Methods for detecting extracellular vesicles

Abstract

To provide a method for simply and accurately detecting extracellular vesicles released from specific cells.SOLUTION: A method for detecting extracellular vesicles comprises the steps of: genetically introducing (a) a polynucleotide encoding a fusion protein between an extracellular vesicle-localizing protein and a detection protein or (b) a polynucleotide capable of separately expressing the extracellular vesicle-localizing protein and the detection protein into a specific cell; culturing the introduced specific cell to release extracellular vesicles; and determining the amount of the extracellular vesicle-localizing protein released by the specific cell transfected with the (a) and the amount of the extracellular vesicle-localizing protein released by the specific cell transfected with the (b), followed by correcting a determined value in the specific cell transfected with the (a) with a determined value in the specific cell transfected with the (b).SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for detecting extracellular vesicles released from specific cells, and in particular to a method for measuring and detecting proteins localized in the extracellular vesicles. [Background technology]

[0002] It is known that body fluids such as blood and cell culture media contain extracellular vesicles released from cells present in the body fluids or culture media. Extracellular vesicles are colloidal particles covered with a lipid bilayer membrane, and representative examples include exosomes and apoptotic vesicles. In recent years, it has been reported that extracellular vesicles function as a mediator of intercellular communication in the body and are related to diseases such as cancer and physiological phenomena, and research is being conducted to elucidate their physiological functions and apply them to disease testing.

[0003] As a method for detecting extracellular vesicles, a detection method targeting the membrane surface protein of the vesicles has been reported (Patent Document 1). In addition, a method is known in which the vesicles are purified / concentrated from a sample containing extracellular vesicles by ultracentrifugation or the like, and then the proteins present inside the vesicles are detected by mass spectrometry, immunoblotting, Enzyme-Linked Immuno Sorbent Assay (ELISA), or the like to detect the vesicles. However, these detection methods are complicated and highly dependent on the quality of the antibodies used, making it difficult to achieve highly sensitive detection. In addition, when the sample containing extracellular vesicles is a cell culture supernatant, it is necessary to remove extracellular vesicles derived from components contained in the culture medium, such as fetal bovine serum, and it is necessary to optimize the culture conditions for selectively recovering extracellular vesicles released by cultured cells.

[0004] In response to these problems, a method for detecting extracellular vesicles has been reported in which a polynucleotide encoding a fusion protein of CD63, which has been reported to be localized in extracellular vesicles, and a detection protein, nanoluciferase (Nluc), is introduced into cells, and the luciferase activity in the culture supernatant of the cells is measured (Non-Patent Document 1). However, the method described in Non-Patent Document 1 may not be localized in extracellular vesicles, such as being present in the culture supernatant as an aggregated free protein, and the amount of protein localized in extracellular vesicles that is non-selectively encapsulated in the extracellular vesicles cannot be excluded. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2012-508577 [Non-patent literature]

[0006] [Non-Patent Document 1] Scientific Reports,8,2018,14035 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for simply and highly accurately detecting extracellular vesicles released from specific cells. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors conducted extensive research and arrived at the present invention.

[0009] That is, the first aspect of the present invention is (1) introducing into a specific cell a polynucleotide encoding a polypeptide comprising a protein localized in an extracellular vesicle; (2) culturing the introduced specific cells and releasing extracellular vesicles; (3) detecting a protein localized in the released extracellular vesicles, The step (1) is a step of introducing into a specific cell (a) a polynucleotide encoding a fusion protein of a protein localized in extracellular vesicles and a detection protein, or (b) a polynucleotide capable of expressing a protein localized in extracellular vesicles and a detection protein separately; In the detection method, the step (3) is a step of measuring the amount of protein localized in extracellular vesicles released by the specific cells transfected with genes in step (a) and the amount of protein localized in extracellular vesicles released by the specific cells transfected with genes in step (b), and correcting the measured value in the specific cells transfected with genes in step (a) with the measured value in the specific cells transfected with genes in step (b).

