Extracellular vesicle lung cancer biomarker, application thereof and lung cancer screening kit

Abstract

The invention discloses an extracellular vesicle lung cancer biomarker, application thereof and a lung cancer screening kit, and belongs to the technical field of molecular diagnosis. The invention discovers that the expression levels of the extracellular vesicle surface proteins SFTPA1, SCGB1A1, SFTPB and SFTPC are related to the lung cancer for the first time, so that the proteins can be used as biomarkers for screening the lung cancer. Furthermore, based on the biomarker, the invention provides a kit for screening lung cancer, which has the advantages of high accuracy, rapidness, convenience, non-invasiveness and the like, and provides support for early diagnosis of lung cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular diagnosis, and particularly relates to a lung cancer marker of extracellular vesicles and application of the marker and a lung cancer screening kit. BACKGROUND

[0002] According to the 2022 cancer statistics report of the American Cancer Society, lung cancer is the second most common disease in men and women. At present, the clinical lung cancer screening mainly adopts low-dose computed tomography (LDCT), but not all early lung cancers have significant imaging findings. Therefore, imaging combined with biopsy is the gold standard for diagnosing lung cancer. However, most lung cancer biopsy methods are invasive, and patients may be accompanied by delayed pneumothorax, hemothorax, infection, etc. Biopsy needs to be performed by doctors with rich clinical experience and skilled operation, and most primary hospitals cannot carry out it, so it is crucial to develop a lung cancer screening kit.

[0003] Liquid biopsy is a non-invasive or minimally invasive examination of body fluids to diagnose cancer, which can detect and analyze circulating tumor cells, circulating tumor DNA, extracellular vesicles (EVs) released from tumor sites into the circulation, and has become a breakthrough method for cancer detection, dynamic detection of disease progression, and auxiliary treatment selection. Among them, EVs are lipid bilayer membrane structure vesicles existing in various body fluids, which can transport various nucleic acids, proteins, lipids, small molecules and other essential information to realize cell-to-cell communication and information interaction with the extracellular microenvironment, and directly affect cell migration, cell growth, cell differentiation and other processes. EVs produced by tumor cells have been shown to promote tumor growth, promote tumor cell migration, induce immune escape, and enhance drug resistance, so EVs are considered as potential biomarkers for lung cancer diagnosis.

[0004] The high-throughput nanobiochip integrated system (HNCIB) for liquid biopsy is a detection system with high reliability, high sensitivity and high specificity. The detection system has the characteristics of high throughput (up to 384 samples per detection), short detection time (~6h), and small sample size (~90μL) required. The technology uses total internal reflection fluorescence (TIRF) microscopy to detect captured EVs to identify potential biomarkers on the EV membrane. The system can automatically analyze image data, and the HNCIB uses deep learning algorithms for automatic analysis to achieve automatic high-throughput image screening, quantitative analysis of biomarkers, and co-localization of various proteins. Compared with the methods currently recorded in the literature (such as dynamic light scattering, transmission electron microscopy, and Zeta potential measurement), the analysis method is more practical and has a high signal-to-noise ratio. By introducing a rule-based algorithm in the system, the signal quality can be greatly improved, thereby enabling more objective evaluation. Since EVs exist in large quantities in various blood, EVs can be separated and the biomarker information carried by EVs can be detected by using the high-sensitivity and high-specificity HNCIB technology.

[0005] The application creatively proposes that lung cancer patients can be screened by detecting EVs markers SFTPA1, SCGB1A1, SFTPB and SFTPC, and has the advantages of high accuracy, convenience and efficiency. At present, no study has focused on the potential of EVs carrying markers SFTPA1, SCGB1A1, SFTPB and SFTPC in lung cancer diagnosis. In addition, the rapid detection kit for the extracellular vesicle lung cancer screening marker provided by the application has not been reported. SUMMARY

[0006] One object of the application is to provide a set of extracellular vesicle lung cancer markers and a substance for detecting the markers for use in the preparation and / or screening of products for diagnosing or assisting in the diagnosis of lung cancer. A second object of the application is to provide a set of antibodies that can specifically bind to the extracellular vesicle lung cancer markers. Another object of the application is to provide a kit that can be used for diagnosing or assisting in the diagnosis of lung cancer, and has the advantages of high accuracy, convenience and efficiency.

