 202190), a serum substitute (B-27), NAC (N-Acetyl-L-cysteine), nicotinamide, a glutamine substitute (L-alanyl-L-glutamine dipeptide; product name: GlutaMax), a zwitterionic buffer (HEPES), and antibiotics (Penicillin-Streptomycin and Primocin) in Advanced DMEM / F12 medium, but is not limited thereto. Any medium commonly used for AT1 cell or AT2 cell culture can be used without limitation.
[0179] According to one embodiment of the present invention, the type 1 alveolar cells express the Aquaporin 5 (AQP5) gene, and the type 2 alveolar cells express the SFTPC gene. Therefore, by confirming the expression of the AQP5 and SFTPC genes in the airway organoid, the concentration of Rhosin applied to the airway organoid can be adjusted to produce an organoid having a desired ratio of type 1 alveolar cells and type 2 alveolar cells, and therefore, the medium composition of the present invention can be variously utilized to produce an organoid suitable for a desired purpose such as drug screening and cell therapy.
[0180] In this specification, “active ingredient” means an ingredient that exhibits the intended activity alone or can exhibit the intended activity together with a carrier or the like that is inactive in itself.
[0181]
[0182] In addition, the present invention provides, in another aspect, a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition according to the present invention. The kit of the present invention is not particularly limited in type, and any kit commonly used in the art can be used.
[0183] The above kit is not limited to the medium composition according to the present invention, and may include other reagents or devices. For example, it may include a culture plate for culturing target cells or organoids, a reagent for assessing the reprogramming of type 1 alveolar cells into type 2 alveolar cells, and may also include cells or organoids to be cultured.
[0184] The kit according to the present invention may be provided in a single container containing all of the rosin, nutrient mixture, growth factor, or basic medium and other reagents in appropriate volumes and / or forms, or may be provided in separate containers.
[0185] The kit according to the present invention may include an instruction manual describing the procedure for performing the method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells according to the present invention, which will be described later.
[0186]
[0187] In addition, the present invention provides, in another aspect, a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising the step of culturing type 1 alveolar cells in a medium composition according to the present invention and inducing them into type 2 alveolar cells.
[0188] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing an airway organoid (containing type 1 alveolar cells) in a medium composition according to the present invention to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells.
[0189] In the present invention, the method comprises culturing type 1 alveolar cells or airway organoids in the medium composition to induce reprogramming of type 1 alveolar cells into type 2 alveolar cells, and the culturing may be performed for 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 6 to 10 days, 7 to 10 days, 1 to 7 days, 2 to 7 days, 3 to 7 days, 1 to 3 days, 2 to 3 days, 1 to 2 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, and according to one embodiment of the present invention, airway organoids or type 1 alveolar cells may be cultured for 2 days or 7 days in the medium composition containing Rhosin at a concentration of 5 or 10 μM, but is not limited thereto. The above airway organoid is an airway organoid containing type 1 alveolar cells.
[0190] The present invention can reprogram type 1 alveolar cells into type 2 alveolar cells by culturing type 1 alveolar cells in a medium composition according to the present invention, and specifically, by culturing airway organoids containing type 1 alveolar cells in a medium composition according to the present invention, type 1 alveolar cells in the airway organoids can be reprogrammed into type 2 alveolar cells, thereby easily securing type 2 alveolar cells, which are difficult to secure in sufficient numbers, and by reprogramming while culturing the organoids themselves rather than culturing single cells, type 2 alveolar cells can be secured in a state in which physiological activity is more similar to that of actual tissues.
[0191]
[0192] [Reprogramming of Type 1 Pneumocytes into Type 2 Pneumocytes Using Dasatinib]
[0193] The present inventors completed the present invention by confirming that AT1 cells were reprogrammed into AT2 cells as a result of treating airway organoids with Dasatinib, a tyrosine kinase inhibitor.
[0194] Specifically, according to one embodiment of the present invention, when airway organoids were produced (cultured) in a medium mixed (added, treated) with Dasatinib compared to airway organoids cultured in a medium commonly used for airway organoid production (culture), it was confirmed that type 1 alveolar cells (AT1 cells) decreased and type 2 alveolar cells (AT2 cells) increased, thereby confirming that Dasatinib reprograms type 1 alveolar cells into type 2 alveolar cells.
[0195]
[0196] Hereinafter, the present invention will be described in detail.
[0197]
[0198] The present invention provides a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising Dasatinib as an active ingredient, wherein the reprogramming of type 1 alveolar cells (AT1 cells) into type 2 alveolar cells (AT2 cells) can be induced by adding (treating, mixing) Dasatinib to airway organoids being cultured in the medium.
[0199] The term “Dasatinib” in this specification refers to a signal transduction inhibitor among targeted therapeutic agents, which inhibits the proliferation of tumor cells by inhibiting the action of tyrosine kinase, and is a therapeutic agent used to treat chronic myelogenous leukemia and acute lymphoblastic leukemia.
[0200] In the present invention, the dasatinib is present in an amount of 0.1 to 50 μM, 0.1 to 40 μM, 0.1 to 30 μM, 0.1 to 20 μM, 0.1 to 10 μM, 0.1 to 5 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, 0.5 to 10 μM, 0.5 to 7 μM, 0.5 to 5 μM, 0.5 to 3 μM, 0.5 to 1 μM, 1 to 10 μM, 1 to 8 μM, 1 to 5 μM, 1 to 3 μM, 3 to 5 μM, 5 to 8 μM, 5 to 10 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 It may be included at a concentration of μM, 8 μM, 9 μM, or 10 μM, and according to one embodiment of the present invention, the dasatinib may be included at a concentration of 1 μM or 5 μM relative to the total composition, but is not limited thereto, and may be included at a concentration in an appropriate range that can reprogram AT1 cells into AT2 cells without inducing cell death.
[0201] According to one embodiment of the present invention, the dasatinib is characterized by reprogramming type 1 alveolar cells into type 2 alveolar cells.
[0202] As used herein, the term "reprogramming" refers to a method of converting a specific cell into a desired cell by controlling the global gene expression pattern of the specific cell. Specifically, reprogramming in the present invention refers to a method of converting a specific cell into a cell with completely different characteristics by artificially manipulating the specific cell, and for the purpose of the present invention, the reprogramming may be performed by treating type 1 alveolar cells with dasatinib. For example, reprogramming may include, but is not limited to, dedifferentiation of cells, direct reprogramming (direct conversion), or direct transdifferentiation.
[0203] The term “trans-differentiation” in this specification, also called lineage reprogramming, refers to the transformation of mature somatic cells into other mature somatic cells without passing through an intermediate pluripotent or progenitor cell stage (Graf, T.; Enver, T. (2009). "Forcing cells to change lineages". Nature 462 (7273): 587-594).
[0204] That is, transdifferentiation is the conversion of one type of mature cell into another type of mature cell, and the conversion can be confirmed by detecting the morphology of each cell or the expression of specific genes. In the present invention, the differentiation into type 2 alveolar cells can be confirmed through the expression of LysoTracker, which is specifically expressed in type 2 alveolar cells.
[0205] In this specification, the term “direct reprogramming” is differentiated from the technology of producing induced pluripotent stem cells with pluripotency through a reprogramming process, and is a technology that directly induces conversion into a desired target cell through reprogramming culture. Conventional somatic cell nuclear transfer has the disadvantage of requiring the use of oocytes, which makes it less applicable than other cell reprogramming technologies. In addition, since induced pluripotent stem cell reprogramming technology inherently passes through pluripotent stem cells, there is the disadvantage that the presence of undifferentiated cells and the assurance of safety must be verified. However, the present invention is expected to provide an alternative that can overcome the problems of the above technology, such as production time, cost, efficiency, and safety, by directly producing type 2 alveolar cells, which are target cells, from type 1 alveolar cells through direct reprogramming. For the purposes of the present invention, direct reprogramming may be used interchangeably with direct dedifferentiation, direct differentiation, direct conversion, direct transdifferentiation, and transdifferentiation. In the present invention, direct reprogramming may mean direct dedifferentiation or cross-differentiation into type 2 alveolar cells.
[0206] As used herein, the term "medium" refers to a mixture of nutrients that enable the growth, survival, expansion, or differentiation of cells or organoids in vitro, and includes all suitable conventional media used in the art. The type of medium and culture conditions can be selected based on the type of cell or organoid within the skill of the art.
[0207] In the present invention, the medium composition may additionally include a basal medium for cell or organoid culture containing a carbon source, a nitrogen source, and trace elements, and the basal medium may include, for example, DMEM (Dulbecco's Modified Eagle Medium), Ham's F12, DMEM / F12, and Advanced DMEM / F12. According to one embodiment of the present invention, the basic medium may be Advanced DMEM / F12, and preferably, it may be a medium for culturing airway organoids containing a Wnt agonist (R-Spondin-1), a mitogenic growth factor (FGF-7 and FGF-10), a TGF-β inhibitor (Noggin and A83-01), a ROCK inhibitor (Y-27632), a p38 MAPK inhibitor (SB 202190), a serum substitute (B-27), NAC (N-Acetyl-L-cysteine), nicotinamide, a glutamine substitute (L-alanyl-L-glutamine dipeptide; product name: GlutaMax), a zwitterionic buffer (HEPES), and antibiotics (Penicillin-Streptomycin and Primocin) in the Advanced DMEM / F12 medium, but is not limited thereto. Any medium commonly used for AT1 cell or AT2 cell culture can be used without limitation.
[0208] According to one embodiment of the present invention, the type 1 alveolar cells are characterized by expressing the Aquaporin 5 (AQP5) gene. Therefore, by confirming the expression of the AQP5 gene in the airway organoid, the concentration of dasatinib treated to the airway organoid can be adjusted to produce organoids having a desired ratio of type 1 alveolar cells and type 2 alveolar cells. Therefore, the medium composition of the present invention can be variously utilized to produce organoids suitable for desired purposes such as drug screening and cell therapy.
[0209] In this specification, “active ingredient” means an ingredient that exhibits the intended activity alone or can exhibit the intended activity together with a carrier or the like that is inactive in itself.
[0210]
[0211] In addition, the present invention provides, in another aspect, a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition according to the present invention. The kit of the present invention is not particularly limited in type, and any kit commonly used in the art can be used.
[0212] The above kit is not limited to the medium composition according to the present invention, and may include other reagents or devices. For example, it may include a culture plate for culturing target cells or organoids, a reagent for assessing the reprogramming of type 1 alveolar cells into type 2 alveolar cells, and may also include the target cells or organoids to be cultured.
[0213] The kit according to the present invention may be provided in a single container containing dasatinib, a nutrient mixture, a growth factor, or a basal medium and other reagents in appropriate volumes and / or forms, or may be provided in separate containers.
[0214] The kit according to the present invention may include an instruction manual describing the procedure for performing the method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells according to the present invention, which will be described later.
