Methods for diagnosing bladder cancer

Abstract

The present invention identifies a substance present in urine microvesicles of a bladder cancer patient to thereby construct a method that assists early and exact diagnosis of bladder cancer. This assistant method comprises concentrating microvesicles contained in urine derived from a subject patient, and determining whether the patient has bladder cancer or not depending on the amount of a marker protein present in the microvesicles.

Description

[Technical Field]

[0001] This invention relates to the detection of cancer cell-specific extracellular vesicles (medium / large extracellular vesicles) present in the urine of bladder cancer patients and to a method for diagnosing bladder cancer using these vesicles. [Background technology]

[0002] Cancer, recognized as a leading cause of death in Japan since the 1980s, is also among the leading causes of death worldwide. Despite advances in medical diagnostic and treatment techniques, it is still difficult to say that diagnostic or treatment methods are fully established. Bladder cancer, in particular, sees an estimated hundreds of thousands of diagnoses worldwide each year, with 20,000 new cases diagnosed in Japan. Approximately one-third of these cases are thought to be potentially invasive or metastatic at the time of diagnosis, and it has the highest incidence and mortality rates among various urinary tract cancers (renal pelvis, ureter, bladder) (Non-patent Literature 1).

[0003] Bladder cancer is caused by the malignant transformation of bladder tissue, including urothelium, and histologically, urothelial carcinoma accounts for 90% of all cases. Of these cases, 70% are superficial and non-invasive. Approximately 75% of patients diagnosed with transitional cell carcinoma present with superficial tumors that can be treated by transurethral resection. The recurrence rate of superficial disease exceeds 60%, and less than 30% of recurrent bladder tumors progress to invasive disease (Non-patent Literature 2). Therefore, in addition to the importance of early detection of patients with or suspected of having bladder cancer, lifelong surveillance is considered crucial for treatment success.

[0004] The vast majority of patients with bladder cancer seek medical attention after experiencing macroscopic or microscopic hematuria, or other concerning urinary symptoms such as frequent urination and dysuria. However, there is no accurate and easy way to identify the presence of early-stage bladder cancer, and a definitive diagnosis of bladder cancer requires a combination of procedures.

[0005] As an initial screening method, urine cytology, which involves microscopic examination of cells derived from a urine sample, may be used. Urine cytology allows for the examination of detached cells for the presence of specific cell surface antigens, nuclear morphology, gene expression, or other biological markers. While useful for cases of high-grade malignant tumors, it has limitations in its sensitivity to detect other low-grade tumors (Non-Patent Literature 3). Furthermore, the accuracy of urine cytology is problematic because the degree to which bladder cancer cells are released into the urine varies greatly depending on the timing of urine collection and the stage of the disease. In addition, subjectivity cannot be ruled out in the interpretation of the results, and the results can fluctuate. Therefore, cytology is not ideal for screening and surveillance of bladder cancer.

[0006] For patients exhibiting the above symptoms and suspected of having bladder cancer, the most reliable test is considered to be a definitive diagnosis performed by cystoscopy under local or general anesthesia, followed by solid tissue biopsy if necessary. However, these symptoms are often caused by conditions such as urinary tract infections or benign prostatic hyperplasia, and such invasive tests that place a significant burden on patients must be performed even on patients who do not have bladder cancer. Therefore, there is a need for less invasive screening tests using highly accurate biomarkers.

[0007] Commonly known methods for measuring tumor markers for urological cancers include NMP22 (Nuclear Matrix Protein 22), BTA (Bladder Tumor Antigen), and cytokeratin 8 and 18. However, while these methods have relatively higher sensitivity than cytology, they have been reported to produce a high rate of false positives (Non-Patent Literature 4, Non-Patent Literature 5, Non-Patent Literature 6).

[0008] Therefore, methods for detecting bladder cancer patients using existing biomarkers are not sufficient as diagnostic markers for bladder cancer, and do not provide guidance for their application in the early diagnosis of bladder cancer. Consequently, a bladder cancer diagnostic marker that enables early diagnosis and has high sensitivity and specificity has yet to be found.

[0009] In recent years, it has been proposed to use micromembrane fractions separated from cells as liquid biopsies, a non-invasive diagnostic material, to determine disease status and the presence or absence of disease (Patent Documents 1 and 2). Such micromembrane fractions are broadly classified into exosomes, microvesicles, etc., depending on their size, and the use of all of them is attracting attention. Among them, extracellular vesicles (microvesicles) are known to play an extremely important role in tumor invasion (Non-Patent Document 7), and extracellular vesicles present in urine have been reported to be involved in diseases such as diabetic nephropathy (Non-Patent Document 5), so their clinical usefulness is expected. Extracellular vesicles are present in large quantities in human body fluids compared to circulating tumor cells (CTCs) in the blood, and it has been suggested that they play an extremely important role in tumor invasion and malignancy (Non-Patent Documents 7 and 8).

[0010] In particular, knowledge is accumulating regarding methods for extracting and detecting the micromembrane fraction in urine. For example, it is known that urine contains large amounts of Tamm-Horsfall protein (uromodulin) (hereinafter abbreviated as THP), which forms high-molecular-weight polymers. To separate these high-molecular-weight polymers from the micromembrane fraction, a method is known in which the high-molecular-weight polymers are decomposed in advance by reduction treatment before extracting exosomes (Non-Patent Document 9). In addition, a method is known in which microvesicles in the urine of healthy individuals are concentrated and characterized using a flow cytometer (Patent Document 3). [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Patent No. 5838963 [Patent Document 2] Japanese Patent Publication No. 2015-91251 [Patent Document 3] Japanese Patent Publication No. 2019-215342 [Non-patent literature]

[0012] [Non-Patent Document 1] Jemal et al., Cancer statistics. CA Cancer J Clin. 57; 43-66, 2007 [Non-Patent Document 2] Zieger et al., Long-term follow-up of noninvasive bladder tumours(stage Ta): recurrence and progression. BJU Int. 85; 824-8, 2000 [Non-Patent Document 3] Wiener et al. Accuracy of urinary cytology in the diagnosis of primary and recurrent bladder cancer. Acta Cytol. 37(2); 163-9, 1993 [Non-Patent Document 4] Takashi Munehisa et al., Bladder cancer markers (urinary BTA, urinary NMP22). Clinical Laboratory Guide 2001-2002. 945-7, 2001. [Non-Patent Document 5] Takashi M et al., Use of diagnostic categories in urinary cytology in comparison with the bladder tumor antigen(BTA) test in bladder cancer patients. Int Urol Nephrol. 31; 189-96, 1999 [Non-Patent Document 6] Hideyuki Akaza. Clinical study of urinary NMP (nuclear matrix protein 22) in urothelial carcinoma (Part 1) - Usefulness of urinary NMP22 sensitivity testing and follow-up in bladder cancer. Cancer and Chemotherapy 24; 829-36, 1997. [Non-Patent Document 7] James W et al. Regulated delivery of molecular cargo to invasive tumour-derived microvesicles. Nat Commun. 6(6919), 2015

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0013] As described above, urine can be a non-invasive and effective clinical test material in the examination of bladder cancer. However, there is no test that can diagnose bladder cancer early and accurately using biomarkers contained in urine as an indicator. Therefore, in the present invention, attention is paid to bladder cancer-specific extracellular vesicles contained in urine, particularly microvesicles (medium / large extracellular vesicles) with a diameter of 0.1 to 1 μm, and substances present in urinary microvesicles of bladder cancer patients are identified, and the object of the present invention is to construct a method for assisting in the early and accurate diagnosis of bladder cancer.

[0014] In the present specification, extracellular vesicles refer to 100nmIt means medium / large extracellular vesicles that do not contain the following exosomes or small extracellular vesicles, and includes microvesicles. Hereinafter, the case of using microvesicles as an example of extracellular vesicles will be described, but the present invention is not limited thereto. Also, in this specification, separation shall be used synonymously with fractionation, detection, etc. [Means for Solving the Problems]

[0015] As a result of intensive studies to solve the above problems, the inventors were able to identify proteins specifically present in microvesicles in urine collected from bladder cancer patients by shotgun proteomics analysis. Some of the identified proteins were further analyzed, and microvesicles specifically present in bladder cancer that were positive for anti-human CD66a antibody, anti-human CD66c antibody, and / or anti-human CD66e antibody (hereinafter, these may be collectively referred to as anti-human CD66a / c / e antibody) were newly discovered. Furthermore, by calculating the abundance ratio of this anti-human CD66a / c / e antibody-positive microvesicle, a method capable of assisting in the diagnosis of bladder cancer with low invasiveness, early and accurate diagnosis was established. Using this method, dozens of patient samples (bladder cancer patients, non-bladder cancer patients, healthy individuals) were used to verify this method, and it was shown that microvesicles positive for CD66a / c / e are useful for the diagnosis / inspection application of bladder cancer patients. It is also suggested that proteins other than CD66a / c / e identified by shotgun proteomics analysis are useful for the diagnosis / inspection application of bladder cancer patients.

