Method for concentrating tetraspanin-10-positive extracellular vesicles, and method for detecting cancer or predicting cancer prognosis

Abstract

Provided is a method for concentrating tetraspanin-10-positive extracellular vesicles, the method comprising: a step for obtaining an extracellular-vesicle-containing sample from a sample derived from the body fluid of a subject; a step for separating this extracellular-vesicle-containing sample into at least two fractions by size-exclusion chromatography; and a step for recovering the fraction containing the tetraspanin-10-positive extracellular vesicles.

Description

Method for concentrating tetraspanin 10-positive extracellular vesicles and method for detecting cancer or predicting cancer prognosis

[0001] The present disclosure relates to methods for enriching tetraspanin 10-positive extracellular vesicles and methods for detecting cancer or predicting the prognosis of cancer.

[0002] Extracellular vesicles (EVs), typified by exosomes, are a collective term for vesicles with a diameter of approximately 100 nm surrounded by a lipid bilayer membrane that are produced by cells in vivo or in culture and secreted extracellularly. Extracellular vesicles express tetraspanins (TSPANs) and adhesion molecules on their membranes, and contain nucleic acids such as mRNA and miRNA, skeletal proteins, proteins such as various enzymes, lipids, metabolic products, and the like. Thus, extracellular vesicles maintain the molecular structure of the cells that produce them. For example, extracellular vesicles derived from cancer cells have the same molecular structure as the cancer cells. Therefore, extracellular vesicles are expected to be useful as blood diagnostic materials for cancer.

[0003] However, extracellular vesicles derived from cancer cells in blood are diluted with body fluids (for example, about 5 L for a 60 kg human), resulting in a very low concentration. It is said that the number of extracellular vesicles in human blood is about 1 trillion / mL, and if a subject has cancer, only a small amount of extracellular vesicles derived from cancer cells will be present in the total extracellular vesicles. Therefore, there is a need for a method that can concentrate extracellular vesicles derived from cancer cells and easily detect cancer with high accuracy.

[0004] Extracellular vesicles secreted by cancer cells are known to have a variety of molecular compositions. Therefore, to identify the surface antigens of extracellular vesicles, proteome analysis of extracellular vesicles from colorectal cancer patients was performed (see Non-Patent Document 1). This proteome analysis revealed that tetraspanin family members such as CD9, CD63, and CD151 are expressed as surface antigens on extracellular vesicles from colorectal cancer patients. However, no specific markers for cancer cell-derived extracellular vesicles have yet been identified.

[0005] Atsushi Ikeda et al. , Mol Cancer Res. , 2021, May: 19(5), p. 834-846

[0006] An object of the present disclosure is to provide a method for concentrating tetraspanin 10-positive extracellular vesicles, which can easily and efficiently concentrate tetraspanin 10-positive extracellular vesicles.

[0007] Means for solving the above problems are as follows: <1> A method for concentrating tetraspanin 10-positive extracellular vesicles, comprising: obtaining a sample containing extracellular vesicles from a sample derived from a subject's body fluid; separating the sample containing extracellular vesicles into two or more fractions by size exclusion chromatography; and recovering fractions containing tetraspanin 10-positive extracellular vesicles. <2> A method for detecting cancer or predicting the prognosis of cancer, comprising: obtaining a sample containing extracellular vesicles from a sample derived from a subject's body fluid; separating the sample containing extracellular vesicles into two or more fractions by size exclusion chromatography; and contacting each of the two or more fractions separated in the separation step with a substance for detecting tetraspanin 10, and determining whether or not a fraction containing tetraspanin 10-positive extracellular vesicles is present.

[0008] According to the present disclosure, a method for concentrating tetraspanin 10-positive extracellular vesicles can be provided, which allows for simple and efficient concentration of tetraspanin 10-positive extracellular vesicles.

[0009] Figure 1A shows the results of analyzing tetraspanin 10 mRNA expression in various normal tissues based on FANTOM5 published in THE HUMAN PROTEIN ATLAS. The vertical axis shows the TSPAN10 mRNA expression level (normalized tags per million). Figure 1B shows the results of analyzing tetraspanin 10 mRNA expression in various normal blood cells based on FANTOM5 published in THE HUMAN PROTEIN ATLAS. The vertical axis shows the TSPAN10 mRNA expression level (normalized tags per million). Figure 1C shows the results of analyzing tetraspanin 10 mRNA expression in various cancer tissues based on data published in The Cancer Genome Atlas (TCGA). The vertical axis shows the amount of TSPAN10 mRNA expression (normalized tags per million). Figure 2 shows the detection results of subjects 1 and 2 in Test Example 1 by ELISA. The vertical axis shows absorbance at a wavelength of 450 nm. The horizontal axis shows fraction number. Figure 3 shows a transmission electron microscope (TEM) image of extracellular vesicles in the serum of a stage III colon cancer patient in Test Example 1. The arrow indicates a gold colloid label. The scale bar is 100 nm. FIG. 4 shows the results of analyzing the expression of TSPAN10 protein and p53 protein in extracellular vesicles of cancer patients based on the database published in the Te-EVs Proteome Library in Test Example 2. The vertical axis shows the relative expression level of p53 protein, and the horizontal axis shows the relative expression level of TSPAN10 protein. FIG. 5 shows the results of analyzing the expression of tetraspanin 10 mRNA and MUC16 mRNA in various cancer tissues based on data published by TCGA in Test Example 3. The vertical axis shows the expression level (FPKM) of TSPAN10 mRNA or MUC16 mRNA. FIG. 6A shows the receiver operating characteristic (ROC) curve as a result of analytical method validation by ELISA using anti-TSPAN10 antibody and anti-MUC16 antibody in Test Example 4. The vertical axis shows specificity, and the horizontal axis shows sensitivity.Figure 6B is a diagram showing a receiver operating characteristic (ROC) curve as a result of analytical method validation by ELISA using anti-CD9 antibody and anti-MUC16 antibody in Test Example 4. The vertical axis indicates specificity, and the horizontal axis indicates sensitivity. Figure 7 is a diagram showing the results of ELISA using anti-TSPAN10 antibody and anti-MUC16 antibody for serum samples from the control group and pancreatic cancer stage III group in Test Example 5. The vertical axis indicates absorbance at a wavelength of 450 nm. Figure 8 is a diagram showing a receiver operating characteristic (ROC) curve as a result of analytical method validation by ELISA in Test Example 6. The vertical axis indicates sensitivity, and the horizontal axis indicates specificity.

[0010] (Method for concentrating tetraspanin 10-positive extracellular vesicles) The method for concentrating tetraspanin 10-positive extracellular vesicles of the present disclosure includes a step of obtaining a sample containing extracellular vesicles from a body fluid-derived sample of a subject (hereinafter, sometimes referred to as a "sample preparation step"), a step of separating the sample containing extracellular vesicles into two or more fractions by size exclusion chromatography (hereinafter, sometimes referred to as a "separation step"), and a step of recovering fractions containing tetraspanin 10-positive extracellular vesicles (hereinafter, sometimes referred to as a "recovery step"), and may further include other steps as necessary. Note that the method for concentrating tetraspanin 10-positive extracellular vesicles of the present disclosure is performed in vitro.

[0011] In the present disclosure, "tetraspanin 10-positive extracellular vesicles" means that tetraspanin 10 or a mutant thereof is expressed in the extracellular vesicles. That is, it means that at least one of tetraspanin 10 protein or a mutant thereof is expressed on the surface of the extracellular vesicles, and DNA encoding tetraspanin 10, its gene product, or a mutant thereof is expressed in the extracellular vesicles. In this specification, positive may be indicated by "+" and negative may be indicated by "-".

[0012] Tetraspanin (TSPAN) family proteins are a family of membrane proteins that span the cell membrane four times and are cell surface receptors present on the cell membrane. Tetraspanin family proteins form complexes with adhesion molecules such as integrins, and different types of tetraspanins also form complexes with each other. In humans, 33 types of tetraspanin family protein members are known, including CD9, CD63, CD81, CD82, and CD151. However, tetraspanin 10 (also known as oculospanin) is a different protein from these.

[0013] An example of a human tetraspanin 10 protein is GeneBank Accession Number AAH21923.1 (235 amino acids). Other examples of human tetraspanin 10 proteins include Q9H1Z9 (Y218H) (355 amino acids) and Q9H1Z9 (R187H) (355 amino acids) in the protein database Uniport (https: / / www.uniprot.org / ). GeneBank Accession Number AAH21923.1 consists of the amino acid sequence represented by SEQ ID NO: 1.

[0014] In addition to Q9H1Z9 and Q9H1Z9, mutants of tetraspanin 10 proteins include, for example, those having an amino acid sequence in which one or two or more amino acid residues are deleted, added, inserted, and / or substituted in the amino acid sequence shown in GeneBank Accession No. AAH21923.1, Q9H1Z9, or Q9H1Z9, or any of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 5 described below. Specific examples include those having an amino acid sequence that has 95% or more sequence identity, 90% or more sequence identity, 85% or more sequence identity, 80% or more sequence identity, 75% or more sequence identity, or 70% or more sequence identity to the amino acid sequence shown in GeneBank Accession No. AAH21923.1, Q9H1Z9, or Q9H1Z9, or any of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 5 described below. Among these, those having an amino acid sequence with 80% or more sequence identity to the amino acid sequence shown in GeneBank accession number: AAH21923.1, Q9H1Z9, or Q9H1Z9, or any of SEQ ID NOs: 2 to 5 described below, are preferred, those having an amino acid sequence with 85% or more sequence identity are more preferred, those having an amino acid sequence with 90% or more sequence identity are even more preferred, and those having an amino acid sequence with 95% or more sequence identity are particularly preferred.