[0007] In a first aspect, the application provides a use of an extracellular vesicle biomarker in the preparation and / or screening of products for diagnosing or assisting in the diagnosis of lung cancer.

[0008] The extracellular vesicle biomarker is selected from one or a combination of two or more of SFTPA1, SCGB1A1, SFTPB and SFTPC.

[0009] Preferably, the extracellular vesicle biomarker is selected from a combination of SFTPA1, SCGB1A1, SFTPB and SFTPC.

[0010] In a second aspect, the present application provides use of a substance for detecting an extracellular vesicle biomarker in the manufacture and / or screening of a product for diagnosing or aiding in the diagnosis of lung cancer.

[0011] The product includes but is not limited to a reagent, a kit, a chip, a test paper, a membrane strip or a detection platform.

[0012] The extracellular vesicle biomarker is selected from one or a combination of two or more of SFTPA1, SCGB1A1, SFTPB and SFTPC.

[0013] Preferably, the extracellular vesicle biomarker is selected from a combination of SFTPA1, SCGB1A1, SFTPB and SFTPC.

[0014] The substance for detecting the extracellular vesicle biomarker includes any reagent required for detecting the protein content or gene expression level of the extracellular vesicle biomarker by RT-PCR, RT-qPCR, biochip detection, Southern blotting, in situ hybridization or immunohistochemistry.

[0015] In some embodiments of the present application, the substance for detecting the extracellular vesicle biomarker is a reagent for detecting the protein content of the extracellular vesicle biomarker by immunohistochemical techniques. The immunohistochemical techniques include but are not limited to immunofluorescence and immunoenzyme labeling.

[0016] In a specific embodiment, the present application detects the protein content of the extracellular vesicle biomarker by immunofluorescence, and the detection reagent includes a fluorescent group-labeled antibody, polypeptide, protein or nucleic acid molecule that specifically binds to the extracellular vesicle biomarker. The fluorescent group is selected from one of FITC, FAM, CY3, CY5, JOE and ROX.

[0017] In a specific embodiment, the present application detects the protein content of the extracellular vesicle biomarker by immunoenzyme labeling, and the detection reagent includes an enzyme-labeled antibody, polypeptide, protein or nucleic acid molecule that specifically binds to the extracellular vesicle biomarker. The enzyme is selected from one of horseradish peroxidase (HRP), alkaline phosphatase (ALP) and β-galactosidase (β-Gal).

[0018] In a third aspect, the present application provides an antibody for detecting an extracellular vesicle biomarker, wherein the antibody is a fluorescent group-labeled or enzyme-labeled antibody selected from at least one of the following:

[0019] a) an antibody that specifically binds to the extracellular vesicle biomarker SFTPA1;

[0020] b) an antibody that specifically binds to the extracellular vesicle biomarker SCGB1A1;

[0021] c) an antibody that specifically binds to the extracellular vesicle biomarker SFTPB;

[0022] d) an antibody that specifically binds to the extracellular vesicle biomarker SFTPC.

[0023] In the most preferred embodiment of the present application, the antibody is a combination of the antibodies of a) - d) that are labeled with a fluorescent group or an enzyme.

[0024] The antibody that specifically binds to the extracellular vesicle biomarker of the present application is a monoclonal antibody prepared by a person skilled in the art according to conventional techniques.

[0025] In a fourth aspect, the present application provides a kit comprising the antibody of the third aspect of the present application.

[0026] Preferably, the kit further comprises a CD9, CD63 or CD81 capture antibody for capturing extracellular vesicles.

[0027] Preferably, the kit further comprises other conventional reagents required for immunofluorescence detection technology, chemiluminescence detection technology or enzyme-linked immunosorbent assay (ELISA), including but not limited to CD9, CD63 or CD81 capture antibody, chemiluminescence solution, color developing solution, etc.

