Exosomes and anticancer agent

Abstract

The purpose of the present invention is to provide a novel therapeutic agent that has excellent anticancer action.　The present invention relates to: exosomes that are characterized by high expression of at least one type of miRNA selected from the group that consists of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p; and an anticancer agent that contains the exosomes.

Description

Exosomes and anticancer drugs

[0001] The present invention relates to exosomes and anticancer agents containing the same.

[0002] Cancer has been the leading cause of death in Japan since 1981, and by 2022, approximately one in four people will die from cancer. It is estimated that approximately one million people were diagnosed with cancer in 2019, and approximately one in two people will develop cancer in their lifetime. By age group, cancer accounts for approximately 10% of deaths among children and approximately 36% of deaths among people aged 20 to 64. According to the "2022 Vital Statistics (Definitive Figures)" published by the Ministry of Health, Labor and Welfare in September 2023, the number of deaths due to cancer (malignant neoplasms (tumors)) will be 385,797, accounting for 24.6% of all deaths. Furthermore, the number of cancer deaths is expected to continue to increase due to the increase in elderly cancer patients caused by the rapid increase in the elderly population. Therefore, the development of new anticancer drugs with excellent efficacy is desired.

[0003] Mesenchymal stem cells are multipotent progenitor cells that were first isolated from bone marrow by Friedenstein (1982) (see Non-Patent Document 1). Mesenchymal stem cells have been shown to exist in various tissues, such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected to be a new treatment method for various intractable diseases (see Patent Documents 1 and 2). Recently, it has been discovered that cells with equivalent functions exist in interstitial cells of adipose tissue, placenta, umbilical cord, fetal membrane, etc. Therefore, mesenchymal stem cells are sometimes referred to as mesenchymal stromal cells.

[0004] The effects of mesenchymal stem cells and mesenchymal stem cell-derived exosomes on tumors have been investigated, and both enhancing effects (see Non-Patent Documents 2 to 4) and suppressing effects have been reported.

[0005] JP 2012-157263 A JP 2012-508733 A

[0006] Science, 1999, 284,pp.143-147Cell Death and Disease, 2018, 9, 218J. Experimental & Clinical Cancer Research, 2019, 38, 62Molecular Therapy: Oncolytics, 2021, 20, pp.132-146

[0007] In view of the above circumstances, an object of the present invention is to provide a novel therapeutic agent having excellent anticancer activity.

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that exosomes that highly express specific genes have a strong anti-cancer effect, and completed the present invention. According to the present invention, a novel therapeutic agent with a strong anti-cancer effect can be provided. That is, the gist of the present invention is as follows.

[0009] [1] An exosome characterized by high expression of at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p. [2] The exosome according to [1], which is derived from mesenchymal stem cells. [3] The exosome according to [2], wherein the mesenchymal stem cells are derived from umbilical cord tissue or adipose tissue. [4] An anticancer agent containing the exosome according to any one of [1] to [3]. [5] The anticancer agent according to [4], which increases the expression of TGF-β2 or GRN in a subject by administering it to the subject. [6] A method for treating cancer, comprising the step of administering to a patient an effective amount of exosomes highly expressing at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p. [7] The method of treatment according to [6], wherein the exosomes are derived from mesenchymal stem cells. [8] The method of treatment according to [7], wherein the mesenchymal stem cells are derived from umbilical cord tissue or adipose tissue. [9] Use of exosomes characterized by high expression of at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p in the manufacture of an anticancer agent.

[10] The use according to [9], wherein the exosomes are derived from mesenchymal stem cells.

[11] The use according to

[10] , wherein the mesenchymal stem cells are derived from umbilical cord tissue or adipose tissue.

[0010] According to the present invention, a highly effective anticancer agent can be provided. The anticancer agent of the present invention has a high tumor suppressing effect and therefore exhibits excellent therapeutic effects.

