Cancer treatment using extracellular secretions

Abstract

This disclosure provides cancer treatment using extracellular secretions. In specific embodiments, this disclosure provides cancer treatment using a combination of extracellular secretions and an ascorbic acid component. This disclosure provides uses, methods, combinations or compositions for cancer treatment. In one embodiment, the cancer may be breast cancer (including triple-negative breast cancer lacking estrogen receptor, progesterone receptor, and HER2). Mammals to be treated include humans, mice, rats, cats, and dogs.

Description

[Technical Field] 【0001】 This disclosure relates to the treatment of cancer using extracellular secretions. More specifically, this disclosure relates to the treatment of cancer using a combination of extracellular secretions and ascorbic acid components. [Background technology] 【0002】 Cell-based therapies such as regenerative medicine using iPS cells and CAR-T therapy are being developed. However, cell-based treatments have problems such as the risk of cancer. [Overview of the project] [Means for solving the problem] 【0003】 The inventors of this invention discovered that extracellular secretions are useful in treating cancer, and thus completed the present invention. 【0004】 This disclosure provides, for example, the following items: (Item 1) A combination for treating cancer, containing extracellular secretions and ascorbic acid components. (Item 2) The aforementioned cancer is any combination of the above items, including breast cancer or cervical cancer. (Item 3) The aforementioned cancer is any combination of the above items, including breast cancer. (Item 4) The aforementioned cancer is any combination of the above items, including triple-negative breast cancer. (Item 5) Any combination of the above items for treating humans or non-human mammals. (Item 6) A combination of any of the above items, containing the ascorbic acid component in a ratio of approximately 3 to approximately 100 mg per 1 mL of the extracellular secretion. (Item 7) The combination of any of the above items, wherein the ascorbic acid component is ascorbic acid, its derivative, or its salt. (Item 8) The combination of any of the above items, wherein the extracellular secretion is obtained from the culture supernatant of mesenchymal stem cells. (Item 9) The combination of any of the above items, wherein the extracellular secretion contains small extracellular vesicles (sEVs) at a concentration of about 6×10 10 cells / mL or more. (Item 10) A composition for treating cancer, comprising an extracellular secretion, which is characterized by being used in combination with an ascorbic acid component. (Item 11) A composition for treating cancer, comprising an ascorbic acid component, which is characterized by being used in combination with an extracellular secretion. [Advantages of the Invention] 【0005】 The present disclosure provides a novel treatment for cancer. [Brief Description of the Drawings] 【0006】 [Figure 1] Shows the effects of combinations of extracellular secretion (ES) and ascorbic acid (AA) or reduced glutathione (GSH) on MCF-7 cells. The vertical axis indicates the cell count, and the horizontal axis indicates the respective medium conditions on day 0 and day 6 of culture. [Figure 2] Microscopic images of MCF-7 cells cultured with a combination of extracellular secretion (ES) and ascorbic acid (AA) or reduced glutathione (GSH). From top to bottom, the images are at 24 hours, 96 hours, and 120 hours after the start of culture. [Figure 3] Microscopic images of MCF-7 cells cultured with a combination of extracellular secretion (ES) and ascorbic acid (AA) or reduced glutathione (GSH). From top to bottom, the images are at 24 hours, 96 hours, and 120 hours after the start of culture. [Figure 4] This graph shows the effects of extracellular secretions (ES) and combinations of ascorbic acid (AA) or reduced glutathione (GSH) on HeLa cells. The vertical axis represents cell number, and the horizontal axis represents the respective culture medium conditions at day 0 and day 6 of culture. [Figure 5] These are microscopic images of HeLa cells cultured with extracellular secretions (ES) and a combination of ascorbic acid (AA) or reduced glutathione (GSH). From top to bottom, the images are from 24 hours, 96 hours, and 120 hours after the start of culture. [Figure 6] These are microscopic images of HeLa cells cultured with extracellular secretions (ES) and a combination of ascorbic acid (AA) or reduced glutathione (GSH). From top to bottom, the