阅读说明：本技术 Ctsf在非小细胞肺癌诊断中的应用 (Application of CTSF (cytokine induced plasma) in non-small cell lung cancer diagnosis ) 是由 王琪 魏嵩 刘雯雯 徐明鑫 李恩成 于 2021-09-14 设计创作，主要内容包括：本发明涉及生物技术、生物医药领域,具体涉及CTSF在非小细胞肺癌诊断中的应用。在一种实施方式中,所述CTSF的表达量在患者体内升高,在晚期患者体内进一步升高。(The invention relates to the fields of biotechnology and biomedicine, in particular to application of CTSF in non-small cell lung cancer diagnosis. In one embodiment, the expression level of CTSF is increased in patients and further increased in patients at an advanced stage.)

1. The application of a reagent for detecting the expression level of CTSF in preparing a product for diagnosing lung cancer, wherein the expression level of CTSF is increased in a patient body;

preferably, the diagnosis includes diagnosing early stage lung cancer, differentiating early and late stage lung cancer;

preferably, the lung cancer is non-small cell lung cancer;

preferably, the early stage lung cancer is stage I-IIIA in TNM staging;

preferably, the advanced stage comprises four stages of distant target organ-free metastasis, lung cancer brain metastasis, lung cancer liver metastasis, lung cancer bone metastasis.

2. The use of claim 1, wherein said expression level comprises mRNA expression level and/or protein expression level.

3. The use of claim 2, wherein the agent includes, but is not limited to, the agents used in the following methods: immunodetection, in situ hybridization, RT-PCR, real-time quantitative PCR or chip detection.

4. The use of claim 3, wherein the reagent is a reagent used in an immunoassay;

preferably, the immunoassay includes, but is not limited to: enzyme-linked immunosorbent assay, radioimmunoassay, fluoroimmunoassay, immunohistochemistry, chemiluminescence immunoassay, and electrochemiluminescence immunoassay;

preferably, the immunoassay is an enzyme-linked immunosorbent assay and/or an immunohistochemical assay.

5. The use of claim 4, wherein the product for early diagnosis of lung cancer comprises an antibody that specifically binds to CTSF.

6. The use according to claim 5, wherein the early diagnosis lung cancer product comprises at least one of the following components used in protein experiments: blocking solution, antibody diluent, washing buffer solution, chromogenic stop solution and standard substance for preparing a standard curve.

7. A kit for diagnosing lung cancer, which comprises a reagent for detecting the expression level of CTSF in a sample of a subject;

preferably, the reagent for detecting the expression level of CTSF in a sample of a subject is the reagent for detecting the expression level of CTSF as described in any one of claims 1-6;

preferably, the lung cancer is non-small cell lung cancer;

preferably, the diagnosis includes diagnosing early stage lung cancer, differentiating early and late stage lung cancer;

preferably, the early stage lung cancer is stage I-IIIA in TNM staging;

preferably, the advanced stage comprises four stages of no distant target organ metastasis, lung cancer brain metastasis, lung cancer liver metastasis, lung cancer bone metastasis.

8. The kit of claim 7, wherein the sample includes, but is not limited to, blood, nasal epithelial cells, tissue, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusions, amniotic fluid, bladder irrigation fluid, and bronchoalveolar lavage fluid;

preferably, the sample is blood and/or tissue.

9.A system for diagnosing lung cancer, the system comprising computing means for determining whether a subject is diseased based on the CTSF expression level;

preferably, the diagnosis includes diagnosing early stage lung cancer, differentiating early and late stage lung cancer;

preferably, the early stage lung cancer is stage I-IIIA in TNM staging;

preferably, the advanced stage comprises four stages of no distant target organ metastasis, lung cancer brain metastasis, lung cancer liver metastasis, lung cancer bone metastasis;

preferably, the lung cancer is non-small cell lung cancer.

10. Use of the kit of claim 7 or 8, the system of claim 9 for the preparation of a product for the diagnosis of lung cancer;

preferably, the diagnosis includes diagnosing early stage lung cancer, differentiating early and late stage lung cancer;

preferably, the lung cancer is non-small cell lung cancer.

Technical Field

The invention relates to the fields of biotechnology and biomedicine, in particular to application of CTSF in non-small cell lung cancer diagnosis.