[0011] Figure 1 shows the results of investigating the antitumor effect of exosomes of the present invention on A549-LUC2 (in vitro). Figure 2 shows the antitumor effect of exosomes of the present invention on A549-LUC2 (in vivo). Figure 3 shows the antitumor effect of exosomes of the present invention on A549-LUC2 (in vivo). Figure 4 shows the antitumor effect of exosomes of the present invention on A549-LUC2 (in vivo, photographs). Figure 5-1 is a heat map showing changes in gene expression in A549-LUC2 cells upon treatment with exosomes of the present invention. Figure 5-2 is a heat map showing changes in gene expression in mouse cells that accumulate in A549-LUC2 cells upon treatment with exosomes of the present invention. Figure 6-1 is a volcano plot showing changes in gene expression in A549-LUC2 cells upon treatment with exosomes of the present invention. Figure 6-2 is a Volcano plot showing changes in gene expression in mouse cells accumulating in A549-LUC2 upon treatment with the exosomes of the present invention. Figure 7-1 is a diagram showing the results of core analysis using gene expression data obtained in Example 4. Figure 7-2 is a diagram showing the results of core analysis using gene expression data obtained in Example 4. Figure 8 is a heat map for evaluating the similarity of gene patterns possessed by exosomes derived from various cells. Figure 9 is a diagram visualizing the interaction between miRNA and its target gene. Figure 10 is a Volcano plot for visually representing the magnitude and statistical significance of changes in gene expression.

[0012] The anticancer agent of the present invention will be described in detail below.

[0013] The anticancer agent of the present invention is characterized by comprising exosomes in which at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p is highly expressed. The anticancer agent of the present invention is also characterized by increasing the expression of TGF-β2 or GRN (Granulin) in the body upon administration.

[0014] In the present invention, "high expression" of the miRNA in exosomes means that the content of the miRNA is at least two times, more preferably at least three times, even more preferably at least five times, and particularly preferably at least ten times that of conventional exosomes. Here, conventional exosomes refer to exosomes obtained by ultracentrifugation from a culture supernatant obtained by culturing mesenchymal stem cells in a serum-free medium for 2 to 5 days.

[0015] The phrase "administration of the anticancer agent of the present invention increases the expression of TGF-β2 or GRN in the body" means that administration of the anticancer agent of the present invention to a subject increases the expression of TGF-β2 or GRN protein and / or gene in the blood, tumor tissue, tissue without tumor, etc. compared to before administration.

[0016] Exosomes in which at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p is highly expressed have the effect of suppressing the proliferation of cancer cells and causing tumor regression. Therefore, by containing such exosomes, the anticancer agent of the present invention exhibits excellent therapeutic effects against cancer.

[0017] <Exosomes> In the present invention, exosomes are endoplasmic reticulum derived from cellular endosomes, and are vesicles released from cells that are small enough to be visible under an electron microscope. The specific size of exosomes is an average particle diameter of 1 nm to 1,000 nm, preferably 10 nm to 500 nm, and more preferably 30 nm to 200 nm. Here, the average particle diameter refers to the average diameter of each particle measured by dynamic light scattering or electron microscopy. The exosomes may have a lipid bilayer surrounding biomolecules. Furthermore, the exosomes may include, for example, membrane particles, membrane vesicles, microvesicles, nanovesicles, microvesicles (average particle diameter 30 to 1,000 nm), exosome-like vesicles, ectosome-like vesicles, ectosomes, and / or exovesicles. Exosomes derived from different cell types can also be distinguished based on their intracellular origin, exosome density in sucrose, shape, sedimentation rate, lipid composition, protein markers, and mode of secretion (i.e., signal-induced or spontaneous (constitutive)). For example, exosomes can be fractionated to 1.0-1.5 g / mL, preferably 1.1-1.3 g / mL, by density gradient centrifugation.

[0018] Exosomes contain any of phosphatidylserine, cholesterol, sphingomyelin, and ceramide as their constituent lipids. The exosomes of the present invention contain various proteins and RNAs derived from secretory cells, including endosome-derived proteins, proteins involved in intracellular transport, and cell membrane-derived proteins, as well as lipids derived from the cell membrane of secretory cells and the endosomal membrane. Examples of endosome-derived proteins include ESCRTs and TSG101; examples of proteins involved in intracellular transport include Rab and GTPase; examples of cell membrane-derived proteins include CD9, CD63, and CD81; and examples of endosomal membrane-derived lipids include cholesterol and sphingomyelin.