images are from 24 hours, 96 hours, and 120 hours after the start of culture. [Figure 7] This graph shows the cell count on day 6 of MDA-MB468 cells (human triple-negative breast cancer) cultured with extracellular secretions (ES) and ascorbic acid (AA). The vertical axis shows the relative cell count in each condition, with the cell count in the control condition (without additional drug additions) set to 1, and the horizontal axis shows the respective culture medium conditions. [Modes for carrying out the invention] 【0007】 The present invention will be described below with reference to illustrative examples, as necessary, with reference to the accompanying drawings. Throughout this specification, singular expressions should be understood to include the concept of their plural forms unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In case of any conflict, this specification (including definitions) shall prevail. 【0008】 (definition) In this specification, “extracellular secretions” (sometimes abbreviated as “ES”) refers to substances produced by cells that are released outside the cell, or compositions containing such secretions. Typically, extracellular secretions are the supernatant of a cell culture or its components (extracellular vesicles, proteins, etc.) that do not contain cells themselves. Extracellular secretions are also obtained by removing certain components (extracellular vesicles, proteins, etc.) from the supernatant of a cell culture. 【0009】 In this specification, “extracellular vesicles” (sometimes abbreviated as “EVs”) refer to vesicles smaller than cells that are located outside the cell and surrounded by a lipid membrane. Extracellular vesicles typically contain proteins (e.g., membrane proteins), nucleic acids, and lipids. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. Extracellular vesicles typically have a diameter of about 50 to 5000 nm. In this specification, sEVs refer to extracellular vesicles with a particle size of about 50 to 600 nm. The particle size of extracellular vesicles can be measured, for example, by nanotracking. 【0010】 In this specification, "mesenchymal stem cells" are cells that have at least one of the following characteristics: (1) expression of specific cell membrane markers CD73, CD90, and CD105; (2) lack of expression of CD11b, CD14, CD34, CD45, CD19, CD79a, and HLA-DR; and (3) three germ layer pluripotency (the ability to differentiate into osteoblasts, chondrocytes, and adipocytes). Mesenchymal stem cells can be obtained from bone marrow, adipose tissue, teeth, etc., and can also be differentiated from pluripotent cells. 【0011】 In this specification, “ascorbic acid component” refers to 3,4-dihydroxy-5-(1,2-dihydroxyethyl)furan-2(5H)-one (ascorbic acid) or its derivatives or salts thereof. The ascorbic acid component typically has the following structure [ka] A compound (also called vitamin C), including its stereoisomers and racemates. Examples of derivatives of ascorbic acid include ester derivatives, ether bond derivatives, etc. The derivatives of ascorbic acid may be precursors of ascorbic acid that are metabolized in the body of mammals such as humans to produce ascorbic acid. Examples of ester derivatives include esters with saturated or unsaturated linear or branched fatty acids (C 2-24 etc.) such as ascorbyl palmitate or ascorbyl stearate, phosphate esters, etc. Examples of ether bond derivatives include ascorbic acid 2-glucoside, ethyl ascorbic acid, glyceryl ascorbic acid, bisglyceryl ascorbic acid, etc. 【0012】 Derivatives of ascorbic acid may include, for example, compounds represented by the following formulas: 【Chemical formula】 In the formula, The bond represented by the wavy line 【Chemical formula】 represents a single bond or a double bond, R 1 is NR A R B , OR A or halogen, R 2 is hydrogen, OR C or halogen, R 3 and R 4 are each independently hydrogen, pentose, hexose, disaccharide (e.g., sucrose), C(=O)R D or C 0-4 aliphatic chain - R D where, R A and R B are each independently hydrogen or R', or or R A and R BThey may come together to form a 5-10 member monocyclic or bicyclic aromatic heterocycle substituted with 0-4 substituents R'. R C is hydrogen or P(=O)(OR X )2, R X Each of them independently consists of hydrogen or (C 0-4 It is an aliphatic chain)-phenyl, R D Each of these is independently a hydrogen atom and a saturated or unsaturated linear or branched carbon atom substituted with 0 to 4 substituents R''. 