[0010] A second aspect of the present invention is the detection method according to the first aspect, wherein the protein localized in extracellular vesicles is a four-transmembrane protein.

[0011] A third aspect of the present invention is the detection method according to the first or second aspect, wherein the polynucleotide capable of expressing the protein localized in extracellular vesicles and the detection protein separately is a polynucleotide encoding a polypeptide in which a self-cleaving peptide is inserted between the protein localized in extracellular vesicles and the detection protein.

[0012] A fourth aspect of the present invention is a method for optimizing purification of extracellular vesicles released by specific cells based on the results obtained by the detection method described in any one of the first to third aspects.

[0013] The present invention will be described in detail below.

[0014] In the present invention, extracellular vesicles refer to lipid-covered vesicles with a diameter of 1 nm to 1 μm that are released by cells, regardless of whether they are active or passive. Examples include exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, and apoptotic vesicles (Nature Reviews, 9, 2009, 581-593). It has been reported that extracellular vesicles are generally composed of lipids and proteins with different compositions from the cells that released them (BioScience, 65, 2015, 783-797).

[0015] In the present invention, the origin of the extracellular vesicles is not particularly limited. For example, body fluids, cell suspensions, culture fluids and culture supernatants after cell culture, and tissue cell lysates are included. Among these, body fluids and culture supernatants after cell culture are preferred. Examples of body fluids include blood components such as whole blood, serum, plasma, blood components, various blood cells, blood clots, and platelets, as well as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymphatic fluid, bone marrow fluid, tissue fluid, saliva, gastric juice, synovial fluid, pleural effusion, bile, ascites, and amniotic fluid. Among these, blood components are preferred, and may be treated with anticoagulants such as citric acid, heparin, and EDTA.

[0016] In the present invention, the protein localized in the extracellular vesicles is not particularly limited. Examples include 4-transmembrane proteins (Transmembrane 4 superfamily, Tetraspanin) such as CD63, CD9, and TM4SF1 (Transmembrane 4 Superfamily Member 1), as well as integrins and cadherins. Among these, it is preferable to use 4-transmembrane proteins as proteins localized in the extracellular vesicles.

[0017] In the present invention, the detection protein is not particularly limited, and may be appropriately selected in consideration of the simplicity of the experiment and the required sensitivity. Examples include luminescent proteins such as luciferase and β-galactosidase, fluorescent proteins such as GFP (green fluorescent protein), RFP (red fluorescent protein) and DsRED (Discosoma red fluorescent protein), and epitope tag peptides such as FLAG tag, MYC tag, His tag and V5 tag. Among them, luciferase (Nluc) derived from Pseudomonas niger is preferred because it can cause a substrate to emit light in an ATP-independent manner, has a wide dynamic range of detection, is highly sensitive, and has a smaller molecular weight than luciferase and GFP derived from fireflies and Renilla reniformis.

[0018] When designing a fusion protein of a protein localized in extracellular vesicles and a detection protein, there is no particular limitation on the fusion method. For example, a linker sequence may be inserted between the proteins after matching the codon frames, or the proteins may be fused directly without insertion. However, it is preferable to insert an oligonucleotide encoding a linker composed of glycine and serine, such as a GS linker (e.g., SEQ ID NO: 10), because it improves the flexibility of the expressed fusion protein. In addition, there is no particular limitation on the arrangement of the protein localized in extracellular vesicles and the detection protein, and the protein localized in extracellular vesicles may be arranged on the N-terminus side and the detection protein on the C-terminus side, respectively, or the reverse arrangement may be used. Furthermore, in order to improve the detection sensitivity, a plurality of these proteins may be arranged in tandem.