[0016] That is, the present invention provides the following: [1] A method for assisting in determining whether a target patient has bladder cancer by concentrating microvesicles contained in urine derived from the target patient and based on the abundance of marker proteins in the microvesicles. [2] Observation of the abundance of marker proteins in the microvesicles indicates that the microvesicles contain IGGH1, CRABP2, PLG, AHSG, HP, IGGH2, ANXA2, IGHM, SERPINA1, CFB, TF, NME2, C4BPA, A2M, TACSTD2, FGG, C4B, F2, C3, IGHA1, CP, SERPINA3, CA1, CAPN5, APOB, FAM129B, C9, TMSB10, C1QB, IGLC6, IGKC, CEACAM7, UPK3A, CEACAM5, ARHGDIB, SRC, FGB, The method of [1] is characterized by detecting the abundance of at least one of C1QC, LRG1, UPK3BL, DEFA3, SERPIND1, FN1, SDCBP2, APOA1, ITIH4, AGRN, SERPINF1, PDLIM1, ANXA9, PSCA, HBA1, SLC2A1, ITIH2, CYSRT1, SERPINC1, PGLYRP2, SPRR1A, PROS1, RAB27B, UGDH, MARCKS, EFHD2, APOL1, CD66a, CD66b, CD66c, CGM2, or CD66e. [3] The method according to [1] or [2], characterized in that the amount of CD66a, CD66b, CD66c, CGM2, or CD66e in the microvesicles is calculated by the ratio (A / B) of microvesicles (A) expressing CD66a, CD66b, CD66c, CGM2, and CD66e to microvesicles (B) expressing CD10, CD13, and CD26. [4] The method according to [1] or [2], characterized in that the amount of CD66a, CD66b, CD66c, CGM2, or CD66e in the microvesicles is calculated by the ratio (A / C) of microvesicles (A) expressing CD66a, CD66b, CD66c, CGM2, and CD66e to microvesicles (C) expressing CD10, CD13, CD26, and MUC1. [5] Any of the methods [1] to [4] that indicates a patient has bladder cancer, wherein the amount or ratio of marker protein in urinary microvesicles derived from the patient is higher than the amount or ratio of marker protein in urinary microvesicles derived from the control. [6] A step of measuring the amount of CD66a / b / c / e or CGM2 present in urinary microvesicles, and if the measured amount of CD66a / b / c / e or CGM2 is greater than that of the control, urine A method for assisting the determination of whether or not a person has bladder cancer, comprising the step of determining that a substance originates from a bladder cancer patient. [7] Any of the methods [1] to [6] for observing the course of treatment for bladder cancer or determining the effectiveness of treatment for bladder cancer, by comparing the amount of protein present in a first sample taken from the patient with the amount of protein present in a second sample taken from the patient after the treatment period. [8] Any method of [1] to [7] further comprising the step of correlating as indicating superficial bladder cancer, invasive stage 1 bladder cancer, or invasive stage 2-3 bladder cancer.

[0017] Furthermore, the present invention includes, A method for diagnosing bladder cancer by measuring (or detecting) marker proteins in the microvesicles. A method for measuring (or detecting) marker proteins in microvesicles for the diagnosis of bladder cancer. An in vitro diagnostic method for bladder cancer, characterized by measuring (or detecting) marker proteins in the microvesicles. The use of an antibody capable of detecting marker proteins in the microvesicles in the manufacture of a bladder cancer diagnostic kit. A method for measuring (or detecting) marker proteins in microvesicles in order to provide information necessary for the diagnosis of bladder cancer. It includes. [Effects of the Invention]

[0018] The present invention provides a simple and specific method for separating and observing microvesicles specifically present in the urine of bladder cancer patients, enabling a less invasive, early, and accurate diagnosis of bladder cancer in first-time patients. Furthermore, it is expected to improve diagnostic performance (improved clinical sensitivity and reduced missed diagnoses) compared to conventional urine cytology tests in monitoring the prognosis (presence or absence of metastasis) after treatment such as surgery or chemotherapy in bladder cancer patients. In addition, it is expected that observing the fraction obtained by this invention will enable estimation of disease, drug administration effects, or other medical conditions in the subject. [Brief explanation of the drawing]

[0019] [Figure 1] This is a histogram of particle size distribution measured using scattered light and fluorescence NTA for fractions extracted from bladder cancer patients. [Figure 2] This is a histogram of particle size distribution measured using scattered light and fluorescence NTA for fractions extracted from healthy individuals. [Figure 3] This figure shows the percentage of particle diameters representing 10%, 50%, and 90% of the fractions extracted from healthy individuals and bladder cancer patients, measured using scattered light and trend NTA. [Figure 4] This Venn diagram shows the number of proteins detected by shotgun proteomics analysis of extracted microvesicle fractions in a pool of bladder cancer patients and a pool of healthy individuals. [Figure 5] This table shows the results of enrichment analysis of 585 proteins detected only in bladder cancer patients. Note that those highlighted in light gray (regulated exocytosis, hemostasis, and transport of small molecules in the Description column) were also detected in similar analyses of healthy individuals. [Figure 6] This graph shows the results of principal component analysis performed on the data from bladder cancer patients and healthy individuals, illustrating up to the second principal component. [Figure 7] This figure was obtained by performing OPLS-DA analysis based on the results from bladder cancer patients and healthy individuals, respectively. [Figure 8] This heatmap shows the quantitative results of protein measurements performed individually on the enriched microvesicle fraction (bladder cancer patients and healthy individuals) using shotgun proteomics analysis. [Figure 9] Figure 8 is a heatmap showing the profiles of all detected CEACAM family (CD66) proteins among the proteins shown. [Figure 10] This figure shows the estimated size of observed microvesicles in the urine of bladder cancer patients, obtained by unfolding CD66a / c / e-positive microvesicles using side-scattered light (SSC) size-validating beads and histograms. [Figure 11] The images above show flow cytometry observations of urinary microvesicles from bladder cancer patients stained with anti-human CD66a, anti-human CD66c, and anti-human CD66e antibodies, and the graph below shows the proportion of CD66c and e relative to CD66a (set at 100%), and the proportion of extracellular vesicles that merge with CD66a. [Figure 12] These are flow cytometry images of urinary microvesicles from bladder cancer patients stained with anti-human CD66b antibody and anti-human CD66a / c / e antibody. [Figure 13] These are flow cytometry images of urinary microvesicles stained with anti-human CD66a / c / e antibodies from bladder cancer patients, non-bladder cancer patients, and healthy individuals. [Figure 14] This is an explanatory diagram showing a method for targeting microvesicles that are positive for CD66a / c / e. It is a flow cytometry gating method that selects only CD66a / c / e-positive microvesicles from an overall observation. [Figure 15] This is a flow cytometry image of the overall micromembrane fraction in the observed image, showing MUC1-positive (CD66a / c / e negative), multipeptidase-positive (CD10, CD13, CD26 positive, CD66a / c / e negative), and CD66a / c / e-positive microvesicles unfolded with Annexin 5 and side-scattered light. [Figure 16]This figure illustrates that the CD66a / c / e-positive microvesicle fraction in bladder cancer patients is different from multipeptidase-positive (CD10, CD13, CD26-positive) microvesicles. [Figure 17] The CD66a / c / e-positive microvesicle fraction in bladder cancer patients is also MUC1-positive. This diagram illustrates that CD66a / c / e-positive microvesicles are secreted from cancerous urothelial cells that have transformed from normal cells. [Figure 18] This figure shows a list of all microvesicles observed by flow cytometry in bladder cancer patients, non-bladder cancer patients, and healthy individuals (percentage of each fraction in the overall observed image). [Figure 19] Percentage comparison of flow cytometry observations of CD66a / c / e positive fractions and multipeptidase positive fractions (10 bladder cancer patients, 4 non-bladder cancer patients, and 9 healthy individuals). [Figure 20] This figure compares the ratio of CD66a / c / e-positive fractions to multipeptidase-positive fractions, and the ratio of CD66a / c / e-positive fractions to the combined multipeptidase-positive fraction and MUC1-positive fraction (10 bladder cancer patients, 4 non-bladder cancer patients, and 9 healthy individuals). [Figure 21] This figure shows negative / positive graphs and ROC analysis performed on bladder cancer patients and other patients, based on the CD66a / c / e positive fraction, the ratio of the CD66a / c / e positive fraction to the multipeptidase positive fraction, and the ratio of the CD66a / c / e positive fraction to the combined multipeptidase positive fraction and MUC1 positive fraction. [Figure 22] This figure clearly shows the cutoff line (the peak of the ROC analysis) at which the sensitivity and specificity of the CD66a / c / e positive fraction, the ratio of the CD66a / c / e positive fraction to the multipeptidase positive fraction, and the ratio of the CD66a / c / e positive fraction to the combined multipeptidase positive fraction and MUC1 positive fraction have a good balance of sensitivity and specificity and provide the best diagnostic performance. [Figure 23]This figure illustrates a method for concentrating microvesicles using a 10 mL sample volume, and demonstrates that the number of detectable CD66a / c / e positive fractions can be increased compared to using a 0.8 mL sample volume. [Figure 24] This figure compares the results of urine cytology (negative and positive) with the results obtained using this method. A urine sample that tested negative for bladder cancer in urine cytology can be tested positive using this method, allowing for the detection of cancer. [Modes for carrying out the invention]

[0020] The embodiments of the present invention will be described in detail below, but the methods of use are not limited thereto.

