JP2021188970A - Method for examining lung cancer based on analysis of protein composite body in extracellular vesicle - Google Patents

Abstract

To provide a method and a kit for examining a lung cancer more accurately.SOLUTION: The present invention relates to a method for examining a lung cancer, including the step of analyzing a composite body of CHL1 in a film of an extracellular vesicle in a blood sample of a subject and such a CHL1 partner protein as Caspase-14, and also relates to a lung cancer inspection kit including an anti-CHL1 antibody and a reagent which detects the CHL1 partner protein.SELECTED DRAWING: Figure 4C

Description

The present invention relates to a method for testing lung cancer.

Tumor markers in biological samples, especially in serum, are used as an auxiliary diagnostic method for cancer such as cancer screening, as a material for determining prognosis after surgery, and as a material for determining therapeutic effect. CYFRA21-1, CEA, SCC, SLX, CA125, etc. have been used as lung cancer markers, but the sensitivity and specificity are not high, and lung cancer markers with better sensitivity and specificity are desired. rice field.

On the other hand, the present inventors reported that the level of CHL1 (Close homolog of L1), which is a cell adhesion molecule, is increased in lung cancer patients, and that CHL1 can be used as a lung cancer marker (Patent Document 1 and Non-Patent Document 1). ).

JP 2019-190883

Biochem Biophys Res Commun. 2018 Jul 2; 501 (4): 982-987.

As mentioned above, it was clarified that the test using CHL1 as a marker is useful for diagnosing lung cancer, but there was room for improvement in order to obtain a more accurate diagnostic index.
Therefore, it is an object of the present invention to provide a method and a test kit for more accurately testing lung cancer.

The present inventors have conducted diligent studies to solve the above problems. As a result, we found that the complex of CHL1 and its partner protein was significantly increased in the membrane of extracellular vesicles (EV) contained in the blood sample derived from lung cancer patients, and the blood sample of the subject was used. We have found that lung cancer can be accurately tested by analyzing the complex using the complex, and have completed the present invention.

That is, the gist of the present invention is as follows.
[1] A method for examining lung cancer, which comprises a step of analyzing a complex of CHL1 and a CHL1 partner protein in the membrane of extracellular vesicles contained in a blood sample of a subject.
[2] CHL1 partner proteins are Caspase-14, SLC4A1 (Solute Carrier Family 4 Member 1), Thrombospondin 1, Desmoplakin, Lactotransferrin, 14-3-3 protein sigma, Heat shock protein beta-1, Annexin A2, Calmodulin-like protein. 3, Galectin-7, Desmocollin-1, Fatty acid-binding protein 5, Transgelin-2, Annexin A1, Alpha-enolase, Glutathione S-transferase P, Tubulin beta-2A chain, 14-3-3 protein zeta / delta, Tubulin alpha-4A chain, Epiplakin, Polymeric immunoglobulin receptor, Tubulin alpha-1B chain, Serpin B3, Tubulin beta-4B chain, Triosephosphate isomerase, Protein S100-A14, Protein S100-A11, Zymogen granule protein 16 homolog B, Calmodulin-like protein 5, Prelamin-A / C, Elongation factor 1-alpha 1, or Heat shock co
The method for testing lung cancer according to [1], which is a gnate 71 kDa protein.
[3] The method for testing lung cancer according to [1], wherein the CHL1 partner protein is Caspase-14.
[4] A lung cancer test method according to any one of [1] to [3], wherein the analysis is performed by the EMARS method.
[5] A lung cancer test kit containing an anti-CHL1 antibody and a CHL1 partner protein detection reagent.
[6] The kit for lung cancer testing according to [5], wherein the anti-CHL1 antibody is a horseradish peroxidase (HRP) -binding antibody, and the kit contains an EMARS reagent.
[7] The lung cancer test kit according to [5] or [6], which further comprises a reagent for separating extracellular vesicles.

According to the present invention, lung cancer can be examined more accurately, which can contribute to the medical and diagnostic fields.