[0019] The present invention is characterized in that a polynucleotide encoding the above-mentioned fusion protein is introduced into a specific cell, and a polynucleotide capable of expressing a protein localized in extracellular vesicles and a detection protein separately is introduced into another specific cell. There is no particular limitation on the method for producing a transgenic cell capable of expressing a protein localized in extracellular vesicles and a detection protein separately. For example, a specific cell may be transfected with a plasmid for gene recombination in which a polynucleotide encoding a protein localized in extracellular vesicles and a polynucleotide encoding a detection protein are separately arranged downstream of different promoters. Alternatively, a specific cell may be transfected with a plasmid for gene recombination, including a linker nucleotide such as an oligonucleotide encoding a self-cleaving peptide such as an IRES (Internal Ribosome Entry Site) or a 2A peptide, which is inserted between a polynucleotide encoding a protein localized in extracellular vesicles and a polynucleotide encoding a detection protein. Among them, it is preferable to use a polynucleotide encoding a polypeptide in which a self-cleaving peptide is inserted between a protein localized in extracellular vesicles and a detection protein as the polynucleotide to be inserted into the plasmid, since it is possible to construct a recombinant plasmid almost equivalent to that in which a polynucleotide encoding the fusion protein is used. As an example of a self-cleaving peptide, 2A peptide, P2A (SEQ ID NO: 1), T2A (SEQ ID NO: 2), E2A (SEQ ID NO: 3), F2A (SEQ ID NO: 4), etc. are known, and P2A (SEQ ID NO: 1), which generally has high cleavage activity, is particularly preferred.

[0020] In the present invention, gene introduction into specific cells is not particularly limited as long as the cells can be introduced with the above-mentioned polynucleotide. A gene may be introduced transiently using a commercially available reagent such as Lipofectamine3000 (manufactured by ThermoFisher) or FuGENE (manufactured by Promega), or a stable expression cell of the introduced gene may be established using electroporation or the PiggyBac system (manufactured by System Biosciences). In addition, when the base length of the above-mentioned polynucleotide is a size (e.g., 5 kbp or more) that makes it difficult to insert it into a general animal cell expression plasmid, a polynucleotide encoding a detection protein may be directly inserted into the genomic DNA held by the specific cell using genome editing techniques such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system or TALEN (Transcription Activator-Like Effector Nuclease).

[0021] In the present invention, a gene-transfected cell obtained by introducing a polypeptide encoding a fusion protein of a protein localized in extracellular vesicles and a detection protein into a specific cell (hereinafter, simply referred to as a "fusion protein-expressing cell"), and a gene-transfected cell obtained by introducing a polynucleotide capable of expressing a protein localized in extracellular vesicles and a detection protein separately into a specific cell (hereinafter, also simply referred to as a "non-fusion protein-expressing cell") are measured for the amount of protein localized in the extracellular vesicles released by each of them, and the measurement value in the fusion protein-expressing cell is corrected by the measurement value in the non-fusion protein-expressing cell to detect the extracellular vesicles released by the specific cell. The detection of the protein localized in the extracellular vesicles may be performed based on the detection protein introduced into the specific cell. As an example, when luciferase such as Nluc is used as the detection protein, luciferin may be allowed to act, and detection may be performed based on the presence or absence of luminescence derived therefrom and its intensity. The correction of the measurement value in the fusion protein-expressing cell to the measurement value in the non-fusion protein-expressing cell may be based on the difference or ratio of the absolute values ​​of the measurement values, or may be based on the difference or ratio of the relative values ​​of the measurement values. This correction enables accurate detection of extracellular vesicles released by specific cells.