[0215]
[0216] In addition, the present invention provides, in another aspect, a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising the step of culturing type 1 alveolar cells in a medium composition according to the present invention and inducing them into type 2 alveolar cells.
[0217] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing an airway organoid (containing type 1 alveolar cells) in a medium composition according to the present invention to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells.
[0218] In the present invention, the method comprises culturing type 1 alveolar cells or airway organoids in the medium composition to induce reprogramming of type 1 alveolar cells into type 2 alveolar cells, and the culturing may be performed for 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 6 to 10 days, 7 to 10 days, 1 to 7 days, 2 to 7 days, 3 to 7 days, 5 to 7 days, 5 to 6 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 1 day, 2 days, 3 days, 4 days, or 5 days, and according to one embodiment of the present invention, airway organoids or type 1 alveolar cells may be cultured for 5 days in the medium composition containing Dasatinib at a concentration of 1 μM or 5 μM, but is not limited thereto. The above airway organoid is an airway organoid containing type 1 alveolar cells.
[0219] The present invention can reprogram type 1 alveolar cells into type 2 alveolar cells by culturing type 1 alveolar cells in a medium composition according to the present invention, and specifically, by culturing airway organoids containing type 1 alveolar cells in a medium composition according to the present invention, type 1 alveolar cells in the airway organoids can be reprogrammed into type 2 alveolar cells, thereby easily securing type 2 alveolar cells, which are difficult to secure in sufficient numbers, and by reprogramming while culturing the organoids themselves rather than culturing single cells, type 2 alveolar cells can be secured in a state in which physiological activity is more similar to that of actual tissues.
[0220]
[0221] [Reprogramming of Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using ML141]
[0222] The present inventors completed the present invention by confirming that AT1 cells were reprogrammed into AT2 cells as a result of treating airway organoids with ML141, a Cdc42 / Rac1 GTPase inhibitor.
[0223] Specifically, according to one embodiment of the present invention, when airway organoids were produced (cultured) in a medium mixed (added, treated) with ML141 compared to airway organoids cultured in a medium commonly used for airway organoid production (culture), it was confirmed that type 1 alveolar cells (AT1 cells) decreased and type 2 alveolar cells (AT2 cells) increased, and from this, it was confirmed that ML141 can reprogram type 1 alveolar cells into type 2 alveolar cells.
[0224]
[0225] Hereinafter, the present invention will be described in detail.
[0226]
[0227] The present invention provides a medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, which comprises ML141 as an active ingredient, and the reprogramming of type 1 alveolar cells (AT1 cells) into type 2 alveolar cells (AT2 cells) can be induced by adding (treating, mixing) ML141 to airway organoids being cultured in the medium.
[0228] The term "ML141" used herein refers to an intracellular signal transduction inhibitor that selectively inhibits Cdc42 and Rac1. Cdc42 and Rac1 are known to be involved in actin formation within cells. Furthermore, ML141 increases p38 within cells and induces p38-mediated apoptosis and senescence.
[0229] In the present invention, the ML141 is present in an amount of 1 to 100 μM, 1 to 90 μM, 1 to 80 μM, 1 to 70 μM, 1 to 60 μM, 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, 1 to 10 μM, 1 to 5 μM, 5 to 50 μM, 5 to 40 μM, 5 to 30 μM, 5 to 20 μM, 5 to 10 μM, 8 to 10 μM, 10 to 13 μM, 10 to 15 μM, 9 to 11 μM, 9 to 10 μM, 10 to 11 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or It can be included at a concentration of 10 μM, and according to one embodiment of the present invention, the ML141 can be included at a concentration of 10 μM relative to the entire composition, but is not limited thereto, and can be included at a concentration in an appropriate range that can reprogram AT1 cells into AT2 cells without inducing cell death relative to the entire composition.
[0230] According to one embodiment of the present invention, the ML141 is characterized by reprogramming type 1 alveolar cells into type 2 alveolar cells.
[0231] As used herein, the term "reprogramming" refers to a method of converting a specific cell into a desired cell by controlling the global gene expression pattern of the specific cell. Specifically, reprogramming in the present invention refers to a method of converting a specific cell into a cell with completely different characteristics by artificially manipulating the specific cell, and for the purpose of the present invention, the reprogramming may be performed by treating type 1 alveolar cells with ML141. For example, reprogramming may include, but is not limited to, dedifferentiation of cells, direct reprogramming (direct conversion), or direct transdifferentiation.
[0232] The term “trans-differentiation” in this specification, also called lineage reprogramming, refers to the transformation of mature somatic cells into other mature somatic cells without passing through an intermediate pluripotent or progenitor cell stage (Graf, T.; Enver, T. (2009). "Forcing cells to change lineages". Nature 462 (7273): 587-594).
[0233] That is, transdifferentiation is the conversion of one type of mature cell into another type of mature cell, and the conversion can be confirmed by detecting the morphology of each cell or the expression of specific genes. In the present invention, the differentiation into type 2 alveolar cells can be confirmed through the expression of LysoTracker, which is specifically expressed in type 2 alveolar cells.
[0234] In this specification, the term “direct reprogramming” is differentiated from the technology of producing induced pluripotent stem cells with pluripotency through a reprogramming process, and is a technology that directly induces conversion into a desired target cell through reprogramming culture. Conventional somatic cell nuclear transfer has the disadvantage of requiring the use of oocytes, which makes it less applicable than other cell reprogramming technologies. In addition, since induced pluripotent stem cell reprogramming technology inherently passes through pluripotent stem cells, there is the disadvantage that the presence of undifferentiated cells and the assurance of safety must be verified. However, the present invention is expected to provide an alternative that can overcome the problems of the above technology, such as production time, cost, efficiency, and safety, by directly producing type 2 alveolar cells, which are target cells, from type 1 alveolar cells through direct reprogramming. For the purposes of the present invention, direct reprogramming may be used interchangeably with direct dedifferentiation, direct differentiation, direct conversion, direct transdifferentiation, and transdifferentiation. In the present invention, direct reprogramming may mean direct dedifferentiation or cross-differentiation into type 2 alveolar cells.
[0235] As used herein, the term "medium" refers to a mixture of nutrients that enable the growth, survival, expansion, or differentiation of cells or organoids in vitro, and includes all suitable conventional media used in the art. The type of medium and culture conditions can be selected based on the type of cell or organoid within the skill of the art.
[0236] In the present invention, the medium composition may additionally include a basal medium for cell or organoid culture containing a carbon source, a nitrogen source, and trace elements, and the basal medium may include, for example, DMEM (Dulbecco's Modified Eagle Medium), Ham's F12, DMEM / F12, and Advanced DMEM / F12. According to one embodiment of the present invention, the basic medium may be Advanced DMEM / F12, and preferably, it may be a medium for culturing airway organoids containing a Wnt agonist (R-Spondin-1), a mitogenic growth factor (FGF-7 and FGF-10), a TGF-β inhibitor (Noggin and A83-01), a ROCK inhibitor (Y-27632), a p38 MAPK inhibitor (SB 202190), a serum substitute (B-27), NAC (N-Acetyl-L-cysteine), nicotinamide, a glutamine substitute (L-alanyl-L-glutamine dipeptide; product name: GlutaMax), a zwitterionic buffer (HEPES), and antibiotics (Penicillin-Streptomycin and Primocin) in the Advanced DMEM / F12 medium, but is not limited thereto. Any medium commonly used for AT1 cell or AT2 cell culture can be used without limitation.
[0237] According to one embodiment of the present invention, the type 1 alveolar cells are characterized by expressing the Aquaporin 5 (AQP5) gene. Therefore, by confirming the expression of the AQP5 gene in the airway organoid, the concentration of ML141 applied to the airway organoid can be adjusted to produce organoids having a desired ratio of type 1 alveolar cells and type 2 alveolar cells, and therefore, the medium composition of the present invention can be variously utilized to produce organoids suitable for desired purposes such as drug screening and cell therapy.
[0238] In this specification, “active ingredient” means an ingredient that exhibits the intended activity alone or can exhibit the intended activity together with a carrier or the like that is inactive in itself.
[0239]
[0240] In addition, the present invention provides, in another aspect, a kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition according to the present invention. The kit of the present invention is not particularly limited in type, and any kit commonly used in the art can be used.
[0241] The above kit is not limited to the medium composition according to the present invention, and may include other reagents or devices. For example, it may include a culture plate for culturing target cells or organoids, a reagent for assessing the reprogramming of type 1 alveolar cells into type 2 alveolar cells, and may also include the target cells or organoids to be cultured.
[0242] The kit according to the present invention may be provided in a single container containing ML141, a nutrient mixture, growth factors, or a basal medium and other reagents in appropriate volumes and / or forms, or may be provided in separate containers.
[0243] The kit according to the present invention may include an instruction manual describing the procedure for performing the method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells according to the present invention, which will be described later.
[0244]
[0245] In addition, the present invention provides, in another aspect, a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising the step of culturing type 1 alveolar cells in a medium composition according to the present invention and inducing them into type 2 alveolar cells.
[0246] In addition, the present invention provides a method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing an airway organoid (containing type 1 alveolar cells) in a medium composition according to the present invention to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells.
[0247] In the present invention, the method comprises culturing type 1 alveolar cells or airway organoids in the medium composition to induce reprogramming of type 1 alveolar cells into type 2 alveolar cells, and the culturing may be performed for 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 6 to 10 days, 7 to 10 days, 1 to 7 days, 2 to 7 days, 3 to 7 days, 4 to 7 days, 5 to 7 days, 4 to 6 days, 5 to 6 days, 4 to 5 days, 1 day, 2 days, 3 days, 4 days, or 5 days, and according to one embodiment of the present invention, airway organoids or type 1 alveolar cells may be cultured for 5 days in the medium composition containing ML141 at a concentration of 10 μM, but is not limited thereto. The above airway organoid is an airway organoid containing type 1 alveolar cells.
[0248] The present invention can reprogram type 1 alveolar cells into type 2 alveolar cells by culturing type 1 alveolar cells in a medium composition according to the present invention, and specifically, by culturing airway organoids containing type 1 alveolar cells in a medium composition according to the present invention, type 1 alveolar cells in the airway organoids can be reprogrammed into type 2 alveolar cells, thereby easily securing type 2 alveolar cells, which are difficult to secure in sufficient numbers, and by reprogramming while culturing the organoids themselves rather than culturing single cells, type 2 alveolar cells can be secured in a state in which physiological activity is more similar to that of actual tissues.
[0249]
[0250] [Composition for inhibiting lung cancer metastasis containing ML 141]
[0251] The present inventors completed the present invention by confirming that ML 141 reprograms cells in respiratory tissue into type 2 alveolar cells, thereby changing the shape of the cells to a cubic shape and changing actin into a shape that is not observed in the cytoplasm, thereby effectively inhibiting the metastasis of cancer cells.