[0021] This invention provides a method to assist in the early and accurate diagnosis of bladder cancer using cancer cell-specific microvesicles that are positive for anti-human CD66a / c / e antibodies present in the urine of bladder cancer patients. Cancer cell-specific microvesicles that are positive for anti-human CD66a / c / e antibodies can be characterized by two components: a substance that recognizes human CD66a / c / e and microvesicles, which are particles with a diameter of approximately 0.1 to 1 μm. In this specification, pretreatment includes the meaning of concentration and recovery, and concentration and recovery are sometimes used as nearly synonymous terms.

[0022] In this specification, urine may be used as is after collection, or it may be dissolved or suspended in water, an acidic solution, an alkaline solution, a buffer solution, etc., and further processed as necessary. The acidic solutions, alkaline solutions, and buffer solutions available in this invention can be appropriately selected and used by those skilled in the art.

[0023] The following describes an example of the conditions and diagnostic methods for isolating and observing cancer cell-specific microvesicles that are positive for anti-human CD66a / c / e antibodies using a flow cytometer, but the present invention is not limited to this.

[0024] The pretreatment method for removing impurities and concentrating microvesicles, which can be used in the present invention, is characterized by centrifugal separation, and can be carried out according to the following steps, for example, with reference to the method described in Japanese Patent Application Publication No. 2019-215342.

[0025] The collected urine sample is separated from the cell fraction and debris by low-speed centrifugation, and the supernatant is collected (Step 1). At this time, the low-speed centrifugation conditions can be carried out according to known methods, and the supernatant obtained by centrifugation at 2,000 × g for 20 minutes at room temperature can be used.

[0026] The supernatant obtained in step 1 may be further centrifuged at a low speed to collect the supernatant. This centrifugation step is preferable because it allows for the precipitation of platelet-derived vesicles and apoptotic vesicles. Similar to step 1, the supernatant obtained by centrifugation at 2,000 × g for 20 minutes at room temperature can be collected and used.

[0027] The supernatant obtained in step 1 is centrifuged at high speed to precipitate the microvesicle fraction (step 2). For example, the precipitate obtained by centrifugation at 20,000 × g for 30 minutes at room temperature can be used for this step.

[0028] A buffer containing a reducing agent (for example, 1,4-dithiothreitol (DTT) or tris(2-carboxyethyl)phosphinehydrochloride (TCEP)) is added to the precipitate fraction obtained in step 2, and the mixture is allowed to stand (step 3). This step reduces and cleaves the disulfide bonds in the THP polymer, thereby degrading the THP polymer.

[0029] Step 4 involves high-speed centrifugation of the sample to which the reducing agent was added in Step 3, to concentrate the microvesicles and remove the reducing agent. This high-speed centrifugation allows for the concentration of the microvesicle fraction and the removal of impurities. For this step, the precipitate obtained by centrifugation at 20,000 × g for 30 minutes at room temperature can be used. Step 4 may be repeated as needed. Performing high-speed centrifugation multiple times is preferable as it removes impurities and increases the purity of the microvesicles.

[0030] The reducing agent to be added is added for the purpose of cleaving disulfide bonds, so any agent capable of cleaving disulfide bonds is acceptable. For example, a person skilled in the art can appropriately select and use from among those commonly used as reducing agents for protecting the SH group of proteins, cleaving disulfide bonds, etc., such as dithiothreitol, 2-mercaptoethanol, 2-mercaptoetiramine hydrochloride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), cysteine ​​hydrochloride, tributylphosphine (TBP), iodoacetamide, glutathione, and hydrazine. It is preferable that these reducing agents do not affect the measurement of microvesicles and, more preferably, do not destabilize the lipid bilayer. These reducing agents may be used individually or in combination of two or more.

[0031] Several pretreatment methods are possible as one embodiment of the present invention, in addition to performing the steps described above. For example, the desired microvesicle fraction can be extracted by combining membrane filters with various pore sizes, which are used for filtration sterilization of aqueous solutions and protein-containing solutions. In this case, a method equivalent to microfiltration using membrane filters with a pore size of 0.1 to 10 μm in diameter is preferred. Alternatively, a method may be used to isolate only the target microvesicles by using a substance that can specifically bind to the microvesicles, such as a substance that can specifically bind to proteins, lipids, or sugars present on the surface.

[0032] Other sample pretreatment methods include equilibrium density gradient centrifugation, which fractionates the sample by centrifugation along with a density gradient solute; immunological capture, which uses antibodies specific to the surface antigens of the micromembrane fraction to collect the micromembrane fraction by binding it to various carriers; size exclusion chromatography (gel filtration), which collects fractions that elute faster than soluble proteins; phospholipid affinity, which uses carriers that bind to the membrane components of the micromembrane fraction in the presence of metal ions; and polymer precipitation, which mixes a high molecular weight polymer with the micromembrane fraction and precipitates the target micromembrane fraction. Those skilled in the art can appropriately select and implement these methods, but these methods can also be implemented as alternatives to steps 1 to 4 above by adding a reducing agent to the sample.

[0033] Hereinafter, samples that have been concentrated and contaminants removed using these methods may be referred to as processed urine samples.

[0034] Next, we will describe an example of observation conditions using a flow cytometer specifically designed for microvesicles, but the present invention is not limited to this.

[0035] The flow cytometer that can be used in this invention is not limited to a specific instrument, and any flow cytometer employing any flow cell, such as a quartz cuvette type or a jet-in-air type, can be used.

[0036] The observation method using a flow cytometer available in this invention can be carried out, for example, according to the following steps.

[0037] (1) Staining of microvesicles In this step, a reducing agent is added, and the concentrated urine sample is mixed with a staining reagent to stain the microvesicles (Step 5-A). More specifically, the microvesicles are stained by mixing the urine sample with a staining reagent, for example, a fluorescently labeled antibody specific to the surface antigen. Those skilled in the art can use a standard staining method, and if necessary, the conditions can be appropriately considered and implemented. To target multiple antigens in the microvesicles, multicolor differentiation may be performed by staining with various labeled antibodies. For example, Annexin 5, a protein that binds to phosphatidylserine (PS), a membrane component of microvesicles, in the presence of metal ions, can be used.

[0038] Other substances that can specifically bind to microvesicles include, for example, any substances that can bind to proteins, lipids, or sugars present on the surface of microvesicles. The step of reacting a substance that can bind to such proteins, lipids, or sugars with microvesicles is performed both when characterizing cancer cell-specific microvesicles present in urine and when characterizing microvesicles derived from normal tissue present in urine.

[0039] Examples of proteins present on the surface of microvesicles in urine include CD235a, CD59, CD44, CD33, CD45, CD144, CD66a, CD66b, CD66c, CD66e, CD41, CD61, EP-CAM, CD324, CD14, CD81, CD31, CD274, CD63, Alix, CD105, CD133, CD279, CD15, TSG101, CD20, CD249, CD5, CD10, CD26, CD273, CD9, MUC1, CD13, CD146, CD62E, Annexin5, or combinations thereof.

[0040] For example, it is preferable to use a fraction in which microvesicles containing various membrane peptidases, such as those found in the urine of healthy individuals, are all positive for CD10, CD13, and CD26, and to use a combination of these with microvesicles derived from epithelial cells, such as those positive for Annexin 5 and CD227 (MUC1), as this allows for more accurate separation. On the other hand, it is preferable to use microvesicles in the urine of bladder cancer patients that are positive for CD66a, CD66c, CD66e, and MUC1 on their surface, and to use them for differential diagnosis, assessment, and observation of disease progression in bladder cancer patients.

[0041] Other substances that can specifically bind to microvesicles include, for example, substances having binding affinity to proteins, enzyme substrates, coenzymes, regulators, substances that specifically bind to receptors, lectins, sugars, glycoproteins, antigens, antibodies or their antigen-binding fragments, hormones, neurotransmitters, phospholipid-binding proteins, proteins containing pleckstrin homology (PH) domains, cholesterol-binding proteins, or combinations thereof. Antigen-binding fragments include antigen-binding sites, and may include, for example, single-domain antibodies, Fab, Fab', or scFv.