[0019] Other proteins and fatty acids contained in exosomes of the present invention include, for example, IL-10, HGF (Hepatocyte Growth Factor), pelargonic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid, palmitic acid, isopalmitic acid, margaric acid, isoheptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, nonadecylic acid, isononadecylic acid, arachidic acid, 11Z-eicosenoic acid, dihomo-γ-linolenic acid, arachidonic acid, erucic acid, 13Z, 16Z-docosadienoic acid, 13Z, 16Z, 19Z-docosadienoic acid, acid, adrenic acid, clupanodonic acid, etc., preferably IL-10, HGF, pelargonic acid, lauric acid, myristic acid, pentadecanoic acid, isopentadecanoic acid, palmitic acid, isopalmitic acid, margaric acid, isoheptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, arachidic acid, 11Z-eicosenoic acid, arachidonic acid, erucic acid, 13Z, 16Z-docosadienoic acid, and the like, more preferably IL-10, HGF, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, isoheptadecanoic acid acid, stearic acid, oleic acid, linoleic acid, nonadecylic acid, erucic acid, 13Z,16Z-docosadienoic acid, and particularly preferred are IL-10, HGF, palmitic acid, margaric acid, stearic acid, oleic acid, 13Z,16Z-docosadienoic acid, and the like, and the exosomes of the present invention preferably contain these.

[0020] The exosomes of the present invention may be those contained in cells, those contained in cell culture supernatant, or exosomes isolated from cells or cell culture supernatant. The isolated exosomes may contain cells such as interstitial cells (mesenchymal stem cells), or may contain a medium such as a culture medium for interstitial cells (mesenchymal stem cells).

[0021] The origin of the exosomes in the present invention is not particularly limited and may be mammalian cells such as human, horse, cow, goat, sheep, pig, dog, cat, rabbit, mouse, or rat; microorganisms; or plants such as rice. Exosomes can be collected from culture supernatant obtained by culturing mesenchymal stem cells, or from plants such as rice containing exosomes, or from microorganisms. Furthermore, the exosomes in the present invention are not limited to those produced by living organisms and may be artificial endoplasmic reticulum such as liposomes.

[0022] When the exosomes of the present invention are derived from mammals as described above, examples of the derived cells include mesenchymal stem cells, liver-derived cells, fibroblasts, epithelial cells, myoblasts, pluripotent stem cells, and plant stem cells. Of these, mesenchymal stem cells and pluripotent stem cells are preferred, and mesenchymal stem cells are more preferred.

[0023] Hereinafter, a case where the exosomes of the present invention are derived from mesenchymal stem cells will be described.

[0024] When the cells are mesenchymal stem cells, the exosomes contained in the anticancer agent of the present invention can be recovered from the culture supernatant obtained by culturing the mesenchymal stem cells. For example, mesenchymal stem cells are cultured in a culture vessel until they reach a subconfluent or confluent state, and the medium is replaced with new medium. The cells are then cultured for an additional 1 to 5 days, and the culture supernatant is recovered. Exosomes can be obtained by separating them from the culture supernatant of mesenchymal stem cells.

[0025] The anticancer agent of the present invention may contain, in addition to the exosomes, mesenchymal stem cells themselves that have the ability to encapsulate or secrete the exosomes.

[0026] (Mesenchymal stem cells) In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more types of cells belonging to the mesenchymal system (bone cells, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) and can proliferate while maintaining this ability. The term mesenchymal stem cells used in the present invention refers to the same cells as interstitial cells and does not particularly distinguish between the two. They may also be simply referred to as mesenchymal cells. Examples of tissues that contain mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. Mesenchymal stem cells in the present invention include those derived from adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc., and among these, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells are preferred, with adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells being more preferred.

[0027] The species of mesenchymal stem cells in the present invention include humans, horses, cows, goats, sheep, pigs, dogs, cats, rabbits, mice, and rats. The mesenchymal stem cells in the present invention may be derived from the same species as the subject (subject) to be treated, or may be derived from a different species.

[0028] Mesenchymal stem cells may be cells provided by, for example, PromoCell, Inc., Lonza, Inc., Biological Industries, Inc., Veritas, Inc., R&D Systems, Inc., and Corning, Inc., or may be cells prepared by methods well known to those skilled in the art. Furthermore, mesenchymal stem cells may be primary cells isolated from donor tissue or established cell lines.