[0252]
[0253] In this specification, “lung cancer” refers to a general term for malignant tumors that occur in the lungs, and can be divided into primary lung cancer in which cancer cells arise from the tissues that make up the lungs themselves, and metastatic lung cancer in which cancer cells arise from other organs and then spread to the lungs through blood vessels or lymphatic vessels.
[0254] In this specification, “metastasis” means the movement of a tumor from its initial site of occurrence to another site in the host, and for the purposes of the present invention, the metastasis may mean the movement and proliferation of a tumor that originated in the lung to another site in the body.
[0255] In this specification, "ML 141" refers to one of the compounds belonging to the intracellular signal transduction inhibitor. ML 141 is known to selectively inhibit Cdc42 and Rac1, which are known to be involved in the formation of actin within cells, and is also known to induce p38-mediated cell death (apoptosis) and cell senescence by increasing p38 within cells. The IUPAC name is 4-[3-(4-methoxyphenyl)-5-phenyl-3,4-dihydropyrazol-2-yl]benzenesulfonamide. In the present specification, the ML 141 may be included at a concentration of 1 to 500 μM, 1 to 400 μM, 1 to 300 μM, 1 to 200 μM, 1 to 100 μM, 1 to 90 μM, 1 to 80 μM, 1 to 70 μM, 1 to 60 μM, 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, 1 to 10 μM, 1 to 5 μM relative to the entire composition, and according to one embodiment of the present invention, the ML 141 may be included at a concentration of 10 μM relative to the entire composition, but is not limited thereto, and may be included at an appropriate concentration range capable of inhibiting metastasis of lung cancer without inducing death of normal cells relative to the entire composition.
[0256] The above ML 141 can be used in the form of a pharmaceutically acceptable salt, and as a salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The acid addition salt can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc.; non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc.; organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, These include phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate.
[0257] In this specification, “reprogramming”, also called reprogramming, refers to a general method of converting a specific cell into a desired cell by controlling the global gene expression pattern of the specific cell. Specifically, in the present invention, reprogramming means a method of converting a specific cell into a cell with completely different characteristics by artificially manipulating the specific cell, and for the purpose of the present invention, the reprogramming may be to induce conversion into type 2 alveolar cells by treating ML 141 to respiratory organ tissue. For example, reprogramming may include, but is not limited to, dedifferentiation of cells, direct reprogramming (direct conversion), or direct transdifferentiation.
[0258] In this specification, the “pharmaceutical composition” may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized as being for human use. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and may include a mixture of buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injections, and may include a base, excipients, lubricants, preservatives, etc. for topical administration. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing it with the pharmaceutically acceptable carrier described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated and used in the form of sugar-coated tablets, gels, pills, powders, granules, suppositories, external preparations, solutions, suspensions, sustained-release preparations, slurries, etc. Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.
[0259] The route of administration of the pharmaceutical composition according to the present invention is not limited to these, but oral or parenteral administration is preferred, and includes, for example, oral, intravenous, intramuscular, intraarticular, intrasynovial, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, intradermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, rectal, intrasternal, intralesional, intracranial, etc.
[0260] The dosage of the pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated, and may be appropriately selected by those skilled in the art, although it may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, and may be administered at 0.0001 to 500 mg / kg or 0.001 to 500 mg / kg per day. The administration may be administered once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way.
[0261] In this specification, the term “food composition” refers to various foods, such as beverages, gum, tea, vitamin complexes, health supplements, etc., and may be used in the form of pills, powders, granules, infusions, tablets, capsules, or beverages. The food composition includes a health functional food composition. At this time, the amount of ML 141 in the food or beverage may generally be added in an amount of 0.01 to 30 wt% of the total food weight in the case of the food composition of the present invention, and in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 mL in the case of a health beverage composition.
[0262] The food composition of the present invention has no particular limitations on the components added other than the ML 141 as an essential component, and may include conventional food additives used in the art, such as natural carbohydrates, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, various nutrients, vitamins, minerals (electrolytes), etc. Examples of the natural carbohydrates include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; polysaccharides, such as dextrin, cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of the flavoring agent include natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) that can be advantageously used. Examples of flavoring agents include honey, D-mannitol, maltitol solution, and krill concentrate. Additionally, the composition may contain pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. These ingredients may be used independently or in combination. The proportions of these additives are not critical, but are typically selected from a range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
[0263] In this specification, “prevention” means any act of inhibiting metastasis of lung cancer or delaying its onset by administering a composition according to the present invention.
[0264] In this specification, “improvement” means any act in which the symptoms of lung cancer metastasis are improved or beneficially changed by administration of the composition according to the present invention.
[0265] In this specification, “inhibiting” means any act of inhibiting or delaying metastasis of lung cancer, improving or beneficially changing the symptoms of lung cancer metastasis, or treating metastasis by administering a composition according to the present invention.
[0266] In this specification, “subject” refers to a subject to which the composition of the present invention can be administered, and there is no limitation on the subject.
[0267] In this specification, when a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless specifically stated otherwise.
[0268] In this specification, the terms "about," "substantially," and the like are used to mean values or values close to values when manufacturing and material tolerances inherent to the meanings stated are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that state precise or absolute values to aid understanding of the present invention. Furthermore, in this specification, the terms "step of ~" or "step of ~" do not mean "step for ~."
[0269]
[0270] [Composition for inhibiting lung cancer metastasis containing dasatinib]
[0271] The present inventors completed the present invention by confirming that dasatinib reprograms cells in respiratory tissue into type 2 alveolar cells, thereby changing the shape of the cells to cuboid and changing actin into a form that is not observed in the cytoplasm, thereby effectively inhibiting the metastasis of cancer cells.
[0272]
[0273] In this specification, “lung cancer” refers to a general term for malignant tumors that occur in the lungs, and can be divided into primary lung cancer in which cancer cells arise from the tissues that make up the lungs themselves, and metastatic lung cancer in which cancer cells arise from other organs and then spread to the lungs through blood vessels or lymphatic vessels.
[0274] In this specification, “metastasis” means the movement of a tumor from its initial site of occurrence to another site in the host, and for the purposes of the present invention, the metastasis may mean the movement and proliferation of a tumor that originated in the lung to another site in the body.
[0275] In this specification, "Dasatinib" is a compound belonging to a signal transduction inhibitor among targeted therapeutic agents, which inhibits the action of tyrosine kinase to suppress the proliferation of tumor cells and is a therapeutic agent used in the treatment of chronic myelogenous leukemia, acute lymphoblastic leukemia, etc. The IUPAC Name is N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazole carboxamide monohydrate. In the present specification, the Dasatinib may be included in a concentration of 0.1 to 500 μM, 0.1 to 400 μM, 0.1 to 300 μM, 0.1 to 200 μM, 0.1 to 100 μM, 0.1 to 90 μM, 0.1 to 80 μM, 0.1 to 70 μM, 0.1 to 60 μM, 0.1 to 50 μM, 0.1 to 40 μM, 0.1 to 30 μM, 0.1 to 20 μM, 0.1 to 10 μM, 0.1 to 5 μM, 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM relative to the entire composition, and according to one embodiment of the present invention, the Dasatinib may be included in a concentration of 0.1 to 500 μM, 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM relative to the entire composition. It may be included at a concentration of, but is not limited to, 1 μM or 5 μM, and may be included at a concentration in an appropriate range capable of inhibiting metastasis of lung cancer without inducing death of normal cells compared to the entire composition.
[0276] The above Dasatinib can be used in the form of a pharmaceutically acceptable salt, and as a salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The acid addition salt can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc.; non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc.; organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, These include phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate.
[0277] In this specification, “reprogramming”, also called reprogramming, refers to a general method of converting a specific cell into a desired cell by controlling the global gene expression pattern of the specific cell. Specifically, in the present invention, reprogramming means a method of converting a specific cell into a cell with completely different characteristics by artificially manipulating the specific cell, and for the purpose of the present invention, the reprogramming may be to induce conversion into type 2 alveolar cells by treating respiratory organ tissue with dasatinib. For example, reprogramming may include, but is not limited to, dedifferentiation of cells, direct reprogramming (direct conversion), or direct transdifferentiation.
[0278] In this specification, the “pharmaceutical composition” may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized as being for human use. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and may include a mixture of buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injections, and may include a base, excipients, lubricants, preservatives, etc. for topical administration. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing it with the pharmaceutically acceptable carrier described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated and used in the form of sugar-coated tablets, gels, pills, powders, granules, suppositories, external preparations, solutions, suspensions, sustained-release preparations, slurries, etc. Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.
[0279] The route of administration of the pharmaceutical composition according to the present invention is not limited to these, but oral or parenteral administration is preferred, and includes, for example, oral, intravenous, intramuscular, intraarticular, intrasynovial, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, intradermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, rectal, intrasternal, intralesional, intracranial, etc.
[0280] The dosage of the pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated, and may be appropriately selected by those skilled in the art, although it may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, and may be administered at 0.0001 to 500 mg / kg or 0.001 to 500 mg / kg per day. The administration may be administered once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way.
[0281] In this specification, the term “food composition” refers to various foods, such as beverages, gum, tea, vitamin complexes, health supplements, etc., and may be used in the form of pills, powders, granules, infusions, tablets, capsules, or beverages. The food composition includes a health functional food composition. At this time, the amount of Dasatinib in the food or beverage may generally be added in an amount of 0.01 to 30 wt% of the total food weight in the case of the food composition of the present invention, and in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 mL in the case of a health beverage composition.
[0282] The food composition of the present invention has no particular limitations on the components added other than the essential component, Dasatinib, and may include conventional food additives used in the art, such as natural carbohydrates, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, various nutrients, vitamins, minerals (electrolytes), etc. Examples of the natural carbohydrates include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; polysaccharides, such as dextrin, cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of the flavoring agent include natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) that can be advantageously used. Examples of flavoring agents include honey, D-mannitol, maltitol solution, and krill concentrate. Additionally, the composition may contain pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. These ingredients may be used independently or in combination. The proportions of these additives are not critical, but are typically selected from a range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
[0283] In this specification, “prevention” means any act of inhibiting metastasis of lung cancer or delaying its onset by administering a composition according to the present invention.
[0284] In this specification, “improvement” means any act in which the symptoms of lung cancer metastasis are improved or beneficially changed by administration of the composition according to the present invention.
[0285] In this specification, “inhibiting” means any act of inhibiting or delaying metastasis of lung cancer, improving or beneficially changing the symptoms of lung cancer metastasis, or treating metastasis by administering a composition according to the present invention.
[0286] In this specification, “subject” refers to a subject to which the composition of the present invention can be administered, and there is no limitation on the subject.
[0287] In this specification, when a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless specifically stated otherwise.