[0042] (2) Addition of IgG In this step, IgG fluorescently labeled to detect THP polymers that could not be removed by the reduction treatment in the observed image is mixed in and reacted with the THP polymer (Step 5-B). In the previous step, the treated urine sample and the staining reagent are mixed to obtain a mixture, or the treated urine sample before mixing with the staining reagent is mixed with fluorescently labeled IgG and reacted (Step 5-B). The origin of the IgG is not particularly limited; for example, mouse IgG, human IgG, rat IgG, rabbit IgG, goat IgG, bovine IgG, etc., can be used. Similar to the staining process for microvesicles, this can be carried out according to standard methods by those skilled in the art. For example, it can be carried out using allophycocyanin (APC)-labeled mouse IgG, the Mix-n-Stain APC Antibody Labeling kit (Biotium), and a standard protocol, allowing it to stand at room temperature for 30 minutes.

[0043] (3) Flow cytometer settings The voltage and threshold of the photomultiplier tubes for forward and side-scattered light are adjusted to focus a group of particles with a specific diameter into the observation area. When setting these parameters and other conditions, it is preferable to perform measurements on unstained and stained samples to determine the voltage settings and sheath flow velocity that satisfy the detection sensitivity for detecting each target.

[0044] Those skilled in the art can explore and appropriately set the parameters of a flow cytometer to find the optimal conditions. For example, the flow rate can be set to 12 μL / min, the forward scattered light voltage to 381, and the side scattered light voltage to 340, with the fluorescence intensity threshold set to 200 for each detection sensitivity. The wavelength and voltage of the excitation light (Ex.) and fluorescence detection filter (Em.) for each fluorescent substance can be appropriately selected and set according to the respective fluorescent substance used.

[0045] The particle size in the observation area is estimated using the flow cytometer parameters set above. Measurements can be performed according to standard methods using polystyrene beads of uniform size (e.g., SPHEROTM Nano Polystyrene Size Standard Kit, Spherotech). To estimate the particle size in the observation area, it is preferable that the particle sizes of the polystyrene beads are, for example, 0.22 μm, 0.45 μm, 0.88 μm, and 1.35 μm.

[0046] The diameter of the observation area is not particularly limited as long as it includes the microvesicles, but for example, it is preferably about 1 μm or less, more preferably 100 nm to 1 μm, and even more preferably 200 nm to 1 μm. This is especially preferable when observing these minute particles because the side-scattered light has higher resolution than the forward-scattered light and can be used for verifying the size of the particles.

[0047] Furthermore, this process is not limited to being performed between the staining process and the IgG addition process and the measurement / separation process described below. For example, the staining process, IgG addition process, and measurement / separation process can be performed after setting the parameters, or, once the parameters have been set, the series of steps can be repeatedly performed without setting the parameters for each measurement.

[0048] (4) Measurement and separation by flow cytometry Data is acquired using a flow cytometer. If multicolor measurement is performed, leakage correction is performed for fluorescence that leaks from each fluorescence to detectors other than the one assigned to it, and the measurement results are obtained under those conditions (step 6).

[0049] (5) Removal of aggregated microvesicles and complexes with IgG capture fraction (Step 6-A) Microvesicles may aggregate, but to exclude aggregated microvesicles from the observed image, you can mark and gate out those where 1) the pulse width value of the side-scattered light is significantly higher than the group, or 2) both the forward-scattered light and side-scattered light plots show significantly higher values ​​than the group. This is particularly effective when microvesicles aggregate after preprocessing.

[0050] Furthermore, to remove immune complexes with the IgG capture fraction, the positive population that reacts to fluorescently labeled IgG can be marked and gated out. This step is preferable because it allows for the exclusion of THP polymers that could not be completely removed by the reduction treatment from the observed image by marking and gated out the positive population that reacts to fluorescently labeled IgG.

[0051] Furthermore, the above-mentioned steps of removing aggregated microvesicles and removing immune complexes captured by IgG may be performed in either order, or both steps may be performed simultaneously. A person skilled in the art can appropriately select the order according to the environment in which the process is carried out.

[0052] (6) Characterization of the obtained microvesicle population (Step 6-B) Microvesicles detected using multicolor imaging can be separated and characterized into their respective cellular microvesicle populations through a gating process. This process allows for confirmation that the separated microvesicles are indeed from urine.

[0053] The present invention enables the fractionation and characterization of microvesicles in urine, and these characterized fractions can be further separated using a cell sorter. The specific contents (nucleic acids, metabolites, proteins, lipids) of these separated fractions can be observed and applied clinically. The separated fractions may also be used for frequency analysis of each microvesicle population or for expression level analysis of target molecules.

[0054] Characterization techniques that aid in the early and accurate diagnosis of bladder cancer can be performed by combining the particle size of microvesicles with the detection of one or more proteins present on the microvesicle surface. The detection of proteins present on the microvesicle surface may be a single protein or a combination of multiple proteins. The combination of proteins can be identified by first confirming the reactivity of urinary microvesicles from bladder cancer patients, non-bladder cancer (urinary tract disease) patients, and healthy individuals, and then narrowing down the selection to protein combinations that exhibit reactivity specific to bladder cancer patients.

[0055] A person skilled in the art can appropriately select and implement methods for analyzing proteins contained in concentrated microvesicles to search for markers that can aid in the early and accurate diagnosis of bladder cancer. For example, it is preferable to identify them using methods such as shotgun proteomics, two-dimensional electrophoresis, or FCM-MS. For instance, concentrated microvesicles from the urine of bladder cancer patients, non-bladder cancer (urinary tract disease) patients, and healthy individuals may be observed by immunofluorescence staining, and proteins that are observed in very high concentrations only in the urine of bladder cancer patients may be identified. Proteins found in urinary microvesicles of bladder cancer patients include IHG1, CRABP2, PLG, AHSG, HP, IHG2, ANXA2, IGHM, SERPINA1, CFB, TF, NME2, C4BPA, A2M, TACSTD2, FGG, C4B, F2, C3, IGHA1, CP, SERPINA3, CA1, CAPN5, APOB, FAM129B, C9, TMSB10, C1QB, IGLC6, IGKC, CEACAM7, UPK3A, CEACAM5, ARHGDIB, SRC, FGB, C1QC, LRG1, UPK3BL, DEFA3, SERPIND1, FN1, SDCBP2, APOA1, ITIH4, AGRN, SERPINF1, PDLIM1, ANXA9, PSCA, HBA1, SLC2A1, ITIH2, CYSRT1, SERPINC1, PGLYRP2, SPRR1A, PROS1, RAB27B, UGDH, MARCKS, EFHD2, APOL1, etc. are considered and can be used as markers derived from microvesicles specific to bladder cancer patients. These markers may be used individually or in combination of multiple types. Using multiple types in combination is preferable because it is expected to improve the detection accuracy specific to bladder cancer patients. In particular, microvesicles that are positive for at least one of CD66a, CD66c, or CD66e (preferably two or more of CD66a, CD66c, or CD66e, and especially preferably CD66a, CD66c, and CD66e) as proteins present in large quantities only in the urine of bladder cancer patients can be extracted and used, but are not limited thereto.

[0056] Furthermore, diagnostic accuracy can be improved by identifying and excluding proteins that exhibit specific reactivity in urinary microvesicles derived from non-bladder cancer (urinary tract disease) patients and healthy individuals. Such proteins include microvesicles that are positive for at least one of CD10, CD13, or CD26 (preferably two or more of CD10, CD13, or CD26, particularly preferably CD10, CD13, and CD26) (in addition, more preferably negative for CD66a, CD66c, and CD66e), which are thought to originate from the renal tubules and can be used as microvesicles contained in the urine of healthy individuals. For example, by calculating the ratio (A / B) of microvesicles (A) that are positive for CD66a, CD66c, and / or CD66e (particularly preferably positive for CD66a, CD66c, and CD66e) and microvesicles (B) that are positive for CD10, CD13, and / or CD26 (particularly preferably positive for CD10, CD13, and CD26) (and more preferably negative for CD66a, CD66c, and CD66e), diagnostic accuracy can be significantly improved. In this specification, for example, a microvesicle positive for CD66a, CD66c, and / or CD66e means a microvesicle that is positive for at least one of CD66a, CD66c, or CD66e, or a microvesicle that is positive for two or more of CD66a, CD66c, or CD66e, or a microvesicle that is positive for CD66a, CD66c, and CD66e.

[0057] In addition to the above-mentioned microvesicle populations that are positive for CD10, CD13, and CD26, microvesicles that are positive for CD227 (MUC1) may also be extracted and used in combination with the above-mentioned proteins. Since MUC1-positive microvesicles are thought to be highly likely to originate from urothelial cells, it is preferable to extract MUC1-positive microvesicles and use them in combination with the above-mentioned CD10, CD13, and CD26-positive microvesicles to extract the microvesicle population to be excluded, as this improves accuracy. For example, by calculating the ratio (A / C) of microvesicles (A) that are positive for CD66a, CD66c, and / or CD66e (particularly preferably positive for CD66a, CD66c, and CD66e) and microvesicles (C) that are positive for CD10, CD13, CD26, and / or MUC1 (particularly preferably positive for CD10, CD13, CD26, and MUC1) (and more preferably negative for CD66a, CD66c, and CD66e), diagnostic accuracy can be significantly improved.