1-24 It is an aliphatic chain, a 5-6 membered alicyclic ring substituted with 0-4 substituents R'', or a 5-6 membered aromatic ring substituted with 0-4 substituents R''. Each R' independently represents a halogen, =O, or a saturated or unsaturated linear or branched carbon substituted with 0 to 4 substituents R''. 1-24 Aliphatic chains, or (C) substituted with 0-4 substituents R'' 0-4 It is an aliphatic chain-(5-6 member aromatic ring), R'' independently consists of a hydroxyl group, a halogen, a cyano group, and C 1-4 aliphatic chain, OC 1-4 aliphatic chain or O-(C 0-4 It is an aliphatic chain-aromatic ring with 5-6 members. 【0013】 Specific ascorbic acid derivatives may include, for example, the following compounds: [ka] (Bn = benzyl, Ph = phenyl) 【0014】 Examples of salts include, but are not limited to, salts with sodium, potassium, magnesium, calcium, or ammonia. 【0015】 In this specification, “treatment” includes both therapy and prevention. In this specification, “therapy” means, with respect to a disease or disorder, preventing the worsening of such disease or disorder when such a condition has occurred, preferably maintaining the current condition, more preferably reducing it, and even more preferably eliminating it, and includes exerting a symptom-improving or preventive effect on the patient’s disease or one or more symptoms associated with the disease. In this specification, “prevention” means, with respect to a disease or disorder, preventing such a condition from occurring before it occurs. 【0016】 In this specification, the term "approximately" refers to plus or minus 10% of the indicated value unless otherwise specified. When "approximately" is used for temperature, it refers to plus or minus 5°C of the indicated temperature; when "approximately" is used for pH, it refers to plus or minus 0.5 of the indicated pH. 【0017】 (Preferred embodiment) Preferred embodiments of the Disclosure are described below. The embodiments provided below are provided for a better understanding of the Disclosure, and it will be understood that the scope of the Disclosure should not be limited to the descriptions below. Accordingly, it will be obvious that those skilled in the art can make appropriate modifications within the scope of the Disclosure, taking into consideration the descriptions herein. It will also be understood that the embodiments of the Disclosure below can be used individually or in combination. 【0018】 (For anti-cancer use) This disclosure provides cancer treatment using extracellular secretions. In specific embodiments, this disclosure provides cancer treatment using a combination of extracellular secretions and an ascorbic acid component. While this specification primarily describes cancer treatment using extracellular secretions, various embodiments are also contemplated as inventions, including the use of extracellular secretions in cancer treatment, methods of cancer treatment including the step of administering extracellular secretions, and combinations or compositions for cancer treatment containing extracellular secretions. Descriptions of one embodiment, such as a method, apply similarly to other embodiments. 【0019】 In one embodiment, cancers to be treated include malignant and benign tumors (for example, primary, metastatic or recurrent breast cancer, prostate cancer, pancreatic cancer, stomach cancer, lung cancer, colorectal cancer (colon cancer, rectal cancer, anal cancer), esophageal cancer, duodenal cancer, head and neck cancer (tongue cancer, pharyngeal cancer, laryngeal cancer, thyroid cancer), brain tumors, schwannomas, neuroblastomas, gliomas, non-small cell lung cancer, small cell lung cancer, liver cancer, kidney cancer, bile duct cancer, uterine cancer (endometrial cancer, cervical cancer), ovarian cancer, bladder cancer, skin cancer, hemangiomas, malignant lymphomas, malignant melanomas, bone tumors, angiofibromas, retinal sarcomas, penile cancers, pediatric solid tumors, Kaposi's sarcoma, maxillary sinus tumors, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, uterine fibroids, osteoblastoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, leukemia, and other tumors). In one embodiment, the cancer may be breast cancer (including triple-negative breast cancer lacking estrogen receptor, progesterone receptor, and HER2). In one embodiment, the cancer to be treated is characterized by elevated Stat3 expression and includes cancer cells exhibiting Stat3 expression levels approximately 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, or greater than the average Stat3 expression levels (e.g., mRNA count, protein amount) in normal cells in the organ with cancer or in cells in the same organ of a healthy subject without cancer. 