[0015] Among these, the mutant tetraspanin 10 protein preferably comprises an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5, and more preferably consists of an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5. SEQ ID NO: 2: SCVKYLIFLSNFPFSLLGLLALAIGLWGLAVKGSLGSDLGGPLPAADPMLG SEQ ID NO: 3: SCVKYLIFLSNFPFSLLGLLALAIGLWGLAVKGSLGSDLGGPLPTDPMLG SEQ ID NO: 4: WGLAVKGSLGSDLGGPLPTDP SEQ ID NO: 5: GPLQDSLEHTLRVAIAHYQDDPDLRFLLDQVQLGLRCCGAASYQDWQQNLYFNCSSPGVQACSLPASCIDPREDGASVNDQCGFGVLRLDADAAQRVVYLEGCGPPLRRWLRANLAASGGYAIAVVLLQGAELLLAARLLGALAARSGAAYGPGAHGEDRAGPQSPSPGAPPAAKPARG

[0016] An example of a DNA sequence encoding human tetraspanin 10 is GeneBank Accession No. 83882.

[0017] Examples of variants of the DNA sequence encoding tetraspanin 10 include those in which one or more deoxyribonucleic acid bases have been deleted, added, inserted, and / or substituted in GeneBank Accession No. 83882. Specific examples include deoxyribonucleic acid sequences having 95% or more sequence identity, 90% or more sequence identity, 85% or more sequence identity, 80% or more sequence identity, 75% or more sequence identity, and 70% or more sequence identity to GeneBank Accession No. 83882. Among these, deoxyribonucleic acid sequences having 80% or more sequence identity to GeneBank Accession No. 83882 are preferred, deoxyribonucleic acid sequences having 85% or more sequence identity are more preferred, deoxyribonucleic acid sequences having 90% or more sequence identity are even more preferred, and deoxyribonucleic acid sequences having 95% or more sequence identity are particularly preferred.

[0018] Examples of RNA sequences encoding human tetraspanin 10 include GeneBank accession number NM_001290212 (1,840 bp) consisting of the base sequence represented by SEQ ID NO: 6, and GeneBank accession number NM_031945.5 (1,887 bp) consisting of the base sequence represented by SEQ ID NO: 7.

[0019] Examples of variants of RNA sequences encoding tetraspanin 10 include those in which one or more ribonucleic acid bases have been deleted, added, inserted, and / or substituted in GeneBank Accession No. NM_001290212 or NM_031945.5. Specific examples include ribonucleic acid sequences having 95% or more sequence identity, 90% or more sequence identity, 85% or more sequence identity, 80% or more sequence identity, 75% or more sequence identity, and 70% or more sequence identity to GeneBank Accession No. NM_001290212 or NM_031945.5. Among these, ribonucleic acid sequences having 80% or more sequence identity to GeneBank Accession Number: NM_001290212 or NM_031945.5 are preferred, ribonucleic acid sequences having 85% or more sequence identity are more preferred, ribonucleic acid sequences having 90% or more sequence identity are even more preferred, and ribonucleic acid sequences having 95% or more sequence identity are particularly preferred.

[0020] The RNA sequence encoding tetraspanin 10 is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include mRNA, ncRNA, tRNA, RNAi, siRNA, shRNA, miRNA, and RISC.

[0021] <Sample Preparation Step> The sample preparation step is a step of obtaining a sample containing extracellular vesicles from a subject's body fluid-derived sample. Note that the sample containing extracellular vesicles includes not only tetraspanin 10-positive extracellular vesicles but also tetraspanin 10-negative extracellular vesicles.

[0022] <<Subject>> The subject is preferably a human, but is not limited thereto, and can also be used for mammals other than humans, such as monkeys, mice, rats, hamsters, dogs, cats, rabbits, cows, and pigs.

[0023] The subject's condition is preferably a subject who is currently suffering from or has previously suffered from cancer. Examples of subjects who are currently suffering from cancer include subjects who have already been diagnosed with cancer by another method (e.g., conventionally known cancer diagnostic methods such as ultrasound, mammography, and MRI (magnetic resonance imaging)), and subjects currently undergoing cancer treatment. Examples of subjects who have previously suffered from cancer include subjects who have undergone known cancer treatments such as surgical resection, radiation therapy, and chemotherapy.

[0024] In this disclosure, "cancer" refers to a condition that originates from cells that make up the body, grows uncontrollably, and abnormally spreads to the surrounding area, or metastasizes or invades other parts of the body, causing serious damage to life. In this disclosure, "cancer" includes "carcinoma."

[0025] The type of cancer for subjects who are currently suffering from or have previously suffered from cancer is not particularly limited, and examples include breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, stomach cancer, lung cancer, thyroid cancer, uterine cancer (including uterine cancer and cervical cancer), ovarian cancer, malignant melanoma, kidney cancer, liver cancer, epithelial cancer, rectal cancer, colon cancer, papillary renal cell carcinoma, head and neck squamous cell carcinoma, serous cystadenocarcinoma, chromophobe renal cell carcinoma, prostate cancer, lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, renal non-clear cell carcinoma, hepatocellular carcinoma, testicular germ cell tumor, pancreatic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, and ovarian cancer. Intestinal adenocarcinoma, prostate cancer, brain cancer, skin cancer, bone cancer, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemia, lymphoma, myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, basal cell carcinoma, bladder cancer, brain tumors (glioma, central nervous system atypical teratoid rhabdoid tumor, central nervous system embryonal tumor, astrocytoma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, ependymoma, intermediate pineal parenchymal tumor, supratentorial tumor) primary tumor, and pineoblastoma), bronchial tumor, Burkitt lymphoma, carcinoma of unknown primary site, carcinoid tumor, childhood cancer, chordoma, chronic myeloproliferative disorder, cutaneous T-cell lymphoma, endocrine pancreatic islet cell tumor, endometrial cancer, olfactory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastrointestinal stromal cell tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, hairy cell leukemia, cardiac cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, Langerhans cell tumor Histocytosis, laryngeal cancer, lip cancer, malignant fibrous histiocytoma, Merkel cell carcinoma, Merkel cell skin cancer, mesothelioma, metastatic squamous cell neck cancer of occult primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloproliferative neoplasm, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-melanoma skin cancer, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, papillomatosis, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuroembryonic tumor,Primary central nervous system (CNS) cancers include primary hepatocellular carcinoma, respiratory tract cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, T-cell lymphoma, thymic carcinoma, thymoma, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureteral cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0026] Among these, the type of cancer is preferably a solid cancer, and more preferably at least one type selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, lung cancer, thyroid cancer, uterine cancer, ovarian cancer, malignant melanoma, kidney cancer, liver cancer, epithelial cancer, rectal cancer, colon cancer, papillary renal cell carcinoma, head and neck squamous cell carcinoma, serous cystadenocarcinoma, chromophobe renal cell carcinoma, prostate cancer, lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial cancer, renal non-clear cell carcinoma, hepatocellular carcinoma, testicular germ cell tumor, pancreatic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, and colorectal adenocarcinoma.

[0027] There are no particular limitations on the stage of cancer in a subject who is currently suffering from or has previously suffered from cancer. In the present disclosure, the "stage" of cancer refers to a stage based on the TMN classification.

[0028] The TNM classification determines the stage by combining three factors: "T (Tumor)," i.e., the size of the lump; "N (Lymph Note)," i.e., the state of metastasis to the lymph nodes; and "M (Metastasis)," i.e., metastasis to other organs.

[0029] The present inventors analyzed FANTOM5, which is published in THE HUMAN PROTEIN ATLAS (an open access database by Atlas Antibodies, https: / / www.proteinatlas.org / about / download, the entire contents of which are incorporated herein by reference), and found that, among normal human tissues, tetraspanin 10 is highly expressed only in the retina (see FIG. 1A ). They also found that it is not expressed in normal human blood cells (see FIG. 1B ).

[0030] Furthermore, the present inventors have found, based on data from The Cancer Genome Atlas (TCGA) (an open access database, https: / / www.cancer.gov / ccg / research / genome-sequencing / tcga, the contents of which are incorporated herein by reference in their entireties), that tetraspanin 10 is expressed in human cancer tissues of many types, regardless of the cancer type (see Figure 1C).

[0031] The retina is an environment isolated from circulating blood by the blood-retinal barrier, and retina-derived tetraspanin 10 is unlikely to be expressed in the blood. Therefore, if the subject is currently suffering from cancer or has previously suffered from cancer, tetraspanin 10 in a sample derived from the subject's body fluid is thought to be derived from cancer cells.

[0032] <<Sample Derived from Body Fluid>> The sample derived from body fluid is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include peripheral blood, serum, plasma, ascites, lymph, cerebrospinal fluid, saliva, bone marrow, urine, synovial fluid, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, amniotic fluid, breast milk, etc. These may be used alone or in combination of two or more.

[0033] The body fluid-derived sample may be collected at any time of day. The method for collecting the body fluid-derived sample is not particularly limited and can be appropriately selected from known methods depending on the type of sample to be collected.

[0034] The sample derived from a body fluid may be the body fluid itself, or may be a sample obtained by subjecting the body fluid to some kind of treatment.

[0035] The method for treating the body fluid-derived sample is not particularly limited and can be appropriately selected from known methods depending on the type of body fluid, and examples include separation treatment, purification treatment, drug addition treatment, etc. These may be performed alone or in combination of two or more. For example, when the body fluid is a blood sample, serum may be separated by a known method.

[0036] Among these, the body fluid-derived sample is preferably a blood sample such as peripheral blood, serum, or plasma, and more preferably serum.

[0037] The method for treating the body fluid-derived sample is not particularly limited, and can be appropriately selected from known methods depending on the type of sample to be collected.

[0038] <<Extracellular Vesicles>> Examples of the extracellular vesicles include circulating microvesicles (cMVs), microvesicles, exosomes, nanovesicles, dexosomes, blebs, vesicles, prostasomes, microparticles, intraluminal vesicles, membrane fragments, intraluminal endosomal vesicles, endosome-like vesicles, exocytosis vehicles, endosomal vesicles, endosomal vesicles, apoptotic bodies, multivesicular bodies, secretory vesicles, phospholipid vesicles, liposomal vesicles, argosomes, texasomes, secretosomes, trellosomes, melanosomes, oncosomes, exocytosis vehicles, etc. The body fluid-derived sample may contain one type of these vesicles alone or two or more types.