[0058] When using samples from patients exhibiting hematuria, blood-derived microvesicles may be present, reducing the proportion of CD66a / c / e-positive and multipeptidase-positive microvesicles. Therefore, in such cases, a correction may be made to identify true cancer cell-derived microvesicles in the flow cytometry observation. Such a correction is preferable because it allows for highly accurate diagnostic assistance according to the present invention. For example, CD235a (Glycoprotein A)-positive microvesicles expressed on human erythrocytes and erythroid progenitor cells can be used for the correction. Furthermore, those skilled in the art can appropriately set criteria for determining appropriate combinations.

[0059] The apparatus used to carry out the present invention is not particularly limited, as long as it is capable of accurately observing particle sizes suitable for microvesicles and measuring specific surface antigens. For example, JVC Kenwood's ExoCounter( JVC A Kenwood product can be used. ExoCounter specifically detects exosomes by sandwiching them between a disc and nanobead antibodies in a surface antigen-specific manner. The particle size can be characterized by limiting the size to those that fit within the grooves (260 nm) on the disc surface. This is preferable because it allows for the direct measurement of exosomes in bodily fluid samples without the need for isolation and purification steps.

[0060] In addition to the above, other measurement techniques capable of separating and observing microvesicles include, for example, the NanoSight (Malvern Panalytical) nanotracking particle size analyzer, which can be used in combination with NTA (Nano Tracking Analysis) and FFF (Field Flow Fractionation) techniques. NTA allows for real-time observation of the Brownian motion of nanoparticles in a liquid on a PC screen, while FFF techniques perform separation within a thin flow channel. Due to the special geometric shape of this channel, the flow becomes a laminar flow with a radial cross-section, and by acting perpendicular to this laminar flow, a separation force is generated, allowing for the separation of minute particles such as microvesicles by size, thus enabling more precise separation and observation. By combining such separation / observation methods with surface antigens that characterize individual microvesicles, microvesicles can be characterized, increasing organ specificity and disease specificity, and making them available for clinical application.

[0061] In a different embodiment of the present invention, it can also be used as a method for monitoring disease progression in a subject or for monitoring disease recurrence in an individual. These methods include a step of separating microvesicles from a urine sample, as well as a profiling step of observing the substances contained in the microvesicles. By observing the profile in a subject individual with a specific medical condition, it can be used, for example, to estimate the presence of a specific disease. For example, by appropriately setting the sampling period for separating microvesicles according to the detection of the target disease, it is possible to obtain a more detailed profile, observe the condition, and assist in diagnosis. This can also be used as a method for monitoring the disease state after drug administration.

[0062] This invention enables the effective use of microvesicles as clinical testing materials. It allows for the simple and specific extraction and observation of microvesicles present in urine, and further allows for the characterization of individual microvesicles (determining their origin from specific cells, tissues, or organs) using surface antigens present on the membrane. As a result, it is expected to enhance the value of this test, enabling diagnostic and testing applications focused on specific organs, tissues, or cells that match specific diseases. Additionally, observation using the fractions obtained by this invention makes it possible to estimate diseases, drug administration effects, or other medical conditions in the subject. [Examples]

[0063] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0064] Example 1: Concentration of microvesicles from the urine of human bladder cancer patients. Approximately 10 mL of urine was collected from healthy individuals and bladder cancer patients. The urine samples were centrifuged at 2,330 × g for 10 minutes at 20°C to obtain the supernatant. This supernatant was then centrifuged again at approximately 2,330 × g for 10 minutes at approximately 20°C to remove urinary lipids, cellular residues, blood-derived components, etc. The supernatant was used in the experiments described below.

[0065] 800 μL of the obtained urine was centrifuged at 18,900 × g for 30 minutes at 20°C. The supernatant was removed from the centrifuged product, and the microvesicle fraction was concentrated in the pellet. To this pellet, 180 μL of phosphate-buffered saline (PBS) containing dithiothreitol (DTT) at a final concentration of 10 mg / mL was added. After stirring by vortexing, the mixture was allowed to stand at 37°C for 10 minutes. After standing, it was centrifuged at 18,900 × g for 30 minutes at 20°C. The microvesicle fraction was concentrated in the pellet from the centrifuged product.

[0066] Example 2: Particle measurement of extracted fractions using a nanoparticle analysis system. The microvesicle fraction was concentrated into a pellet using the same procedure as in Example 1 (except using 10 mL of urine). 100 μL of PBS was added to the pellet, and the resulting 3-fold dilution with PBS was measured using a Nanosight NS300 (Malvern Panalytical). The observation conditions of the instrument are shown in Table 1 (scattered light measurement) and Table 2 (fluorescence measurement). Extracellular vesicles labeled with ExoGlow-NTA Dye (System Biosciences), which specifically binds to intact extracellular vesicle membranes, were also measured by fluorescence NTA (Nano Tracking Analysis). Figures 1 and 2 show the particle size distribution histograms measured by scattered light and fluorescence NTA for fractions extracted from healthy individuals and bladder cancer patients, respectively. Figure 3 also shows the diameters corresponding to 10%, 50%, and 90% of the total particle count observed by scattered light NTA in eight healthy individuals and eight bladder cancer patients, respectively. In this concentration process, fractions with a diameter of 1 μm or larger were almost entirely absent, with the majority distributed in the 200-300 nm diameter range. Furthermore, some of these fractions included cell membranes (10-60%), which, considering their size, are considered "medium-sized extracellular vesicles (microvesicles)." It was also confirmed that there was no difference in particle size distribution between healthy individuals and cancer patients in the extracted extracellular vesicle fraction.

[0067] [Table 1]

[0068] [Table 2]

[0069] Example 3: Proteins detected in the concentrated microvesicle fraction by shotgun proteomics analysis. A. Extraction of the protein fraction from the concentrated microvesicle fraction obtained by urine pretreatment. To identify proteins specifically present in microvesicles in the urine of bladder cancer patients, urine samples from four bladder cancer patients (Patient 1: depth of invasion Tis, urine cytology class III; Patient 2: depth of invasion T2, urine cytology class V; Patient 3: depth of invasion T2, urine cytology class V; Patient 4: depth of invasion Ta, urine cytology class II) and four healthy individuals were used, and the microvesicle fractions were concentrated in the same manner as in Example 1. After quantifying the proteins in each group, samples from the four patients and the four healthy individuals were mixed and pooled to obtain an equivalent amount per patient. Two types of fractions were prepared: one containing microvesicles extracted from each patient and one healthy individual sample.

[0070] To efficiently extract membrane proteins from the concentrated microvesicle fraction, a protein solubilization method using PTS (Phase Transfer Surfactant) was performed. MPEX PTS Reagents for MS (GL Science Inc.) was used as the reagent kit for this principle. In Example 1, 250 μL of reagent B from the kit was added to the concentrated microvesicle fraction pellet, and sonication (power: MAX) was performed 10 times in an ultrasonic homogenizer (Bioruptor: Sonic Bio) with a cycle of 1 minute 30 seconds operation followed by a 30-second interval, at 10°C. After disruption, the fraction was concentrated by repeating 14,000 g × 15 min twice using a centrifugal filter (Amicon ultra 3K, Merck). This fraction was then subjected to a BCA assay (Pierce TMProtein quantification was performed using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc.).

[0071] B. Enzymatic digestion of protein fractions The sample for membrane protein digestion was prepared by quantification using a BCA assay and dissolved in reagent B to a concentration of 7 μg / 30 μL. 1.5 μL of 100 mmol / L dithiothreitol (DTT) was added (for reductive cleavage of disulfide bonds), and the mixture was incubated at room temperature for 30 minutes. 1.5 μL of 550 mmol / L iodoacetamide was added (for carbamide methylation of Cys), and the mixture was incubated in the dark at room temperature for 30 minutes. 116 μL of reagent A from the kit was added, followed by 1.5 μL of trypsin (TPCK-Trypsin (Thermo Fisher Scientific Inc.: Prod#20233)). Protein digestion was performed by incubation overnight at room temperature. 150 μL of reagent C and 1.5 μL of reagent D were added. After addition, the mixture was vortexed for 1 minute, and then centrifuged at 25°C and 15,600 × g for 2 minutes to separate the two phases. Unnecessary solubilizers would settle in the upper phase, so they were removed by pipetting. To remove the remaining reagent C, the mixture was concentrated by centrifugation, and then 50 μL of 5% acetonitrile and 0.1% trifluoroacetic acid (TFA) were added and vortexed.