[0029] In the present invention, the medium used for culturing mesenchymal stem cells is not particularly limited as long as it is a medium that can culture mesenchymal stem cells while maintaining their state in good condition. However, it is preferable that the medium be a medium that can proliferate human mesenchymal stem cells while maintaining their ability to differentiate into bone cells, chondrocytes, and adipocytes.

[0030] The medium used in the present invention may be prepared by adding serum to a basal medium and / or one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. Furthermore, these media may further contain, as necessary, amino acids such as glutamine, sugars such as glucose, metal salts such as sodium chloride and magnesium sulfate, trace metals such as selenium, lipids such as cholesterol and unsaturated fatty acids, vitamins such as pantothenic acid, proteins such as albumin, insulin, transferrin, growth factors, cytokines, polysaccharides, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0031] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixed media thereof.

[0032] The medium used to culture the mesenchymal stem cells used in the present invention is preferably a xeno-free medium that does not contain xenogeneic components such as serum, from the viewpoint of administration to patients. Examples of such media include Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use, manufactured by PromoCell), Mesenchymal Stem Cell Growth Medium XF (Ready-to-use, manufactured by PromoCell), MSCGM Bullet Kit, MSCGM™ Mesenchymal Stem Cell Growth Medium Bullet Kit (manufactured by Lonza), and Xeno-Free Medium for Human Mesenchymal Stem Cells (MSC NutriStem (registered trademark) XF, Biological Examples of such a medium include media provided as pre-prepared media for mesenchymal stem cells (stromal cells), such as MesenCult-ACF Plus (manufactured by Veritas), StemXVivotm Serum-Free Human MSC Expansion Media (manufactured by R&D Systems, Corning), serum-free medium for adipose-derived stem cells (KBM ADSC-4, manufactured by Kohjin Bio), and serum-free medium for mesenchymal stem cells (R:STEM Medium for hMSC High Growth, manufactured by Rohto).

[0033] Examples of the serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, porcine serum, sheep serum, rabbit serum, rat serum, etc. When serum is used, it may be added to the basal medium at 5 v / v % to 15 v / v %, preferably 10 v / v %.

[0034] (Preparation of mesenchymal stem cell culture supernatant) The mesenchymal stem cell culture supernatant of the present invention can be obtained by the following method. Alternatively, the mesenchymal stem cell culture supernatant of the present invention may be obtained by removing unnecessary components from the supernatant by means of dialysis, ultrafiltration, or the like, by fractionating the supernatant using a column or the like, by selecting a fraction using an antibody against a specific molecule, or by centrifugation.

[0035] The medium used to obtain the culture supernatant can be the same as the medium used to culture mesenchymal stem cells. The method for obtaining the culture supernatant is not particularly limited as long as it is a method suitable for culturing each mesenchymal stem cell. For example, the culture supernatant can be obtained by culturing the mesenchymal stem cells at a temperature of 20°C to 37°C in a medium containing 2% to 7% CO. ２ Under environmental conditions, 5% to 21% O ２ In an environment, preferably room temperature to 37°C, 5% CO ２ This method involves culturing mesenchymal stem cells in a suitable environment and obtaining the culture supernatant.

[0036] The mesenchymal stem cell culture supernatant of the present invention may be prepared by contacting mesenchymal stem cells with a medium, and the wash solution obtained by washing mesenchymal stem cells with a medium can also be used as the culture supernatant of the present invention. The contact time between the mesenchymal stem cells and the medium is, for example, within 14 days, preferably within 10 days, more preferably within 7 days, and even more preferably within 5 days. Specifically, it is preferred to culture the mesenchymal stem cells in a subconfluent or confluent state in a culture vessel, replace the medium with new medium, and then further culture for 1 to 5 days, after which the culture supernatant is recovered. The culture to obtain the culture supernatant may be performed by plate culture in which the stem cells are attached to a flask, or by suspension / agitation culture in which the stem cells are attached to microbeads or the like.