Background

According to statistics, about 160 million new cancer patients and about 130 million cancer death patients are counted in China. Cancer is the first cause of death among the many causes of death for residents in big and medium cities of our country, and cancer is the second cause of death among the causes of death in rural areas. Cancer is a frequently occurring and common disease for the middle-aged and the elderly. About 80% -90% of patients with early cancer can be cured; however, there are few patients with advanced cancer who survive more than 5 years after treatment. After the early cancer patient is treated, the survival rate is improved, and the survival quality of the patient is also improved. The mortality rate of cancer patients also decreases. It is well known that the treatment of patients with early stage cancer saves much more manpower, money and time than the treatment of patients with late stage cancer. Therefore, we advocate cancer patients to find, diagnose and treat early. In conditional places, regular health checks or cancer screening (preliminary screening) are better methods for finding early stage cancer.

Lung cancer is one of the most rapidly growing malignancies that threaten human health and life. In many countries, the incidence and mortality of lung cancer have been reported to be significantly higher in recent 50 years, with lung cancer incidence and mortality in men accounting for the first of all malignancies, in women accounting for the second, and mortality accounting for the second. The etiology of lung cancer is not completely clear up to now, and a large amount of data show that a large amount of smoking for a long time has a very close relationship with the occurrence of lung cancer. Existing studies have demonstrated that: the probability of lung cancer of a large number of smokers in a long term is 10-20 times that of non-smokers, and the smaller the smoking starting age is, the higher the probability of lung cancer is. In addition, smoking not only directly affects the health of the user, but also has adverse effects on the health of surrounding people, so that the lung cancer prevalence of passive smokers is obviously increased. The incidence of lung cancer in urban residents is higher than that in rural areas, which may be related to urban atmospheric pollution and carcinogens contained in smoke dust. So no smoking should be advocated and the urban sanitation work should be enhanced.

The clinical manifestations of lung cancer are complex, with symptoms and signs being present, mild and severe, and the morning and evening of the appearance, depending on the site of the tumor, the type of pathology, the presence or absence of metastasis and complications, and the differences in the extent of response and tolerance of the patients. Early lung cancer symptoms are often mild and may even be without any discomfort. Central lung cancer symptoms appear early and severe, while peripheral lung cancer symptoms appear late and mild, even asymptomatic, and are often found during physical examination. The symptoms of lung cancer are roughly classified into: local symptoms, systemic symptoms, extrapulmonary symptoms, infiltrates and metastases.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a molecular marker for early diagnosis of lung cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an application of a reagent for detecting CTSF expression level in preparing a product for diagnosing lung cancer, wherein the expression level of CTSF is increased in a patient body;

in one embodiment, the diagnosis includes diagnosing early stage lung cancer, differentiating early stage lung cancer from late stage lung cancer.

In one embodiment, the lung cancer comprises non-small cell lung cancer, small cell lung cancer.

In one embodiment, the lung cancer is non-small cell lung cancer.

Preferably, the early stage lung cancer of the present invention is stage I-IIIA in TNM staging.

Preferably, the advanced stage of the invention comprises four stages of distant target organ-free metastasis, lung cancer brain metastasis, lung cancer liver metastasis and lung cancer bone metastasis.

In one embodiment, the non-small cell lung cancer comprises squamous cell carcinoma (squamous carcinoma), adenocarcinoma, large cell carcinoma.

In one embodiment, the expression level of CTSF is increased in the patient.

In one embodiment, the method used for the product includes, but is not limited to, the following methods: immunodetection, in situ hybridization, RT-PCR, real-time quantitative PCR or chip detection.

The term "immunoassay" as used herein refers to a method for measuring the content of a substance to be detected in a sample by using the immunological principle and using the substance to be detected as an antigen or an antibody, unless otherwise specified. Preferably, the immunoassay includes, but is not limited to: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluoroimmunoassay, Immunohistochemistry (IHC), chemiluminescence immunoassay, electrochemiluminescence immunoassay, and the like.

In one embodiment, the immunoassay uses ELISA and/or IHC methods.

In one embodiment, the product for early diagnosis of lung cancer using immunodetection comprises at least an antibody that specifically binds to CTSF.

In one embodiment, the product for early diagnosis of lung cancer by RT-PCR comprises at least one pair of primers for specific amplification of CTSF.

In one embodiment, the product for early diagnosis of lung cancer by real-time quantitative PCR comprises at least one pair of primers for specific amplification of CTSF.

In one embodiment, the product for early diagnosis of lung cancer using in situ hybridization comprises at least a probe that specifically hybridizes to a nucleic acid sequence of CTSF.

In one embodiment, the product may further comprise the following components that may be used in protein assays: blocking solution, antibody diluent, washing buffer solution, chromogenic stop solution and standard substance for preparing a standard curve.