[0072] C. Desalting and concentration of peptides GL-Tip SDB (GL Science Inc.) was used to desalt and concentrate the enzyme-digested sample described above. First, the tip was conditioned by adding 20 μL of 80% acetonitrile and 0.1% TFA aqueous solution and centrifugation at room temperature at 3,000 × g for 2 minutes. Then, 20 μL of 5% acetonitrile and 0.1% TFA aqueous solution was added and the column was equilibrated by centrifugation at room temperature at 3,000 × g for 2 minutes. The entire amount of the sample obtained in the procedure described in Example 3B was then added and the peptide was adsorbed onto the column by centrifugation at room temperature at 3,000 × g for 5 minutes. After washing the column by adding 20 μL of 5% acetonitrile and 0.1% TFA aqueous solution and centrifugation at room temperature at 3,000 × g for 2 minutes, the peptide was eluted by adding 50 μL of 80% acetonitrile and 0.1% TFA aqueous solution and centrifugation at room temperature at 3,000 × g for 2 minutes. The peptide-containing solution was dried once using a miVac centrifuge evaporator and then suspended in 25 μL of 0.1% formic acid / 2% acetonitrile aqueous solution.

[0073] D. Mass spectrometry measurements The above samples were subjected to LC-MS / MS analysis using a Fourier transform type orbitrap mass spectrometer (Q-Exactive: Thermo Fisher Scientific Inc.) connected to a nano-LC system (EASY-nLC1000: Thermo Fisher Scientific Inc.). 5 μL of the mass spectrometry sample prepared above was used for the analysis. Acclaim was used as the trap column. RT PepMap100 (Thermo Fisher Scientific Inc.: C18, packing material diameter 3 μm, inner diameter 75 μm, column length 2 cm), as the analytical column, Acclaim RT A PepMapRSLC (Thermo Fisher Scientific Inc.: C18, packing material diameter 2 μm, inner diameter 50 μm, column length 15 cm) was used. Mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was a 0.1% formic acid / acetonitrile. The flow rate was 200 nL / min, and the gradient was measured at 0-40% mobile phase B for 200 minutes, 40-100% mobile phase B for 10 minutes, and 100% mobile phase B for 10 minutes.

[0074] For the MS measurement, the Full MS / dd-MS2 mode was used. After scanning the Full MS, the MS / MS spectrum of the signal with high intensity was acquired. The settings are shown in Table 3.

[0075] [Table 3]

[0076] E. Data Analysis The data obtained was processed using Proteome Discoverer 1.4 software (Thermo Fisher Scientific Inc.), and the database search was performed using the SequestHT algorithm with the Homo sapiens taxonomy catalogued in the UniProt database (UP000005640; October 18, 2020). The search conditions for SequestHT are shown in Table 4.

[0077] [Table 4]

[0078] Furthermore, the obtained MS / MS data were subjected to LFQ (unlabeled quantitative analysis) using the MaxQuant platform (v1.6.6.0). Database searches were performed using the same UniProt database as described above. Protein and peptide identification was performed under the following conditions: false discovery rate (FDR) of 0.01, minimum number of peptides required for protein identification of 1, minimum score of 40 for modified peptides, and no lower limit for unmodified peptides.

[0079] To roughly compare the proteins contained in urinary microvesicles of bladder cancer patients and healthy individuals, samples from four patients and four healthy individuals were analyzed. After quantifying the proteins in each group, the samples were mixed and pooled to obtain equivalent amounts per patient. Proteins detected in both groups were selected from the Proteome Discoverer search results if they had a score of 1.0 or higher. Figure 4 and Table 5 show a list of proteins detected by shotgun proteomics analysis of the extracted microvesicle fractions for the patient pool and healthy individual pool, respectively (1334 types in patients, 1393 types in healthy individuals). Furthermore, protein enrichment analysis using metascape (Tripathi et al., Cell Host & Microbe (2015), 18: 723-735) was performed on proteins detected only in bladder cancer patients (585 types) from each pool. The results are shown in Figure 5. While some functional categories related to cell adhesion, which can be considered a characteristic of cancer, were observed, the most frequently categorized function was ribosome-related functions (protein complexes, translation), such as Eukaryotic Translation Elongation or ribonucleoprotein complex assembly RNA. This may be one characteristic of the proteins contained in microvesicles derived from cancer patients.

[0080] Next, analysis was performed using microvesicles individually extracted from samples of 4 bladder cancer patients (Patient 1: depth of invasion Tis, urine cytology class III, Patient 2: depth of invasion T2, urine cytology class V, Patient 3: depth of invasion T2, urine cytology class V, Patient 4: depth of invasion Ta, urine cytology class II) and 4 healthy individuals. The obtained MS / MS data was subjected to label-free quantification (LFQ) analysis using the MaxQuant platform to calculate the quantification results for each patient. The following statistical analysis was performed using these data. MetaboAnalyst 5.0 was used for the statistical analysis. Principal component analysis was performed on the results of each of the 4 bladder cancer patients and 4 healthy individuals, and the graph showing up to the second principal component is shown in Fig. 6. Patient 2 had a slightly different profile from the other 3 patients, but there was a tendency for a certain difference in the detected protein profiles between the group of 4 bladder cancer patients and the group of 4 healthy individuals. Therefore, orthogonal partial least squares discriminant analysis (OPLS-DA) that enables discriminant analysis between groups was performed. As an evaluation of the model obtained by OPLS-DA, R 2 Y and Q 2 The closer Y is to 1, the better the model (R 2 Y is 0.65 or more, Q 2 If Y is 0.5 or more, it is considered a good model). In the score plot shown in Fig. 7, R 2 X = 0.253, R 2 Y = 0.847, Q 2 Y = 0.608. R 2 Y and Q 2 Since Y was good, the patient group and the healthy group were significantly discriminated. Next, s-plot analysis was performed for the purpose of estimating the components (proteins) important for discriminating the two groups. The components important for discrimination were judged from p[1] and p[corr], and proteins with p[1] of 0.05 or more and p[corr] of 0.6 or more were extracted as components important for discrimination. The list of these extracted proteins is shown in Table 5, and a heat map was created using these (Fig. 8). Note that the heat map shown in Fig. 8 visualizes the obtained numerical values (2 to -2) in shades of red (2 to 0) and blue (0 to -2), and the numerical values before visualization are shown in Table 6-1 (corresponding to the upper half of Fig. 8) and Table 6-2 (corresponding to the lower half of Fig. 8).

[0081] [Table 5-1]

[0082] [Table 5-2]

[0083] [Table 6-1]

[0084] [Table 6-2]

[0085] Table 5 shows proteins that have previously been suggested to be associated with bladder cancer (UPK3A, PSCA, SRC, etc.). These proteins are suggested to be useful when utilizing extracellular vesicles as biomarkers in the urine of bladder cancer patients in the future. Furthermore, CEACAM5 and CEACAM7 were present with high scores in the extracted proteins, suggesting that the CEACAM family (CD66) proteins are the candidate proteins mentioned above. In addition, the profiles of all detected CEACAM family (CD66) proteins were visualized as heatmaps for the patient group and the healthy control group (Figure 9). The heatmap shown in Figure 9 visualizes the obtained values ​​(2 to -2) using shades of red (2 to 0) and blue (0 to -2). The values ​​before visualization are shown in Table 7.

[0086] [Table 7]

[0087] The CEACAM family proteins that could be detected were CEACAM1 (CD66a), CEACAM5 (CD66e), CEACAM6 (CD66c), CEACAM7 (CGM2), and CEACAM8 (CD66b). The expression profiles of these CEACAM proteins differed from patient to patient, and from the perspective of sensitively detecting bladder cancer patients, it was considered preferable to detect a combination of several CEACAM proteins in microvesicles. Proteins detected by shotgun proteomics analysis may be used as surface markers to characterize microvesicles in bladder cancer urine, and their contents may be used as biomarkers for clinical testing and diagnosis.

[0088] Example 4: Observation of microvesicles in the urine of bladder cancer patients using a flow cytometer. A. Immunofluorescence staining of concentrated microvesicles from urine. In Example 1, 60 μL of PBS was added to the pellet containing the concentrated microvesicle fraction, and the microvesicle fraction was dispersed in the solution using a vortex mixer. For this solution, FITC-Annexin5 (Becton Dickinson Biosciences), APC / Cy7anti-human CD10 (Biolegend), Brilliant Violet421anti-human CD13 (Biolegend), PEanti-human CD26 (Biolegend), PerCP / Cy5.5anti-human CD66a / c / e (Biolegend), PerCPanti-human CD66b (Biolegend), Anti-Human CEACAM-1 / CD66a Alexa Fluor 488 Antibody (R&D Company), Anti-Human CEACAM-6 / CD66c Alexa Fluor 750 Antibody (R&D Company), Anti-Human CEACAM-5 / CD66e Alexa Fluor 405 Antibody (R&D Company), Brilliant Violet421 anti-human CD66a / c / e (Biolegend), PE / Cy7 anti-human CD227 MUC1 (Biolegend), APC / Cy7 anti-human CD235a (Biolegend), and APC-labeled mouse IgG (normal mouse IgG purchased from Wako, labeled using the standard protocol included with the Mix-n-Stain APC Antibody Labeling kit from Biotium) were each added in 1 μL and allowed to stand at room temperature for 30 minutes.