[0037] (Isolation of Exosomes) When isolating exosomes, the culture supernatant of mesenchymal stem cells recovered by the above-mentioned method can be obtained by a conventionally known method, such as ultrafiltration, ultracentrifugation, density gradient centrifugation, affinity purification, size exclusion chromatography, separation using various exosome isolation kits, etc. Preferred methods for separating exosomes are ultrafiltration, ultracentrifugation, affinity purification, and size exclusion chromatography. An example of an ultrafiltration method is tangential flow filtration, and systems that perform tangential flow filtration include the Minimate EVO system (PALL Laboratory), TFF-Easy (HansaBioMed Life Sciences Ltd), Ambr (registered trademark) Crossflow (Sartorius), Sartoflow (registered trademark) series (Sartorius), FlexAct (registered trademark) Modular CF (Sartorius), and KrosFlo (registered trademark) TFDF (registered trademark) system (Repligen Corporation).

[0038] The amount of exosomes contained in the anticancer agent of the present invention is, as a concentration in the entire anticancer agent, from 0.001 pg / mL to 100 ug / mL, preferably from 0.005 pg / mL to 100 ug / mL, more preferably from 0.01 pg / mL to 10 ug / mL, and particularly preferably from 0.1 pg / mL to 100 ng / mL. By setting the amount of exosomes contained in the anticancer agent of the present invention within the above numerical range, the anticancer agent of the present invention exhibits excellent anticancer effects.

[0039] The anticancer agent of the present invention may contain other components in addition to exosomes, as long as the effects of the present invention are not impaired. Examples of other components include protective agents such as dimethyl sulfoxide (DMSO) and albumin, antibiotics, sugars, amino acids, vitamins, carriers, excipients, disintegrants, buffers, emulsifiers, stabilizers, preservatives, antiseptics, and physiological saline.

[0040] The species of mesenchymal stem cells that are the target of the composition of the present invention may be any mammal, including, for example, humans, horses, cows, goats, sheep, pigs, dogs, cats, rabbits, mice, rats, and rare animals.

[0041] The anticancer agent of the present invention can be produced by a conventional method by mixing the above-mentioned exosomes with the other components as needed.

[0042] The present invention also includes a method for producing an anticancer agent, characterized by incorporating exosomes in which at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p is highly expressed.

[0043] [Cancer Treatment Method] The present invention also includes a cancer treatment method, which comprises the step of administering to a patient an effective amount of exosomes in which at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p is highly expressed.

[0044] Exosomes in which at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p is highly expressed have the effect of suppressing the proliferation of cancer cells and causing tumor regression. Therefore, the cancer treatment method of the present invention, which includes the step of administering an effective amount of such exosomes to a patient, exhibits excellent therapeutic effects against cancer.

[0045] The exosomes are preferably derived from mesenchymal stem cells, and the mesenchymal stem cells are preferably derived from umbilical cord tissue or adipose tissue.

[0046] The cancer treatment method of the present invention can also be said to be a method comprising the step of administering the anticancer agent of the present invention to a patient. Therefore, the explanation in the "Anticancer Agent" section above also applies to the treatment method of the present invention.

[0047] [Use of specific exosomes in the production of anticancer drugs] The present invention also includes the use of exosomes highly expressing at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p in the production of anticancer drugs. The exosomes are preferably derived from mesenchymal stem cells. Furthermore, the mesenchymal stem cells are preferably derived from umbilical cord tissue or adipose tissue.

[0048] [Exosomes] The present invention also includes exosomes (exosomes) in which at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p is highly expressed. As described above, exosomes in which at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p is highly expressed have a high tumor-suppressing effect, and are therefore suitable for use as anticancer agents. For details of the exosomes of the present invention, the explanation in the section on anticancer agents can be applied.

[0049] The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[0050] Example 1: Preparation of exosomes After obtaining consent from a human donor, subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. To destroy the extracellular matrix and isolate cells, collagenase (Roche diagnostics) was added, and the tissue was dispersed by shaking at 37°C for 90 minutes. Subsequently, the suspension was centrifuged at 800 x g for 5 minutes to obtain a precipitate of stromal vascular cells. The cell precipitate was suspended in serum-free medium for mesenchymal stem cells (RIM medium, RIM-GF MIX (planned for registration in the original drug register), containing EGF, bFGF, albumin, transferrin, and insulin, Rohto). After culturing for 3 days, the cell suspension was centrifuged at 400 x g for 5 minutes. After removing the supernatant, the cells were resuspended in RIM medium and seeded into flasks. The cells were incubated at 37°C for several days in 5% CO ２After several days, the culture was washed with PBS to remove blood cells and residual adipose tissue contained in the culture medium, and mesenchymal stem cells adhered to the plastic container were obtained. The obtained adipose tissue-derived mesenchymal stem cells were cultured in a T25 flask for adherent cells (Thermo Fisher Scientific) at 5000 cells / cm. ２ and cultured in RIM medium for 7-10 days (37°C, 5% CO ２ ) to obtain a culture supernatant. Exosomes were obtained from the culture supernatant by total exosome isolation, ultracentrifugation, and tangential flow filtration (TF). Note that exosomes obtained by total exosome isolation are referred to as TEI, exosomes obtained by ultracentrifugation as UC, and exosomes obtained by tangential flow filtration as EVs.