Schematic diagram of molecular assembly (BiEV) analysis in extracellular vesicles of serum EV. The target EV (tEV) represents an EV to be detected and investigated. Serum EV was purified by combining polymer precipitation and size exclusion chromatography prior to the EMARS reaction. Appropriate fluorescein-labeled BiEV partner molecules were measured by sandwich ELISA in order to measure the amount of BiEV to determine the amount of tEV contained in the sample. Chromatographic fractionation of serum EV derived from EML4-ALK transgenic mice using Sephacryl S-500. Preliminary experiments were performed using blue dextran (indicator of void volume) and mouse serum (indicator of protein elution). The dotted line represents the absorbance of blue dextran. The solid line represents the protein concentration measured using the BCA protein kit. Morphological observation of serum EV using cryo-electron microscopy (photo). The figure on the right of fraction No. 6 is an enlargement of a part of the figure on the left. Scale bar; 200 nm (left figure) and 50 nm (right figure). EMARS product (photo) obtained from serum EV derived from EML4-ALK transgenic mouse. In order to average the experimental results for each group, 10 sera were collected from each of the (WT) group, the mouse (TL) group carrying a large lung tumor, and the mouse (TS) group carrying a small lung tumor. (10 μl each), mixed in equal proportions and subjected to EV purification and EMARS. The EMARS product was purified and concentrated by immunoprecipitation using the anti-fluorescein antibody Sepharose. The obtained sample was subjected to SDS-PAGE analysis using fluorescence detection. “IP” represents an immunoprecipitated sample and “Lys” represents a lysate sample prior to immunoprecipitation. The right column is the same gel as the left column, but with a longer exposure time. Confirmation of CHL1 partner molecule identified by MS proteomics (photo). EMARS products of WT, TL and TS were subjected to immunoprecipitation and Western blot analysis using anti-SLC4A1 antibody (left column). After stripping, the membrane was restained with anti-THBS1 antibody (right column). Arrows represent the detected bands of SLC4A1 and THBS1 (including the expected dimer). Asterisks represent unknown bands (expected to be non-specific or partial fractions). In the measurement of CHL1-SLC4A1 BiEV in serum EV, fluorescein-labeled SLC4A1 was measured using sandwich ELISA. Serum EVs from 12 wild-type mice (white bar graph) and 18 small tumor-carrying EML4-ALK gene-introduced mice (black bar graph) were subjected to EMARS reaction and then subjected to ELISA measurement. EMARS products containing fluorescein-labeled SLC4A1 were added to ELISA plates coated with anti-SLC4A1 antibody. The BiEV index value (SLC4A1) was calculated based on the value of the fluorescein-labeled SLC4A1 of the reference sample. Comparison of BiEV index values for WT and TS. In the Mann-Whitney test, the BiEV index value of TS was significantly higher than the BiEV index value of WT (p = 8.9 × 10 ^-3 ). ROC curve of BiEV index value. The AUC was calculated to be 0.782. Scatter plot of tumor volume vs. BiEV index values. The correlation between the BiEV index value and the amount of lung cancer tumor in TS was evaluated using Spearman's rank correlation coefficient (0.389: p = 0.111). Western blot analysis of SLC4A1 across serum EVs from WT and TS (photo). Serum collected from 12 wild-group mice and 18 EML4-ALK gene-introduced mice carrying small lung tumors were mixed in equal proportions and then subjected to EV purification. Arrows represent the detected bands of SLC4A1. The asterisk is an unknown band (expected to be non-specific or partial fraction). Measurement of CHL1-Caspase-14 BiEV in serum EV derived from lung cancer patients (photo). EMARS products obtained from serum EVs of healthy individuals (H) and lung cancer (LC) patients were purified. 50 μl of serum was recovered from the H and LC groups, used for EV purification, and subjected to the EMARS reaction. In order to average the experimental results for each group, sera obtained from 5 H and 5 LC were separated (10 μl each) and mixed in equal amounts. EMARS products were subjected to SDS-PAGE analysis using fluorescence detection. Confirmation of CHL1 partner molecule identified by MS proteomics (photo). H and LC samples were subjected to Western blot analysis using immunoprecipitation (anti-fluorescein antibody Sepharose) and anti-Caspase-14 antibody, respectively. Arrows indicate bands of detected Caspase-14 protein (including the expected dimer). Measurement of fluorescein-labeled Caspase-14 using sandwich ELISA. Serum EVs obtained from 12 H (white bar graph) and 12 LC (black bar graph) were each subjected to EMARS reaction, and then ELISA measurement was performed. EMARS products containing fluorescein-labeled Caspase-14 were added to ELISA plates coated with anti-Caspase-14 antibody. The "BiEV index value (Caspase-14)" was calculated based on the value of fluorescein-labeled recombinant Caspase-14 prepared using the fluorescein-labeled reagent. These values are shown as the average of three ELISA experiments performed separately using the same sample. An asterisk indicates that the sample has fallen below the detection limit. Comparison of BiEV index values between H and LC. In the Mann-Whitney test, the BiEV index value of LC was significantly higher than the BiEV index value of H (p = 0.019). ROC curve of BiEV index value. The AUC was calculated to be 0.811. Western blot analysis of Caspase-14 of whole serum EV obtained from H and LC (photo). Separated sera from 12 H and LC (2 μl each) were mixed in equal proportions and then the EV was purified using a precipitation protocol. Arrows represent the detected Caspase-14 bands.

The method for testing lung cancer of the present invention comprises a step of analyzing a complex of CHL1 and a CHL1 partner protein in the membrane of extracellular vesicles contained in a blood sample of a subject.

The subject may be any animal that may cause lung cancer, but is preferably a mammal, and more preferably a human.

Lung cancer is classified into non-small cell lung cancer and small cell lung cancer according to histological type. Non-small cell lung cancer is further classified into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In the present invention, lung cancer may be classified into any of the above. Furthermore, lung cancer is not particularly limited in terms of disease site, stage, etc. in the lung, and includes any disease site, stage, etc.

Lung cancer may be EML4-ALK mutant lung cancer. EML4-ALK mutant lung cancer is a type of non-small cell lung cancer. In humans, a slight inversion occurs in the short arm of chromosome 2, resulting in microtubule binding of the intracellular region of the receptor tyrosine kinase ALK. It is a lung cancer characterized by the formation of a new active fusion kinase EML4-ALK fused with the protein EML4 (Reference: Journal of the Japanese Society of Internal Medicine, Vol. 99, No. 4, p155-159). Here, EML4-ALK mutant lung cancer is a type of non-small cell lung cancer and is found in about 4 to 5% of patients with non-small cell lung cancer.

Blood samples include serum, plasma and the like. The serum or plasma may be used as it is, but it may be divided into a precipitate fraction (extracellular vesicle-containing fraction) and a supernatant fraction, and the precipitate fraction may be used.

Extracellular vesicles (EVs) are tiny vesicles with a membrane structure that are secreted by various types of cells. Extracellular vesicles include, for example, exosomes, ectosomes, microvesicles and extracellular vesicles. Preferably, the extracellular vesicles are exosomes. Extracellular vesicles can also be defined by their size. The size of the extracellular vesicle is, for example, 30 to 1000 nm, preferably 50 to 300 nm, and more preferably 80 to 200 nm. The size of the extracellular vesicle can be measured, for example, by a method based on Brownian motion of the extracellular vesicle, a light scattering method, an electric resistance method, or the like. For example, the size of extracellular vesicles is measured by NanoSight (manufactured by Malvern Instruments).