[0089] B. Observation of urinary microvesicles using a flow cytometer Measurements were performed using a BD FACSVerse™ (Becton Dickinson and Company) as a flow cytometer. The measurement procedure and parameter settings are as follows. The sample used was the same as in the example described above. 4A.Fractions stained with various fluorescent dyes were suspended in 750 μL of 10 mmol / L Hepes (pH 7.4), 0.14 mol / L NaCl, and 2.5 mmol / L CaCl2. The flow rate was set to 12 μL / min, the voltage of the forward scattered light was set to 381, and the voltage of the side scattered light was set to 340, with each threshold set to 200. The excitation light (Ex.) and fluorescence detection filter (Em.) wavelengths and voltages for each fluorescent substance were as follows: FITC: Ex. 488nm, Em. 527 / 32nm, voltage 442; PE: Ex. 488nm, Em. 586 / 42nm, voltage 411; PerCP: Ex. 488nm, Em. 700 / 54nm, voltage 556; PE / Cy7: Ex. 488nm, Em. 783 / 56nm, voltage 564.3; APC: Ex. 640nm, Em. 660 / 10nm, voltage 538.2; APC / Cy7: Ex. 640nm, Em. 783 / 56nm, voltage 584.8; Brilliant Violet421: Ex. 405nm, Em. 448 / 45nm, voltage 538.2. To estimate the particle size in the observation area, measurements were performed using polystyrene beads of uniform size (SPHEROTM Nano Polystyrene Size Standard Kit, Spherotech). The polystyrene beads used had particle sizes of 0.25 μm, 0.45 μm, 0.79 μm, and 1.34 μm. The observation target was primarily the area where the observed particle size converged to 1 μm or less at a side-scattered light (SSC) intensity (approximately 5.0 × e04 or less). (Figure 10)

[0090] C. Microvesicles positive for CD66a, c, and e present in the urine of bladder cancer patients To determine whether the CEACAM proteins detected by shotgun proteomics analysis were present in the same microvesicle or in different microvesicles, we used antibodies that specifically recognize CD66a, c, and e (CEACAM1, 6, and 5). The results are shown in Figure 11. Urine samples from nine bladder cancer patients were analyzed using the flow cytometry method described above. In patient 2-1, microvesicles positive for CD66a, c, and e merged almost entirely with microvesicles positive for the other antigens. This patient showed the presence of microvesicles expressing all three antigens simultaneously on their surface. In patient 2-8, numerous microvesicles positive only for CD66a were observed. The positivity rates for each individual antigen (CD66a, c, and e) and the merging rates of these antigens are plotted in a graph for the nine patients (Figure 11). These results suggest that different patients contain different varieties of CD66a, c, and e-positive microvesicles. This suggests the existence of microvesicles that are positive for CD66a, c, and e individually, as well as microvesicles that contain two or three types of this antigen simultaneously.

[0091] Microvesicles possessing D.CD66b (CEACAM8) as a surface antigen We also attempted to analyze CD66b (CEACAM8), which was detected by shotgun proteomics analysis, using flow cytometry. The results are shown in Figure 12. In patient 2, in whom CEACAM8 was detected by shotgun analysis, we observed microvesicles that were also positive for CD66b (CEACAM8) in flow cytometry analysis. Since we also observed populations of these microvesicles that merged with CD66a / c / e, it is thought that they were co-expressing one or more of the antigens.

[0092] E. CD66a / c / e-positive microvesicles that are present in large quantities only in the urine of bladder cancer patients. Microvesicles concentrated from urine using the above method were observed with a flow cytometer. Based on previous results, many microvesicles containing CD66a, c, and e (CEACAM1, 6, 5) were observed in patient urine. Therefore, it was determined that a diagnostic system using antibodies that simultaneously recognize these antibodies is appropriate, and flow cytometry analysis was performed using a detection system with CD66a / c / e-recognizing antibodies. When microvesicles in the urine of bladder cancer patients, non-bladder cancer (urinary tract disease) patients, and healthy individuals were observed, CD66a / c / e-positive microvesicles were observed in a very large number only in the urine of bladder cancer patients (Figure 13). This suggests that CD66a / c / e-positive microvesicles may be useful as a diagnostic marker using the urine of bladder cancer patients.

[0093] Example 5: Characterization of microvesicles in the urine of bladder cancer patients using a flow cytometer. A. Targeting of microvesicles that are positive for CD66a / c / e (Figure 14) Among the observed images, some showed microvesicles condensing and merging together (groups in which all surface antigens stained were positive). To remove these from the observed images, an unfolded image was prepared using pulse width (vertical axis) and pulse area (horizontal axis) in the side-scattered light, and groups that were significantly outside the observed image were excluded. Next, from the unfolded image of side-scattered light and APC mouse IgG, the APC-positive group, i.e., the mouse IgG group that could be captured by the residual THP polymer, was extracted, and the APC-positive group was extracted and excluded from the observed image. Furthermore, from the group that was positive for CD10 and CD13, which are abundant in the urine of healthy individuals, which was excluded (the group that was negative for CD10 and CD13 was gated in), those that were positive for CD66a / c / e were selected (Figure 14).

[0094] B. Overall view of microvesicles in the observed image (Figure 15) Among those that were negative for CD66a / c / e, a group was extracted that was positive for both CD10 and CD13 from the unfolded diagrams of CD10 and CD13, and further a group that was positive for CD26 was selected. This group was designated as CD10, CD13, and CD26 positive microvesicles. CD10, CD13, and CD26 are thought to originate from the renal tubules, and these CD10, CD13, and CD26 positive microvesicles can be used as microvesicles contained in the urine of healthy individuals. In addition, a group that was negative for CD66a / c / e and positive for CD227 (MUC1) was selected. This group was designated as MUC1 positive microvesicles. In the above, an example of a figure obtained by superimposing the characterization of CD66a / c / e positive microvesicles, CD10, CD13, CD26 positive microvesicles, and MUC1 positive microvesicles onto the unfolded diagram of Annexin 5 using lateral scattered light is shown in Figure 15.

[0095] Characterization of microvesicles that test positive for C.CD66a / c / e (Figures 16 and 17) Regarding microvesicles that are positive for CD66a / c / e, it is clear from the unfolded diagrams of CD66a / c / e and CD26 that microvesicles that are positive for CD66a / c / e are different from microvesicles that are positive for CD10, CD13, and CD26 (Figure 16). Furthermore, from the unfolded diagrams of CD66a / c / e and MUC1, microvesicles that are positive for CD66a / c / e may also be microvesicles that are positive for MUC1 (Figure 17). However, in the urine of bladder cancer patients, there are also MUC1-positive microvesicles that are similar to those found in healthy individuals and are negative for CD66a / c / e. As mentioned in the non-patent document Igami et al., 2020. Characterization and function of medium and large extracellular vesicles from plasma and urine by surface antigens and Annexin V. PeerJ Analytical Chemistry 2:e4 doi.org / 10.7717 / peerj-achem.4, CD10, CD13, and CD26-positive microvesicles are likely to originate from the renal tubules, and MUC1-positive microvesicles are likely to originate from urothelial cells. Microvesicles that are positive for CD66a / c / e may be cells that originally originated from urothelial cells that became cancerous, and which secrete microvesicles with CD66a / c / e on their surface as an antigen.

[0096] Example 6: Percentage of each microvesicle observed in the urine of 10 bladder cancer patients, 4 non-bladder cancer (urinary tract disease) patients, and 9 healthy individuals.

[0097] Based on these results, a method was established to concentrate microvesicles in urine and observe the microvesicle fractions of different originating cells using a flow cytometer, based on the proteins present on the surface of each microvesicle. Specifically, these are four types of microvesicles with a diameter of 1 μm or less that are MUC1 positive (CD66a / c / e negative), multipeptidase positive (CD10, CD13, CD26 positive, CD66a / c / e negative), CD235a positive (red blood cell-derived microvesicles, hematuria-derived, CD66a / c / e negative), and CD66a / c / e positive. Figure 18 shows the percentage (%) of these four types of microvesicles observed in the urine of 10 bladder cancer patients, 4 non-bladder cancer (urinary tract disease) patients, and 9 healthy individuals. The proportion (%) of CD66a / c / e-positive microvesicles in the total observed microvesicle population was compared among bladder cancer patients, non-bladder cancer patients, and healthy individuals (Figure 19). Compared to non-bladder cancer patients and healthy individuals, bladder cancer patients showed a significantly increased number of CD66a / c / e-positive microvesicles. Furthermore, multipeptidase-positive microvesicles were significantly decreased in bladder cancer patients compared to the other two groups (Figure 19).