[0039] A method for forming extracellular vesicles is described. Small internal vesicles containing proteins, mRNA, microRNA (miRNA), and other proteins within the cytoplasm are formed inside the cell. The internal vesicles then fuse with the cell membrane and are released to the outside as extracellular vesicles. The released extracellular vesicles travel, for example, via the blood, toward a destination determined by the attachment of specific ligands on their surface. Upon reaching the target cell, the extracellular vesicles either enter the cell via the endocytosis pathway or fuse with the target cell membrane to directly release the extracellular vesicle components, i.e., proteins and other components derived from the cell that formed the extracellular vesicles, into the cytoplasm. The DNA molecules inside the extracellular vesicles represent the entire genome and can reflect the state (e.g., mutation status) of the cancer cell that released the extracellular vesicles.

[0040] The size of the extracellular vesicles is not particularly limited, but the diameter is preferably 10 nm to 2,000 nm, more preferably 20 nm to 1,500 nm, even more preferably 20 nm to 1,000 nm, still more preferably 20 nm to 500 nm, and particularly preferably 50 nm to 150 nm.

[0041] <Separation Step> The separation step is a step of separating the sample containing the extracellular vesicles into two or more fractions by size exclusion chromatography.

[0042] The number of fractions is not particularly limited as long as it is two or more, and can be appropriately selected depending on the type of carrier and the amount of sample containing extracellular vesicles added, etc., but a larger number is preferable in terms of being able to separate tetraspanin 10-positive extracellular vesicles with high accuracy.

[0043] Size exclusion chromatography can be performed by known methods or using commercially available kits, such as an exosome purification column (EVSecond, manufactured by GL Sciences Inc.).

[0044] In the separation step, other separation processes besides size exclusion chromatography may be used in combination, as long as fractions containing tetraspanin 10-positive extracellular vesicles are not excluded. The other separation processes can be appropriately selected from known separation processes for extracellular vesicles, and examples include density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoadsorption capture, affinity purification, affinity capture, affinity selection, immunoassay, ELISA, microfluidics separation, and flow cytometry. These processes may be used alone or in combination.

[0045] <Recovery Step> The recovery step is a step of recovering a fraction containing tetraspanin 10-positive extracellular vesicles.

[0046] A fraction containing tetraspanin 10-positive extracellular vesicles can be suitably identified by the identification step described below.

[0047] The method for recovering fractions containing tetraspanin 10-positive extracellular vesicles is not particularly limited, and examples include methods for recovering one or more fractions containing tetraspanin 10-positive extracellular vesicles.

[0048] <Other Steps> The other steps are not particularly limited as long as they do not impair the effects of the present disclosure, and can be appropriately selected depending on the purpose. Examples of the other steps include a step of collecting a body fluid-derived sample from a subject (hereinafter sometimes referred to as a "collection step"), a step of applying some kind of treatment to the collected body fluid-derived sample (hereinafter sometimes referred to as a "sample treatment step"), a step of identifying a fraction containing tetraspanin 10-positive extracellular vesicles (hereinafter sometimes referred to as an "identification step"), and a step of further concentrating tetraspanin 10-positive extracellular vesicles (hereinafter sometimes referred to as a "concentration step").

[0049] <<Collecting Step>> The collecting step is a step of collecting a body fluid-derived sample from a subject. The collecting step is preferably carried out before the sample preparation step.

[0050] The method for collecting a body fluid-derived sample is not particularly limited and can be appropriately selected depending on the type of body fluid-derived sample. For example, when the body fluid-derived sample is a blood sample, a method of collecting blood by injection can be used.

[0051] <<Identification Step>> The identification step is a step of identifying a fraction containing tetraspanin 10-positive extracellular vesicles. The identification step is preferably performed before the recovery step.

[0052] Specifically, the identification step is a step of contacting each of the two or more fractions separated in the separation step (separation step) with a substance for detecting tetraspanin 10, and identifying fractions containing tetraspanin 10-positive extracellular vesicles.

[0053] The method for identifying a fraction containing tetraspanin 10-positive extracellular vesicles is not particularly limited and can be appropriately selected depending on the type of detection substance, etc. Examples include methods for detecting tetraspanin 10 protein and identifying a fraction containing tetraspanin 10-positive extracellular vesicles, such as ELISA (Enzyme-Linked Immunosorbent Assay), immunostaining, and Western blotting; methods for detecting RNA sequences as gene products of gene sequences encoding tetraspanin 10 and identifying a fraction containing tetraspanin 10-positive extracellular vesicles, such as RNA-seq analysis, RT-PCR (including quantitative RT-PCR), nucleic acid chip analysis, and Northern blotting; and methods for detecting DNA sequences encoding tetraspanin 10 and identifying a fraction containing tetraspanin 10-positive extracellular vesicles, such as PCR, digital PCR, Southern blotting, and microarray. These methods may be used alone or in combination of two or more.

[0054] In the identification step, the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles is determined based on the detection values ​​obtained by these methods.

[0055] -ELISA method- When the ELISA method is used, for example, a method may be used in which the fraction obtained in the separation step is reacted with an anti-TSPAN10 antibody, tetraspanin 10-positive extracellular vesicles in the fraction are captured with the anti-TSPAN10 antibody, and the tetraspanin 10-positive extracellular vesicles are detected by utilizing an enzyme reaction using an enzyme that can be used in a known ELISA method.

[0056] - Western Blotting Method - When the Western blotting method is used, for example, the fraction obtained in the separation step is electrophoresed by SDS-PAGE in a standard manner, and the resultant is transferred to a hydrophobic membrane such as a nylon membrane in a standard manner. This hydrophobic membrane is reacted with an anti-TSPAN10 antibody, and then a secondary antibody labeled with an enzyme such as HRP (horseradish peroxidase) is reacted, and detection is performed by a chemiluminescence method or a color development method that utilizes the enzyme activity.

[0057] -RNA-seq analysis method- When the RNA-seq analysis method is used, for example, cDNA of tetraspanin 10 is prepared from RNA in the fraction obtained in the separation step according to a standard method, and sequence analysis is performed using a next-generation sequencer or the like, and mapping, gene expression analysis, expression level analysis, etc. are performed based on the obtained data to obtain expression level information.

[0058] -RT-PCR Method- When the RT-PCR method is used, for example, cDNA is prepared from RNA in the fraction obtained in the separation step according to a standard method, and a pair of nucleic acid primers (a positive strand that binds to the -strand of the cDNA, and a reverse strand that binds to the +strand) is hybridized to the cDNA so that the target region can be amplified using the cDNA as a template, followed by PCR according to a standard method and detecting the resulting amplified double-stranded DNA. The amplified double-stranded DNA can be detected by performing the PCR using nucleic acid primers that have been labeled in advance with RI or a fluorescent substance, detecting labeled double-stranded DNA produced by the PCR, or by transferring the produced double-stranded DNA to a nylon membrane or the like according to a standard method and hybridizing it with a labeled nucleic acid probe for detection.

[0059] - Nucleic Acid Chip Analysis Method - When the nucleic acid chip analysis method is used, for example, a nucleic acid chip to which a nucleic acid probe (single-stranded or double-stranded) complementary to at least a part of the DNA sequence encoding tetraspanin 10 is attached is prepared, and the resulting double-stranded strand is detected by hybridizing the probe with RNA in the fraction obtained in the separation step or with nucleic acid prepared from the RNA by a standard method.

[0060] - Northern Blotting Method - When the Northern Blotting method is used, for example, a nucleic acid probe complementary to at least a part of the DNA sequence encoding the tetraspanin 10 protein is irradiated with a radioisotope ( ３２ P. ３３Examples of such methods include a method in which a nucleic acid probe is labeled with a labeling substance (RI or a fluorescent substance, etc.) of the nucleic acid probe or a fluorescent substance, and hybridized with mRNA in the fraction obtained in the separation step, which has been transferred to a nylon membrane or the like in a conventional manner, and the resulting double strand formed between the nucleic acid probe and the mRNA in the fraction obtained in the separation step is detected by a radiation detector, a fluorescence detector, or the like, by detecting a signal derived from the labeling substance (RI or a fluorescent substance, etc.) of the nucleic acid probe.

[0061] -PCR Method- When the PCR method is used, for example, a method can be used in which a pair of nucleic acid primers (a positive strand that binds to the -strand of the cDNA and a reverse strand that binds to the +strand) is hybridized to the DNA in the fraction obtained in the separation step or cDNA prepared in a conventional manner from the RNA in the fraction obtained in the separation step as a template so that a target region can be amplified, and the PCR method is performed in a conventional manner, and the resulting amplified double-stranded DNA is detected. Note that the amplified double-stranded DNA can be detected by performing the PCR using nucleic acid primers that have been labeled in advance with RI or a fluorescent substance, detecting labeled double-stranded DNA produced by the PCR, or by transferring the produced double-stranded DNA to a nylon membrane or the like in a conventional manner, and then hybridizing and detecting the double-stranded DNA using a labeled nucleic acid probe.

[0062] Southern blotting method When the Southern blotting method is used, for example, a nucleic acid probe complementary to at least a part of the DNA sequence encoding tetraspanin 10 is irradiated with a radioisotope ( ３２ P. ３３ Examples of such methods include a method in which a nucleic acid probe is labeled with a labeling substance (RI or a fluorescent substance, etc.) of the nucleic acid probe or a fluorescent substance, and hybridized with DNA in a fraction obtained in the separation step that has been transferred to a nylon membrane or the like in a conventional manner, and then the formed double strand between the nucleic acid probe and the DNA in the fraction obtained in the separation step is detected by a radiation detector, a fluorescence detector, or the like by detecting a signal derived from the labeling substance (RI or a fluorescent substance, etc.) of the nucleic acid probe.