[0288] In this specification, the terms "about," "substantially," and the like are used to mean values or values close to values when manufacturing and material tolerances inherent to the meanings stated are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that state precise or absolute values to aid understanding of the present invention. Furthermore, in this specification, the terms "step of ~" or "step of ~" do not mean "step for ~."
[0289] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.
[0290]
[0291] [Reprogramming Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using Rhosin]
[0292]
[0293] [Experimental Method]
[0294]
[0295] 1. Production of patient-derived airway organoids
[0296] Patient-derived airway organoids were produced as schematically shown in Figure 1. Specifically, airway tissue obtained from the patient's lung was washed with phosphate-buffered saline (PBS) solution containing 100 U / mL penicillin-streptomycin, and red blood cells (RBCs) were removed. After finely dissociating the airway tissue, 2 mg / mL collagenase (Roche, 11097113001) was added and incubated at 37°C for 1 hour. The reacted cell solution was filtered, treated with RBC Lysis Buffer (Invitrogen, 00-4333-57), and incubated at room temperature for 2 minutes. After centrifugation and washing, single cells derived from the airway tissue were obtained. 5,000-10,000 airway tissue-derived single cells were suspended in 20 μL of Matrigel (50%) solution and incubated at 37°C for 15 min to form Matrigel domes. Single cells were then embedded within the Matrigel domes, and AO medium, an airway organoid medium, was added. The cells were cultured in an incubator at 5% CO2 and 37°C. Half of the medium was replaced with fresh medium every 2-3 days to confirm organoid formation.
[0297] The specific composition of the AO medium used for the production of airway organoids was as follows: Advanced DMEM / F12 (Invitrogen, 12634-034) supplemented with 500 ng / mL R-Spondin-1 (PeproTech, 120-38), 25 ng / mL FGF-7 (PeproTech, 100-19), 100 ng / mL FGF-10 (PeproTech, 100-26), 100 ng / mL Noggin (PeproTech, 120-10C), 500 nM A83-01 (Tocris, 2939), 5 μM Y-27632 (Abmole, M1817), 500 nM SB 202190 (Sigma, S7067), 2% (v / v) B-27 (Gibco, 17504044), 1.25mM N-Acetyl-L-cysteine (Sigma-Aldrich, A9165), 5mM Nicotinamide (Sigma, N0636), 1% GlutaMax (Invitrogen, 35050061), 10mM HEPES (Gibco, 15630056), 100 U / mL Penicillin-Streptomycin (Gibco, 15140122), 50 μg / mL Primocin (Invivogen, Ant-pm-1).
[0298]
[0299]
[0300]
[0301] 2. Rhosin treatment of organoids
[0302] After producing airway organoids, the Rho inhibitor Rhosin (Sigma-Aldrich (Merck), 555460-M) was mixed into the organoid medium at a predetermined concentration. The Rhosin solvent used was dimethylsulfoxide (DMSO), and among the control groups, the vehicle group was treated with DMSO only. Organoids treated with either Rhosin or DMSO were cultured in an incubator at 5% CO2 and 37°C for a predetermined period of time.
[0303]
[0304] 3. Immunofluorescence staining
[0305] After removing the external organoid medium, the Matrigel dome was washed with phosphate-buffered saline (PBS). The organoids inside the Matrigel dome were fixed with 2% paraformaldehyde (PFA) containing 0.05% glutaraldehyde (Sigma-Aldrich, G6257) at room temperature for 20 minutes. After fixation, the organoids were washed three times with PBS, and the Matrigel dome and organoids were frozen using a cryomold (Sakura Finetek, 4565) and Optimal Cutting Temperature (OCT) (Sakura Finetek, 4583) compound to create OCT blocks. The frozen OCT blocks were sectioned using a Cryocut Microtome (Leica, CM1850) and washed with PBS for 15 minutes at room temperature. After quenching with 0.3 M glycine at room temperature for 20 minutes, the sections were washed with PBS at room temperature for 15 minutes. After blocking with 5% normal goat serum (Vector Laboratories, Inc., S-1000) at room temperature for 1 hour, the sections were treated with the primary antibodies Aquaporin 5 (1:200, abcam, ab92320) and SFTPC (1:100, ThermoFisher Scientific, PA5-102493) overnight at 4°C. After treatment with the primary antibodies, the sections were washed with PBS at room temperature for 15 minutes and treated with the secondary antibody goat anti-rabbit IgG 488 (1:1000, Invitrogen, A-11008) at room temperature for 1 hour. After washing with PBS at room temperature for 15 minutes, the sections were stained with DAPI (Invitrogen, D1306) for 2 minutes at room temperature.After mounting the organoid samples on cover slides, they were photographed using Carl Zeiss LSM800 or LSM900 confocal microscopes (Carl Zeiss).
[0306] To stain AT2 cells with LysoTracker, the medium was removed from cultured organoids, and 50 nM LysoTracker Green DND-26 (Invitrogen, L7526) was added to prewarmed organoid medium at 37°C. After 1 h of treatment in an incubator at 37°C with 5% CO2, the medium containing LysoTracker was removed, and the cells were washed three times with phosphate-buffered saline (PBS). After adding organoid medium again, images were taken using a fluorescence microscope.
[0307]
[0308] [Example]
[0309] Example 1. Identification of AT1 and AT2 cells in airway organoids.
[0310] Since AT2 cells are inherently present in airway organoids even without reprogramming with rhosin, AT1 and AT2 cells within the airway organoids were identified before reprogramming AT1 (Alveolar Type 1) cells into AT2 (Alveolar Type 2) cells. Specifically, as shown in Fig. 2a, Aquaporin 5 (Aqp5) within airway organoids cultured for 20 days confirmed that AT1 cells, the initiating cells for reprogramming into AT2 cells, were present within the airway organoids.
[0311] In addition, as shown in Fig. 2b, the presence of AT2 cells in airway organoids was confirmed using LysoTracker, which specifically stains only AT2 cells among the cells that make up the respiratory tract. As a result, it was confirmed that a certain level of AT2 cells are basically present when airway organoids are cultured for a sufficient period of time even before reprogramming with AT2 cells.
[0312]
[0313] Example 2. Rhosin reprogramming AT1 cells into AT2 cells in airway organoids.
[0314] To reprogram AT1 cells into AT2 cells within airway organoids, the produced airway organoids were treated with Rhosin, a Rho inhibitor, and the presence of AT2 was confirmed using LysoTracker. As a result, as shown in Fig. 3a, when Rhosin was treated at concentrations of 100 μM, 500 μM, and 1 mM for 7 days, a marked increase in AT2 cells was observed in the 100 μM Rhosin group. However, when Rhosin was treated at concentrations of 500 μM and 1 mM, cell death was observed due to high concentrations of Rhosin.
[0315] Furthermore, FACS (Fluorescence-Activated Cell Sorting) analysis was performed on AT2 cells generated after Rhosin treatment in the airway organoids. Specifically, LysoTracker of the control group and the Rhosin 100 μM treatment group + As a result of comparing the ratio of cells (AT2 cells) by FACS, as shown in Fig. 3b, a higher ratio of LysoTracker was observed when Rhosin was treated compared to the control group. + The cells could be observed.
[0316]
[0317] Example 3. Confirmation of Rhosin Conditions Capable of Reprogramming AT1 Cells into AT2 Cells in Airway Organoids
[0318] As examined in Example 2 above, high concentrations of Rhosin rather induced cell death, and the concentration of Rhosin capable of reprogramming AT1 cells into AT2 cells without inducing cell death was confirmed.
[0319] To confirm the specific conditions for AT2 cell reprogramming using Rhosin, airway organoids were treated with lower concentrations of 10 μM, 50 μM, and 100 μM Rhosin compared to Example 3, and then cultured for 2 days. As shown in Fig. 4a, airway organoids proliferated normally and maintained their morphology even after 2 days of reaction with each Rhosin treatment concentration. Therefore, it was decided to confirm whether AT2 cells were generated in airway organoids treated with Rhosin at concentrations below 100 μM.
[0320] Compared to Example 2, which treated with Rhosin for 7 days, reprogramming conditions were confirmed by treating with Rhosin at a concentration of 100 μM or less for a short period of time. Specifically, after treating with 10 μM and 50 μM Rhosin for 2 days, the presence of AT2 cells was confirmed using LysoTracker. As shown in Fig. 4b, it was confirmed that AT2 cells were generated in the airway organoids.
[0321] Furthermore, to confirm the effect of Rhosin at low concentrations, the organoids were treated with 5 μM, 10 μM, and 50 μM Rhosin for 7 days and then stained with LysoTracker. As shown in Fig. 4c, it was confirmed that AT2 cells increased in the airway organoids treated with 5 μM Rhosin.
[0322]
[0323] Example 4. Confirmation of AT2 cell reprogramming using Rhosin in airway organoids.
[0324] LysoTracker is known to specifically stain only AT2 cells among the cells that make up the respiratory tract, but AT2 cell reprogramming using Rhosin was performed using SFTPC, a biomarker of AT2 cells. + Additional validation was performed using cells. Specifically, after treating airway organoids with Rhosin at 5 μM, 10 μM, and 50 μM for 7 days, SFTPC, a biomarker of AT2 cells, was observed. + The cells were confirmed.
[0325] As a result, as shown in Fig. 5, when Rhosin was treated, SFTPC was detected in the same manner as the results of confirming AT2 cells using LysoTracker. + We were able to confirm that the cells had increased.
[0326] When treating tracheal organoids with Rhosin, LysoTracker + SFTPC + It was confirmed that the cells were produced by Rhosin, and from this, it was determined that AT2 cells were produced by Rhosin in the airway organoids.
[0327]
[0328] Example 5. Confirmation of reprogramming from AT1 cells to AT2 cells.
[0329] To confirm that AT2 cells were reprogrammed from AT1 cells, 5 μM Rhosin, which effectively induced AT2 cells in Example 1, was treated to airway organoids, and the remaining AT1 cells were confirmed. As a result, as shown in Fig. 6, the number of cells expressing Aquaporin 5 (AQP5) in airway organoids treated with Rhosin was confirmed to be significantly lower compared to the control group not treated with Rhosin.
[0330]
[0331] [Reprogramming of Type 1 Pneumocytes into Type 2 Pneumocytes Using Dasatinib]
[0332]
[0333] [Experimental Method]
[0334]
[0335] 1. Production of patient-derived airway organoids
[0336] Patient-derived airway organoids were produced as schematically shown in Figure 7. Specifically, airway tissue obtained from the patient's lung was washed with phosphate-buffered saline (PBS) solution containing 100 U / mL penicillin-streptomycin, and red blood cells (RBCs) were removed. After finely dissociating the airway tissue, 2 mg / mL collagenase (Roche, 11097113001) was added and incubated at 37°C for 1 hour. The reacted cell solution was filtered, treated with RBC Lysis Buffer (Invitrogen, 00-4333-57), and incubated at room temperature for 2 minutes. After centrifugation and washing, single cells derived from the airway tissue were obtained. 5,000-10,000 airway tissue-derived single cells were suspended in 20 μL of Matrigel (50%) solution and incubated at 37°C for 15 minutes to form Matrigel domes. Single cells were then embedded within the Matrigel domes, and AO medium, an airway organoid medium, was added. The cells were cultured in an incubator at 5% CO2 and 37°C. Half of the medium was replaced with fresh medium every 2-3 days to confirm organoid formation.