[0051] Example 2: Anticancer effect of exosomes (in vitro) A549-LUC2 (lung cancer cells, manufactured by American Type Culture Collection) suspended in D-PBS (control group) and A549-LUC2 (TEI group, UC group, EV#13 group, and EV#14 group) suspended in D-PBS supplemented with 500.8 pg of exosomes (TEI, UC, and EV#13 group and EV#14 group) obtained by each production method were added to DMEM high glucose (manufactured by Gibco) medium and cultured for one day, after which the medium was replaced and cultured for another day (37°C, 5% CO ２ ) was performed. EV#13 and EV#14 are different exosome lots.

[0052] After the above-mentioned culture, cell proliferation, tumor volume, and tumor weight were measured using WST-8. There was no difference in tumor (lung cancer cell) volume or weight between the control group and the exosome group, and no direct anti-cancer effect of exosomes was observed. The cell proliferation results are shown in Figure 1.

[0053] [Example 3: Anticancer effect of exosomes (in vivo)] Mice (BALB / c Slc-nu / nu, male, 6 weeks old) were administered 5 × 10 exosomes suspended in D-PBS.６ｃｅｌｌｓ A549-LUC2 (lung cancer cells, manufactured by American Type Culture Collection) (control group), 5 × 10 cells suspended in D-PBS containing 500.8 pg of exosomes obtained by tangential flow filtration (TF) prepared in Example 1, ６ｃｅｌｌｓ A549-LUC2 (high dose group) and 5 × 10 cells suspended in D-PBS supplemented with 167 pg of exosomes. ６ｃｅｌｌｓ A549-LUC2 (1 / 3 administration group) and Dulbecco's phosphate-buffered saline (Kohjin Bio Co., Ltd.) were subcutaneously administered (200 μL). At 9, 12, 15, 19, 23, 26, and 30 days after administration, the tumor diameter of the cancer cells was measured using digital calipers, and the tumor volume was calculated (Figure 2). The weight of the cancer cells at 30 days later was measured using an electronic balance (Figure 3). Photographs of the tumors are shown in Figure 4.

[0054] As shown in Figures 2 and 3, a decrease in the volume and weight of cancer cells was observed in the high exosome administration group and 1 / 3 administration group compared to the control group. Furthermore, as shown in Figure 4, it was also confirmed by visual observation that the tumors were smaller in the high exosome administration group and 1 / 3 administration group compared to the control group. These results demonstrate that administration of the exosomes of the present invention suppresses the proliferation of cancer cells.

[0055] [Example 4: RNA-seq Analysis] Each cancer cell obtained in Example 3 was cryopreserved, and total RNA was extracted from the frozen tissue. Subsequently, quality confirmation was performed by electrophoresis to determine the presence or absence of degradation and quantify the nucleic acid concentration. From the total RNA sample obtained from the RNA whose quality was confirmed, mRNA was purified using the Poly-A selection method. A reverse transcription reaction was performed using this mRNA as a template to synthesize single-stranded cDNA. Next, using this single-stranded cDNA as a template, second-strand cDNA was created using nucleoside triphosphates containing dUTP instead of dTTP. Subsequently, both ends of the obtained double-stranded cDNA were blunted and phosphorylated, followed by 3'-dA overhang treatment. After this treatment, a sequencing adapter containing dUTP was ligated. Finally, dUTP in the adapter and second-strand cDNA was selectively cleaved with USER enzyme to obtain a template for a library (strand-specific library) synthesized with single-stranded DNA having directional information.