EV binds to any method known in the art, such as density gradient centrifugation, ultracentrifugation utilizing sucrose gradient, microfiltration, immunochromatography, substances present on the outer membrane of extracellular vesicles. It can be separated by a separation method using a carrier on which an antibody is immobilized and a commercially available kit (for example, ExoQuick ™).

CHL1 (Close homolog of L1) protein is a one-time transmembrane cell adhesion molecule (CAM), also called CALL (cell adhesion L1-like), which is a cell adhesion molecule L1, and its function in nerve cells has been suggested. There is.

The CHL1 protein may be a protein derived from the test subject, but when the test target is a human, the amino acid sequence of SEQ ID NO: 1 is exemplified, and when the test target is a mouse, SEQ ID NO: 2 is exemplified. The amino acid sequence of is exemplified. In addition, as long as it retains the function of the CHL1 protein and forms a complex with the partner protein on the EV membrane, one or several (for example, 1 to 20) amino acids are substituted in the amino acid sequence of SEQ ID NO: 1 or 2. , A protein having an amino acid sequence deleted, inserted or added, or a protein having an amino acid sequence having 90% or more or 95% or more identity with SEQ ID NO: 1 or 2. When a different animal is used as the test target, the homolog protein derived from the animal is the measurement target.

The CHL1 partner protein may be any protein that forms a complex on the membrane of CHL1 and EV and the complex increases in lung cancer. Examples thereof include the proteins shown in Table 1 below. Table 1 shows an example of the human protein sequence of each partner protein (GenBank).
Registration sequence) is listed. However, as shown in the references in Table 1, each partner protein is known, and proteins having various sequence variations other than these sequences are known, and are limited to proteins having these specific sequences. Not done. Preferably, each partner protein has 90% or more or 95% or more sequence identity with the amino acid sequences listed in Table 1.

Preferred examples of CHL1 partner proteins include Caspase-14. Caspase-14 is a member of the caspase family involved in keratinocyte differentiation (J Cell Biol 180 (3): 451-458. 2008, Cancer Res. 58 (22), 5201-5205. 1998) and is listed in Table 1. As shown, it has the sequence of GenBank P31944 (human) (SEQ ID NO: 3) or O89094 (mouse) (SEQ ID NO: 4). Caspase-14 is a protein or SEQ ID NO: 3 or 4 having an amino acid sequence in which one or several (for example, 1 to 20) amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 3 or 4. It may be a protein having an amino acid sequence having 90% or more or 95% or more identity.

Another preferred example of the CHL1 partner protein is SLC4A1. SLC4A1 is a membrane protein responsible for the intracellular and extracellular transport of anions (Biochem. J. 256 (3), 703-712 (
1988), Nature 316 (6025), 234-238 (1985)), eg, GenBank P02730 (human) (SEQ ID NO: 5) or P04919 (mouse (SEQ ID NO: 6)). SLC4A1 is 90% with proteins or SEQ ID NOs: 5 or 6 having an amino acid sequence in which one or several (eg 1 to 20) amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 5 or 6. It may be a protein having an amino acid sequence having the above or 95% or more identity.

Another preferred example of the CHL1 partner protein is Thrombospondin 1 (THBS 1). THBS1 is a glycoprotein involved in cell adhesion (J. Cell Biol. 103 (5), 1635-1648 (1986), Genomics 11 (3), 587-600 (1991)), for example, GenBank P07996 (human). It has the sequence of (SEQ ID NO: 7) or P35441 (mouse) (SEQ ID NO: 8). THBS1 is 90% with proteins or SEQ ID NOs: 7 or 8 having an amino acid sequence in which one or several (eg 1-20) amino acids have been substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 7 or 8. It may be a protein having an amino acid sequence having the above or 95% or more identity.

Analysis method of complex The analysis method of the complex is not particularly limited, and methods known in the fields of biochemistry and genetic engineering can be used. For example, EV purified from a blood sample or a membrane fraction thereof or a membrane fraction thereof or Examples thereof include a method in which these treated products are treated with an anti-CHL1 antibody to separate a complex of CHL1 and a CHL1 partner protein, and further detected with a CHL1 partner protein detection reagent such as an anti-CHL1 partner protein antibody. Methods such as flow cytometry and co-immunoprecipitation are also exemplified.

Each antibody used may be either a monoclonal antibody or a polyclonal antibody, or may be an antibody fragment such as Fab. In addition, each antibody is commonly used in mice, rats, etc.
Rabbit, goat, sheep, bird-derived antibodies, etc. can be used, but the antibody is not limited to these, and any antibody that specifically binds to CHL1 or CHL1 partner protein can be used. As the antibody, a commercially available antibody may be used, or an antibody obtained by a method known to those skilled in the art for producing an antibody may be used.
In order to measure CHL1 on the EV membrane, an antibody that recognizes the extracellular domain of CHL1 is preferable. Specifically, in human CHL1, an antibody that recognizes a part of the extracellular domain represented by the amino acids 1 to 1096 of SEQ ID NO: 1 is preferable.

The anti-CHL1 antibody and the anti-CHL1 partner antibody may be labeled with an enzyme or a dye.
Examples of the labeling substance include enzymes, radioisotopes, fluorescent substances, luminescent substances, colloidal gold and the like. As enzymes, horseradish peroxidase (HRP) and alkaline phosphatase (AP)
), Glucose oxidase (GOD) and the like are more preferable, and an enzyme that generates radicals is preferably used in order to carry out the EMARS method described later, and HRP is particularly preferable.
In addition, fluorescent dyes such as FITC and rhodamine can also be used as the labeling substance.