[0028] In a fifth aspect, the present application provides a method for detecting an extracellular vesicle biomarker without the purpose of diagnosis or treatment, comprising the following steps:

[0029] (1) obtaining a biological sample to be tested;

[0030] (2) taking a biotin and avidin coated biochip, adding a capture antibody for incubation, and washing;

[0031] (3) adding the biological sample to be tested for incubation, and washing;

[0032] (4) adding a detection antibody for incubation, and washing;

[0033] (5) signal detection based on immunofluorescence detection technology, chemiluminescence detection technology or enzyme-linked immunosorbent assay.

[0034] The biological sample is selected from one or a combination of two or more of plasma, serum, oral sputum, alveolar lavage fluid, and pleural effusion.

[0035] In the most preferred detection method of the present application, the present application provides a high-throughput nanobiochip integrated system (HNCIB) based method for detecting the content of biomarkers on the membrane of captured extracellular vesicles using total internal reflection fluorescence (TIRF) microscopy.

[0036] The technical solutions provided by the present application have the following advantages:

[0037] The present application first creatively proposes a set of biomarkers on the surface of the membrane of extracellular vesicles, namely the combination of EV-SFTPA1, EV-SCGB1A1, EV-SFTPB and EV-SFTPC, which can be used for screening and detecting lung cancer, and has the advantages of high accuracy, convenience and efficiency.

[0038] SFTPA1, SCGB1A1, SFTPB and SFTPC have strong tissue specificity and are mainly synthesized by Club cells, alveolar type II cells and lung epithelial cells in the lung. SFTPA1, SFTPB and SFTPC proteins encoded by SFTPA1, SFTPB and SFTPC are mainly present in alveolar surfactant and belong to lung-specific proteins, which have the functions of regulating alveolar surface tension, maintaining alveolar stability and preventing alveolar collapse, and are related to the occurrence and development of many lung diseases. SCGB1A1 plays an important physiological function in the respiratory system and can promote the chemotaxis and aggregation of alveolar macrophages and other immune cells. These proteins closely related to the respiratory system are screened out and related to the occurrence and development of lung cancer. The present application believes that the research on SFTPA1, SCGB1A1, SFTPB and SFTPC helps to further understand the pathogenesis of lung cancer and provides guidance for the treatment and management of lung cancer.

[0039] The present application prepares a lung cancer screening method and a screening kit for the biomarkers, which has the following advantages:

[0040] (1) High diagnostic rate: the high sensitivity and high specificity ensure the accuracy of the diagnostic results and reduce the risk of misdiagnosis and missed diagnosis.

[0041] (2) Convenient and rapid: using the kit for lung cancer diagnosis does not require complex laboratory equipment and professional technical personnel, and only needs simple operation steps to complete the diagnosis process. The diagnostic results can be obtained in a short time, greatly shortening the waiting time of patients and improving the diagnostic efficiency.

[0042] (3) Non-invasive: compared with traditional lung cancer diagnosis methods, the kit does not need to perform puncture sampling, but only needs to obtain samples from the blood of patients for detection. This non-invasive detection method not only reduces the pain of patients, but also reduces the risk of infection and other complications.

[0043] The application proves for the first time that the expression levels of EV-SFTPA1, EV-SCGB1A1, EV-SFTPB and EV-SFTPC are related to lung cancer, and EV-SFTPA1, EV-SCGB1A1, EV-SFTPB and EV-SFTPC can be used as a marker for screening and diagnosing lung cancer, thereby providing a diagnostic kit for lung cancer screening that can be widely promoted. The new type of lung cancer diagnostic kit provides strong support for the early diagnosis and treatment of lung cancer due to its high accuracy, rapid and convenient, non-invasive and other advantages, and is expected to play an important role in clinical practice. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 are four indexes specifically highly expressed in lung cancer screened by bioinformatics technology.

[0045] Figure 2 are the biological functions of SFTPA1, SCGB1A1, SFTPB and SFTPC and the correlation with endocytic vesicles (late endosomes).

[0046] Figure 3 is a schematic diagram of in situ capture and detection of EVs.

[0047] Figure 4 is a schematic diagram of chemiluminescence detection.