[0337] The specific composition of the AO medium used to produce the organoids is as follows:
[0338] Advanced DMEM / F12 (Invitrogen, 12634-034) supplemented with 500 ng / mL R-Spondin-1 (PeproTech, 120-38), 25 ng / mL FGF-7 (PeproTech, 100-19), 100 ng / mL FGF-10 (PeproTech, 100-26), 100 ng / mL Noggin (PeproTech, 120-10C), 500 nM A83-01 (Tocris, 2939), 5 μM Y-27632 (Abmole, M1817), 500 nM SB 202190 (Sigma, S7067), 2% (v / v) B-27 (Gibco, 17504044), 1.25 mM N-Acetyl-L-cysteine (Sigma-Aldrich, A9165), 5 mM Nicotinamide (Sigma, N0636), 1% GlutaMax (Invitrogen, 35050061), 10 mM HEPES (Gibco, 15630056), 100 U / mL Penicillin-Streptomycin (Gibco, 15140122), 50 μg / mL Primocin (Invivogen, Ant-pm-1).
[0339]
[0340]
[0341]
[0342] 2. 오가노이드에 dasatinib 처리
[0343] After generating airway organoids, the tyrosine kinase inhibitor dasatinib (StemCell Technologies, 73082) was mixed into the organoid medium at a predetermined concentration. The dasatinib solvent used was dimethylsulfoxide (DMSO), and the vehicle control group was treated with DMSO alone. Organoids treated with either dasatinib or DMSO were cultured in an incubator at 37°C and 5% CO2 for 5 days.
[0344]
[0345] 3. Immunofluorescence staining
[0346] To stain AT2 cells with LysoTracker, the medium was removed from cultured organoids, and 50 nM LysoTracker Green DND-26 (Invitrogen, L7526) was treated using organoid medium prewarmed to 37°C. After treatment for 1 hour in an incubator at 37°C with 5% CO2, the medium containing LysoTracker was removed, and the cells were washed three times with phosphate-buffered saline (PBS). Nuclei were stained with Hoechst 33342 (ThermoFisher Scientific, 62249) and then washed with phosphate-buffered saline (PBS). After the staining process was completed, the organoid medium was replaced, and images were taken using a fluorescence microscope.
[0347] In addition, after removing the external organoid medium, the Matrigel dome was washed with phosphate-buffered saline (PBS) solution. The organoids inside the Matrigel dome were fixed with 2% paraformaldehyde (PFA) containing 0.05% glutaraldehyde (Sigma-Aldrich, G6257) at room temperature for 20 minutes. After fixation, the organoids were washed three times with PBS, and the Matrigel dome and organoids were frozen using a cryomold (Sakura Finetek, 4565) and Optimal Cutting Temperature (OCT) (Sakura Finetek, 4583) compound to create OCT blocks. The frozen OCT blocks were sectioned using a Cryocut Microtome (Leica, CM1850) and washed with PBS for 15 minutes at room temperature. After quenching with 0.3 M glycine at room temperature for 20 minutes, the sections were washed with PBS at room temperature for 15 minutes. After blocking with 5% normal goat serum (Vector Laboratories, Inc., S-1000) at room temperature for 1 hour, the sections were treated with the primary antibody Aquaporin 5 (1:200, abcam, ab92320) at 4°C overnight. After treatment with the primary antibody, the sections were washed with PBS at room temperature for 15 minutes, and the sections were treated with the secondary antibody goat anti-rabbit IgG 488 (1:1000, Invitrogen, A-11008) at room temperature for 1 hour. After washing with PBS at room temperature for 15 minutes, the sections were stained with DAPI (Invitrogen, D1306) at room temperature for 2 minutes. After mounting the organoid samples on cover slides, they were photographed using Carl Zeiss LSM800 or LSM900 confocal microscopes (Carl Zeiss).
[0348]
[0349] [Example]
[0350] Example 1. Dasatinib induces reprogramming of AT2 cells in airway organoids.
[0351] Dasatinib can induce apoptosis, and we determined the concentration of dasatinib that could reprogram AT2 cells without inducing apoptosis. Specifically, we observed cultures cultured for 5 days after treating airway organoids with dasatinib at concentrations of 1 μM, 5 μM, and 10 μM.
[0352] As a result, as shown in Fig. 8, airway organoids proliferated normally and maintained their morphology even after 5 days of reaction with each dasatinib treatment concentration. Therefore, the generation of AT2 cells was confirmed using LysoTracker in airway organoids treated with 1 μM, 5 μM, and 10 μM dasatinib.
[0353] As a result, as shown in Figure 9, it was confirmed that AT2 cells increased when dasatinib was treated at 1 μM and 5 μM. On the other hand, when treated with the highest concentration of 10 μM, it was confirmed that all cells constituting the organoids died due to cytotoxicity. From the above results, it was confirmed that dasatinib generates AT2 cells under appropriate concentration conditions.
[0354] In addition, when we more specifically examined whether AT2 cells were generated using LysoTracker in airway organoids treated with dasatinib at 1 μM, 5 μM, and 10 μM, as shown in Fig. 10, at the highest concentration of 10 μM, a significant number of organoids died due to high-concentration treatment, but a high proportion of LysoTracker+ cells, i.e., AT2 cells, were observed in the remaining organoids. In addition, it was confirmed that organoids treated with dasatinib at 1 μM and 5 μM showed no abnormalities in culture and that the generation of AT2 cells, which are LysoTracker+ cells, increased.
[0355]
[0356] Example 2. Confirmation of reprogramming from AT1 cells to AT2 cells.
[0357] To confirm that AT2 cells were reprogrammed from AT1 cells, 5 μM dasatinib, which effectively induced AT2 cells in Example 1, was applied to airway organoids, and the remaining AT1 cells were confirmed. As a result, as shown in Fig. 11, the number of cells expressing Aquaporin 5 (AQP5) in airway organoids treated with 5 μM dasatinib was confirmed to be significantly lower compared to the control group not treated with dasatinib.
[0358] AQP5 is an AT1 marker, and from the above results, it was confirmed that dasatinib treatment converted AT1 cells inside the airway organoids into AT2 cells, thereby reducing the number of remaining AT1 cells.
[0359]
[0360] [Reprogramming of Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using ML141]
[0361]
[0362] [Experimental Method]
[0363]
[0364] 1. Production of patient-derived airway organoids
[0365] Patient-derived airway organoids were produced as schematically shown in Figure 12. Specifically, airway tissue obtained from the patient's lung was washed with phosphate-buffered saline (PBS) solution containing 100 U / mL penicillin-streptomycin, and red blood cells (RBCs) were removed. After finely dissociating the airway tissue, 2 mg / mL collagenase (Roche, 11097113001) was added and incubated at 37°C for 1 hour. The reacted cell solution was filtered, treated with RBC Lysis Buffer (Invitrogen, 00-4333-57), and incubated at room temperature for 2 minutes. After centrifugation and washing, single cells derived from the airway tissue were obtained. 5,000-10,000 airway tissue-derived single cells were suspended in 20 μL of Matrigel (50%) solution and incubated at 37°C for 15 minutes to form Matrigel domes. Single cells were then embedded within the Matrigel domes, and AO medium, an airway organoid medium, was added. The cells were cultured in an incubator at 5% CO2 and 37°C. Half of the medium was replaced with fresh medium every 2-3 days to confirm organoid formation.
[0366] The specific composition of the AO medium used to produce the organoids is as follows:
[0367] Advanced DMEM / F12 (Invitrogen, 12634-034) supplemented with 500 ng / mL R-Spondin-1 (PeproTech, 120-38), 25 ng / mL FGF-7 (PeproTech, 100-19), 100 ng / mL FGF-10 (PeproTech, 100-26), 100 ng / mL Noggin (PeproTech, 120-10C), 500 nM A83-01 (Tocris, 2939), 5 μM Y-27632 (Abmole, M1817), 500 nM SB 202190 (Sigma, S7067), 2% (v / v) B-27 (Gibco, 17504044), 1.25 mM N-Acetyl-L-cysteine (Sigma-Aldrich, A9165), 5 mM Nicotinamide (Sigma, N0636), 1% GlutaMax (Invitrogen, 35050061), 10 mM HEPES (Gibco, 15630056), 100 U / mL Penicillin-Streptomycin (Gibco, 15140122), 50 μg / mL Primocin (Invivogen, Ant-pm-1).
[0368]
[0369]
[0370]
[0371] 2. 오가노이드에 ML141 처리
[0372] After generating airway organoids, the Cdc42 / Rac1 GTPase inhibitor ML 141 (Tocris, 4266) was mixed into the organoid medium at a predetermined concentration. The ML141 solvent was dimethylsulfoxide (DMSO), and the vehicle control group was treated with DMSO only. Organoids treated with either ML141 or DMSO were cultured in an incubator at 37°C and 5% CO2 for 5 days.
[0373]
[0374] 3. Immunofluorescence staining
[0375] To stain AT2 cells with LysoTracker, the medium was removed from cultured organoids, and 50 nM LysoTracker Green DND-26 (Invitrogen, L7526) was added to prewarmed organoid medium at 37°C. After 1 h of treatment in an incubator at 37°C and 5% CO2, the medium containing LysoTracker was removed, and the cells were washed three times with phosphate-buffered saline (PBS). Nuclei were stained with Hoechst 33342 (ThermoFisher Scientific, 62249) and then washed with phosphate-buffered saline (PBS). After the staining process was completed, the organoid medium was replaced, and images were taken using a fluorescence microscope.
[0376] Additionally, after removing the external organoid medium, the Matrigel dome was washed with phosphate-buffered saline (PBS) solution. The organoids inside the Matrigel dome were fixed with 2% paraformaldehyde (PFA) containing 0.05% glutaraldehyde (Sigma-Aldrich, G6257) at room temperature for 20 minutes. After fixation, the organoids were washed three times with PBS, and the Matrigel dome and organoids were frozen using a cryomold (Sakura Finetek, 4565) and Optimal Cutting Temperature (OCT) (Sakura Finetek, 4583) compound to create OCT blocks. The frozen OCT blocks were sectioned using a Cryocut Microtome (Leica, CM1850) and washed with PBS for 15 minutes at room temperature. After quenching with 0.3 M glycine at room temperature for 20 minutes, the sections were washed with PBS at room temperature for 15 minutes. After blocking with 5% normal goat serum (Vector Laboratories, Inc., S-1000) at room temperature for 1 hour, the sections were treated with the primary antibody Aquaporin 5 (1:200, abcam, ab92320) at 4°C overnight. After treatment with the primary antibody, the sections were washed with PBS at room temperature for 15 minutes, and the sections were treated with the secondary antibody goat anti-rabbit IgG 488 (1:1000, Invitrogen, A-11008) at room temperature for 1 hour. After washing with PBS at room temperature for 15 minutes, the sections were stained with DAPI (Invitrogen, D1306) at room temperature for 2 minutes. After mounting the organoid samples on cover slides, they were photographed using Carl Zeiss LSM800 or LSM900 confocal microscopes (Carl Zeiss).