In order to detect the complex of CHL1 and CHL1 partner protein more specifically, it is preferable to use the EMARS (Enzyme-Mediated Activation of Radical Sources method) method in the analysis of the complex.
The EMARS method is described in the following literature.
Jiang, S., Kotani, N., Honke, K., et.al., A proteomics approach to the cell-surface
interactome using the enzyme-mediated activation of radical sources reaction. Proteomics 2012,12,54-62
Kotani N, Gu J, Isaji T, Udaka K, Taniguchi N, Honke K. Biochemical visualizatio
n of cell surface molecular clustering in living cells.
Proc. Natl. Acad. Sci. US A. 105 (21): 7405-9 (2008).
Taniguchi, N., Commentary-Searching for partners, Proteomics 2012,12,9-10
Japanese Patent No. 5773405 Japanese Patent No. 4927462

Specifically, the following methods are exemplified as the EMARS method.
(1) An HRP-labeled recognition molecule (anti-CHL1 antibody) is added to EV, and an EMARS reagent, which is a compound containing a radical source such as aryl azide and a labeling substance such as a fluorescent substance, is added.
(2) Radical sources such as aryl azide constituting the EMARS reagent are radicalized in the presence of HRP and attack molecules gathered in a limited range on the EV membrane (within 300 nm near HRP), and CHL1 Labels molecules (partner proteins) that associate with.
(3) After the EMARS reaction, the molecule (partner protein) labeled with the EMARS reagent is identified by an antibody or other protein analysis method.

As the EMARS reagent, a commercially available EMARS reagent (Tokyo Mirai Style) can also be used.

By the EMARS reaction, the CHL1 partner protein (protein forming a complex with CHL1) labeled with the labeling substance may be detected by a known method such as immunoprecipitation, Western blotting, ELISA, and mass spectrometry.
As the detection reagent used for detecting the CHL1 partner protein, a substance that specifically binds to the CHL1 partner protein can be used, and an anti-CHL1 partner protein antibody can be preferably used.
The CHL1 partner protein bound to CHL1 can also be quantified by Western blotting band intensity, ELISA, fluorescence measurement, or the like.

In the method of the invention, "analyzing a complex of CHL1 and a CHL1 partner protein" comprises detecting and / or quantifying the complex.
Lung cancer can be tested based on the presence or abundance of the complex. That is, the method of the present invention can provide data for diagnosing lung cancer.

For example, if a complex is detected, for example, if Caspase-14 bound to CHL1 is detected, the subject can be determined to have or are at high risk for lung cancer. It is also possible to predict the prognosis of lung cancer.
In addition, when the amount of the complex is a certain amount or more, for example, when the amount is 1.5 times or more or twice or more that of a healthy person, the subject is determined to have lung cancer or have a high risk of lung cancer. be able to. When the cutoff value is predetermined and higher than that value, the subject can be determined to have lung cancer or to be at high risk of lung cancer.

The test result by the method of the present invention may be combined with other lung cancer marker tests, imaging tests, and the like.
A doctor can diagnose lung cancer based on the measurement results of the present invention, and based on the results, can formulate a treatment policy such as administration of a lung cancer therapeutic agent or lung cancer surgery.

Another aspect of the invention provides a kit for testing lung cancer comprising an anti-CHL1 antibody and a CHL1 partner protein detection reagent.
The anti-CHL1 antibody and CHL1 partner protein detection reagent (preferably anti-CHL1 partner protein antibody) are as described above.

When performing the EMARS method, anti-CHL1 antibody labeled with a radical generating enzyme such as HRP and EMARS
Reagents may be included.

In addition, the kit of the invention may include reagents for separating EVs from a subject's blood sample.
In addition, reagents such as a standard substance, a labeled secondary antibody, a substrate thereof when the label is an enzyme, and a blocking agent such as BSA can be included. In addition, package inserts describing procedures and diagnostic criteria may be included.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

<Materials and methods>
The C57BL / 6J mice used as animal wild-type mice are self-propagated with a pair of mice purchased from Japan CLEA Co. (Tokyo, Japan) and carry a specific pathogen at 20 to 25 ° C. The animals were bred and maintained in a non-existent state (SPF).
EML4-ALK transgenic mice (Proc Natl Acad Sci 105 (50): 19893-19897.2008) were also bred and maintained in an SPF state at room temperature.
All animals were maintained in an environment of 12 hours in the light period / 12 hours in the dark period, and food and water were freely ingested.

Human serum samples Serum samples for healthy people and lung cancer patients are available at the Medical Genome Center Biobank, the National Center for Geriatrics and Gerontology, and the Japan Agency for Medical. The one obtained from the BioBank Japan Project (J Epidemiol 27 (3): S2-S21, 2017) supported by Research and Development (AMED) was used.

Cell culture and EV isolation
LK2 human squamous cell lung cancer cell line (RIKEN CELL BANK) 5% CO using RPMI 1640 medium (Wako Chemicals) supplemented with 5% fetal bovine serum (FBS; GIBCO) The cells were cultured in humidified air at 37 ° C containing _2. In order to recover EV secreted from LK2 cells, the medium was replaced with ASF medium 104 (Ajinomoto), which is a serum-free medium, and then the cells were replaced with 2 cells.
Incubated continuously for days. The medium fraction obtained by culturing was partially purified by treating the secreted EV with ExoQuick-TC Exosome Precipitation Solution (20 μl / 100 μl serum; System Biosciences) overnight at 4 ° C. Precipitates containing EV were solubilized in 100 μl PBS and then used for further analysis.

Preparation of HRP-binding antibody for EMARS reaction Mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R & D systems) were partially reduced and bound to HRP using the peroxidase labeling kit SH (Dojindo). .. The binding ability of HRP-labeled antibody was evaluated according to the previous report (Cancer Sci 110 (8): 2607-2619, 2019).