[0048] Figure 5 are the expression levels of SFTPA1 protein in plasma EVs of the control group and lung cancer patients detected by TIRF, chemiluminescence and ELISA.

[0049] Figure 6 are the expression levels of SCGB1A1 protein in plasma EVs of the control group and lung cancer patients detected by TIRF, chemiluminescence and ELISA.

[0050] Figure 7 are the expression levels of SFTPB protein in plasma EVs of the control group and lung cancer patients detected by TIRF, chemiluminescence and ELISA.

[0051] Figure 8 are the expression levels of SFTPC protein in plasma EVs of the control group and lung cancer patients detected by TIRF, chemiluminescence and ELISA.

[0052] Figure 9 is the evaluation of the diagnostic efficiency of EV-SFTPA1, EV-SCGB1A1, EV-SFTPB and EV-SFTPC alone and in combination for diagnosing lung cancer and non-lung cancer patients by ROC curve. DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of, rather than all of, the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0054] Extracellular vesicle lung cancer biomarker detection

[0055] Example 1

[0056] Step one: sample collection and pretreatment

[0057] S1: centrifuge the peripheral blood specimen at 820g for 10 minutes at room temperature to obtain plasma;

[0058] S2: centrifuge at 16000g for 10 minutes at 4℃, sub-pack and gradually freeze-preserve (2 hours at 4℃, 30 minutes at 20℃, and preservation at 80℃);

[0059] S3: quickly thaw the frozen sample in a 37℃ water bath, add into an EP tube, and centrifuge at 300g for 5 minutes at room temperature

[0060] S4: carefully transfer the supernatant into a new Eppendorf (EP) tube, centrifuge at 2000g for 10 minutes at room temperature to remove residual cell debris;

[0061] S5: collect the supernatant and transfer into a new EP tube, centrifuge at 12,000g for 30 minutes at 4℃ to remove large vesicles;

[0062] S6: transfer the supernatant into a new EP tube, add an equal volume of phosphate buffered saline (PBS), mix the sample uniformly, and filter with a 0.22μm filter.

[0063] Step two: detection based on immunofluorescence technology

[0064] S1: take out the biotin and avidin coated biochip (well plate), and wash once with PBS;

[0065] S2: cover the plate or stick the sealing plate film, incubate at 400rpm for 30 minutes at room temperature;

[0066] S3: remove the solution, and wash 3 times with PBS;

[0067] S4: take 1μL of CD63 capture antibody, add into 399μL of 1X PBS, and vortex to mix;

[0068] S5: add 100μL of capture antibody diluent into each well, stick the sealing plate film, and plate overnight at 4℃;

[0069] S6: remove solution, wash 3 times with PBS;

[0070] S7: add 300 μL 5% BSA in PBS, block at room temperature for 2 hours (cover or seal the plate film);

[0071] S8: remove solution, wash 3 times with PBS;

[0072] S9: add 100 μL sample, negative and positive controls per well, seal the plate film, and incubate at 37°C for 60 min;

[0073] S10: remove solution, wash 3 times with PBS;

[0074] S11: take 1 μL EV-SFTPA1-FITC, EV-SCGB1A1-FITC, EV-SFTPB-FITC, and EV-SFTPC-FITC detection antibodies, add to 999 μL 5% BSA in PBS, and mix well;

[0075] S12: add 100 μL detection antibody diluent per well, seal the plate film, and incubate at 37°C for 60 min;

[0076] S13: wash the plate 3 times with 350 μL 1X PBST (after each wash solution is soaked for 30 sec, discard it), and shake or tap the plate at the end;

[0077] S14: use TIRF microscopy to collect fluorescence signals, obtain fluorescence intensity related to lung cancer markers SFTPA1, SCGB1A1, SFTPB, and SFTPC, and use deep learning to analyze the data.