[0377]
[0378] [Example]
[0379] Example 1. Reprogramming ML141 into AT2 cells in airway organoids.
[0380] High concentrations of the compound can induce apoptosis, so we determined the concentration of ML141 that could reprogram AT2 cells without inducing apoptosis. Specifically, we observed cultures cultured for 5 days after treating airway organoids with ML141 at 5 μM, 10 μM, and 50 μM.
[0381] As a result, as shown in Fig. 13, even after 5 days of reaction with each ML141 treatment concentration, the airway organoids proliferated normally and maintained their morphology. Therefore, we decided to confirm the generation of AT2 cells using LysoTracker for airway organoids treated with 5 μM, 10 μM, and 50 μM ML141. As a result, as shown in Fig. 14, it was confirmed that AT2 cells increased when ML141 was treated at 5 μM and 10 μM. On the other hand, when treated with the highest concentration of 50 μM, all cells constituting the organoids died due to cytotoxicity. Therefore, it was confirmed that ML141 generates AT2 cells under appropriate concentration conditions.
[0382] In addition, when we more specifically examined whether AT2 cells were generated using LysoTracker in airway organoids treated with ML141 at 5 μM, 10 μM, and 50 μM, as shown in Fig. 15, at the highest concentration of 50 μM, a significant number of organoids died due to stress caused by the treatment with a high concentration of the compound, but a high proportion of LysoTracker+ cells, i.e., AT2 cells, were observed in the remaining organoids. In addition, it was confirmed that organoids treated with ML141 at 10 μM showed no abnormalities in culture and that the generation of AT2 cells, which are LysoTracker+ cells, also increased.
[0383]
[0384] Example 2. Confirmation of reprogramming from AT1 cells to AT2 cells.
[0385] To confirm that AT2 cells were reprogrammed from AT1 cells, 10 μM ML141, which effectively induced AT2 cells in Example 1, was treated to airway organoids, and the remaining AT1 cells were confirmed. As a result, as shown in Fig. 16, the number of cells expressing Aquaporin 5 (AQP5) in organoids treated with 10 μM ML141 was confirmed to be significantly lower compared to the control group not treated with ML141.
[0386] AQP5 is an AT1 marker, and from the above results, it was confirmed that AT1 cells inside the airway organoids were converted into AT2 cells by ML141 treatment, and the number of remaining AT1 cells was reduced.
[0387]
[0388] [Composition for inhibiting lung cancer metastasis containing ML 141]
[0389]
[0390] [Example]
[0391]
[0392] Example 1: Reprogramming AT1 cells into AT2 cells
[0393] Among the various cells that make up the lungs, the cells that perform major functions include alveolar type 1 (AT1) cells, which are adult cells, and alveolar type 2 (AT2) cells, which are stem cells. AT1 cells are cells that make up the alveoli and perform the main function of mediating gas exchange, and AT2 cells are known to secrete surfactant to protect and maintain the alveoli, and as precursor cells, they perform the function of repairing the lungs by producing AT1 cells when epithelial cells are damaged. AT1 and AT2 cells show differences in their internal structure and cell appearance. A representative difference in their internal structure is F-actin. In AT1 cells, actin forms a structure that extends from the inside of the cytoplasm to the outside of the nucleus, giving the nucleus of AT1 cells an oval shape. However, in AT2 cells, actin is not observed in the cytoplasm and does not contact the outside of the nucleus, giving the nucleus of AT2 cells a relatively close to a circle. In addition, the cell shape is cuboidal in the case of AT2 cells, and flat in the case of AT1 cells. In addition, since some studies have reported that AT1 cells have the property of cell migration compared to AT2 cells, the inventors of the present invention predicted that this cell shape would affect the metastasis of lung cancer, and tried to confirm whether reprogramming AT1 cells into AT2 cells in lung cancer tissues has an effect of inhibiting lung cancer metastasis.
[0394] For the primary reprogramming experiment, patient-derived airway organoids were generated. Specifically, airway tissue obtained from the patient's lungs was washed with phosphate-buffered saline (PBS) containing 100 U / mL penicillin-streptomycin, and red blood cells (RBCs) were removed. The airway tissue was then finely dissociated and incubated with 2 mg / mL collagenase (Roche Holding AG) at 37°C for 1 hour. After incubation, the solution was filtered, treated with RBC Lysis Buffer (Invitrogen) for 2 minutes at room temperature, and centrifuged and washed to obtain single cells derived from the airway tissue. A cell suspension of 5,000 to 10,000 airway-derived single cells was prepared using 20 μL of Matrigel (50%) solution, and incubated at 37°C for 15 minutes to form Matrigel domes. Afterwards, the organoid medium, A0 medium (Advanced DMEM / F12 (Invitrogen) supplemented with 500 ng / mL R-Spondin-1 (PeproTech), 25 ng / mL FGF-7 (PeproTech), 100 ng / mL FGF-10 (PeproTech), 100 ng / mL Noggin (PeproTech), 500 nM A83-01 (Tocris), 5 μM Y-27632 (Abmole), 500 nM SB202190 (Sigma), 2% (v / v) B-27 (Gibco), 1.Organoids were generated by adding 25 mM N-Acetyl-L-cysteine (Sigma-Aldrich), 5 mM Nicotinamide (Sigma), 1% GlutaMax (Invitrogen), 10 mM HEPES (Gibco), 100 U / mL Penicillin-Streptomycin (Gibco), 50 μg / mL Primocin (Invivogen)) and replacing half of the medium with fresh medium every 2–3 days in a 5% CO2, 37°C environment. Then, the generated airway organoids were treated by adding ML 141 (Tocris Bioscience™, 4266) to the medium at concentrations of 5, 10, and 50 μM. As a control, airway organoids treated only with DMSO (Dimethylsulfoxide), which was used as a solvent for ML 141, were used. Airway organoids were cultured for 5 days in a 5% CO2, 37°C environment, treated with ML 141 or DMSO, respectively.
[0395] And to confirm whether the airway organoids treated with ML 141 were reprogrammed into AT2 cells, immunofluorescence staining was performed. More specifically, to stain AT2 cells with LysoTracker, the medium was removed from the cultured airway organoids, and LysoTracker Green DND-26 (Invitrogen) was treated at a concentration of 50 nM using organoid medium prewarmed to 37°C, and after reacting for 1 hour in a 5% CO2, 37°C environment, the cells were washed three times with PBS to remove any remaining unstained LysoTracker. The cell nuclei were then stained with Hoechst 33342 (ThermoFisher Scientific) and washed with PBS. After all staining processes were completed, the organoid medium was replaced with fresh organoid medium and observed using a fluorescence microscope. The results are shown in Figure 17.
[0396] As shown in Fig. 17, in the experimental groups treated with ML 141 at concentrations of 5 or 10 μM, it was confirmed that AT2 cells increased as the concentration increased. In the experimental group treated with a concentration of 50 μM, a significant number of cells died due to stress caused by the treatment with a high concentration of the compound, but most of the remaining cells were confirmed to be LysoTracker positive. In other words, it was confirmed that most of them were reprogrammed into AT2 cells. On the other hand, no AT2 cells were observed in the control group treated with DMSO only. Through the above results, it was confirmed that ML 141 induces reprogramming into AT2 cells with stem cell properties in heterogeneous respiratory tissues where various cells coexist.
[0397]
[0398] Example 2: Confirmation of the lung cancer metastasis inhibition effect of ML 141.
[0399] To determine whether ML 141 can inhibit lung cancer metastasis by promoting reprogramming into AT2 cells in respiratory tract tissues, we first created three-dimensional (3D) spheroids using the A549 cell line (The Korean Cell Line Bank; KCLB), which is primarily used as a representative lung cancer model. In the case of a two-dimensional (2D) culture system, cancer cells grow in a single layer, so the actual drug efficacy cannot be determined, and in many cases, different results are shown from in vivo under conditions such as resistance to anticancer drugs and metastasis. Therefore, the inventors of the present invention created 3D spheroids with a form similar to in vivo and conducted experiments. In more detail, A549 (CCL-185) cell line was cultured using culture medium (Advanced DMEM / F-12 (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin / streptomycin (ThermoFisher Scientific)). Cells attached to the plate were detached using 0.25% Trypsin-EDTA (ThermoFisher Scientific) solution and washed three times using PBS. Then, a cell suspension was prepared using fresh culture medium and seeded in a SPL3D Cell Floater round-bottom 96-well plate (SPL Life Sciences) at a density of 2 × 10 4 After dispensing the cells to a concentration of 10 cells / well, the plate was centrifuged at 500xg for 5 minutes to collect the cells in the center of the well, and then cultured in a 5% CO2, 37°C environment for 4 days. The results are shown in Fig. 18.
[0400] As shown in Figure 18, after 4 days, 3D spheroids of lung cancer cell lines measuring approximately 500 μm in diameter were confirmed to have been formed normally. Tumor spheroids measuring 500 to 600 μm in diameter are known to be 3D structures that most closely mimic the state of in vivo tumors, including growth and micrometastasis.
[0401] 3D spheroids generated in the same manner as above were treated with Recombinant Human EGF (epidermal growth factor, PeproTec) at a concentration of 50 ng / mL to induce cell migration and create a metastatic environment for lung cancer. Simultaneously, ML 141 was treated at a concentration of 10 μM and cultured for 6 days in a 5% CO2, 37℃ environment to confirm whether cancer cell migration was inhibited. EGF and ML 141 were mixed in the culture medium and added slowly from the side of the well to avoid damaging the spheroids. The effect of inhibiting cancer cell migration was confirmed by measuring the total size (the sum of the size of the 3D spheroid itself and the distance of the migrated cells) and the maximum distance of the migrated cells excluding the size of the 3D spheroid itself using ImageJ (NIH) and GraphPad Prism 8 (GraphPad Software Inc.). All experiments were repeated at least three times, and the results are expressed as the mean ± standard deviation. Statistical analysis was performed using two-way ANOVA (alpha = 0.05, *p < 0.05, **p < 0.01, ***p<0.001). The results are shown in Figure 19.