Purification of Serum EV and EMARS Reaction Serum EV from mouse and human serum was purified in combination with polymer precipitation and size exclusion chromatography. Mouse serum and blue dextran (Sigma-Aldrich) to determine which fractions should be recovered for EV purification in size exclusion chromatography.
Was placed on a short column packed with 2 ml of Sephacryl S-500 resin (GE Healthcare Life Sciences) and equilibrated with PBS. To avoid non-specific binding of EV to the resin, the resin was pretreated with yeast extract solution (BD Biosciences; 0.1 g / 100 ml H ₂ O, 1 hour). The sample fraction was then eluted with 100 μL PBS / fraction. The protein concentration of each fraction was measured at 562 nm using a micro BCA protein assay kit (Thermo Fisher Scientific) and a VarioSkan Flash microplate reader (Thermo Fisher Scientific). Blue dextran was measured at 300 nm using a VarioSkan Flash microplate reader.

Serum (50-100 μl) was treated with ExoQuick Exosome Precipitation Solution (25.2 μl / 100 μl serum; System Biosciences) on ice for 30 minutes. The precipitate (hereinafter referred to as precipitated EV) was solubilized with 100 μl of PBS, and then 0.25 μg of HRP-binding anti-mouse or human CHL1 antibody was added with gentle stirring. After incubating for 20 minutes at room temperature, the precipitated EV mixture was run on a short column packed with 2 ml Sephacryl S-500 equilibrated with PBS. Stepwise elution was performed by feeding PBS by gravity drop (100 μl PBS / fraction). Fractions No. 6-8 containing EV with HRP-bound CHL1 antibody were collected for each experiment, added to Nanosep® centrifuge device (30K), and then centrifuged at 8,000 g for 5 minutes to produce EV particles. Was concentrated. Wash the concentrated EV particles on the filter by centrifuging once with 100 μl PBS, followed by the EMARS reaction reagent fluorescein-tyramide (FT) reagent (Cancer Sci 110 (8): 2607-2619, 2019). It was added onto the filter with gentle stirring. After incubating for 10 minutes at room temperature, the residual FT reagent was removed by centrifugation at 8,000 g for 5 minutes and then washed by centrifugation once with PBS. 50 mM containing 1% SDS on EMARS-reacted EV protein on filter
30 μl of Tris-HCl (pH 7.4) was added, gently pipetting, and transferred to a new tube for recovery. Approximately 1 ml of Sephadex G-50 (GE Healthcare Life Sciences) equilibrated with 50 mM Tris-HCl (pH 7.4) containing 1% SDS of the recovered sample to remove excess FT reagent.
Was placed on a mini column filled with. The eluted sample (about 30 μl) was used for analysis of EMARS products such as immunoprecipitation, Western blotting, and ELISA described later. When EMARS products were detected directly on SDS-PAGE gels, the gels were measured directly in gel fluorescence detection mode (blue light filter for fluorescein) using a ChemiDoc MP image analyzer (BIO-RAD). Post-exposure gels were stained with Coomassie Brilliant Blue solution for loading control.

Cryo electron microscopy
To image the EV with cryo-electron microscopy, place the grid on a glow discharged holey carbon grid (Quantifoil Mo 2/2, 200 mesh) with 3 μl of sample. Made. Sample LEICA EM GP2
Was embedded in ice at a humidity of 100% and 10 ° C. Images were acquired using a Volta phase plate in linear mode using a FEI Talos Arctica microscope (200 kV) equipped with a Falcon III detector. Dose rate (dose rate) 10e ^- and / pixel / sec, the total irradiation electron dose (total accumulated dose) at 4 seconds to about 40 e ^- has acquired the data as / Å ^2.

Purification and concentration of EMARS products
EMARS products were purified and concentrated according to previously reported using Sepharose conjugated with an anti-fluorescein antibody (Cancer Sci 110 (8): 2607-2619, 2019). Specifically, the pass-through fraction of the G-50 column described above was diluted with 300 μL of NP-40 lysis buffer. Next, 20 μl of the anti-fluorescein antibody Sepharose 20 μl prepared by binding reaction between the anti-fluorescein antibody (600101096; Rockland or 6400-01; Southern Biotech) and NHS activated Sepharose 4 Fast Flow (GE Healthcare Life Sciences) was added to the sample. Then, the mixture was mixed while rotating at 4 ° C. overnight. After washing the resin, add an appropriate solution (reduced SDS sample buffer for Western blotting or 1% SDS solution containing MPEX PTS reagent (GL Science) for MS analysis) to Sepharose beads and heat at 95 ° C for 5 minutes. , The fluorescein-labeled molecule was eluted from the resin.

Proteomics analysis of EMARS products using mass spectrometry Proteomics analysis was performed using nano liquid chromatography / electrospray ionization mass spectrometry (nano LC-ESI-MS / MS). Specifically, a peptide sample of the EMARS product obtained from serum EV was purified in the same manner as described above and injected into a nano-UHPLC device (Bruker Daltonics). A maXis-4G-CPR (Bruker Daltonics) mass spectrometer equipped with a nano-ESI source was used for mass spectrometry. Magic C18AQ UHPLC NanoTrap column (particle size 5 μm, pore size 200)
An L-column ODS (0.1 × 150 mm, particle size 3 μm, Ceri) with Å) connected upstream was used. The peptide was eluted from the column in 50 minutes with a gradient of 10% to 35% using acetonitrile. The eluted peptides were directly electrosprayed into a spectroscope and MS / MS spectra were acquired in data dependent mode.

For proteomics analysis of BiEV in human serum EV, peptide samples of EMARS products prepared in the same manner as mouse samples were injected into Ultimate 3000 RSLC nano (Thermo Fisher Scientific). Mass spectrometry was performed using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) equipped with a nano ESI source. A NIKKYO nano HPLC capillary column (3 μm C18, 75 μm ID x 120 mm; Nikkyo Technos Co., Ltd.) with a C18 PepMap100 column (300 μm ID x 5 mm; Thermo Fisher Scientific) connected upstream was used. The peptide was eluted with acetonitrile using a gradient of 4% -35% in 97 minutes. The eluted peptides were directly electrosprayed and introduced into the spectroscope for precursor ion scanning and data-dependent MS / MS scanning.