[0078] Example 2

[0079] Step one of Example 1;

[0080] Step two: detection based on chemiluminescence technology

[0081] S1: take out the biotin and avidin coated biochip (well plate), and wash once with PBS;

[0082] S2: cover or seal the plate film, rotate at 400 rpm, and incubate at room temperature for 30 min;

[0083] S3: remove solution, wash 3 times with PBS;

[0084] S4: take 1 μL CD9 capture antibody, add to 399 μL 1X PBS, and vortex well;

[0085] S5: Add 100 μL of capture antibody dilution to each well, seal the plate, and incubate at 4°C overnight;

[0086] S6: Remove the solution and wash 3 times with PBS;

[0087] S7: Add 300 μL of 5% BSA in PBS to each well, and incubate at room temperature for 2 hours (cover the plate or seal the plate with film);

[0088] S8: Remove the solution and wash 3 times with PBS;

[0089] S9: Add 100 μL of sample, negative control, and positive control to each well, seal the plate, and incubate at 37°C for 60 minutes;

[0090] S10: Remove the solution and wash 3 times with PBS;

[0091] S11: Take 1 μL of EV-SFTPA1-HRP, EV-SCGB1A1-HRP, EV-SFTPB-HRP, and EV-SFTPC-HRP detection antibody, add to 999 μL of 5% BSA in PBS, and mix well;

[0092] S12: Add 100 μL of detection antibody dilution to each well, seal the plate, and incubate at 37°C for 60 minutes;

[0093] S13: Wash the plate 3 times with 350 μL of 1X PBST (soak for 30 seconds after each wash and discard), and shake or tap the plate at the end;

[0094] S14: Add 100 μL of chemiluminescence solution to each well, shake the plate for 5 seconds, and incubate at room temperature in the dark for 5 minutes;

[0095] S15: Read the plate using a chemiluminescence instrument.

[0096] Example 3

[0097] Step one of Example 1;

[0098] Step two: detection based on ELISA technology

[0099] S1: Take out the biotin and avidin coated biochip (well plate), and wash once with PBS;

[0100] S2: Cover the plate or seal the plate with film, incubate at 400 rpm for 30 minutes at room temperature;

[0101] S3: Remove the solution and wash 3 times with PBS, and shake or tap the plate at the end;

[0102] S4: Take 1 μL of CD9 capture antibody, add to 399 μL of 1X PBS, and mix well by vortexing;

[0103] S5: Add 100 μL of capture antibody dilution to each well, seal the plate membrane, and incubate at 4℃ overnight.

[0104] S6: Remove the solution and wash 3 times with PBS.

[0105] S7: Add 300 μL of 5% BSA PBS, and seal the plate membrane at room temperature for 2 hours (cover plate or seal plate membrane).

[0106] S8: Remove the solution and wash 3 times with PBS.

[0107] S9: Add 100 μL of sample, negative control and positive control to each well, seal the plate membrane, and incubate at 37℃ for 60 min.

[0108] S10: Remove the solution and wash 3 times with PBS.

[0109] S11: Take 1 μL of EV-SFTPA1-HRP, EV-SCGB1A1-HRP, EV-SFTPB-HRP and EV-SFTPC-HRP detection antibodies, add to 999 μL of 5% BSA PBS, and mix well.

[0110] S12: Add 100 μL of detection antibody dilution to each well, seal the plate membrane, and incubate at 37℃ for 60 min.

[0111] S13: Wash the plate 3 times with 350 μL of 1X PBST (after each wash solution is soaked for 30 sec, discard), and shake or tap the plate at the last time.

[0112] S14: Add 50 μL of TMB color developing solution to each well, and incubate at room temperature for 15 min in the dark.

[0113] S15: Add 50 μL of termination solution to each well in time (pipette), shake the plate for 10 sec, and read the plate at 450 / 620 nm with an enzyme marker.