In one embodiment, a SYBR Green polymerase chain reaction system, a primer pair for amplifying a molecular marker gene and a housekeeping gene (internal reference gene) may also be included in the product; the SYBR Green polymerase chain reaction system comprises: PCR enzyme, PCR buffer solution, dNTPs and SYBR Green fluorescent dye.

In one embodiment, the product may further comprise reagents for visualizing the amplicons to which the primers correspond; RNA extraction reagent; a reverse transcription reagent; a cDNA amplification reagent; preparing a standard substance for a standard curve; a positive control; and (5) a negative control product.

In one embodiment, the product for early diagnosing lung cancer by using a chip comprises: protein chips and gene chips; wherein, the protein chip at least comprises an antibody which is specifically combined with the CTSF, and the gene chip at least comprises a probe which is specifically hybridized with a nucleic acid sequence of the CTSF.

In one embodiment, the specific antibody comprises a monoclonal antibody, a polyclonal antibody. Antibodies specific for the aforementioned markers include intact antibody molecules, any fragment or modification of an antibody (e.g., chimeric antibodies, scFv, Fab, F (ab') 2, Fv, etc., so long as the fragment retains the ability to bind to the aforementioned marker.

In one embodiment the specific antibody may be labelled with a radioisotope, an enzyme, a fluorescent molecule, a chemiluminescent reagent.

In one embodiment, the specific antibody may be labeled with a secondary antibody (second antibody) labeled with a radioisotope, an enzyme, a fluorescent molecule, or a chemiluminescent reagent.

In one embodiment, the enzyme label comprises an alkaline phosphatase (ALP) label, a horseradish peroxidase (HRP) label.

In one embodiment, the probes/probes may be commercially available or self-prepared.

In one embodiment, the probe may be DNA, RNA, DNA-RNA chimeras, PNA or other derivatives. The length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and at most, usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.

The detection principle of the gene chip used in the invention can be, but is not limited to, any one of the following:

(1) optical signal labeling is carried out on a probe-target specific affinity reaction combination or a hybrid positioned at different positions on the surface of a solid phase, and then fluorescence, chemical or biological luminescence is generated by excitation and detection is carried out;

in one embodiment, the material of the solid phase can be glass slide, silicon wafer, quartz, polyvinylidene fluoride membrane, nitrocellulose membrane, magnetic bead, plastic bead, or enzyme-linked immunosorbent assay plate or strip, among others;

(2) by detecting the speed of movement or mobility of the labeled or unlabeled probe or target in a liquid phase to which a voltage is applied, i.e., electrophoretic techniques or other techniques resulting from the combination of electrophoretic techniques with other techniques;

(3) performing chromatographic analysis by means of probes, targets or probe-target complexes and their different affinities for labels;

(4) combining a molecule or a partial structure of the molecule with redox property, a metal complex and a probe, a target or a probe-target complex positioned and fixed on different positions or electrodes on the surface of a solid phase, and carrying out redox reaction under the action of an external proper mode or proper voltage, so that electrons of a reducing molecule or group are transferred to an oxidizing molecule or group to form an electron current which is transmitted to a detection system through the electrodes for measurement, wherein the measured current intensity is proportional to the amount of hybridized and labeled double-stranded nucleic acid;

(5) various kinds of identification, detection and investigation of other aspects of the nucleic acid molecule reflected thereby by measurement of the magnitude of electron flow conducted through the nucleic acid molecule by utilizing the difference in conductivity properties between the double-stranded nucleic acid and the single-stranded nucleic acid, or the difference in conductivity properties between a double-stranded nucleic acid molecule formed by hybridization of nucleic acids whose sequences are perfectly complementary paired and a double-stranded nucleic acid molecule having mismatched base pairs;

(6) detection techniques that convert electrons or charge transfer reactions or electron currents occurring on nucleic acid molecules or by substances with redox properties into other detectable or exploitable forms;

(7) the probe or the target molecule is directly or indirectly labeled with a substance having redox properties, and the labeled substance is brought close to the surface of the electrode to cause an electron transfer reaction, thereby detecting a biological or chemical reaction occurring on the surface of the electrode.

In one embodiment, the solid surface of the present invention comprises an organic material or an inorganic material.

In one embodiment, the solid phase surface of the present invention can be glass, metal, plastic, etc. and their derivatives; the glass derivative may be amino glass slide, aldehyde glass slide, epoxy glass slide, polyamino acid coated glass slide, etc.

In one embodiment, sample sources for early diagnosis of lung cancer include, but are not limited to, blood, nasal epithelial cells, tissue, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid, and bronchoalveolar lavage fluid.

In a particular embodiment of the invention, the source of the sample for early diagnosis of lung cancer is blood and/or tissue.