[0063] --Substance for detecting tetraspanin 10-- The substance for detecting tetraspanin 10 is not particularly limited as long as it can detect tetraspanin 10, and is preferably a substance that can detect, for example, a DNA sequence encoding tetraspanin 10, a gene sequence encoding tetraspanin 10 or its gene product, or a mutant thereof. Examples of gene products of a gene sequence encoding tetraspanin 10 include an RNA sequence, a protein, and an amino acid sequence encoding the protein.

[0064] The form of the detection substance is not particularly limited and can be appropriately selected depending on the detection method, etc. Examples include nucleic acids, DNA molecules, RNA molecules, antibodies, antibody fragments, aptamers, peptoids, zDNA, peptide nucleic acids (PNA), locked nucleic acids (LNA), lectins, peptides, dendrimers, membrane protein labeling substances, chemical substances, etc. These may be used alone or in combination of two or more.

[0065] ---Substance for detecting tetraspanin 10 protein--- A specific example of a detection substance for detecting tetraspanin 10 protein (hereinafter, sometimes referred to as a "substance for detecting tetraspanin 10 protein") is an antibody capable of binding to tetraspanin 10.

[0066] The antibody capable of binding to tetraspanin 10 is not particularly limited as long as it can detect tetraspanin 10 protein and does not impair the effects of the present disclosure, and examples thereof include polyclonal antibodies, monoclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, etc. that can bind to tetraspanin 10 protein. Among these, the antibody capable of binding to tetraspanin 10 is preferably an antibody that recognizes an amino acid sequence represented by any one of SEQ ID NOs: 2 to 5 as an epitope sequence.

[0067] The method for obtaining the antibody capable of binding to tetraspanin 10 is not particularly limited and can be appropriately selected depending on the purpose. Examples include a method in which tetraspanin 10 protein is administered as an antigen to an animal (e.g., rabbit, chicken, pig, mouse, rat, etc.) and the antibody is obtained by purifying the serum obtained from the animal by a known method, and a method in which a commercially available product is used.

[0068] Examples of commercially available antibodies capable of binding to tetraspanin 10 include tetraspanin 10 polyclonal antibody (manufactured by Proteintech), anti-TSPAN10 antibody (SAB2102589, rabbit host antibody, manufactured by Sigma), etc. These may be used alone or in combination of two or more.

[0069] ---Substance for detecting nucleic acid encoding tetraspanin 10--- The detection substance (hereinafter, may be referred to as "nucleic acid detection substance") for detecting a nucleic acid sequence (RNA sequence or DNA sequence) encoding tetraspanin 10 is not particularly limited as long as it does not impair the effects of the present disclosure, but is preferably at least one type selected from the group consisting of a nucleic acid primer capable of specifically hybridizing to a nucleic acid sequence encoding tetraspanin 10 and a nucleic acid probe capable of specifically hybridizing to tetraspanin 10.

[0070] In the present disclosure, the term "capable of specifically hybridizing" refers to, for example, a nucleic acid primer or nucleic acid probe having a sequence complementary to the full length or a part of the nucleic acid sequence encoding tetraspanin 10, or a nucleic acid primer or nucleic acid probe capable of hybridizing under stringent conditions to the full length or a part of the nucleic acid sequence encoding tetraspanin 10. These may be used alone or in combination of two or more.

[0071] In the present disclosure, "stringent conditions" means that hybridization is carried out in a hybridization solution having 5xSSC or an equivalent salt concentration at a temperature of 37°C to 42°C for about 12 hours, followed by pre-washing with 5xSSC or a solution having an equivalent salt concentration, followed by washing with 1xSSC or a solution having an equivalent salt concentration. To achieve higher stringency, washing can be carried out in 0.1xSSC or a solution having an equivalent salt concentration.

[0072] The length of the nucleic acid of the nucleic acid detection substance is not particularly limited and can be appropriately selected depending on the purpose.

[0073] When the nucleic acid detection substance is a nucleic acid primer used in a PCR method, the sequence capable of specifically hybridizing with a DNA sequence encoding tetraspanin 10 is preferably 50 mer or less, more preferably 30 mer or less, and even more preferably 15 mer to 25 mer. The nucleic acid primer serving as the nucleic acid detection substance may contain a sequence that does not encode tetraspanin 10 or that does not hybridize with a DNA sequence encoding tetraspanin 10.

[0074] When the nucleic acid detection substance is a nucleic acid probe used in RT-PCR, the sequence capable of specifically hybridizing with a DNA sequence encoding tetraspanin 10 is preferably 50 mer or less, more preferably 30 mer or less, and even more preferably 15 mer to 25 mer. The nucleic acid probe serving as the nucleic acid detection substance may contain a sequence that does not encode tetraspanin 10 or that does not hybridize with a DNA sequence encoding tetraspanin 10. Furthermore, it is preferable that one end of the nucleic acid probe serving as the nucleic acid detection substance is labeled with a fluorescent dye and the other end is labeled with a quencher for the fluorescent dye.

[0075] --Other detection substances-- In the identification step, detection substances other than the tetraspanin 10 detection substance may be used in combination. The other detection substances are not particularly limited and can be appropriately selected depending on the type of cancer to be targeted for enrichment of tetraspanin 10-positive extracellular vesicles, and examples include a PCNA detection substance that detects PCNA (proliferating cell nuclear antigen), a marker for extracellular vesicles. These substances may be used alone, or two or more types may be used in combination.

[0076] Detection of tetraspanin 10-positive extracellular vesicles using the other detection substance may be carried out simultaneously with detection of tetraspanin 10-positive extracellular vesicles using the tetraspanin 10 detection substance, or may be carried out separately from detection of tetraspanin 10-positive extracellular vesicles using the tetraspanin 10 detection substance. When detection of tetraspanin 10-positive extracellular vesicles using the other detection substance is carried out separately from detection of tetraspanin 10-positive extracellular vesicles using the tetraspanin 10 detection substance, there are no particular limitations on the timing of detection of tetraspanin 10-positive extracellular vesicles using the other detection substance, and it can be selected appropriately depending on the type of the other detection substance.

[0077] ---PCNA Detecting Substance--- The PCNA detecting substance is a marker for extracellular vesicles, and is therefore preferably used before detection of tetraspanin 10-positive extracellular vesicles with a tetraspanin 10 detecting substance.

[0078] The PCNA detection substance is not particularly limited as long as it can detect PCNA, and is preferably a substance that can detect, for example, a DNA sequence encoding PCNA, a gene sequence encoding PCNA or its gene product, or a mutant thereof, etc. Examples of gene products of a gene sequence encoding PCNA include an RNA sequence, a protein, and an amino acid sequence encoding the protein.

[0079] The detection substance may be labeled with any labeling agent, such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, biotin, a magnetic label, an enzyme, a chemiluminescent probe, a metal particle, a non-metallic colloid particle, a polymer dye particle, a dye molecule, a dye particle, an electrochemically active species, a semiconductor nanocrystal, another nanoparticle (e.g., a quantum dot, a gold particle, etc.), or a fluorophore.

[0080] Examples of luminescent substances include green fluorescent protein (GFP) or variants thereof (eg, cyan fluorescent protein, yellow fluorescent protein, etc.), luciferase, and the like.

[0081] Examples of fluorescent substances include rare earth chelates (e.g., europium chelates, etc.), rhodamine, FITC, 5-carboxyfluorescein, 6-carboxyfluorescein, TAMRA, dansyl, Lissamine, cyanine, phycoerythrin, Texas Red, Cy3, Cy5, dapoxyl, NBD, Cascade Yellow, dansyl, PyMPO, pyrene, 7-dimethylaminocoumarin-3-carboxylic acid or other coumarin derivatives, Marina Blue, Pacific Blue, Cascade Blue, 2-anthracenesulfony, PyMPO, 3, Examples of the fluorescent dye include 4,9,10-perylene-tetracarboxylic acid, 2,7-difluorofluorescein, 5-carboxyfluorescein, Texas Red-X, Alexa Fluor 430, 5-carboxytetramethylrhodamine (5-TAMRA), 6-carboxytetramethylrhodamine (6-TAMRA), BODIPYFL, bimane, and Alexa Fluor 350, 405, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, 750, or derivatives thereof.

[0082] The radioisotope is not particularly limited and can be appropriately selected from known isotopes, for example: ３ H. １１ C. １４ C. １８ F. ３２ P. ３５ S. ６４ Cu,６８ Ga, ８６ Y. ９９ Tc, １１１ In, １２３ I, １２４ I, １２５ I, １３１ I, １３３ Xe, １７７ Lu, ２１１ At, ２１３ Bi, etc.

[0083] <<Concentration Step>> The concentration step is a step of further concentrating tetraspanin 10-positive extracellular vesicles. The concentration step is preferably carried out after the recovery step.

[0084] The method for concentrating tetraspanin 10-positive extracellular vesicles is not particularly limited and can be appropriately selected depending on the purpose. For example, a method in which the separation treatment in the separation step is repeated can be mentioned.

[0085] Alternatively, tetraspanin 10-positive extracellular vesicles may be enriched using a commercially available kit. Examples of commercially available kits include an exosome flow cytometry kit (PS Capture ＴＭ Examples include the Exosome Flow Cytometry Kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0086] <Applications> According to the method for concentrating tetraspanin 10-positive extracellular vesicles of the present disclosure, tetraspanin 10-positive extracellular vesicles that are not expressed in normal tissues or blood cells but are specifically expressed in cancer can be concentrated simply and efficiently, and therefore the method can be suitably used in methods for detecting cancer or predicting the prognosis of cancer.