[0402] As shown in Fig. 19, in the positive control group treated with EGF, cells were confirmed to have migrated about 150 μm, whereas in the experimental group treated with ML 141, cells were confirmed to have migrated about 100 μm even after 6 days, indicating that cell migration was inhibited. Considering that cells migrated about 50 μm in the negative control group treated with DMSO only, it was confirmed that cell migration was inhibited by about 50% when treated with ML 141.
[0403] In addition, immunofluorescence staining was performed to confirm the cellular characteristics of 3D spheroids after treatment with ML 141. More specifically, after removing the medium outside the 3D spheroids, the 3D spheroids were gently washed with phosphate-buffered saline (PBS) and fixed for 20 minutes at room temperature using a 2% paraformaldehyde solution supplemented with 0.05% glutaraldehyde (Sigma-Aldrich). After fixation, the spheroids were washed three times with PBS and blocked for 1 hour with 5% normal goat serum (Vector Laboratories, Inc.). Then, each 3D spheroid was treated with primary antibodies and incubated at 4°C. The primary antibodies used were Aquaporin 5 antibody (AQP5, 1:200, Abcam), SFTPC antibody (1:25, Invitrogen), and HT2-280 antibody (1:150, Terrace Biotech). After culturing for 16 hours, the cells were washed with PBS to completely remove all unbound antibodies, and then treated with the secondary antibody, goat anti-rabbit IgG 488 (1:1000, Invitrogen), at room temperature for 1 hour. To observe the actin structure, Alexa Fluor 680 anti-beta Actin antibody (1:200, Abcam) was treated at room temperature for 1 hour. After removing all unbound antibodies with PBS, the nuclei were stained with DAPI (Invitrogen) at room temperature for 2 minutes, and the spheroid samples were mounted on cover slides and photographed using Carl Zeiss LSM900 confocal microscopes (Carl Zeiss). The results are shown in Figure 20.
[0404] As shown in Fig. 20, it was confirmed that AT2 cells (SFTPC+, HT2-280+) were significantly increased in the experimental group treated with ML 141. In addition, when examining the structure of actin, when metastasis was inhibited by treatment with ML 141, actin was not evenly formed in the cytoplasm and transitioned to an epithelial state in the epithelial-mesenchymal transition (EMT), whereas in the spheroids in which metastasis was induced by treatment with EGF, actin was evenly formed and transitioned to a mesenchymal state for metastasis.
[0405] Through the above results, it was confirmed that ML 141 of the present invention can suppress cancer cell metastasis by reprogramming cells in respiratory organ tissue into AT2 cells, thereby changing the cell shape to cuboid and the form in which actin is not observed in the cytoplasm. Therefore, it was confirmed that by using ML 141, not only can the metastasis of lung cancer be effectively suppressed, the recurrence rate of lung cancer be lowered and the cure rate increased, but also the survival rate of lung cancer can be increased, significantly enhancing the therapeutic effect of lung cancer.
[0406]
[0407] [Composition for inhibiting lung cancer metastasis containing dasatinib]
[0408]
[0409] [Example]
[0410] Example 1: Reprogramming AT1 cells into AT2 cells
[0411] Among the various cells that make up the lungs, the cells that perform major functions include alveolar type 1 (AT1) cells, which are adult cells, and alveolar type 2 (AT2) cells, which are stem cells. AT1 cells are cells that make up the alveoli and perform the main function of mediating gas exchange, and AT2 cells are known to secrete surfactant to protect and maintain the alveoli, and as precursor cells, they perform the function of repairing the lungs by producing AT1 cells when epithelial cells are damaged. AT1 and AT2 cells show differences in their internal structure and cell appearance. A representative difference in their internal structure is F-actin. In AT1 cells, actin forms a structure that extends from the inside of the cytoplasm to the outside of the nucleus, giving the nucleus of AT1 cells an oval shape. However, in AT2 cells, actin is not observed in the cytoplasm and does not contact the outside of the nucleus, giving the nucleus of AT2 cells a relatively close to a circle. In addition, the cell shape is cuboidal in the case of AT2 cells, and flat in the case of AT1 cells. In addition, since some studies have reported that AT1 cells have the property of cell migration compared to AT2 cells, the inventors of the present invention predicted that this cell shape would affect the metastasis of lung cancer, and tried to confirm whether reprogramming AT1 cells into AT2 cells in lung cancer tissues has an effect of inhibiting lung cancer metastasis.
[0412] For the primary reprogramming experiment, patient-derived airway organoids were generated. Specifically, airway tissue obtained from the patient's lungs was washed with phosphate-buffered saline (PBS) containing 100 U / mL penicillin-streptomycin, and red blood cells (RBCs) were removed. The airway tissue was then finely dissociated and incubated with 2 mg / mL collagenase (Roche Holding AG) at 37°C for 1 hour. After incubation, the solution was filtered, treated with RBC Lysis Buffer (Invitrogen) for 2 minutes at room temperature, and centrifuged and washed to obtain single cells derived from the airway tissue. A cell suspension of 5,000 to 10,000 airway-derived single cells was prepared using 20 μL of Matrigel (50%) solution, and incubated at 37°C for 15 minutes to form Matrigel domes. Afterwards, the organoid medium, A0 medium (Advanced DMEM / F12 (Invitrogen) supplemented with 500 ng / mL R-Spondin-1 (PeproTech), 25 ng / mL FGF-7 (PeproTech), 100 ng / mL FGF-10 (PeproTech), 100 ng / mL Noggin (PeproTech), 500 nM A83-01 (Tocris), 5 μM Y-27632 (Abmole), 500 nM SB202190 (Sigma), 2% (v / v) B-27 (Gibco), 1.Organoids were generated by adding 25 mM N-Acetyl-L-cysteine (Sigma-Aldrich), 5 mM Nicotinamide (Sigma), 1% GlutaMax (Invitrogen), 10 mM HEPES (Gibco), 100 U / mL Penicillin-Streptomycin (Gibco), and 50 μg / mL Primocin (Invivogen)) and replacing half of the medium with fresh medium every 2–3 days in a 5% CO2, 37°C environment. Then, the generated airway organoids were treated by adding dasatinib (StemCell Technologies, 73082) to the medium at concentrations of 1, 5, and 10 μM. As a control, airway organoids treated only with DMSO (Dimethylsulfoxide), which was used as a solvent for dasatinib, were used. Airway organoids treated with dasatinib or DMSO were cultured for 5 days in a 5% CO2, 37°C environment.
[0413] And to confirm whether the airway organoids treated with dasatinib were reprogrammed into AT2 cells, immunofluorescence staining was performed. More specifically, to stain AT2 cells with LysoTracker, the medium was removed from the cultured airway organoids, and LysoTracker Green DND-26 (Invitrogen) was treated at a concentration of 50 nM using organoid medium prewarmed to 37°C, and after reacting for 1 hour in a 5% CO2, 37°C environment, the cells were washed three times with PBS to remove any remaining unstained LysoTracker. The cell nuclei were then stained with Hoechst 33342 (ThermoFisher Scientific) and washed with PBS. After all staining processes were completed, the organoid medium was replaced with fresh organoid medium and observed using a fluorescence microscope. The results are shown in Figure 21.
[0414] As shown in Figure 21, in the experimental groups treated with dasatinib at concentrations of 1 or 5 μM, it was confirmed that AT2 cells increased as the concentration increased. In the experimental group treated with a concentration of 10 μM, a significant number of cells died due to stress caused by treatment with a high concentration of the compound, and reprogramming into AT2 cells could not be confirmed. On the other hand, in the control group treated with DMSO only, AT2 cells were not observed. Through the above results, it was confirmed that dasatinib induces reprogramming into AT2 cells with stem cell properties in heterogeneous respiratory tissues where various cells coexist.
[0415]
[0416] Example 2: Confirmation of the effect of dasatinib on lung cancer metastasis.
[0417] To determine whether dasatinib can inhibit lung cancer metastasis by promoting reprogramming into AT2 cells in respiratory tract tissue, we first created three-dimensional (3D) spheroids using the A549 cell line (The Korean Cell Line Bank; KCLB), which is primarily used as a representative lung cancer model. In the case of a two-dimensional (2D) culture system, cancer cells grow in a single layer, so the actual drug efficacy cannot be determined, and in many cases, different results are shown from in vivo under conditions such as resistance to anticancer drugs and metastasis. Therefore, the inventors of the present invention created 3D spheroids with a form similar to in vivo and conducted experiments. In more detail, A549 (CCL-185) cell line was cultured using culture medium (Advanced DMEM / F-12 (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin / streptomycin (ThermoFisher Scientific)). Cells attached to the plate were detached using 0.25% Trypsin-EDTA (ThermoFisher Scientific) solution and washed three times using PBS. Then, a cell suspension was prepared using fresh culture medium and seeded in a SPL3D Cell Floater round-bottom 96-well plate (SPL Life Sciences) at a density of 2 × 10 4 After dispensing the cells to a concentration of 10 cells / well, the plate was centrifuged at 500xg for 5 minutes to collect the cells in the center of the well, and then cultured in a 5% CO2, 37°C environment for 4 days. The results are shown in Fig. 22.
[0418] As shown in Figure 22, after 4 days, 3D spheroids of lung cancer cell lines measuring approximately 500 μm in diameter were confirmed to have been formed normally. Tumor spheroids measuring 500 to 600 μm in diameter are known to be 3D structures that most closely mimic the state of in vivo tumors, including growth and micrometastasis.
[0419] 3D spheroids generated in the same manner as above were treated with Recombinant Human EGF (epidermal growth factor, PeproTec) at a concentration of 50 ng / mL to induce cell migration and create a metastatic environment for lung cancer. Simultaneously, dasatinib was treated at a concentration of 5 μM and cultured for 6 days in a 5% CO2, 37℃ environment to confirm whether cancer cell migration was inhibited. EGF and dasatinib were mixed in the culture medium and added slowly from the side of the well to avoid damaging the spheroids. The effect of inhibiting cancer cell migration was confirmed by measuring the total size (the sum of the size of the 3D spheroid itself and the distance of the migrated cells) and the maximum distance of the migrated cells excluding the size of the 3D spheroid itself using ImageJ (NIH) and GraphPad Prism 8 (GraphPad Software Inc.). All experiments were repeated at least three times, and the results are expressed as the mean ± standard deviation. Statistical analysis was performed using two-way ANOVA (alpha = 0.05, *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001). The results are shown in Figure 23.
[0420]
[0421] As shown in Figure 23, in the positive control group treated with EGF, cells were confirmed to have migrated approximately 150 μm, whereas in the experimental group treated with dasatinib, cells migrated less than 100 μm even after 6 days, confirming that cell migration was inhibited. This was confirmed to be a significant inhibition of lung cancer metastasis, similar to the case of the negative control group treated with only DMSO.