【0020】 Examples of animals that can be treated include mammals such as humans, mice, rats, cats, and dogs. In one embodiment, the treated animal is a human. In another embodiment, the treated animal is a pet (dog, cat, etc.). In another embodiment, extracellular secretions derived from cells of the same species as the treated animal may be used. 【0021】 (Dosage / Dosage) In one embodiment, the extracellular secretion is administered to the subject in doses ranging from about 5 to about 100 mL per dose, for example, about 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 mL, or a range between two of these values. In a typical embodiment, the extracellular secretion is administered to the subject in doses ranging from about 30 mL per dose. In one embodiment, the extracellular secretion is administered to the subject in doses ranging from about 0.1 to about 2 mL per body weight of the subject, for example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.2, about 1.5, about 1.7, about 2 mL, or a range between two of these values. 【0022】 In one embodiment, the ascorbic acid component is administered to a subject in doses ranging from approximately 3 to approximately 300 g at a time, for example, approximately 3, approximately 5, approximately 7, approximately 10, approximately 15, approximately 20, approximately 30, approximately 40, approximately 50, approximately 70, approximately 100, approximately 150, approximately 200, approximately 250, approximately 300 g, or a range between two of these values. In one embodiment, the ascorbic acid component is administered to a subject in doses ranging from approximately 50 to approximately 5000 mg per body weight at a time, for example, approximately 50, approximately 70, approximately 100, approximately 150, approximately 200, approximately 300, approximately 400, approximately 500, approximately 700, approximately 1000, approximately 1500, approximately 2000, approximately 3000, approximately 4000, approximately 5000 mg, or a range between two of these values. In a typical embodiment, the ascorbic acid component is administered by intravenous infusion at a dose of approximately 100 to 1000 mg per body weight of the subject. 【0023】 In one embodiment, the extracellular secretion and ascorbic acid component are administered in a proportion of about 1 to about 200 mg of the ascorbic acid component per 1 mL of extracellular secretion, for example, about 1, about 2, about 3, about 4, about 5, about 7, about 10, about 15, about 20, about 30, about 40, about 50, about 70, about 100, about 120, about 150, about 200 mg, or a range between two of these values. For ascorbic acid components that are ascorbic acid derivatives and / or salts, the above dosage may be calculated based on the amount of ascorbic acid contained therein or derived therefrom. 【0024】 The amounts of extracellular secretions and ascorbic acid components may vary depending on the target of treatment or the nature of the cancer, but those skilled in the art can determine them by standard clinical techniques based on the description herein. In some cases, in vitro assays may be used to assist in identifying the optimal dosage range. The exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease or condition, and should be determined according to the judgment of the attending physician and the circumstances of each patient. The dosing interval is not particularly limited, but may be administered once or twice every 1, 7, 14, 21, or 28 days, or once or twice every two of these intervals. The dosage, dosing interval, and method of administration may be appropriately selected depending on the age, weight, symptoms, etc. of the subject. 【0025】 The routes of administration for extracellular secretions and ascorbic acid components may include, for example, intravenous (e.g., intravenous infusion), intradermal, subcutaneous, intramuscular, intraperitoneal, intrathecal, intraventricular, intraparenchymal, and epidural administration. Local administration is also possible, but even localized cancers can be treated by intravenous administration. 【0026】 Extracellular secretions and ascorbic acid components may be administered simultaneously or at different times. When extracellular secretions and ascorbic acid components are administered simultaneously, they may be administered mixed or separately. 【0027】 In addition to treatment with extracellular secretions and ascorbic acid components, further treatment with other drugs and / or therapies may be performed. For example, typically, other drugs and / or therapies for the treatment of cancer or its complications may be combined. 