[0022] Based on the detection results of the present invention, the purification method of extracellular vesicles released by specific cells can be optimized. In addition, the purification method of extracellular vesicles is not particularly limited. For example, the method using ultracentrifugation is generally the most orthodox, but other methods that can simply sediment and recover a large amount of extracellular vesicles can be exemplified. For example, by combining ultracentrifugation with a density gradient method or a sucrose cushion method, extracellular vesicles with a higher degree of purification can be obtained. However, ultracentrifugation requires a high centrifugal force, and if it affects the subsequent process, it is necessary to appropriately utilize other methods such as size exclusion chromatography and affinity methods listed below. Size exclusion chromatography can also be exemplified. Although this method can highly purify extracellular vesicles, it takes time to process multiple samples, and the eluted extracellular vesicle fraction is more diluted, so it may be necessary to concentrate the sample before input or after elution. Furthermore, the affinity method has the advantage of easily concentrating extracellular vesicles, but there is a possibility of selectively purifying only a subtype of extracellular vesicles that have a specific marker from among all extracellular vesicles. Another example is the polymer precipitation method, which has the advantage of easily recovering relatively intact extracellular vesicles. Taking into account the characteristics of the various purification methods described above, an appropriate method can be selected from ultracentrifugation, size exclusion chromatography, affinity methods targeting molecules localized in extracellular vesicle membranes such as cell membrane phosphatidylserine (PS) and CD9, and polymer precipitation methods, depending on the experimental system and equipment.

[0023] There is no particular limitation on the optimal selection method for the above-mentioned purification method. For example, extracellular vesicles in which a fusion protein of a protein localized in extracellular vesicles and a detection protein is localized are purified for spiking. After spiking the above-mentioned purified extracellular vesicles into a mock sample, purification of each extracellular vesicle is performed, and the most suitable extracellular vesicle purification method may be selected based on the amount of detection protein among the finally purified fractions.

[0024] It may also be used to examine the conditions for each step of a specific purification method. For example, in the purification step of extracellular vesicles, conditions for washing buffers and suspensions with different salt concentrations, surfactants, and sugar concentrations may be prepared, and the most suitable purification step and reagents may be selected using the amount of detection protein in the final purified fraction as an indicator.

[0025] Furthermore, it may be used to investigate the cause of inaccurate recovery during purification of extracellular vesicles. For example, for substrates of experimental equipment such as tubes and devices used in experiments that are suspected of non-specific adsorption, the substrates are recovered using a solution containing a surfactant. This can be used to investigate the cause of non-specific adsorption of the detection protein obtained to the substrate with the highest remaining rate. Effect of the Invention

[0026] The present invention provides a method for detecting vesicles, comprising the steps of (1) introducing a polynucleotide encoding a polypeptide containing a protein localized in extracellular vesicles into specific cells, (2) culturing the introduced specific cells to release extracellular vesicles, and (3) detecting the protein localized in the released extracellular vesicles, characterized in that the step (1) is a step of introducing into the specific cells (a) a polynucleotide encoding a fusion protein of a protein localized in extracellular vesicles and a detection protein, or (b) a polynucleotide capable of individually expressing the protein localized in extracellular vesicles and the detection protein, and the step (3) is a step of measuring the amount of protein localized in extracellular vesicles released by the specific cells transfected with genes in (a) and the amount of protein localized in extracellular vesicles released by the specific cells transfected with genes in (b), and correcting the measured value in the specific cells transfected with genes in (a) by the measured value in the specific cells transfected with genes in (b).

[0027] According to the present invention, extracellular vesicles released from specific cells can be detected simply and accurately based on the proteins localized in the vesicles. The method of the present invention is expected to be useful not only for functional analysis of proteins localized in extracellular vesicles and elucidation of the physiological functions of extracellular vesicles, but also for application as a standard for calibration curves used in disease testing and the like. [Brief description of the drawings]

[0028] [Figure 1] Map of the plasmid used for gene transfer into specific cells in the Examples. [Diagram 2] Results showing localization of recombinant proteins expressed in specific cells to extracellular vesicles. EXAMPLES

[0029] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0030] Example 1 Preparation of gene-transfected cancer cells (1) Design of plasmid for gene transfer A recombinant plasmid was constructed by inserting a polynucleotide encoding a polypeptide shown in Table 1 (i) to (iv) downstream of the EF1α promoter (SEQ ID NO: 5) of an animal cell expression plasmid having an EF1α promoter (pBApo-EF1α Pur DNA [product code: 3244], manufactured by Takara Bio Inc.). The polynucleotide encoding nanoluciferase (Nluc, SEQ ID NO: 9) was prepared by using the Nluc reporter gene inserted into the NanoLuc Vector (manufactured by Promega).