[0056] Sequencing was performed using an Illumina NovaSeq 6000. To verify the quality of the sequences, FastQC software was used to calculate the quality score of the sequence reads. Next, Trimmomatic software was used to trim the sequence reads based on preset values. This removed low-quality reads and adapter sequences. The mapping results were used to calculate the mapping rate of the reads to the reference genome. This evaluated how well the reads aligned to the reference genome. FeatureCounts software was used to calculate the number of reads mapped to known exon regions and count the mapped fragments. Furthermore, FPKM, FPKM-UQ, and TPM values ​​were calculated. These values ​​represent the expression level of each gene. Stats software was used to perform hierarchical clustering of each sample based on the read count, FPKM, or TPM values. This allowed us to evaluate the similarity of gene expression patterns between samples. The software was also used to create heat maps for each sample (Figure 5-1: Human / Figure 5-2: Mouse). The heat maps used Z-scores to represent the change in expression level of each gene. Volcano plots were also created to visually represent the magnitude and statistical significance of gene expression changes. Graphpad software was used to create these plots (Figure 6-1: Human / Figure 6-2: Mouse). The differential expression analysis between the A549-only and MSC-EV-treated groups was visualized using Volcano plots. The x-axis represents the change in expression level, with genes with increased expression levels to the right (red, black in black-and-white drawings) and genes with decreased expression levels to the left (blue, white in black-and-white drawings) of 0. Meanwhile, the y-axis represents statistical significance, with increasing significance at the top. Therefore, genes with large expression variation ratios and high statistical significance are located in the upper right or upper left of the plot.

[0057] Example 5: IPA Analysis Using the gene expression data obtained in Example 4, core analysis was performed using QIAGEN Ingenuity Pathway Analysis (IPA) software. After uploading each gene expression data and setting the specified parameters, the analysis was performed. Using the obtained results, gene expression patterns and pathway changes were analyzed, and the effect of exosome administration on gene expression in cancer cells was interpreted (Figure 7-1: Human / Figure 7-2: Mouse).

[0058] Using RNA-seq data, we investigated known pathways, upstream factors, and their association with toxicity and disease. We found that upstream factors targeting the molecules or genes shown in Figures 7-1 and 7-2 were highly expressed. By integrating the analysis results of upstream regulatory factors and downstream disease / function, we were able to understand how fluctuations in upstream regulatory factors affect downstream pathways via molecules within the dataset. Each line in the figure represents a connection between related molecules. Setting the layout to "Radial" during analysis revealed that the molecule located at the center of all genes and molecules was the overall key factor.

[0059] Example 6: miRNA Analysis: miRNA was extracted from the exosomes obtained by tangential flow filtration (TF) prepared in Example 1 using a miRNA purification kit (QIAGEN). Library preparation was performed using the Small RNA-Seq Library Prep Kit to generate libraries for the NextSeq next-generation sequencer according to the following steps. Next, sequencing was performed using a NextSeq500 (Illumina). Analysis was performed using the program bcl2fastq (Illumina), creating fastq files from the obtained reads, compressing them into .gz format, and storing them on a DVD. Hierarchical clustering was performed using the expression levels of each gene. This allowed for evaluation of the similarity of gene expression patterns between samples. Additionally, a heat map of each sample was drawn using the same software (Figure 8). The heat map used Z scores, which represent the change in expression level of each gene. We also visualized the interactions between miRNAs and their target genes using miRNet. Volcano plots were created to visually represent the magnitude and statistical significance of gene expression changes (Figures 9 and 10). Graphpad software was used to create these plots.

[0060] It was revealed that administration of exosomes increased the expression of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p.

[0061] According to the present invention, a highly effective anticancer agent can be provided. The anticancer agent of the present invention has a high tumor suppressing effect and therefore exhibits excellent therapeutic effects.

Claims

1. An exosome characterized by high expression of at least one miRNA selected from the group consisting of miR-3168, miR-92a-1-5p, miR-7-5p, and miR-146b-5p.

2. The exosome according to claim 1, which is derived from mesenchymal stem cells.

3. The exosome according to claim 2, wherein the mesenchymal stem cells are derived from umbilical cord tissue or adipose tissue.

4. An anticancer agent containing the exosome described in any one of claims 1 to 3.

5. The anticancer agent according to claim 4, which, when administered to a subject, increases the expression of TGF-β2 or GRN in the subject.

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