[0087] (Method for detecting cancer or predicting cancer prognosis) The method for detecting cancer or predicting cancer prognosis of the present disclosure comprises the steps of obtaining a sample containing extracellular vesicles from a body fluid-derived sample of a subject (hereinafter sometimes referred to as a "sample preparation step"), separating the sample containing extracellular vesicles into two or more fractions by size exclusion chromatography (hereinafter sometimes referred to as a "separation step"), and contacting each of the two or more fractions separated in the separation step with a substance for detecting tetraspanin 10 and determining the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles (hereinafter sometimes referred to as a "determination step"), and may further comprise other steps as necessary. Note that the method for detecting cancer or predicting cancer prognosis of the present disclosure is preferably performed in vitro.

[0088] In the present disclosure, "prognosis" refers to the outcome of a subject. The outcome of a subject is an outcome due to the effects of the cancer that the subject has. In the present disclosure, "poor prognosis" means at least one selected from the group consisting of a cancer that is prone to metastasis or infiltration, a cancer that is prone to recurrence, and a cancer-related subject that is likely to die from the cancer. If the cancer prognosis is predicted to be poor based on the biomarker, the subject can consider or select a treatment method for the cancer, such as surgical resection, radiation therapy, or chemotherapy. In addition, in the present disclosure, "good prognosis" means at least one selected from the group consisting of a cancer that is localized without metastasis or infiltration, a cancer that grows slowly, and a cancer-related subject that is unlikely to die from the cancer.

[0089] <Sample Preparation Step> The sample preparation step is a step of obtaining a sample containing extracellular vesicles from a subject's body fluid-derived sample. The sample preparation step can be performed in the same manner as the sample preparation step of the method for concentrating tetraspanin 10-positive extracellular vesicles of the present disclosure, and therefore detailed description thereof will be omitted.

[0090] <Separation step> The separation step is a step of separating a sample containing extracellular vesicles into two or more fractions by size exclusion chromatography. The separation step can be performed in the same manner as the separation step in the method for concentrating tetraspanin 10-positive extracellular vesicles of the present disclosure, and therefore detailed description thereof will be omitted.

[0091] <Determining step> The determining step is a step of contacting each of the two or more fractions separated in the separating step with a substance for detecting tetraspanin 10, and determining the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles.

[0092] The method for determining the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles is not particularly limited and can be appropriately selected depending on the type of detection substance, etc. Examples include methods in which tetraspanin 10 protein is detected and a fraction containing tetraspanin 10-positive extracellular vesicles is identified, such as ELISA (Enzyme-Linked Immunosorbent Assay), immunostaining, and Western blotting; methods in which an RNA sequence as a gene product of a gene sequence encoding tetraspanin 10 is detected and a fraction containing tetraspanin 10-positive extracellular vesicles is identified, such as RNA-seq analysis, RT-PCR (including quantitative RT-PCR), nucleic acid chip analysis, and Northern blotting; and methods in which a DNA sequence encoding tetraspanin 10 is detected and a fraction containing tetraspanin 10-positive extracellular vesicles, such as PCR, Southern blotting, and microarray. These methods may be used alone or in combination of two or more.

[0093] In the determination step, the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles is determined based on the values ​​detected by these methods.

[0094] The substance for detecting tetraspanin 10 can be the same as the substance for detecting tetraspanin 10 used in the identification step of the method for enriching tetraspanin 10-positive extracellular vesicles of the present disclosure.

[0095] <Determination Step> The determination step is a step of determining that the subject is suffering from cancer when a fraction containing tetraspanin 10-positive extracellular vesicles is present in the determination step.

[0096] <Other Steps> The other steps in the method for detecting cancer or the method for predicting the prognosis of cancer are not particularly limited and can be appropriately selected depending on the purpose. Examples of the other steps include a determination step, a step of collecting a body fluid-derived sample from a subject (hereinafter may be referred to as a "collection step"), a step of applying some kind of treatment to the collected body fluid-derived sample (hereinafter may be referred to as a "sample treatment step"), a step of analyzing the detection value or quantification value obtained after the determination step using known analysis software (hereinafter may be referred to as an "analysis step"), and a step of determining a necessary treatment method depending on the detection result of tetraspanin 10 (hereinafter may be referred to as a "treatment method determination step").

[0097] <<Determination step>> The determination step can be broadly divided into a "first mode" in which the subject is determined to have cancer if a fraction containing tetraspanin 10-positive extracellular vesicles is present in the determining step; a "second mode" in which the subject is determined to have cancer or to have a poor prognosis if the detected level of tetraspanin 10-positive extracellular vesicles is higher than a reference value; and a "third mode" in which the expression level of tetraspanin 10 in the subject is compared with the expression level of tetraspanin 10 in healthy subjects.

[0098] First Aspect A first aspect of the determination step is the method for detecting cancer, wherein the subject is determined to be suffering from cancer if a fraction containing tetraspanin 10-positive extracellular vesicles is present in the determining step. The determination step of the first aspect is performed after the determining step.

[0099] Furthermore, the determination step of the first aspect may be a step of determining that the subject is not affected by cancer when a fraction containing tetraspanin 10-positive extracellular vesicles is not present in the method for detecting cancer.

[0100] In addition, in patients suffering from cancer, the tetraspanin 10-positive extracellular vesicles may be detected even after treatment such as surgery or chemotherapy. Therefore, in the method for predicting the prognosis of cancer, the determination step of the second aspect or the determination step of the third aspect described below is preferably used.

[0101] - Second Aspect - In a second aspect of the assessment step, when a fraction containing tetraspanin 10-positive extracellular vesicles is present in the determining step and the detected level of tetraspanin 10-positive extracellular vesicles is higher than a reference value, the subject is assessed to be suffering from cancer or to have a poor prognosis. The assessment step of the second aspect is performed after the determining step.

[0102] Furthermore, the determination step of the second aspect may be a step of determining that the subject is not suffering from cancer or that the prognosis of the subject is good when a fraction containing tetraspanin 10-positive extracellular vesicles is present in the determining step and the detection value of tetraspanin 10 is lower than a reference value.

[0103] The reference value is not particularly limited, as long as it is a value that can determine the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles in a sample derived from a subject's body fluid, and can be determined by a known method. Specific examples of methods for determining the reference value include receiver operating characteristic curve (ROC) curve, discriminant analysis, mode method, Kittler method, 3σ method, and p-tile. The reference value is not particularly limited, and examples that can be used include sensitivity, specificity, positive predictive value, and negative predictive value.

[0104] -Third Aspect- A third aspect of the determination step is carried out by comparing the expression level of tetraspanin 10 in a subject with the expression level of tetraspanin 10 in a healthy subject. Specifically, the flow from the sample preparation step onwards will be described below.

[0105] When the method for detecting cancer or predicting cancer prognosis includes the determination step of the third aspect, the method includes: (a1) obtaining a sample containing extracellular vesicles from a body fluid-derived sample of a subject; (a2) separating the sample containing extracellular vesicles obtained in (a1) into two or more fractions by size exclusion chromatography; (a3) ​​contacting each of the two or more fractions separated in (a2) with a substance for detecting tetraspanin 10 to determine whether or not a fraction containing tetraspanin 10-positive extracellular vesicles is present; (a4) quantifying tetraspanin 10 in fraction F containing tetraspanin 10-positive extracellular vesicles when a fraction containing tetraspanin 10-positive extracellular vesicles is present in (a3); (b1) obtaining a sample containing extracellular vesicles from a body fluid-derived sample of a healthy subject; and (b2) separating the sample containing extracellular vesicles obtained in (b1) into two or more fractions by size exclusion chromatography. (b3) quantifying tetraspanin 10 in fraction f, which corresponds to fraction F in (a4), from among the fractions separated in (b2); and (b4) comparing the quantitative value A of tetraspanin 10 in fraction F with the quantitative value B of tetraspanin 10 in fraction f, and determining that the subject is suffering from cancer or has a poor prognosis if quantitative value A > quantitative value B.

[0106] The (a1) is the sample preparation step, the (a2) is the separation step, and the (a3) ​​is the determination step.

[0107] (b1) can be performed in the same manner as (a1) above, except that the sample derived from the subject's body fluid in (a1) is replaced with a sample derived from the body fluid of a healthy individual. Here, the "healthy individual" is preferably a person who is known to be free of cancer and has not developed cancer, as determined by conventionally known cancer diagnostic methods such as ultrasound, mammography, and MRI (magnetic resonance imaging), and more preferably a person who has no history of cancer among their blood relatives.

[0108] (b2) can be performed in the same manner as (a1) except that the sample containing extracellular vesicles obtained in (a1) is changed to the sample containing extracellular vesicles obtained in (b1).

[0109] In (b3), "fraction f corresponding to fraction F in (a4)" refers to a case where the method for preparing a sample containing extracellular vesicles from a body fluid-derived sample is the same in (a1) and (b1), the number of fractions separated by size exclusion chromatography is the same in (a2) and (b2), and if fraction F determined to contain tetraspanin 10-positive extracellular vesicles in (a3) ​​is the nth fraction, the nth fraction separated in (b2) is fraction f. As a specific example, if the number of fractions separated by size exclusion chromatography in (a2) and (b2) is 12, and fraction F determined to contain tetraspanin 10-positive extracellular vesicles in (a3) ​​is the 8th fraction, the 8th fraction separated in (b2) is fraction f. Fraction F and fraction f may be multiple fractions. When the number of fractions separated by size exclusion chromatography in (a2) and (b2) is 12, and fraction F determined to contain tetraspanin 10-positive extracellular vesicles in (a3) ​​is the 8th to 10th fractions, the 8th to 10th fractions separated in (b2) are designated as fraction f.

[0110] In (a4) and (b4), the quantification of tetraspanin 10 in fraction F or fraction f is usually carried out by measuring the content of tetraspanin 10 in fraction F or fraction f. From the viewpoint of accurate quantification, it is preferable that the quantification method in (a4) and the quantification method in (b4) are carried out by the same method.