In another aspect, the present invention provides a kit for diagnosing whether a subject has lung cancer, the kit comprising a reagent for detecting the expression level of CTSF.

Preferably, the diagnosis includes diagnosing early stage lung cancer, differentiating early and late stage lung cancer.

Preferably, the lung cancer is non-small cell lung cancer.

Preferably, the early stage lung cancer is stage I-IIIA in TNM staging.

Preferably, the advanced stage comprises four stages of no distant target organ metastasis, lung cancer brain metastasis, lung cancer liver metastasis, lung cancer bone metastasis.

In another aspect, the present invention provides a system for diagnosing lung cancer, the system comprising computing means for determining whether a subject is ill based on CTSF expression;

preferably, the diagnosis includes diagnosing early stage lung cancer, differentiating early and late stage lung cancer;

preferably, the system comprises an input device for inputting the CTSF expression amount.

In one embodiment, the system may further comprise an output device for outputting the diagnosis result.

In one embodiment, the system may further comprise a detection device for detecting the amount of expression of mRNA and/or protein.

In one embodiment, the system further includes an evaluation result transmission unit that can transmit the evaluation result of the subject to an information communication terminal device that can be referred to by the patient or the medical staff.

In one embodiment, the kit may further comprise reagents for detecting other disease markers.

On the other hand, the invention also provides application of the kit and the system in preparation of products for early diagnosis of lung cancer.

In another aspect, the present invention provides a method for early diagnosis of whether a subject has lung cancer, the method comprising detecting the expression level of CTSF in the subject.

Implementation of the "method, system for early diagnosis of lung cancer" described herein may include performing or completing selected tasks manually, automatically, or a combination thereof.

Moreover, according to actual instrumentation and equipment of embodiments of the method, system of the present invention, a number of selected tasks could be implemented by hardware, by software, or by firmware, or by a combination thereof using an operating system.

Drawings

FIG. 1 is a graph showing the results of quality control detection of mass spectrometry data; A. mass precision distribution of a mass spectrometer, relationship between protein coverage and molecular weight, and a quantitative RSD (relative standard deviation) distribution box diagram of protein among repeated samples.

FIG. 2 is a graph of the results of proteomic screening of candidate proteins; A. differential protein number identified in proteomics, b. differential protein volcano plot, C: serological test results for candidate proteins.

FIG. 3 is a graph of the results of protein expression in serological validation cohorts; ctsf, b.fbln1, c.akr1b10.

Figure 4 is a ROC curve for markers in differentiating between early stage lung cancer and healthy controls.

FIG. 5 is a ROC curve for markers in differentiating between early and late stage lung cancer.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

The apparatus and reagents used in the present invention are shown in tables 1 and 2 below:

TABLE 1 Instrument used in the invention

Name of instrument	Production company	Producing area
			High-speed refrigerated centrifuge	Beckman Coulter	Germany
Low-speed centrifuge	Beijing times Beili	China
			Medical centrifuge (palm type)	Jiangsu Xinkang medicine	China
A super-low temperature refrigerator at minus 80℃,	Thermo Scientific	USA
			Strata X C18	Phenomenex	USA
Chromatographic column Agilent 300 extended C18	Agilent	China
			EASY-nLC 1000 ultra-high performance liquid phase system	Thermo Scientific	USA

TABLE 2 reagents used in the invention

General procedure

Proteomics sequencing

(1) Preparation of cell secretion

Culturing lung cancer high brain transfer cell line PC9-BrM3 and its parent cell line PC9 in 1640 culture medium containing 10% fetal calf serum, 100U/mL penicillin and 100U/mL streptomycin at 37 deg.C and 5% CO₂. When the cell density reaches about 85% of the area of the culture dish, the culture medium is discarded and replaced by a serum-free 1640 culture medium for continuous culture for 24 hours. After collecting the cell secretion, the cells were removed by centrifugation at 1000g for 5 minutes at 4 ℃ and the supernatant was collected. The supernatant was centrifuged at 12000g for 5 minutes at 4 ℃ and collectedSupernatant, in order to remove cell debris. 150ml of supernatant is collected from each of the high brain transfer cell line PC9-BrM3 and the parent cell line PC9, and the supernatant is frozen at-80 ℃ in 50ml portions in 3 portions.

(2) Protein extraction

First, the sample was taken out from a-80 ℃ refrigerator and left to thaw in a room temperature environment. After the sample is completely thawed, the sample is centrifuged at 12000g at 4 ℃ for 10 minutes to remove solid impurities in the sample. Then, the centrifuged supernatant was transferred to an ultrafiltration centrifuge tube (millipore), and the supernatant was concentrated by centrifugation at 5000g at 4 ℃ to 0.5 mL. Finally, it was replaced once with 8M urea and protein concentration was determined by BCA kit.