[0114] The present application screens four indexes, i.e., SFTPA1, SCGB1A1, SFTPB and SFTPC, which are specifically highly expressed in lung cancer, from gene expression data and corresponding detection information obtained from TCGA (The Cancer Genome Atlas) database. Figure 1 The biological functions of SFTPA1, SCGB1A1, SFTPB and SFTPC are related to endocytic vesicles (late endosomes). Figure 2

[0115] ​​​​​​​​​​​​​​​​​​​​​​​​​​In order to detect the specificity of the EV lung cancer screening marker SFTPA1, SCGB1A1, SFTPB, SFTPC rapid detection kit, 30 non-lung cancer patients' plasma is selected as the control group, and 30 lung cancer patients' plasma is selected as the experimental group. According to the detection method of the extracellular vesicle biomarker provided by the application, the detection results are as shown in Figure 5 、 6 、7、8, in the ELISA, chemiluminescence and immunofluorescence TIRF technology platform test, the protein expression amount of EV-SFTPA1, EV-SCGB1A1, EV-SFTPB, EV-SFTPC of the lung cancer patient plasma is higher than that of the control group, and the lung cancer patient and the healthy person can be distinguished.

[0116] The expression data of EV-SFTPA1, EV-SCGB1A1, EV-SFTPB, EV-SFTPC are analyzed by using the pROC package of R (4.2.1), and then the ROC curve is visualized and drawn by using ggplot2 AUC curve, and the area under the ROC curve (Area Under Curve, AUC) is used for the evaluation of the diagnostic efficiency of the lung cancer marker. The results show that, compared with the single diagnosis of the four markers, the diagnostic efficiency of EV-SFTPA1, EV-SCGB1A1, EV-SFTPB, EV-SFTPC combined diagnosis of lung cancer is higher. Figure 9 )

[0117] Finally, it should be pointed out that: the above embodiments are only used to illustrate the technical solutions of the application, and not to limit them; although the application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the application.

Claims

1. Use of an extracellular vesicle biomarker selected from one or more than two combinations of SFTPA1, SCGB1A1, SFTPB, SFTPC in the preparation and / or screening of a product for diagnosing or aiding in the diagnosis of lung cancer.

2. Use according to claim 1, characterized in that, The extracellular vesicle biomarker is selected from a combination of SFTPA1, SCGB1A1, SFTPB and SFTPC.

3. Use of a substance for detecting an extracellular vesicle biomarker selected from one or more than two combinations of SFTPA1, SCGB1A1, SFTPB, SFTPC in the preparation and / or screening of a product for diagnosing or aiding in the diagnosis of lung cancer.

4. Use according to claim 3, characterized in that, The product includes a reagent, a kit, a chip, a test paper, a membrane strip or a detection platform.

5. Use according to claim 3, characterized in that, The substance for detecting an extracellular vesicle biomarker includes any reagent required for detecting the protein content or gene expression level of an extracellular vesicle biomarker by RT-PCR, RT-qPCR, biochip detection, Southern blotting, in situ hybridization or immunohistochemistry.

6. A kit for detecting an extracellular vesicle biomarker, the kit comprising an antibody, the antibody being a fluorescent group or enzyme-labeled antibody of at least one of the following: a) an antibody specifically binding to the extracellular vesicle biomarker SFTPA1; b) an antibody specifically binding to the extracellular vesicle biomarker SCGB1A1; c) an antibody specifically binding to the extracellular vesicle biomarker SFTPB; d) an antibody specifically binding to the extracellular vesicle biomarker SFTPC.

7. The kit of claim 6, wherein The antibody is a combination of the antibodies of a)-d) labeled with a fluorescent group or an enzyme.

8. The kit of claim 7, wherein The kit further comprises a CD9, CD63 or CD81 capture antibody for capturing extracellular vesicles.

9. The kit of claim 7, wherein The kit further comprises other conventional reagents required for immunofluorescence detection technology, chemiluminescence detection technology or enzyme-linked immunosorbent assay.

10. A method for detecting an extracellular vesicle biomarker for non-diagnostic or therapeutic purposes, the method comprising the following steps: (1) obtaining a biological sample to be tested; (2) taking a biotin and avidin-coated biochip, adding a capture antibody for incubation, and washing; (3) adding a biological sample to be tested for incubation, and washing; (4) adding a detection antibody for incubation, and washing; (5) signal detection based on immunofluorescence detection technology, chemiluminescence detection technology or enzyme-linked immunosorbent assay; The biological sample is selected from one or more than two combinations of plasma, serum, oral sputum, alveolar lavage fluid and pleural effusion.

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