[0111] When detecting tetraspanin 10 protein in (a3), for example, the content of tetraspanin 10 may be quantified by mass or concentration, or may be quantified based on the luminescence intensity of a substrate, etc. In this specification, the term "concentration" is not limited to absolute concentration but also includes relative concentration, mass per unit volume, raw data measured to determine absolute concentration, etc.

[0112] Furthermore, when detecting an RNA sequence as a gene product of a gene sequence encoding tetraspanin 10 in (a3) ​​above, the content of tetraspanin 10 may be quantified by the number of RNA copies or reads, or may be quantified based on fluorescence intensity or the like.

[0113] In (a4) and (b4), the method for quantifying tetraspanin 10 in fraction F or fraction f is not particularly limited as long as it is a method that can quantify tetraspanin 10, and can be appropriately selected depending on the method for determining the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles in (a3), and tetraspanin 10 can be quantified by a conventional method from the detected value of tetraspanin 10 in (a3).

[0114] For example, when determining the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles in (a3) ​​above, a method may be used in which a standard sample, such as a solution containing tetraspanin 10 at a known concentration, is used to perform a test in the same manner as the method for determining the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles in (a3) ​​above, and a calibration curve is drawn in advance to quantify the tetraspanin 10 concentration in the fraction; or a method in which a fraction is mixed with a predetermined amount of a tetraspanin 10 standard prepared in several stages, and the tetraspanin 10 concentration in the fraction is quantified by the internal standard method.

[0115] The step (b4) may be a step of comparing a quantitative value A of tetraspanin 10 in fraction F with a quantitative value B of tetraspanin 10 in fraction f, and determining that the subject is not affected with cancer or that the prognosis of the subject is good if quantitative value A≦quantitative value B is satisfied.

[0116] There are no particular limitations on the method for comparing the quantitative value A of tetraspanin 10 in fraction F with the quantitative value B of tetraspanin 10 in fraction f, and the method can be appropriately selected depending on the method for determining the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles and the method for quantifying tetraspanin 10. For example, in the case of an ELISA method, a higher absorbance at 450 nm finally detected can be determined to indicate a higher quantitative value of tetraspanin 10.

[0117] There are no particular restrictions on the quantitative value A and the quantitative value B as long as they satisfy the relationship of quantitative value A > quantitative value B. However, the quantitative value A is preferably 1.05 times or more, more preferably 1.1 times or more, even more preferably 1.5 times or more, and particularly preferably 2 times or more, of the quantitative value B.

[0118] In the determination step of the third aspect, the amounts of protein or RNA in the fractions F and f may be normalized with the amount of protein or mRNA derived from a housekeeping gene such as GAPDH, β2-microglobulin, or β-actin before comparing the quantitative values ​​A and B of tetraspanin 10.

[0119] <Sample Collection Step> The sample collection step is a step of collecting the body fluid-derived sample from the subject before the sample preparation step. When the determination step is performed in the third aspect, the sample collection step is a step of collecting the body fluid-derived sample from the subject or the healthy subject before (a1) and (b1).

[0120] The method for collecting the body fluid sample is not particularly limited and can be appropriately selected from known methods depending on the type of body fluid. For example, when the body fluid is a blood sample, a method of collecting the sample with a syringe can be used.

[0121] <Sample Treatment Step> The sample treatment step is a step of applying some kind of treatment to the body fluid-derived sample collected in the sample collection step.

[0122] The method for treating the body fluid-derived sample is not particularly limited and can be appropriately selected from known methods depending on the type of body fluid, and examples include separation treatment, purification treatment, drug addition treatment, etc. These may be performed alone or in combination of two or more. For example, when the body fluid is a blood sample, serum may be separated by a known method.

[0123] <Analysis Step> The analysis step is a step of analyzing the detected or quantified value of tetraspanin 10 obtained after the determination step using known analysis software.

[0124] In the analyzing step, when the determining step is carried out in the first or second embodiment, the detected value of tetraspanin 10 obtained in the determining step is analyzed. In addition, in the analyzing step, when the determining step is carried out in the third embodiment, the quantitative values ​​obtained in (a4) and (b3) are analyzed. Therefore, the analysis software used in the analyzing step can be appropriately selected depending on the detected value or quantitative value obtained in the determining step.

[0125] <Treatment method determination step> The treatment method determination step is a step of determining a necessary treatment method depending on the determination result in the determination step. Here, the "determination result" in the treatment method determination step includes the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles in the determination step of the first embodiment, the tetraspanin 10 detection value in the determination step of the second embodiment, and the quantification value in the determination step of the third embodiment.

[0126] For example, if it is determined in the determination step that a fraction containing tetraspanin 10-positive extracellular vesicles is present or if it is determined that the subject is suffering from cancer, it may be decided to perform surgical resection of the cancer, to administer radiation therapy and / or chemotherapy, or to administer a combination of these treatments.

[0127] Furthermore, if the prognosis of the subject is determined to be poor, it may be decided to perform or continue surgical resection of the cancer, to perform or continue radiation therapy and / or chemotherapy, to conduct follow-up after surgical resection of the cancer, to conduct follow-up after radiation therapy and / or chemotherapy, to conduct follow-up while continuing radiation therapy and / or chemotherapy, the duration of these follow-up observations, etc.

[0128] Furthermore, if it is determined in the determination step that there is no fraction containing tetraspanin 10-positive extracellular vesicles, or if it is determined that the subject is not suffering from cancer, or if it is determined that the prognosis of the subject is good, it may be decided to perform follow-up observation, and the duration of such follow-up observation, etc.

[0129] <Applications> According to the method for detecting cancer or the method for predicting cancer prognosis disclosed herein, it is possible to specifically, sensitively, simply, and quickly detect whether or not a subject is suffering from cancer or whether or not the cancer prognosis is poor, and it is also possible to detect cancer at an early stage. Therefore, the method for detecting cancer or the method for predicting cancer prognosis of the present disclosure can be suitably used, for example, as a diagnostic method for diagnosing whether a patient is affected with cancer, a predictive method for predicting whether or not a patient is likely to develop cancer, a method for observing the effect of a treatment in a patient undergoing cancer treatment (e.g., surgery, radiotherapy, etc.) (i.e., the effect of the treatment is observed when the value of the tetraspanin 10 decreases after the treatment compared to before the treatment), a monitoring method for following up on the presence or absence of recurrence (including metastasis) after cancer treatment (i.e., there is a possibility of recurrence when the value of the tetraspanin 10 increases during follow-up compared to immediately after the treatment), a pharmacodynamic method for analyzing the effect of a drug or compound (including existing drugs and clinical trials of new drugs, etc.) on cancer (i.e., the effect of the treatment by the drug is observed when the value of the tetraspanin 10 decreases after the administration of the drug compared to before the administration), a method for predicting the effect of a specific treatment (i.e., the effect of the specific treatment is observed when the value of the tetraspanin 10 decreases after the start of the specific treatment compared to before the start of the treatment), and a companion diagnostic method for selecting a cancer treatment method.

[0130] The treatment is not particularly limited and can be appropriately selected from known cancer treatments, and examples include watchful waiting, surgical resection, radiation therapy (including external radiation therapy and internal radiation therapy), radioactive iodine (I-125), palladium, iridium, hormone therapy, leuprolide, goserelin, buserelin, antiandrogens, flutamide, bicalutamide, megestrol acetate, nilutamide, ketoconazole, aminoglutethimide, gonadotropin-releasing hormone (GnRH), estrogen, cryotherapy, chemotherapy, biological therapy, ultrasound irradiation, proton beam irradiation, and methods targeting HER2 such as Herceptin.

[0131] The present disclosure will be specifically explained below using test examples, but the present disclosure is not limited to these test examples.

[0132] (Test Example 1: Concentration of tetraspanin 10-positive extracellular vesicles) <Acquisition of serum samples> Serum samples from two patients with colon cancer at stage III according to the TNM classification (subject 1 and subject 2) were obtained.

[0133] <Separation of Extracellular Vesicles> The serum sample was purified using an exosome purification column (EVSecond, manufactured by GL Sciences Inc.) according to the product protocol. Specifically, a packing material was added to the column, mixed thoroughly, and then the storage solution was removed. Next, 700 μL of fetal bovine serum (FBS) for cell culture was added to the column for blocking. After removing the FBS, PBS was added to the column and washed three times. Next, 300 μL of the serum sample was loaded, and 100 μL of PBS was added to elute, which was repeated 12 times to obtain elution fractions 1 to 12.

[0134] <Identification of fractions containing tetraspanin 10-positive extracellular vesicles> - Preparation of anti-TSPAN10 antibody solution - An anti-TSPAN10 antibody solution was prepared by diluting an anti-TSPAN10 antibody (TSPAN10 polyclonal antibody, manufactured by Proteintech) in PBS (manufactured by Nacalai Tesque, Inc.) to 300 ng / 50 μL. The epitope sequence recognized by the anti-TSPAN10 antibody is the amino acid sequence represented by SEQ ID NO: 2.

[0135] - Preparation of biotinylated detection antibody solution A - A biotinylated anti-TSPAN10 antibody (polyclonal rabbit anti-human OCSP / TSPAN10 antibody (Biotin, aa74-123, WB) LS-C453227, manufactured by LSBio) was diluted in PBS to 100 ng / 100 μL to prepare a biotinylated detection antibody solution.

[0136] - Preparation of biotinylated detection antibody solution B - Anti-TSPAN10 antibody (SAB2102589, rabbit host antibody, manufactured by Sigma) was prepared using an antibody / protein labeling kit (Biotin Labeling Kit-NH ２The antibody was labeled with biotin using a biotin-containing antibody (manufactured by Dojindo Laboratories, Inc.) to obtain a biotinylated detection antibody solution B. The epitope sequence recognized by the anti-TSPAN10 antibody is the amino acid sequence represented by SEQ ID NO:3.