Raw data from Proteome Discoverer ver. 2.2 software (Thermo Fisher Scientific)
) Was used for analysis. The parameters are as follows: Cys alkylation: iodoacetamide Digestion: Trypsin, Species: Homo sapiens or Mus musculus, FDR (false discovery rate): strict
1%, relaxed 5%.

Western Blotting Analysis To perform Western blot analysis, the sample solubilized in reduced SDS sample buffer (purified EV, etc.) or the sample eluted from the concentrated purified resin described above should be adjusted according to the molecular weight of each candidate protein. Was subjected to 6-12% SDS-PAGE using typical SDS running buffer or Rapid Running Buffer Solution (Nacalai tesque). The gel was then blotting to Immobilon®-P PVDF Membrane (Millipore). After blocking with 5% skim milk solution, the transferred membrane was reacted with the following primary antibody: anti-fluorescein antibody (600101096; Rockland; 0.2 μg / ml), anti-mouse and human CHL1 antibody (AF2147 and MAB2126; R & D systems). 1 μg / ml), HRP-binding anti-mouse and human CHL1 antibody (above), anti-α2 integrin antibody (ab133557; Abcam; 1 μg / ml), anti-β1 integrin antibody (610467; BD transduction laboratories; 0.25 μg / ml) , Anti-FGFR3 antibody (c-15; Santa cruz; 0.2 μg / ml 5% BSA-PBS), Anti-TSG101 antibody (EXOABTSG101-1; System Biosciences; 1: 500), Anti-CD63 antibody (EXOAB-CD63A-1; System) Biosciences; 1: 500), anti-CD5L antibody (AF2834; R & D systems; 0.4 μg / ml), anti-Pregnancy Zone Protein (PZP) antibody (PAG324Ra01; CLOUD-CLONE; 0.5 μg / ml), anti-SLC4A1 antibody (18566-1) -AP; PROTEINTECH; 0.6 μg / ml), anti-thrombospondin 1 (THBS1) antibody (PAA611Hu01; CLOUD-CLONE; 0.5 μg / ml)
And anti-Caspase-14 antibody (MAB8215; R & D systems; 0.5 μg / ml). The reaction with the primary antibody was carried out at room temperature for 1 hour to overnight. Then the appropriate secondary antibodies are HRP-bound anti-mouse IgG (Promega; 1: 3000-5000), HRP-bound anti-rabbit IgG (Promega; 1: 3000-5000), HRP-bound anti-goat IgG (Santa Cruz; 1: 3000-5000), Rabbit or Mouse TrueBlot®: Anti-rabbit or mouse IgG HRP (Rockland; 1: 1000) or HRP-bound anti-rat IgG (Santa Cruz; 1: 3000-5000) reacted at room temperature for 1 hour. rice field. After treatment with the antibody, the membrane was reacted with Immobilon Western Chemiluminescent HRP Substrate (Millipore). Exposing the membrane, ChemiDoc
It was analyzed by MP Image analyzer (BIO-RAD). Pre-stained Protein as a molecular weight marker
Markers, Broad Range (Nacalai Tesque) was used. SDS-PAGE gels were stained with Coomassie Brilliant Blue solution as a loading control. When stripping the membrane, the membrane was treated with a stripping solution (Wako Chemicals) at room temperature for 20 minutes, reblocked and restained with the appropriate antibody.

ELISA of CHL1 protein in serum or EV
The amount of CHL1 in mouse serum was measured by the direct ELISA method using HRP-binding anti-mouse and human CHL1 antibody according to the previous report (Biochem Biophys Res Commun 501 (4): 982-987, 2018). When measuring the amount of human CHL1 in serum and serum EV by sandwich ELISA, an ELISA plate in which the above-mentioned diluted serum or precipitated serum EV is coated with an anti-human CHL1 antibody (10143-MM05; Sino Biological) as a supplementary antibody. After addition to, it was detected with an HRP-bound anti-human CHL1 antibody (above). Relative index values (CHL1) for measuring CHL1 levels in mouse serum were determined based on the values of the reference sample described above (Biochem Biophys Res Commun 501 (4): 982-987, 2018). In the case of human CHL1 ELISA, recombinant human CHL1 partial protein (10143-H08H; Sino Biological)
Was used as a reference.

Fluorescein-labeled protein ELISA
Anti-mouse and human SLC4A1 antibody (18566-1-AP; PROTEINTECH; 1 μg / ml PBS) on a 96-well ELISA plate (# 3369; CORNING) to measure fluorescein-labeled SLC4A1 or Caspase-14 using sandwich ELISA. Or anti-human Caspase-14 antibody (H00023581-M01; Abnova)
(0.6 μg / ml PBS) was coated overnight at 4 ° C. After blocking with a 1% BSA-PBS (5% skim milk-PBS for Caspase-14) solution at 37 ° C. for 1 hour, add the corresponding EMARS product containing fluorescein-labeled SLC4A1 or fluorescein-labeled Caspase-14. 37 37
Incubated at ° C for 1 hour. Gently wash each well with PBST (0.05% tween-PBS solution) and then _{follow the manufacturer's instructions using the peroxidase labeling kit NH 2} (Dojindo) to anti-fluorescein antibody (600101096; It was treated with an HRP-binding anti-fluorescein antibody (1 μg / ml) prepared from Rockland) at 37 ° C. for 1 hour. When detecting the SLC4A1 molecule itself, use the HRP-binding anti-SLC4A1 antibody prepared according to the manufacturer's instructions using the recombinant human SLC4A1 polypeptide (CSBEP021663HU; CAUABIO THECHNOLOGY) and the Zenon rabbit IgG HRP labeling kit (Thermo Fisher Scientific). did. Color was developed by reacting SureBlue / TMB peroxidase substrate (100 μl / well; Sera care) at room temperature for an appropriate period of time (3-10 minutes). After stopping the reaction with 2M HCl solution, the absorbance (OD 450 nm) was measured using a Varioskan Flash microplate reader (Thermo Fisher Scientific). A reference sample of fluorescein-labeled SLC4A1 (100 μl) was prepared as an EMARS product obtained from 100 μl serum of a large tumor-carrying EML4-ALK gene-introduced mouse (30-week-old male). In order to offset the difference between the ELISA plates, the relative value based on the value of the reference sample was calculated as the “BiEV index value”. The "BiEV index value (SLC4A1)" used in this study indicates how many microliters correspond to the above-mentioned reference sample. In the case of fluorescein-labeled Caspase-14, "BiEV index value (Caspase-14)" based on the fluorescein-labeled recombinant Caspase-14 protein (11856-H07E; Sino Biological) labeled with NHS-fluorescein (Thermo Fisher Scientific). "It was used. Caspase-14 protein (2.5 μg) and NHS-fluorescein (8 μg)
) Was mixed in 20 μl PBS and then incubated at room temperature for 50 minutes. To remove excess NHS-fluorescein, the reaction mixture was run on a mini-column packed with about 0.5 ml of Sephadex G-50 (GE Healthcare Life Sciences).