【0028】 (extracellular secretions) In one embodiment, the extracellular secretion contains the culture supernatant or components of mesenchymal stem cells. Therefore, the extracellular secretion may contain components of the culture medium used to cultivate the cells (e.g., salts, buffers, growth factors, vitamins, minerals, cytokines, etc.). In one embodiment, the extracellular secretion does not contain cells. Cells can be easily removed from the culture supernatant using a filter or the like. 【0029】 In one embodiment, extracellular secretions are characterized by small extracellular vesicles (sEVs). Excessive mTOR activation can be suppressed by small extracellular vesicles (sEVs). In one embodiment, extracellular secretions are approximately 1 × 10⁻⁶ 10 pcs / mL or more, approximately 2×10 10 pcs / mL or more, approximately 5×10 10 pcs / mL or more, approximately 5×10 10 pcs / mL or more, approximately 1×10 11 More than 5 cells / mL or approximately 5 x 10 11 More than 5 cells / mL and / or approximately 5 × 10 10 pcs / mL or less, approximately 1×10 11 pcs / mL or less, approximately 2×10 11 pcs / mL or less, approximately 5×10 11 Less than 1 x 10 cells / mL or approximately 1 x 10 12 Contains small extracellular vesicles (sEVs) of less than 1 / mL. 【0030】 In one embodiment, the small-sized extracellular vesicles (sEVs) in the extracellular secretion have a mode particle size of approximately 90–130 nm, 90–120 nm, 90–115 nm, 90–110 nm, 100–130 nm, 100–120 nm, 100–115 nm, 100–110 nm, 105–130 nm, 105–120 nm, or 105–115 nm. 【0031】 In one embodiment, the small extracellular vesicles (sEVs) in the extracellular secretion have a particle size distribution with a full width at half maximum of approximately 40–90 nm, 50–80 nm, 55–75 nm, or 60–70 nm. 【0032】 In one embodiment, the extracellular vesicles in the extracellular secretion of the present invention may have a proportion of small-sized extracellular vesicles (sEVs) of approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, approximately 97% or more, or approximately 99% or more of the total extracellular vesicles. 【0033】 (Preparation of extracellular secretions) In one embodiment, the extracellular secretion may be prepared from the culture supernatant of mesenchymal stem cells. In one embodiment, the mesenchymal stem cells are human cells. In one embodiment, the mesenchymal stem cells are derived from adipocytes, nerve cells, epithelial cells, endothelial cells, hematopoietic cells, connective tissue cells, muscle cells, osteocytes, chondrocytes, germline cells, or iPS cells, preferably derived from adipocytes. 【0034】 Cell culture conditions can affect the components of extracellular secretions (such as extracellular vesicles). In one embodiment, the extracellular secretions of the present disclosure are prepared from the culture supernatant of mesenchymal stem cells cultured at pH levels of approximately 7.0–10.0, 7.0–9.5, 7.0–9.0, 7.0–8.5, 7.5–10.0, 7.5–9.5, 7.5–8.5, 8.0–10.0, 8.0–9.5, 8.0–9.0, 8.0–8.5, 8.5–10.0, or 8.5–9.5. In one embodiment, the culture medium for mesenchymal stem cells comprises Tris hydrochloride buffer, HEPES buffer, or sodium bicarbonate buffer, preferably sodium bicarbonate buffer. In one embodiment, the culture medium for mesenchymal stem cells contains magnesium (e.g., about 0.001 mM to about 1 mM, e.g., about 0.001 mM, about 0.01 mM, about 0.1 mM, about 1 mM, etc.). 【0035】 To prepare extracellular secretions from cell culture supernatant, operations such as integration and distribution of the collected culture supernatant, freezing, and filtering can be performed. 【0036】 In one embodiment, cell culture supernatant can be separated into an extracellular vesicle fraction and a supernatant fraction. The fraction containing a higher concentration of extracellular vesicles compared to the original cell culture supernatant is called the extracellular vesicle fraction, and the fraction containing a lower concentration of extracellular vesicles is called the supernatant fraction. Methods such as centrifugation, sucrose density gradient ultracentrifugation, tangential flow filtration, and size exclusion chromatography can be used for separation. The extracellular secretions of this disclosure may be provided as a combination of the extracellular vesicle fraction and the supernatant fraction. In one embodiment, the extracellular vesicle fraction contains extracellular vesicles or small-sized extracellular vesicles (sEVs) at concentrations of at least about 110%, at least about 120%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 500%, at least about 700%, at least about 1000%, at least about 1500%, or at least about 2000% of the cell culture supernatant. In one embodiment, the supernatant fraction contains extracellular vesicles or small-sized extracellular vesicles (sEVs) at concentrations of up to approximately 90%, up to approximately 80%, up to approximately 50%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 7%, up to approximately 5%, up to approximately 2%, up to approximately 1.5%, or up to approximately 1% of the cell culture supernatant. 