JP7661767B2 - Methods for detecting extracellular vesicles - Google Patents

Description

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

Body fluids such as blood and cell culture media are known to contain extracellular vesicles that are 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.

As a method for detecting extracellular vesicles, a detection method targeting the membrane surface proteins 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 immunosorbent 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 highly sensitive detection difficult. 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.

In response to these problems, a method has been reported for detecting extracellular vesicles by introducing into cells a polynucleotide encoding a fusion protein of CD63, which has been reported to be localized in extracellular vesicles, and the detection protein nanoluciferase (Nluc) and measuring the luciferase activity in the culture supernatant of the cells (Non-Patent Document 1). However, the method described in Non-Patent Document 1 has the possibility that the fusion protein is not localized in extracellular vesicles, for example, it is present in the culture supernatant as an aggregated free protein, and it is not possible to eliminate the amount of protein localized in extracellular vesicles that is non-selectively encapsulated in the extracellular vesicles.

Special Publication No. 2012-508577

Scientific Reports, 8, 2018, 14035

The objective of the present invention is to provide a method for easily and accurately detecting extracellular vesicles released from specific cells.

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

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 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 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;
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).

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

A third aspect of the present invention is the detection method according to the first or second aspect, in which the polynucleotide capable of expressing the protein localized in the 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 the extracellular vesicles and the detection protein.

A fourth aspect of the present invention is 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 the first to third aspects.

The present invention is described in detail below.

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, whether actively or passively. 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 a different composition from the cells from which they were released (BioScience, 65, 2015, 783-797).

In the present invention, the origin of the extracellular vesicles is not particularly limited. Examples include body fluids, cell suspensions, culture fluids and culture supernatants after cell culture, and tissue cell lysates. Of 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 lymph fluid, bone marrow fluid, tissue fluid, saliva, gastric juice, synovial fluid, pleural effusion, bile, ascites, and amniotic fluid. Of these, blood components are preferred, and may be treated with an anticoagulant such as citric acid, heparin, or EDTA.

In the present invention, there is no particular limitation on the proteins localized in extracellular vesicles. Examples include tetramembrane proteins (Transmembrane 4 superfamily, Tetraspanin) such as CD63, CD9, and TM4SF1 (Transmembrane 4 Superfamily Member 1), as well as integrin and cadherin. Among these, it is preferable to use tetramembrane proteins as proteins localized in extracellular vesicles.

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 independently of ATP, has a wide dynamic range of detection, is highly sensitive, and has a smaller molecular weight than luciferase and GFP derived from fireflies and sea pansies.

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 this improves the flexibility of the expressed fusion protein. There is also 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, 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.

In the present invention, a polynucleotide encoding the above-mentioned fusion protein is introduced into a specific cell, and a polynucleotide capable of expressing a protein localized in an extracellular vesicle 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 an extracellular vesicle 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 an extracellular vesicle 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 IRES (Internal Ribosome Entry Site) or an oligonucleotide encoding a self-cleaving peptide such as 2A peptide, inserted between the polynucleotide encoding a protein localized in an extracellular vesicle and the polynucleotide encoding a detection protein. Among these, 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 this allows the construction of a recombinant plasmid that is almost equivalent to that in which a polynucleotide encoding the fusion protein is used. Examples of self-cleaving peptides, such as 2A peptides, include P2A (SEQ ID NO: 1), T2A (SEQ ID NO: 2), E2A (SEQ ID NO: 3), and F2A (SEQ ID NO: 4), and P2A (SEQ ID NO: 1) is particularly preferred, as it generally has high cleavage activity.

In the present invention, gene introduction into a specific cell is not particularly limited as long as the cell can be introduced with the above-mentioned polynucleotide. A gene may be introduced transiently using a commercially available reagent such as Lipofectamine 3000 (manufactured by ThermoFisher) or FuGENE (manufactured by Promega), or a stable expression cell of the introduced gene may be established using the electroporation method or the PiggyBac system (manufactured by System Biosciences). Furthermore, if the base length of the aforementioned polynucleotide is such that it is difficult to insert it into a general animal cell expression plasmid (e.g., 5 kbp or more), the polynucleotide encoding the detection protein may be directly inserted into the genomic DNA of a specific cell using genome editing techniques such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system or TALEN (Transcription Activator-Like Effector Nuclease).

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 the gene-transfected cells, 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 from it and its intensity. The correction of the measurement value in the fusion protein-expressing cell by 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 allows for highly accurate detection of extracellular vesicles released by specific cells.

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 as described above, an appropriate method may be selected from ultracentrifugation, size exclusion chromatography, affinity methods targeting molecules localized in extracellular vesicle membranes such as cell membrane phosphatidylserine (PS) and CD9, polymer precipitation, etc., depending on the experimental system and equipment.

There is no particular limitation on the optimal selection method for the purification methods shown above. 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 appropriate extracellular vesicle purification method may be selected using the amount of detection protein among the final purified fractions as an indicator.

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

Furthermore, it may be used to investigate the cause of inaccurate recovery of extracellular vesicles during purification. For example, for substrates of experimental equipment such as tubes and devices used in experiments that are suspected of non-specific adsorption, they 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.

The present invention is 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 expressing the protein localized in extracellular vesicles and the detection protein separately, 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), respectively, 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).

The present invention makes it possible to easily and accurately detect extracellular vesicles released by specific cells 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, etc.

Map of the plasmid used for gene transfer into specific cells in the Examples. Results showing localization of recombinant proteins expressed in specific cells to extracellular vesicles.

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

Example 1 Preparation of gene-introduced cancer cells (1) Design of plasmid for gene introduction Recombinant plasmids were constructed by inserting polynucleotides encoding the polypeptides 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 the Nluc reporter gene inserted into NanoLuc Vector (manufactured by Promega).

(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 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 15% (v/v) FBS (fetal bovine serum).

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

(2-3) After one day of culture, a plasmid into which a polynucleotide encoding the 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).

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

(1-2) The CM was further centrifuged at 3000 G 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 16000 G 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 16000 G 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").

(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").

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

(2-2) 100 μL of the reaction solution (a solution of the attached substrate diluted 50-fold with the attached buffer) included with the Nano-Glo Luciferase Assay System (Promega) was added to the wells containing each fraction.

(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.

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 MV fraction showing the highest percentage of luciferase activity and the SP fraction showing the lowest percentage of luciferase activity (Table 2(i) and Figure 2(i)). Nluc and TM4SF1 expressed in the gene-transfected cancer cells in (i) above are fused, and therefore are localized in the same location. In other words, the above results indicate that TM4SF1 is localized in the MV fraction.

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 being high in the SP fraction and low in the MV fraction and EX fraction (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 separately, and that the expressed Nluc is present in large amounts in the SP fraction, regardless of the localization position of TM4SF1 (MV fraction). Furthermore, by measuring the luciferase activity in the SP fraction, noise localized as a free protein can be measured.

In addition, since 23.95±8.49% of the luciferase activity was found in the MV fraction in the culture supernatant of the gene-transfected cancer cells (ii) above, it can be seen that when Nluc is overexpressed, it is non-selectively localized in the MV fraction. In other words, by combining the results of the gene-transfected cancer cells (i) above with the results of the gene-transfected cancer cells (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.

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.

Claims (4)

(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). The detection method according to claim 1, wherein the protein localized in extracellular vesicles is a four-transmembrane protein. The detection method according to claim 1 or 2, wherein the polynucleotide capable of expressing the protein localized in the 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 the extracellular vesicles and the detection protein. 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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