<Results and discussion>
EMARS reaction of serum EV derived from EML4-ALK gene-introduced mouse It was confirmed by Western blot analysis using CD63 (protein expressed on EV membrane) that EV could be crudely purified from mouse serum (not shown). .. A large amount of CHL1 was detected in serum EV derived from EML4-ALK transgenic mice (not shown).

A typical overview of BiEV analysis in serum EV is shown in FIG. EMARS products derived from precipitated serum EV (crude purified EV) were detected in the CHL1 probe (+) sample, but the EMARS probe (-)
Was not detected in the sample. This suggests that the CHL1 probe responded adequately to EMARS and that serum factors did not elicit a non-specific EMARS response. However, fluorescein-labeled EMARS products contained so many serum proteins that size exclusion chromatography was also performed. Sephacryl S-500 Fraction No. fractioned with resin
6-8 had a limited amount of serum protein contamination and were most suitable for serum EV recovery (Fig. 2A). Cryo-electron microscopy shows that samples of fractions No. 6-8 show a large number of spherical particles with a size of several tens of nanometers together with a lipid bilayer (Fig. 2B), each fraction having a diameter of about 20. It contained an EV of ~ 100 nm. No obvious serum protein contamination appeared (Fig. 2B).

Serum-derived mice: wild-type (WT) mice, EML4-ALK-introduced mice with large tumors (TL; tumor weight> 0.15 g) and EML4- with small tumors (TS; tumor weight of 0.1 g or less) Serum was divided into 3 groups according to the ALK gene-introduced mice. To obtain averaged experimental results in multiple mice, 10 μl of serum from 10 mice in each group was mixed in equal proportions and used in the next experiment.
EMARS products were detected in TL mouse-derived samples, but not in WT and TS mice (Fig. 2C). The band pattern of the WT sample was similar to that of the TS sample (Fig. 2C). TSG101 and CD63 molecules are present in the TL sample to some extent compared to the other two samples, which means that the large tumor tissue has a certain amount of cEV (cancer EV) expressing CHL1. The secretion suggests that a signal of EMARS product was seen in the TL sample.

Identification of CHL1 Partner Molecules in Serum cEV Due to the low amount of labeled molecules in serum cEVs of small tumor-carrying mice, it may be difficult to detect specific BiEVs from serum cEVs. Candidates for CHL1 BiEV partners suitable for tumor screening in EML4-ALK transgenic mice were investigated by mass spectrometric (MS) proteomics using both TL and TS EMARS products and four candidate molecules (CD5L). , PZP, THBS1 and SLC4A1) were selected.

Western blot analysis with anti-CD5L antibody showed a moderate band at about 60 kDa in the TL sample, but no clear band in the other two samples. After stripping, the same membrane was restained with anti-PZP antibody. High concentrations of PZP (approximately 130 kDa) were detected in the WT sample. This result suggests that some CHL1 BiEVs are also expressed in serum EVs derived from WT mice.

In contrast, Western blot analysis with anti-SLC4A1 and anti-thrombospondin 1 (THBS1) antibodies confirmed moderate bands in both TL and TS samples, but not in WT samples. (Fig. 2D). Therefore, it was considered that the measurement of CHL1-SLC4A1 BiEV in serum EV could be an effective index in cEV screening of EML4-ALK gene-introduced mice.

Measurement of CHL1-SLC4A1 BiEV using sandwich ELISA The amount of CHL1 in serum has been identified as an important indicator for EML4-ALK transgenic mice carrying moderately large lung cancer (Non-Patent Document 1). However, this was insufficient to be an indicator of EML4-ALK transgenic mice carrying small cancer tumors.
CHL1-SL using sandwich enzyme-linked immunosorbent assay (ELISA) to determine if CHL1-SLC4A1 BiEV in serum EV is elevated in EML4-ALK transgenic mice, especially TS mice.
Measurements of fluorescein-labeled SLC4A1 indicating the amount of C4A1 BiEV were performed.