【0037】 In a typical embodiment, the extracellular secretion of the present disclosure is not an exosome concentrate such as a purified exosome preparation, but a liquid that removes cells, extracellular matrix and their fragments from a cell culture, while retaining most of the other components of the cell culture (including extracellular vesicles) in an amount equivalent to that of the original cell culture. 【0038】 (Combination or composition) In one embodiment, the Disclosure provides a combination or composition comprising the extracellular secretions and / or ascorbic acid components of the Disclosure. Each element constituting the combination may be a composition. The composition may include, for example, isotonic agents, thickeners, sugars, sugar alcohols, preservatives, bactericides, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizers, antioxidants, and the like. 【0039】 The combinations or compositions described herein may be provided in various forms. Compositions containing extracellular secretions and / or ascorbic acid components may be in the form of injections, reconstituted powders (e.g., lyophilized agents), etc., but are preferably in the form of solutions. Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers. The aqueous solutions for injection may also contain, for example, physiological saline, sugar (e.g., trehalose), NaCl, or NaOH. In preferred embodiments, the compositions can be formulated as pharmaceutical compositions adapted for administration to humans according to known methods. Such compositions can be administered by injection. Typically, compositions for injection are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include solubilizers and local anesthetics such as lidocaine to relieve pain at the injection site. Generally, the components may be supplied separately or mixed together in unit dose forms, and can be supplied as lyophilized powders or water-free concentrates in sealed containers such as ampoules or sachets indicating the amount of the activator. When administering the composition by injection, it is also possible to dispense it using an injection bottle containing sterile-grade water or saline solution. When administering the composition by injection, it is also possible to provide an ampoule of sterile water or saline solution for injection so that the components can be mixed before administration. The composition may have a pH of, for example, about 5.0 to 9.0, about 5.5 to 8.5, or about 6.0 to 8.0. 【0040】 Combinations or compositions of the present disclosure may be provided as kits. In one embodiment, the present disclosure provides a drug pack or kit comprising one or more containers filled with one or more components that may be added to the compositions of the present disclosure. Optionally, such containers may also include information indicating authorization by a government agency for manufacture, use or sale for human administration, in a form prescribed by the government agency that regulates the manufacture, use or sale of pharmaceutical or biological products. 【0041】 (Other embodiments) The present invention has been described above with reference to preferred embodiments for ease of understanding. The present invention will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Examples] 【0042】 (Example 1: Preparation of extracellular secretions) Adipose-derived mesenchymal stem cells were thawed, and after centrifugation (800 rpm, 5 minutes), the supernatant was discarded. Fresh cell culture medium was then added, and the cells were cultured. The pH was adjusted to approximately 8.0 with sodium bicarbonate, and the culture supernatant was obtained after removing the cells themselves and cell debris. This supernatant was used as the extracellular secretion. In the following examples, unless otherwise specified, the term "extracellular secretion" refers to the entire culture supernatant from which extracellular vesicles have not been separated. 