[0422] In addition, immunofluorescence staining was performed to confirm the cellular characteristics of 3D spheroids after treatment with dasatinib. More specifically, after removing the medium outside the 3D spheroids, the 3D spheroids were gently washed with phosphate-buffered saline (PBS) and fixed for 20 minutes at room temperature using 2% paraformaldehyde solution supplemented with 0.05% glutaraldehyde (Sigma-Aldrich). After fixation, the spheroids were washed three times with PBS and blocked for 1 hour with 5% normal goat serum (Vector Laboratories, Inc.). Each 3D spheroid was then treated with primary antibodies and incubated at 4°C. The primary antibodies used were Aquaporin 5 antibody (AQP5, 1:200, Abcam), SFTPC antibody (1:25, Invitrogen), and HT2-280 antibody (1:150, Terrace Biotech). After culturing for 16 hours, the cells were washed with PBS to completely remove all unbound antibodies, and then treated with the secondary antibody, goat anti-rabbit IgG 488 (1:1000, Invitrogen), at room temperature for 1 hour. To observe the actin structure, Alexa Fluor 680 anti-beta Actin antibody (1:200, Abcam) was treated at room temperature for 1 hour. After removing all unbound antibodies with PBS, the nuclei were stained with DAPI (Invitrogen) at room temperature for 2 minutes, and the spheroid samples were mounted on cover slides and photographed using Carl Zeiss LSM900 confocal microscopes (Carl Zeiss). The results are shown in Figure 24.
[0423] As shown in Fig. 24, it was confirmed that AT2 cells (SFTPC+, HT2-280+) were significantly increased in the experimental group treated with dasatinib. In addition, when examining the structure of actin, when metastasis was inhibited by dasatinib treatment, actin was not formed evenly in the cytoplasm and transitioned to an epithelial state in the epithelial-mesenchymal transition (EMT). On the other hand, in spheroids in which metastasis was induced by EGF treatment, actin was formed evenly and transitioned to a mesenchymal state for metastasis.
[0424] Through the above results, it was confirmed that dasatinib of the present invention can suppress cancer cell metastasis by reprogramming cells in respiratory tissue into AT2 cells, thereby changing the cell shape to cuboid and preventing actin from being observed within the cytoplasm. Therefore, it was confirmed that the use of dasatinib can not only effectively suppress lung cancer metastasis, thereby reducing the recurrence rate of lung cancer and increasing the cure rate, but also significantly increase the survival rate of lung cancer, thereby enhancing the therapeutic effect of lung cancer.
[0425]
[0426] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.
[0427] [Reprogramming Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using Rhosin]
[0428] The present invention relates to a composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, including rhosin as an active ingredient. According to the present invention, by reprogramming AT1 cells into AT2 cells using rhosin, it is possible to efficiently secure AT2 cells, which were difficult to secure in sufficient numbers. In addition, rather than culturing single cells, by culturing the organoids themselves and reprogramming AT1 cells into AT2 cells within the organoids, it is expected that cells having physiological activity more similar to that of actual tissues can be secured, and thus the industrial applicability thereof is recognized.
[0429]
[0430] [Reprogramming of Type 1 Pneumocytes into Type 2 Pneumocytes Using Dasatinib]
[0431] The present invention relates to a composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, including Dasatinib as an active ingredient. According to the present invention, by reprogramming AT1 cells into AT2 cells using Dasatinib, it is possible to efficiently secure AT2 cells, which were difficult to secure in sufficient numbers. In addition, rather than culturing single cells, the organoids themselves are cultured, and by reprogramming AT1 cells into AT2 cells within the organoids, it is expected that cells having physiological activity more similar to that of actual tissues can be secured, and thus the industrial applicability thereof is recognized.
[0432]
[0433] [Reprogramming of Type 1 Pleoblast Cells into Type 2 Pleoblast Cells Using ML141]
[0434] The present invention relates to a composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, including ML141 as an active ingredient. According to the present invention, by reprogramming AT1 cells into AT2 cells using ML141, it is possible to efficiently secure AT2 cells, which were difficult to secure in sufficient numbers. In addition, rather than culturing single cells, the organoids themselves are cultured, and by reprogramming AT1 cells into AT2 cells within the organoids, it is expected that cells having physiological activity more similar to that of actual tissues can be secured, and thus the industrial applicability thereof is recognized.
[0435]
[0436] [Composition for inhibiting lung cancer metastasis containing ML 141]
[0437] It was confirmed that the composition containing ML 141 according to the present invention as an active ingredient reprograms cells in respiratory organ tissue into type 2 alveolar cells, thereby changing the cell shape to a cubic shape and a shape in which actin is not observed in the cytoplasm, thereby inhibiting the metastasis of cancer cells through changes in the cytoskeleton. Therefore, it is expected that the use of ML 141 can effectively inhibit the metastasis of lung cancer, thereby lowering the recurrence rate of lung cancer, and thereby increasing the cure rate and survival rate of lung cancer, thereby significantly enhancing the therapeutic effect of lung cancer, and thus its industrial applicability is recognized.
[0438]
[0439] [Composition for inhibiting lung cancer metastasis containing dasatinib]
[0440] The composition comprising dasatinib as an active ingredient according to the present invention was confirmed to inhibit cancer cell metastasis by reprogramming cells in respiratory tissue into type 2 alveolar cells, thereby changing the cell shape to a cubic shape and a shape in which actin is not observed in the cytoplasm, i.e., through changes in the cytoskeleton. Therefore, it is expected that the use of dasatinib will effectively inhibit lung cancer metastasis, thereby reducing the recurrence rate of lung cancer, and thereby increasing the cure rate and survival rate of lung cancer, thereby significantly enhancing the therapeutic effect of lung cancer, and thus its industrial applicability is recognized.
Claims
1. A medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising rhosin as an active ingredient.
2. In paragraph 1, A medium composition, characterized in that the above rosin is included in a concentration of 1 to 500 μM relative to the total composition.
3. In paragraph 1 or 2, A medium composition characterized in that the type 1 alveolar cells express the Aquaporin 5 gene and the type 2 alveolar cells express the SFTPC gene.
4. A kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition of any one of claims 1 to 3.
5. A method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a step of culturing type 1 alveolar cells in the medium composition of paragraph 1 and inducing them into type 2 alveolar cells.
6. In paragraph 5, A method characterized in that the above culturing is performed for 1 to 10 days.
7. In paragraph 5 or 6, A method characterized in that the type 1 alveolar cells express the Aquaporin 5 gene and the type 2 alveolar cells express the SFTPC gene.
8. A method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising a step of culturing an airway organoid in the medium composition of paragraph 1 to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells. Rhosin for inducing reprogramming of type 9.1 alveolar cells into type 2 alveolar cells; or use of the composition of claim 1; 10. A medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising dasatinib as an active ingredient.
11. In paragraph 10, A medium composition, characterized in that the above dasatinib is included at a concentration of 0.1 to 50 μM relative to the total composition.
12. In paragraph 10 or 11, A medium composition characterized in that the above type 1 alveolar cells express the Aquaporin 5 gene.
13. A kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition of any one of items 10 to 12.
14. A method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a step of culturing type 1 alveolar cells in the medium composition of item 10 and inducing them into type 2 alveolar cells.
15. In paragraph 14, A method characterized in that the above culturing is performed for 1 to 10 days.
16. In paragraph 14 or 15, A method characterized in that the above type 1 alveolar cells express the Aquaporin 5 gene.
17. A method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising a step of culturing the airway organoid in the medium composition of item 10 to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells. Dasatinib for inducing reprogramming of type 18.1 alveolar cells into type 2 alveolar cells; or the composition of claim 10; Use.
19. A medium composition for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising ML141 as an active ingredient.
20. In paragraph 19, A medium composition characterized in that the above ML141 is included at a concentration of 1 to 100 μM relative to the total composition.
21. In paragraph 19 or 20, A medium composition characterized in that the above type 1 alveolar cells express the Aquaporin 5 gene.
22. A kit for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a medium composition of any one of items 19 to 21.
23. A method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells, comprising a step of culturing type 1 alveolar cells in the medium composition of item 19 and inducing them into type 2 alveolar cells.
24. In paragraph 23, A method characterized in that the above culturing is performed for 1 to 10 days.
25. In paragraph 23 or 24, A method characterized in that the above type 1 alveolar cells express the Aquaporin 5 gene.
26. A method for inducing reprogramming of type 1 alveolar cells into type 2 alveolar cells in an airway organoid, comprising the step of culturing the airway organoid in the medium composition of item 19 to induce type 1 alveolar cells in the airway organoid into type 2 alveolar cells. Use of ML141; or the composition of claim 19; for inducing reprogramming of type 27.1 alveolar cells into type 2 alveolar cells.
28. A pharmaceutical composition for inhibiting metastasis of lung cancer, comprising ML 141 as an active ingredient.
29. In paragraph 28, A pharmaceutical composition characterized in that the above pharmaceutical composition induces reprogramming into type 2 alveolar cells.
30. In paragraph 28 or 29, A pharmaceutical composition, characterized in that the above lung cancer is non-small cell lung cancer.
31. In any one of paragraphs 28 to 30, A pharmaceutical composition characterized in that the pharmaceutical composition further comprises a carrier, excipient or diluent in addition to the active ingredient. A food composition for improving or preventing metastasis of lung cancer, comprising 32.ML 141 as an active ingredient. A method for inhibiting metastasis of lung cancer, comprising a step of administering to a subject a pharmaceutically effective amount of a composition containing 33.ML 141 as an active ingredient.
34. Use of a composition containing ML 141 as an active ingredient for inhibiting lung cancer metastasis. 35.ML 141 For the purpose of producing a drug used to suppress metastasis of lung cancer.
36. A pharmaceutical composition for inhibiting metastasis of lung cancer, comprising dasatinib as an active ingredient.
37. In paragraph 36, A pharmaceutical composition characterized in that the above pharmaceutical composition induces reprogramming into type 2 alveolar cells.
38. In paragraph 36 or 37, A pharmaceutical composition, characterized in that the above lung cancer is non-small cell lung cancer.
39. In any one of paragraphs 36 to 38, A pharmaceutical composition characterized in that the pharmaceutical composition further comprises a carrier, excipient or diluent in addition to the active ingredient.
40. A food composition for improving or preventing metastasis of lung cancer, comprising dasatinib as an active ingredient.
41. A method for inhibiting metastasis of lung cancer, comprising a step of administering to a subject a pharmaceutically effective amount of a composition containing dasatinib as an active ingredient.
42. Use of a composition comprising dasatinib as an active ingredient for inhibiting lung cancer metastasis.
43. Use of dasatinib for producing a drug used to suppress metastasis of lung cancer.
Citation Information
Patent Citations
A composition for preventing or treating lung cancer comprising extracts of thallus of lichens, atranorin or salt thereof as an active ingredient
KR1020170082762A