The BiEV index values (SLC4A1) of WT mice and EML4-ALK gene-introduced mice are summarized in Fig. 3A. The serum CHL1 level of small tumor-carrying mice was not significantly different from that of WT mice, but the BiEV index value (SLC4A1) of EML4-ALK gene-introduced mice tended to be higher than that of WT mice as a whole. (Fig. 3A). The mean value of the BiEV index value (SLC4A1) of WT mice was 0.1457 (standard deviation [SD] = 0.0679), and the mean value of EML4-ALK transgenic mice was 0.3162 (SD = 0.1979). A p-value of 8.9 × 10 ^-3 indicates that the difference in the amount of CHL1-SLC4A1 BiEV between WT mice and EML4-ALK transgenic mice is significant (Fig. 3B). The area under the curve (AUC) and cutoff value based on the ROC curve of each BiEV index value (SLC4A1) were calculated to be 0.782 and 0.238, respectively (Fig. 3C).
.. In addition, Spearman's rank correlation coefficient showed a correlation between the BiEV index value (SLC4A1) and the tumor volume (Fig. 3D). This suggests that cEV expressing CHL1-SLC4A1 BiEV was not secreted so much in the small tumor state, and as a result, a correlation was found that was somewhat proportional to the tumor size. In contrast to the increase in CHL1-SLC4A1 BiEV in EML4-ALK transgenic mice, the average SLC4A1 protein expression in whole-serum EV of EML4-ALK transgenic mice was decreased. Was observed (Fig. 3E).

BiEV analysis and measurement in lung cancer patients
Since BiEV was found to be useful for screening serum EV in mice, similar analysis was performed on serum EV obtained from healthy individuals and lung cancer (LC) patients. As in the model mouse experiment, sera collected from healthy individuals (H) and LC patients were mixed in equal proportions and used in the next experiment. EMARS products obtained using the CHL1 EMARS probe were detected in both serum EVs (Fig. 4A). To confirm whether the fluorescein-labeled SLC4A1 identified in the model mouse experiment can be detected in human LC samples, Western blot analysis and ELISA quantification of SLC4A1 were performed on the EMARS products obtained from H and LC. rice field. It was suggested that suitable BiEV could be identified in human samples.

MS proteomics identified potential BiEV partners for CHL1 in patients with lung cancer. The results are shown in Table 1.

From immunoprecipitation and Western blot analysis, Caspase-14 (J Cell Biol 180 (3): 451-8. 2008), a member of the caspase family involved in keratinocyte differentiation, is the partner of CHL1 BiEV in LC-derived serum EV. It was found to be detected in large quantities (Fig. 4B: dimer and monomer). Fluorescein-labeled Caspase-14 was also detected in the EMARS product of EV secreted from LK2 LC cells, suggesting that CHL1-Caspase-14 BiEV is a typical indicator of EV secreted from LC cells. ing. Therefore, the measurement of CHL1-Caspase-14 BiEV in serum EV was considered to be a suitable index for cEV screening in LC.

To measure fluorescein-labeled Caspase-14 in EMARS products using sandwich ELISA, recombinant Caspase-14 was used to make a reference material for fluorescein-labeled Caspase-14. The Caspase-14 ELISA system was suitable for sensitive measurement of fluorescein-labeled Caspase-14 reference material, with a calibration curve correlation coefficient of 0.9878. BiEV index values (Caspase-14) were set based on measurements of fluorescein-labeled Caspase-14 reference material (ng / mL) to compare the results of each ELISA experiment between different plates.

BiEV index values (Caspase-14) obtained from EVs of H and LC sera are summarized in Fig. 4C. The EV of LC tended to be higher than the EV of H as a whole. The average value of the BiEV index value (Caspase-14) of EV of H was 1.065 (SD = 1.168), and the average value of EV of LC was 3.707 (SD = 2.829). p
The value of 0.019 indicates that the difference in the amount of CHL1-Caspase-14 BiEV between the EV of H and the EV of LC was significant (Fig. 4D). The AUC and cutoff values obtained based on the ROC curve of each BiEV index value were 0.811 and 1.889, respectively (Fig. 4E). No increase in total protein expression of Caspase-14 molecules throughout serum EV was observed between H and LC serum EV (Fig. 4F).

As described above, lung cancer can be efficiently detected by analyzing the complex of CHL1 and CHL1 partner protein in the method of the present invention, and in particular, the complex of CHL1 and Caspase-14 in the serum EV membrane is used for lung cancer diagnosis. It turned out to be very good as an indicator for.

Claims (7)

A method for testing lung cancer, which comprises the step of analyzing a complex of CHL1 present in the membrane of extracellular vesicles present in a blood sample of a subject and a CHL1 partner protein. CHL1 partner proteins are Caspase-14, SLC4A1 (Solute Carrier Family 4 Member 1)
, Thrombospondin 1, Desmoplakin, Lactotransferrin, 14-3-3 protein sigma, Heat shock protein beta-1, Annexin A2, Calmodulin-like protein 3, Galectin-7, Desmocollin-1, Fatty acid-binding protein 5, Transgelin-2 , Annexin A1, Alpha-enolase, Glutathione S-transferase P, Tubulin beta-2A chain, 14-3-3 protein zeta / delta, Tubulin alpha-4A chain, Epiplakin, Polymeric immunoglobulin receptor, Tubulin alpha-1B chain, Serpin B3 , Tubulin beta-4B chain, Triosephosphate isomerase, Protein S100-A14, Protein S100-A11, Zymogen granule protein 16 homolog B, Calmodulin-like
The method for testing lung cancer according to claim 1, wherein the protein is protein 5, Prelamin-A / C, Elongation factor 1-alpha 1, or Heat shock cognate 71 kDa protein. The method for testing lung cancer according to claim 1, wherein the CHL1 partner protein is Caspase-14. The lung cancer testing method according to any one of claims 1 to 3, wherein the analysis is performed by the EMARS (Enzyme-Mediated Activation of Radical Sources) method. Lung cancer testing kit containing anti-CHL1 antibody and CHL1 partner protein detection reagent. The kit for lung cancer testing according to claim 5, wherein the anti-CHL1 antibody is a horseradish peroxidase (HRP) -binding antibody, and the kit contains an EMARS reagent. The lung cancer testing kit according to claim 5 or 6, further comprising a reagent for separating extracellular vesicles.

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