【0043】 Using a NanoSight NS300 (Malvern, UK) CMOS camera, a violet laser (405nm, <65mW), and analysis software: NTA3.4, the size of extracellular extracellular space (EV) in extracellular secretions was measured. The EV in extracellular secretions was approximately 6.15 × 10⁶. 10 Total EVs of cells / mL, approximately 6.1 × 10⁻⁶ 10 The sample contained sEVs (with particle sizes of 50-600 nm) at a concentration of 1 / mL, with a mode particle size of approximately 108 nm and a full width at half maximum of approximately 66 nm. The majority of the EVs were sEVs. The culture supernatant can be separated into extracellular vesicles (EVs) and the supernatant by centrifugation. In another experiment, it was confirmed that the supernatant of extracellular secretions acts as an accelerator, activating the mTOR and MAPK pathways, while EVs act as a brake, suppressing the activated mTOR and MAPK pathways to some extent. It has also been confirmed that extracellular secretions can reduce cellular senescence. 【0044】 (Example 2: Cancer treatment using a combination of extracellular secretions and antioxidants) Based on findings from treatments using extracellular secretions, it was hypothesized that a combination of extracellular secretions and antioxidants could effectively treat cancer, and therefore a study was conducted. Ascorbic acid and reduced glutathione were tested as antioxidants. 【0045】 (Materials and experimental procedure) ·Cell culture MCF-7 cells (human mammary gland cancer cells) and HeLa cells (human cervical cancer cells) were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin at 5% CO2 and 37°C. The cultured cells were placed in 24-well plates in 1x10⁶ wells. 4 The seeds were seeded to achieve a cell / well ratio. 【0046】 • Preparation of test media L-ascorbic acid (Sigma A5960) was dissolved in water at a concentration of 500 mmol / L and filtered through a 0.22 μm filter. Reduced glutathione (Sigma G6013) was dissolved in water at a concentration of 150 mmol / L and filtered through a 0.22 μm filter. Extracellular secretions were added to Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin to a final concentration of 5%, L-ascorbic acid to a final concentration of 1 mmol / L, and reduced glutathione to a final concentration of 10 mmol / L. The medium was replaced with these added test media the day after cell seeding, and the cells were cultured in this state for 5 days (120 hours) at 5% CO2 and 37°C. 【0047】 • Cell counting Five days after replacing the culture medium with the test medium, the cell count was measured. The culture medium in the 24-well plate was removed, washed with PBS, and the cells were detached from the wells with trypsin / EDTA solution and collected. The collected cells were collected by centrifugation, suspended in culture medium, and the cell count was measured using a hemocytometer. 【0048】 • Cell observation, photography Each cell was observed morphologically and photographed using a KEYENCE BZ-X710 inverted fluorescence phase-contrast microscope. A 10x lens was used. 【0049】 (result) The test results for MCF-7 cells are shown in Figures 1-3, and the test results for HeLa cells are shown in Figures 4-6. Compared to the control group, extracellular secretions (ES) alone showed an inhibitory effect on both types of cancer cells, but the combination with ascorbic acid (AA) further enhanced the cancer-suppressing effect. On the other hand, the antioxidant reduced glutathione (GSH) did not enhance the cancer-suppressing effect when added to extracellular secretions (ES). From this, it is considered that the combination of extracellular secretions and ascorbic acid is particularly favorable for cancer suppression. 【0050】 (Example 3: Treatment of Triple-Negative Breast Cancer) Since the above examples confirmed that the combination of extracellular secretions and ascorbic acid is effective in suppressing breast cancer, we then tested its effect, particularly against highly malignant triple-negative breast cancer. 【0051】 MDA-MB-468, a well-known cell model for human triple-negative breast cancer, was passed through two generations and then plated in 24-well plates (1x10⁶). 4 Cells were seeded to a concentration of 5% per well. The day after seeding, extracellular secretions were added to a final concentration of 5%, and L-ascorbic acid was added to a final concentration of 100 μmol / L. Cells were cultured for 6 days and then observed. The following four conditions were tested: control condition with no additional drugs (Control), extracellular secretions only (ES), ascorbic acid only (AA 100 μM), and combination of extracellular secretions and ascorbic acid (ES+AA). 