[0137] -Preparation of anti-TSPAN10 antibody-immobilized immunoplate- An anti-TSPAN10 antibody solution was immobilized on an immunoplate (surface treatment: MaxiSorp ＴＭ 50 μL / well of the immunoplate was added to a 500 μL / well plate (manufactured by Thermo Fisher Scientific) and left to stand at 25° C. for 2 hours to immobilize the anti-TSPAN10 antibody on the immunoplate. Next, a blocking buffer (SuperBlock ＴＭ Blocking Buffer (manufactured by Thermo Fisher Scientific) was added at 300 μL / well and allowed to stand at 25° C. for 30 minutes. Thereafter, the blocking buffer was removed, and 400 μL / well of PBS was added and then removed, which was repeated three times for washing.

[0138] - Detection by ELISA - Next, 20 μL / well of eluted fractions 1 to 12 of subject 1 or eluted fractions 1 to 11 of subject 2, 60 μL / well of PBS, and 20 μL / well of HBR-1 (Heterophilic Blocking Reagent 1, manufactured by Scantibodies Laboratory, Inc.) were added, and the wells were rotated at 300 rpm for 1 minute at 4°C using a mixer (Eppendorf ThermoMixer® C, manufactured by Eppendorf), followed by leaving the wells to stand for 1 minute. This process was repeated for 6 hours. Next, the solution containing the eluted fractions was removed from the wells, and 400 μL of PBS was added and then removed, repeating this process three times for washing. Next, 100 μL of biotinylated detection antibody solution A was added to the washed wells, and the wells were rotated at 300 rpm for 1 minute using a mixer (Eppendorf ThermoMixer® C, manufactured by Eppendorf) at 25°C, followed by leaving the wells to stand for 1 minute. This process was repeated for 3 hours. Next, biotinylated detection antibody solution A was removed from the wells, and 400 μL of PBS was added and then removed, repeating this process three times for washing. Next, a poly-HRP label (Streptavidin Poly-HRP40 Conjugate, Horseradish Peroxidase (Fitzgerald Industries International, Inc.) diluted 50,000-fold with PBS was added at 100 μL / well. Next, the solution containing the poly-HRP label was removed from the wells, and 400 μL of PBS was added and then removed three times for washing. Next, TMB substrate (SureBlue ＴＭ 100 μL / well of TMB 1-Component Microwell Peroxidase Substrate (manufactured by Sera Care) was added and reacted at 25°C for 5 minutes, after which the reaction was stopped with 2 M hydrochloric acid. Thereafter, absorbance was measured at a wavelength of 450 nm using a microplate reader (Epoch 2, manufactured by Agilent Technologies).

[0139] The detection results by ELISA are shown in Figure 2. As a result, it was confirmed that elution fractions 8 to 10 in both cases contained high concentrations of tetraspanin 10-positive extracellular vesicles.

[0140] <Enrichment of tetraspanin 10-positive extracellular vesicles> Elution fractions 8 to 12 from the two cases were pooled and then analyzed using an exosome flow cytometry kit (PS Capture ＴＭ Tetraspanin 10-positive extracellular vesicles were enriched using an exosome flow cytometry kit (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0141] <Detection of tetraspanin 10-positive extracellular vesicles> 3 μg of biotinylated detection antibody solution B, 250 μL, which is half the amount of the concentrate obtained by concentrating elution fractions 8 to 12, and beads (PS Capture ＴＭ The beads were mixed with 60 μL of an exosome flow cytometry kit and reacted overnight at 4 ° C. After the reaction, the beads were washed once with 1 mL of PBS, and streptavidin-labeled 5 nm gold colloid particles (Streptavidin-5 nm Gold Conjugate, manufactured by CYTODIAGNOSTICS) were added and reacted overnight at 4 ° C. After the reaction, the beads were washed once with 1 mL of PBS, and the tetraspanin 10-positive extracellular vesicles captured on the beads were eluted with the Exosome Elution Buffer included in the exosome purification kit (MagCapture™ Exosome Isolation Kit PS Ver. 2, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0142] The eluted TSPAN10-positive extracellular vesicles were observed using a transmission electron microscope (TEM) (H-7600, Hitachi, Ltd.) and images are shown in Figure 3. The scale bar indicates 100 nm, and the arrow indicates the gold colloid label. This confirmed that tetraspanin 10-positive extracellular vesicles can be enriched from the serum of cancer patients.

[0143] (Test Example 2: Analysis of TSPAN10 and p53 expression in extracellular vesicles) <Analysis of TSPAN10 protein and p53 protein expression in extracellular vesicles> Using Te-EVs Proteome Library (https: / / repository.jppostdb.org / entry / JPST000867, the contents of which are incorporated herein by reference in their entirety), the expression of TSPAN10 protein and p53 protein was analyzed in a total of 34 extracellular vesicles from 17 cancer patients (two samples per case).

[0144] The analysis results of the expression of TSPAN10 protein and p53 protein in extracellular vesicles of cancer patients are shown in Figure 4. As a result, it was found that the expression levels of TSPAN10 protein and p53 protein correlated.

[0145] (Test Example 3: Analysis of TSPAN10 mRNA and MUC16 mRNA Expression in Cancer Tissues) Based on the TCGA data, the following cancer types were classified according to the TNM classification: skin cutaneous melanoma (n=481), head and neck squamous cell carcinoma (n=499), cervical cancer (n=291), uterine cancer (n=541), ovarian cancer (n=373), breast cancer (n=1,075), and prostate cancer (n=1,076). Adenocarcinoma (n=494), lung squamous cell carcinoma (n=494), lung adenocarcinoma (n=500), thyroid carcinoma (n=501), bladder cancer (n=406), kidney renal clear cell carcinoma (n=528), hepatocellular carcinoma (n=365), testicular cancer (n=134), pancreatic cancer The expression levels of TSPAN10 mRNA and MUC16 mRNA were analyzed in human colon cancer (n=176), stomach cancer (n=354), rectal cancer (n=159), and colon cancer (n=438).

[0146] The analysis results of the expression levels of TSPAN10 mRNA and MUC16 mRNA are shown in Figure 5. Expression of both TSPAN10 mRNA and MUC16 mRNA was observed in all cancer tissues.

[0147] (Test Example 4: Validation of analytical method for various cancers using TSPAN10 and MUC16) <Acquisition of serum samples> A total of 71 serum samples were obtained from subjects. Of these, 12 were sera from healthy individuals. Hereinafter, the healthy individuals may be referred to as the "control group." In addition, 2 samples were sera from head and neck cancer patients, 14 samples were sera from esophageal cancer patients, 10 samples were sera from gastric cancer patients, 12 samples were sera from lung cancer patients, 12 samples were sera from thyroid cancer patients, 1 sample was sera from uterine cancer patients, 1 sample was sera from ovarian cancer patients, 1 sample was sera from melanoma patients, 1 sample was sera from renal cancer patients, and 5 samples were sera from liver cancer patients. Hereinafter, the various cancer patients may be collectively referred to as the "cancer group."

[0148] <Detection by ELISA 1> Using an anti-TSPAN10 antibody-immobilized immunoplate prepared by the same method as the method for preparing the anti-TSPAN10 antibody-immobilized immunoplate in Test Example 1, detection by ELISA was performed in the same manner as in Test Example 1, except that in the detection by ELISA in Test Example 1, elution fractions 1 to 12 of subject 1 or elution fractions 1 to 11 of subject 2 were replaced with serum samples from the control group and the pancreatic cancer stage III group, and the biotinylated detection antibody solution was changed to a biotinylated anti-MUC16 antibody prepared as follows.

[0149] - Preparation of biotinylated anti-MUC16 antibody solution - A biotinylated anti-MUC16 antibody (biotinylated anti-CA125 mouse monoclonal antibody (clone M11), Elecsys (registered trademark) CA 125M, manufactured by Roche) was diluted in PBS to 100 ng / 100 μL to prepare a biotinylated anti-MUC16 antibody solution.

[0150] <Detection by ELISA Method 2> - Preparation of Anti-CD9 Antibody Solution - An anti-CD9 antibody solution was prepared by diluting an anti-CD9 antibody (anti-CD9, monoclonal antibody (1K), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in PBS to 100 ng / 100 μL.

[0151] - Preparation of anti-CD9 antibody-immobilized immunoplate - The anti-CD9 antibody solution was immobilized on an immunoplate (surface treatment: MaxiSorp ＴＭThe anti-CD9 antibody solution was added to a plate (Thermo Fisher Scientific) at 50 μL / well and left to stand at 25° C. for 2 hours to immobilize the anti-CD9 antibody solution on the immunoplate. Next, a blocking buffer (SuperBlock ＴＭ Blocking Buffer (manufactured by Thermo Fisher Scientific) was added at 300 μL / well and allowed to stand at 25° C. for 30 minutes. Thereafter, the blocking buffer was removed, and 400 μL / well of PBS was added and then removed, which was repeated three times for washing.

[0152] - Detection by ELISA - Using an anti-CD9 antibody-immobilized immunoplate, detection by ELISA was performed in the same manner as in Test Example 1, except that in the detection by ELISA in Test Example 1, elution fractions 1 to 12 of subject 1 or elution fractions 1 to 11 of subject 2 were replaced with serum samples from the control group and the pancreatic cancer stage III group, and the biotinylated detection antibody solution was replaced with the biotinylated anti-MUC16 antibody prepared in Detection 1 by ELISA.

[0153] <Results> The receiver operating characteristic (ROC) curves obtained as a result of analytical method validation by ELISA using anti-TSPAN10 antibody and anti-MUC16 antibody are shown in Figure 6A. The receiver operating characteristic (ROC) curves obtained as a result of analytical method validation by ELISA using anti-CD9 antibody and anti-MUC16 antibody are shown in Figure 6B.

[0154] The area under the ROC curve (AUC) of the ELISA assay using anti-TSPAN10 antibody and anti-MUC16 antibody was 0.715, with a 95% confidence interval of 0.556-0.875. The cutoff value (0.450) was determined using Youden's index from the ROC curve, and the results of the number of people classified as "positive" and "negative" by ELISA are shown in Table 1 below.