[0031] [Table 1]

[0032] (2) Cell culture and gene transfer (2-1) Human prostate cancer cells (PC-3 cells) were cultured at 37°C in a 5% CO2 environment using Ham's F-12K (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 15% (v / v) FBS (fetal bovine serum).

[0033] (2-2) After the medium used in (2-1) was placed in a 6-well plate at 2 mL / well, 5 × 10 PC-3 cells cultured in (2-1) were added. 5 The cells were seeded and suspended at 16 cells / well.

[0034] (2-3) After culturing for one day, a plasmid into which a polynucleotide encoding a polypeptide shown in (i) to (iv) was inserted was added at 0.5 μg / well, and gene transfer was performed using a gene transfer reagent (Lipofectamine 3000, Thermo Fisher Scientific) to obtain cells capable of expressing the polypeptide shown in (i) to (iv) (gene-transfected cancer cells).

[0035] Example 2 Measurement of extracellular vesicles released from gene-transfected cancer cells (1) Purification of extracellular vesicles (1-1) After culturing each of the gene-introduced cancer cells obtained in Example 1 for an additional 3 days, the entire culture supernatant (about 2 mL) was collected. After removing floating cells by centrifugation at 300 G at room temperature for 10 minutes, 1.5 mL of the supernatant was collected (culture supernatant, hereinafter also referred to as "CM").

[0036] (1-2) The CM was further centrifuged at 3000G for 10 minutes at 4°C to remove cell debris, and 1.2 mL of the supernatant was collected. The collected supernatant was further centrifuged at 16000G for 60 minutes at 4°C, and 1 mL of the supernatant was transferred to another tube (centrifugation procedure A). The remaining precipitate was suspended in 1 mL of PBS (Phosphate Buffered Saline) and washed by centrifugation at 16000G for 60 minutes at 4°C, and 1 mL of the supernatant was removed. 0.2 mL of the suspension containing the remaining precipitate was used as a microvesicle fraction (hereinafter also referred to as "MV fraction").

[0037] (1-3) 1 mL of the supernatant transferred to the tube in centrifugation step A in (1-2) was mixed with 1 mL of PBS and ultracentrifuged at 2,590,000 G for 70 minutes at 4°C. 1.8 mL of the supernatant was transferred to another tube and used as the free protein fraction (hereinafter also referred to as the "SP fraction"). Meanwhile, 0.2 mL of the suspension containing the remaining precipitate was used as the exosome fraction (hereinafter also referred to as the "EX fraction").

[0038] (2) Measurement of luminescence (luciferase activity) (2-1) Each of the fractions (CM, MV, SP, and EX fractions) prepared in (1) was placed in a 96-well plate for luminescence measurement at 100 μL / well.

[0039] (2-2) A reaction solution provided with the Nano-Glo Luciferase Assay System (Promega) (a solution prepared by diluting the provided Substrate 50-fold with the provided Buffer) was added at 100 μL / well to the wells containing each fraction.

[0040] (2-3) After 3 minutes of reaction at room temperature, the amount of luminescence was measured using a luminometer (Infinite Lumi, manufactured by TECAN), and this measurement was used as the activity of luciferase (Nluc) expressed in the gene-transduced cancer cells. The relative amount of luminescence was calculated when the amount of luminescence in CM was set to 100%, and the amount of luminescence (luciferase activity) of each fraction was compared.