【0052】 (result) Figure 7 shows a comparison of cell counts on day 6. The vertical axis represents the relative cell count in each condition, with the cell count in the control condition (without additional drug addition) set to 1. Adding only extracellular secretions (ES) or only ascorbic acid (AA 100uM) resulted in an increase in cell count compared to the control. However, adding a combination of extracellular secretions and ascorbic acid (ES+AA) resulted in a decrease in cell count compared to the control condition (without additional drug addition). This suggests that a synergistic cancer-suppressing effect can be obtained when a combination of extracellular secretions and ascorbic acid is added to triple-negative breast cancer cells. 【0053】 (Example 4: Impact on Stat3) We investigated the effects of extracellular secretions on Stat3, which is known to be associated with cancer. 【0054】 MCF-7 cells (known to have elevated Stat3 expression) were cultured in the same manner as in Example 2, and extracellular secretions were added in the same manner as in Example 2. Three days after seeding, the cells were harvested, and Western blotting was performed for Stat3. 【0055】 As a result, it was found that the expression level of Stat3 protein was suppressed in cells to which extracellular secretions were added, compared to untreated controls. 【0056】 Stat3 is known to play an important role in the growth process of cancer, including promoting cell proliferation and suppressing apoptosis. It is also known that Stat3 activation is promoted by reactive oxygen species (ROS) (Autophagy. 2010 Nov;6(8):1125-38.). Ascorbic acid can remove ROS by reduction. Therefore, although we do not wish to be bound by any particular theory, it is conceivable that the combination of extracellular secretions and ascorbic acid suppresses Stat3 by reducing both ROS and Stat3 protein levels, thereby exhibiting an inhibitory effect on cancer cell proliferation. For this reason, in addition to the specific examples shown above, the combination of extracellular secretions and ascorbic acid disclosed herein may be particularly effective in treating cancers characterized by Stat3 activation. [Industrial applicability] 【0057】 This disclosure provides a novel cancer treatment.

Claims

[Claim 1] A combination for treating cancer comprising an extracellular secretion and an ascorbic acid component, wherein the extracellular secretion is obtained from the culture supernatant of adipose-derived mesenchymal stem cells, and the ascorbic acid component is ascorbic acid or its phosphate ester or a salt thereof. [Claim 2] The combination according to claim 1, comprising the extracellular secretion and the ascorbic acid component as active ingredients. [Claim 3] The combination according to claim 1 or 2, wherein the ascorbic acid component is ascorbic acid or a salt thereof. [Claim 4] The combination according to claim 1 or 2, wherein the cancer includes breast cancer or cervical cancer. [Claim 5] The combination according to claim 1 or 2, wherein the cancer includes breast cancer. [Claim 6] The combination according to claim 1 or 2, wherein the cancer includes triple-negative breast cancer. [Claim 7] A combination according to claim 1 or 2 for treating a human or non-human mammal. [Claim 8] The combination according to claim 1 or 2, comprising the ascorbic acid component in a ratio of about 3 to about 100 mg per 1 mL of the extracellular secretion. [Claim 9] The extracellular secretions amount to approximately 6 × 10 １０ The combination according to claim 1 or 2, comprising small extracellular vesicles (sEVs) of a certain size per mL or more. [Claim 10] A composition for treating cancer, comprising an extracellular secretion, characterized in that it is used in combination with an ascorbic acid component as an active ingredient, wherein the extracellular secretion is obtained from the culture supernatant of adipose-derived mesenchymal stem cells, and the ascorbic acid component is ascorbic acid or its phosphate ester or a salt thereof. [Claim 11] A composition for treating cancer, comprising an ascorbic acid component as an active ingredient, characterized in that it is used in combination with an extracellular secretion, wherein the extracellular secretion is obtained from the culture supernatant of adipose-derived mesenchymal stem cells, and the ascorbic acid component is ascorbic acid or its phosphate ester or a salt thereof.

Images