[0155] The area under the ROC curve (AUC) of the ELISA assay using anti-CD9 and anti-MUC16 antibodies was 0.572, with a 95% confidence interval of 0.394-0.75. The cutoff value (0.391) was determined using Youden's index from the ROC curve, and the results of the number of people classified as "positive" and "negative" by ELISA are shown in Table 2 below.

[0156]

[0157]

[0158] From the results in Tables 1 and 2, the sensitivity, specificity, positive predictive value, negative predictive value, and prevalence were calculated by the ELISA method using the following formula 1, formula 2, formula 3, formula 4, and formula 5, respectively. The results are shown in Table 3. Sensitivity (%) = [cancer group and ELISA detection positive] / [total of cancer group] × 100 (formula 1) Specificity (%) = [control group and ELISA detection negative] / [total of control group] × 100 (formula 2) Positive predictive value (%) = [cancer group and ELISA detection positive] / [total of ELISA detection positive] × 100 (formula 3) Negative predictive value (%) = [control group and ELISA detection negative] / [total of ELISA detection negative] × 100 (formula 4) Prevalence (%) = [cancer group total] / [total of cancer group total] × 100 (formula 5)

[0159] These results show that the area under the ROC curve (AUC) obtained by ELISA using anti-TSPAN10 antibody and anti-MUC16 antibody was larger than the area under the ROC curve (AUC) obtained by ELISA using anti-CD9 antibody and anti-MUC16 antibody, indicating that the anti-TSPAN10 antibody can specifically detect cancer compared to the anti-CD9 antibody.

[0160] (Test Example 5: Validation of analytical method for pancreatic cancer using TSPAN10 and MUC16) <Acquisition of serum samples> A total of 16 serum samples were obtained from subjects. Of these, 8 samples were sera from healthy individuals (hereinafter sometimes referred to as the "control group"), and 8 samples were sera from patients with stage III pancreatic cancer according to the TNM classification (hereinafter sometimes referred to as the "pancreatic cancer stage III group").

[0161] <Detection by ELISA> In the ELISA detection of Test Example 4, the serum samples were changed to serum samples from the control group and the pancreatic cancer stage III group, and only biotinylated anti-MUC16 antibody was used as the biotinylated detection antibody solution, and detection by ELISA was performed in the same manner as in Test Example 4.

[0162] <Results> The detection results by ELISA are shown in Figure 7. As a result of the Mann-Whitney U test, the p value between the control group and the pancreatic cancer stage III group was 0.005, indicating a significant difference.

[0163] The receiver operating characteristic (ROC) curve resulting from the validation of the analytical method using the ELISA method is shown in Figure 8. In Figure 8, the area under the ROC curve (AUC) was 0.891, and the 95% confidence interval was 0.726-1. The cutoff value (0.650) was determined using Youden's index from the ROC curve, and the results of the number of people classified as "positive" and "negative" by ELISA detection are shown in Table 4 below.

[0164]

[0165] From the results in Table 4, the sensitivity, specificity, positive predictive value, negative predictive value, and prevalence were determined in the same manner as in Test Example 4, except that in Formulas 1 to 5 in Test Example 4, the group was changed to the pancreatic cancer stage III group, and the results are shown in Table 5. It is known that the positive rate for stage III pancreatic cancer of CA125 in serum, a representative existing tumor marker, is 70% (see Takemori Yasuhiro et al., Journal of the Japanese Society of Gastroenterology, 1987, Vol. 84, No. 10, pp. 2386-2392, https: / / doi.org / 10.11405 / nishoshi1964.84.2386).

[0166]

[0167] Examples of aspects of the present disclosure include the following. <1> A method for concentrating tetraspanin 10-positive extracellular vesicles, comprising the steps of: obtaining a sample containing extracellular vesicles from a body fluid-derived sample of a subject; separating the sample containing the extracellular vesicles into two or more fractions by size exclusion chromatography; and recovering fractions containing tetraspanin 10-positive extracellular vesicles. <2> The method for concentrating tetraspanin 10-positive extracellular vesicles according to <1>, wherein the body fluid-derived sample is at least one selected from the group consisting of peripheral blood, serum, plasma, ascites, lymph, cerebrospinal fluid, saliva, bone marrow, urine, synovial fluid, tissue fluid, sweat, tears, sputum, nasal discharge, amniotic fluid, and breast milk. <3> The method for concentrating tetraspanin 10-positive extracellular vesicles according to <1> or <2>, wherein the subject is currently suffering from or has previously suffered from cancer. <4> The method for concentrating tetraspanin 10-positive extracellular vesicles according to any one of <1> to <3>, wherein the tetraspanin 10-positive extracellular vesicles are extracellular vesicles derived from cancer cells. <5> The method for concentrating tetraspanin 10-positive extracellular vesicles according to <3> or <4>, wherein the cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, lung cancer, thyroid cancer, uterine cancer, ovarian cancer, malignant melanoma, kidney cancer, liver cancer, epithelial cancer, rectal cancer, colon cancer, papillary renal cell carcinoma, head and neck squamous cell carcinoma, serous cystadenocarcinoma, chromophobe renal cell carcinoma, prostate cancer, lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, renal non-clear cell carcinoma, hepatocellular carcinoma, testicular germ cell tumor, pancreatic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, and colorectal adenocarcinoma. <6> The method for concentrating tetraspanin 10-positive extracellular vesicles according to any one of <1> to <5>, further comprising a step of contacting each of the two or more fractions separated in the separating step with a substance for detecting tetraspanin 10, and identifying the fraction containing tetraspanin 10-positive extracellular vesicles.<7> The method for enriching tetraspanin 10-positive extracellular vesicles according to <6>, wherein the detection substance is at least one selected from the group consisting of an antibody capable of binding to tetraspanin 10, a nucleic acid primer capable of specifically hybridizing to tetraspanin 10, and a nucleic acid probe capable of specifically hybridizing to tetraspanin 10. <8> The method for enriching tetraspanin 10-positive extracellular vesicles according to <7>, wherein the epitope sequence of the antibody capable of binding to tetraspanin 10 is an amino acid sequence set forth in any of SEQ ID NOs: 2 to 5. <9> A method for detecting cancer or predicting the prognosis of cancer, comprising the steps of: obtaining a sample containing extracellular vesicles from a body fluid-derived sample of a subject; separating the sample containing extracellular vesicles into two or more fractions by size exclusion chromatography; and contacting each of the two or more fractions separated in the separation step with a detection substance for tetraspanin 10, and determining the presence or absence of a fraction containing tetraspanin 10-positive extracellular vesicles. <10> The method according to <9>, further comprising a step of determining that the subject is suffering from cancer if a fraction containing tetraspanin 10-positive extracellular vesicles is present in the determining step.

[0168] This international application claims priority based on Japanese Patent Application No. 2024-047137, filed on March 22, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. A method for concentrating tetraspanin 10-positive extracellular vesicles, comprising the steps of: obtaining a sample containing extracellular vesicles from a subject's body fluid-derived sample; separating the sample containing extracellular vesicles into two or more fractions by size exclusion chromatography; and recovering fractions containing tetraspanin 10-positive extracellular vesicles.

2. The method for concentrating tetraspanin 10-positive extracellular vesicles according to claim 1, wherein the body fluid-derived sample is at least one selected from the group consisting of peripheral blood, serum, plasma, ascites, lymph, cerebrospinal fluid, saliva, bone marrow, urine, synovial fluid, tissue fluid, sweat, tears, sputum, nasal discharge, amniotic fluid, and breast milk.

3. The method for concentrating tetraspanin 10-positive extracellular vesicles according to claim 1 or 2, wherein the subject is a subject who is suffering from or has previously suffered from cancer.

4. A method for concentrating tetraspanin 10-positive extracellular vesicles according to any one of claims 1 to 3, wherein the tetraspanin 10-positive extracellular vesicles are extracellular vesicles derived from cancer cells.

5. The method for concentrating tetraspanin 10-positive extracellular vesicles according to claim 3 or 4, wherein the cancer is at least one selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, lung cancer, thyroid cancer, uterine cancer, ovarian cancer, malignant melanoma, kidney cancer, liver cancer, epithelial cancer, rectal cancer, colon cancer, papillary renal cell carcinoma, head and neck squamous cell carcinoma, serous cystadenocarcinoma, chromophobe renal cell carcinoma, prostate cancer, lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, renal non-clear cell carcinoma, hepatocellular carcinoma, testicular germ cell tumor, pancreatic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, and colorectal adenocarcinoma.

6. A method for concentrating tetraspanin 10-positive extracellular vesicles according to any one of claims 1 to 5, further comprising the step of contacting each of the two or more fractions separated in the separation step with a substance for detecting tetraspanin 10, and identifying the fraction containing the tetraspanin 10-positive extracellular vesicles.

7. The method for concentrating tetraspanin 10-positive extracellular vesicles described in claim 6, wherein the detection substance is at least one selected from the group consisting of an antibody capable of binding to tetraspanin 10, a nucleic acid primer capable of specifically hybridizing to tetraspanin 10, and a nucleic acid probe capable of specifically hybridizing to tetraspanin 10.

8. A method for concentrating tetraspanin 10-positive extracellular vesicles according to claim 7, wherein the epitope sequence of the antibody capable of binding to tetraspanin 10 is an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 5.

9. A method for detecting cancer or predicting the prognosis of cancer, comprising the steps of: obtaining a sample containing extracellular vesicles from a body fluid-derived sample of a subject; separating the sample containing extracellular vesicles into two or more fractions by size exclusion chromatography; and contacting each of the two or more fractions separated in the separation step with a substance for detecting tetraspanin 10, and determining whether or not a fraction containing tetraspanin 10-positive extracellular vesicles is present.

10. The method according to claim 9, further comprising determining that the subject is suffering from cancer if a fraction containing tetraspanin 10-positive extracellular vesicles is present in the determining step.

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