[0041] The results are shown in Table 2 and Figure 2. (i) The percentages of luciferase activity in the MV fraction, EX fraction, and SP fraction in the culture supernatant of cells expressing TM4SF1-Nluc fusion protein were 65.09±8.15%, 18.03±6.68%, and 11.65±2.68%, respectively, with the percentage of luciferase activity being the highest in the MV fraction and the lowest in the SP fraction (Table 2(i) and Figure 2(i)). Since Nluc and TM4SF1 expressed in the gene-transfected cancer cells in (i) above are fused, their localization is consistent. In other words, the above results indicate that TM4SF1 is localized in the MV fraction.

[0042] On the other hand, (ii) the percentages of luciferase activity in the MV fraction, EX fraction, and SP fraction in the culture supernatant of cells expressing TM4SF1-Nluc non-fusion protein were 23.95 ± 8.49%, 19.25 ± 4.85%, and 36.99 ± 11.36%, respectively, with the percentage of luciferase activity in the SP fraction being high and the percentages of luciferase activity in the MV fraction and EX fraction being low (Table 2 (ii) and Figure 2 (ii)). It can be seen that Nluc and TM4SF1 expressed in the gene-transfected cancer cells of (ii) above are expressed individually, and the expressed Nluc is present in large amounts in the SP fraction regardless of the localization position of TM4SF1 (MV fraction). By measuring the luciferase activity in the SP fraction, the noise localized as a free protein can be measured.

[0043] In addition, since the luciferase activity in the MV fraction was 23.95±8.49% in the culture supernatant of the gene-transduced cancer cells in (ii) above, it can be seen that Nluc is non-selectively localized in the MV fraction when overexpressed. In other words, by combining the results of the gene-transduced cancer cells in (i) above and the results of the gene-transduced cancer cells in (ii) above, it is possible to measure the luciferase activity of the MV fraction, which corresponds to the background noise, and to more accurately quantify the extracellular vesicles in which TM4SF1 is localized.

[0044] In addition, when CD63 and CD9, which have been reported to be localized on extracellular vesicles, were evaluated in gene-transfected cancer cells expressing fusion proteins with Nluc, (iii) the percentages of luciferase activity in the MV fraction, EX fraction, and SP fraction in the culture supernatant of cells expressing the CD63-Nluc fusion protein were 49.12±7.44%, 25.26±6.46%, and 20.13±4.80%, respectively, and (iv) the percentages of luciferase activity in the MV fraction, EX fraction, and SP fraction in the culture supernatant of cells expressing the CD9-Nluc fusion protein were 63.63±8.98%, 20.28±4.81%, and 16.18±2.56%, respectively, indicating that all of these are localized in the MV fraction, similar to TM4SF1.

[0045] [Table 2]

Claims

1. (1) transfecting a specific cell with a polynucleotide encoding a polypeptide comprising a protein localized in extracellular vesicles; (2) culturing the introduced specific cells to release extracellular vesicles; (3) A method for detecting the released extracellular vesicles, comprising the step of detecting a protein localized in the extracellular vesicles, The step (1) is a step of genetically introducing into a specific cell (a) a polynucleotide encoding a fusion protein of a protein localized in extracellular vesicles and a detection protein, or (b) a polynucleotide capable of expressing the protein localized in extracellular vesicles and the detection protein separately; The detection method, wherein the step (3) is a step of measuring the amount of protein localized in extracellular vesicles released by the specific cells transfected with genes in step (a) and the amount of protein localized in extracellular vesicles released by the specific cells transfected with genes in step (b), and correcting the measured value in the specific cells transfected with genes in step (a) with the measured value in the specific cells transfected with genes in step (b).

2. The detection method according to claim 1, wherein the protein localized in extracellular vesicles is a four-transmembrane protein.

3. The detection method according to claim 1 or 2, wherein the polynucleotide capable of expressing the protein localized in extracellular vesicles and the detection protein separately is a polynucleotide encoding a polypeptide in which a self-cleaving peptide is inserted between the protein localized in extracellular vesicles and the detection protein.

4. A method for optimizing the purification of extracellular vesicles released by specific cells based on the results obtained by the detection method described in any one of claims 1 to 3.

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