[0098] Example 7: Effect of a diagnostic index combining the proportion of microvesicles that are positive for CD66a / c / e, MUC1, and multipeptidases. Since microvesicles that are positive for CD66a / c / e appear specifically in the urine of cancer patients, the degree of their increase can serve as a direct diagnostic indicator. However, microvesicles that are positive for MUC1 (negative for CD66a / c / e) or positive for multiple peptidases (positive for CD10, CD13, CD26, and negative for CD66a / c / e) tend to decrease in the urine of cancer patients across the entire observed range. This result suggests that incorporating ratios and differences between these parameters could lead to more accurate diagnostic indicators. In fact, when the ratio of the CD66a / c / e-positive fraction (A) to the multiple peptidase-positive fraction (B) (A / B), and the ratio of the CD66a / c / e-positive fraction (A) to the combined multiple peptidase-positive fraction and MUC1-positive fraction (C) (A / C) were quantified and compared between healthy individuals, non-bladder cancer patients, and bladder cancer patients, significant differences were confirmed in each group (Figure 20). Furthermore, when the subjects were divided into bladder cancer patients and others (non-bladder cancer + healthy individuals), ROC curves were evaluated for 1) CD66a / c / e alone (A), 2) the ratio of CD66a / c / e positive fraction (A) to multipeptidase positive fraction (B) (A / B), and 3) the ratio of CD66a / c / e positive fraction (A) to multipeptidase positive fraction and MUC1 positive fraction combined (C) (A / C). The AUC value was highest for 3) the ratio of CD66a / c / e positive fraction to multipeptidase positive fraction and MUC1 positive fraction combined (Figure 21). These findings suggest that combining microvesicles that are positive for CD66a / c / e in the observed image of bladder cancer patients with microvesicles that are also commonly found in healthy individuals may provide a more accurate diagnostic indicator. 1) CD66a / c / e alone, 2) the ratio of the CD66a / c / e positive fraction to the multipeptidase positive fraction, and 3) the ratio of the combined CD66a / c / e positive fraction, multipeptidase positive fraction, and MUC1 positive fraction. For each pattern, the best cutoff line, from the viewpoint of both sensitivity and specificity, was clearly shown along with numerical graphs obtained from actual healthy individuals and bladder cancer patients (Figure 22). Each cutoff line is the same as the cutoff value used when interpreting the test results shown in the lower part of Figure 21.

[0099] Example 8: An example of a simple pretreatment measurement method using a larger urine volume. When collecting urine from bladder cancer patients, it is often possible to obtain a urine volume of 10 mL or more. By using this entire volume as the test material, the density of detectable microvesicles can be increased, providing advantages in conducting the test. As a urine pretreatment step, a centrifugal filtration device was used to extract the microvesicle fraction from a 10 mL urine volume (Figure 23-1). The advantages in conducting the test are demonstrated by comparing the microvesicle fractions extracted from 10 mL and 0.8 mL. Figure 23-2 shows the observed images for each fraction, with the observation time on a flow cytometer set to the same value. When the flow rate of the flow cytometer was set to 12 μL / min, it took 10 seconds to gather 10,000 particles in the observed image of the 10 mL urine volume fraction. When the 0.8 mL urine volume fraction was observed under the same conditions, only about 200 particles could be observed in 10 seconds (Figure 23-2). From these results, it can be said that "the test time can be shortened". Furthermore, since a larger number of particles dissolve in the final buffer solution when extracting a 10 mL urine volume, the scale of the overall particle picture observed can be increased to some extent within an appropriate observation time for the test. Figure 23-3 shows an example where the total number of particles in the observed picture is 100,000. Under the conditions described above, it was possible to observe 100,000 particles in about 1 minute. When the total number of particles in the observed picture was 10,000 in a 0.8 mL urine volume extract, there were 68 CD66a / c / e positive fractions, whereas when the total number of particles in the observed picture was 100,000, there were 269 CD66a / c / e positive fractions (Figure 23-3). Since changing the urine volume does not affect the ratio of positive fractions to the total observed picture, it is thought that increasing the number of target particles leads to improved sensitivity when using the particle concentration of the positive fraction as the target unit of a measurement system.

[0100] Example 9: Comparative study of urine cytology and the present method using urine from bladder cancer patients. In urine samples from 20 bladder cancer patients, the results of urine cytology (classes 2-5) and the percentage of CD66a / c / e positive fractions in the total sample were compared using this method. The cutoff value for the CD66a / c / e positive fraction was 0.81%, as previously mentioned. The breakdown of urine cytology results from the 20 bladder cancer patients was as follows: (Class 2: 5 cases, Class 3: 4 cases, Class 4: 2 cases, Class 5: 9 cases). Class 3 was considered inconclusive, meaning the presence of a malignant tumor could not be definitively determined, while Class 2 was considered not malignant. Using this method, 8 out of the 9 cases with Class 2 or 3 urine cytology results were identified as positive. This suggests the usefulness of utilizing this method in addition to conventional tests, and also its potential as a standalone test (Figure 24). [Industrial applicability]

[0101] This invention focuses on microvesicles in urine that are positive for CD66a / c / e, and by utilizing these in various measurement methods, bladder cancer screening can be dramatically improved compared to current methods using urine cytology and cystoscopy. Specifically, it is a non-invasive diagnosis using urine, and it has the potential to surpass the clinical sensitivity of urine cytology, which is non-invasive but has a clinical sensitivity of about 40% (with many missed diagnoses). In actual clinical settings, this test is considered effective in diagnosing the presence or absence of bladder cancer using the urine of first-time patients, and in monitoring the prognosis (presence or absence of metastasis) after treatment such as surgery or chemotherapy in bladder cancer patients. Furthermore, by utilizing the quantitative parameters of microvesicles contained in urine other than CD66a / c / e positive microvesicles (microvesicles that are MUC1 positive or multipeptidase positive), the diagnostic accuracy of bladder cancer screening can also be improved, and a multi-assay that can simultaneously measure these would be useful in actual testing.

[0102] Furthermore, according to the present invention, by adjusting the urine collection conditions, a larger volume of urine can be subjected to the testing conditions, and by taking advantage of the benefits of urine as a test sample (it is possible to collect a large amount of urine non-invasively from the patient), a more accurate test can be performed.

[0103] In the future, it is conceivable that microvesicles that become CD66a / c / e positive using a simpler method that allows for the control of particle size could be utilized for early cancer detection, daily management of individual transitions from healthy to pre-disease (pre-disease) states, and other clinical applications as preventive and predictive markers in health checkups. The clinical value of micromembrane fractions can be enhanced by combining them with other biomarkers contained in urine.

Claims

1. A method to assist in determining whether a patient has bladder cancer by concentrating microvesicles with a diameter of 200 nm to 1 μm contained in the urine of a target patient, and determining whether the target patient has bladder cancer based on the amount of marker protein present in the microvesicles, The method is characterized in that the observation of the amount of marker protein in the microvesicle is performed by detecting the amount of at least one of CD66a, CD66b, CD66c, CGM2, or CD66e in the microvesicle.

2. The method according to claim 1, characterized in that the amount of CD66a, CD66b, CD66c, CGM2, or CD66e in the microvesicles is observed by calculating the ratio (A / B) of microvesicles (A) expressing CD66a, CD66b, CD66c, CGM2, and / or CD66e to microvesicles (B) expressing CD10, CD13, and / or CD26.

3. The method according to claim 1, characterized in that the amount of CD66a, CD66b, CD66c, CGM2, or CD66e in the microvesicles is observed by calculating the ratio (A / C) of microvesicles (A) expressing CD66a, CD66b, CD66c, CGM2, and / or CD66e to microvesicles (C) expressing CD10, CD13, CD26, and / or MUC1.

4. The method according to any one of claims 1 to 3, wherein the amount or ratio of marker protein in urinary microvesicles derived from the target patient is higher than the amount or ratio of marker protein in urinary microvesicles derived from the control, indicating that the target patient has bladder cancer.

5. A method for assisting the determination of whether or not a person has bladder cancer, comprising the steps of: measuring the amount of CD66a / b / c / e or CGM2 present in microvesicles with a diameter of 200 nm to 1 μm contained in urine; and determining that the urine originates from a bladder cancer patient if the measured amount of CD66a / b / c / e or CGM2 is greater than that of a control.

6. The method according to any one of claims 1 to 5, for observing the course of treatment for bladder cancer or for determining the effectiveness of treatment for bladder cancer, by comparing the amount of protein present in a first sample taken from the target patient with the amount of protein present in a second sample taken from the target patient after the treatment period.

7. The method according to any one of claims 1 to 6, further comprising the step of correlating the findings as indicating superficial bladder cancer, invasive stage 1 bladder cancer, or invasive stage 2-3 bladder cancer.

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