(3) Enzymolysis of pancreatin

First, an equal amount of protein was taken from each sample, an appropriate amount of standard protein was added, and the volume was adjusted to be uniform with a lysis solution. Then, trichloroacetic acid (TCA) was slowly added to a final concentration of 20%, the solution was mixed well by vortex mixer, and allowed to stand at 4 ℃ for 2 hours. 4500g were centrifuged for 5 minutes, the supernatant discarded, and the pellet washed 2-3 times with pre-cooled acetone. After the pellet was air-dried, the pellet was resuspended by adding triethylammonium bicarbonate buffer (TEAB) to a final concentration of 200mM and the pellet was broken up by sonication. Mixing the raw materials in a ratio of 1: 50 (protease: protein, m/m) trypsin was added and the mixture was subjected to enzymatic hydrolysis overnight. Thereafter, Dithiothreitol (DTT) was added to a final concentration of 5mM, and the mixture was reduced at 56 ℃ for 30 minutes. Finally, Iodoacetamide (IAA) was added to a final concentration of 11mM and incubated for 15 minutes at room temperature under exclusion of light.

(4) TMT mark

The peptide fragment sample solution obtained by the enzymolysis of the pancreatin is desalted by Strata X C18(Phenomenex) and then is frozen and dried in vacuum. Peptide fragments were solubilized using TEAB at a concentration of 0.5M. Finally, the peptide fragments were labeled according to the protocol of the TMT kit as follows: thawing the labeled reagent, dissolving the thawed labeled reagent in acetonitrile, mixing the solution with the peptide fragments, incubating the mixture at room temperature for 2 hours, mixing the peptide fragment solutions of all samples into one part after a labeling experiment, and finally desalting and vacuum freeze-drying the mixture.

(5) HPLC fractionation

The peptide fragment was fractionated by reverse phase HPLC at high pH using Agilent 300 extended C18(5 μm particle size, 4.6mm inner diameter, 250mm length) as a chromatographic column. The specific operation method comprises the following steps: the peptide fragment gradient is 8-32% acetonitrile, the pH value is 9, 60 components are separated in 60 minutes, then the peptide fragments are combined into 14 components, and the combined components are subjected to vacuum freeze drying and then are subjected to subsequent operation.

(6) Liquid chromatography-mass spectrometry

Dissolving the peptide fragment with liquid chromatography mobile phase A (aqueous solution containing 0.1% formic acid and 2% acetonitrile), and separating with EASY-nLC 1000 ultra performance liquid system. The liquid phase gradient is set as follows: 0-20 minutes, 8% -22% mobile phase B (aqueous solution containing 0.1% formic acid and 90% acetonitrile); 20-33 minutes, 22% -35% of mobile phase B; 33-37 minutes, 35% -80% of mobile phase B; 37-40 minutes, 80% mobile phase B, flow rate maintained at 600 nL/min. The peptide fragments are separated by an ultra-high performance liquid phase system, injected into an NSI ion source for ionization, and then analyzed by Q active Plus mass spectrometry. The ion source voltage is set to 2.2kV, and the peptide fragment parent ion and the secondary fragment thereof are detected and analyzed by using a high-resolution Orbitrap. The scanning range of the primary mass spectrum is set to be 400-1500m/z, and the scanning resolution is set to be 70,000; the secondary mass spectral scan range was then fixed with a starting point of 100m/z and the secondary scan resolution was set to 17,500.

The data acquisition mode uses a data-dependent scanning (DDA) procedure, i.e., after a primary scan, the first 20 peptide fragment parent ions with the highest signal intensity are selected to enter the HCD collision cell in sequence, and are fragmented using 30% of the fragmentation energy, and secondary mass spectrometry is also performed in sequence.

To improve the effective utilization of the mass spectra, the Automatic Gain Control (AGC) was set to 5E4, the signal threshold was set to 63000ions/s, the maximum injection time was set to 80ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scans of the parent ions.

(7) Database search

Secondary mass spectral data were retrieved using Maxquant (v1.5.2.8). The search parameter settings are as follows:

1. selecting the database as Homo _ sapiens _9606_ SP _20191115(20380 sequences);

2. adding a reverse library for calculating false positive rate (FDR) caused by random matching;

3.a common pollution library is added for eliminating the influence of pollution protein in the identification result;

4. the enzyme cutting mode is set as Trypsin/P;

5. the number of missed cutting sites is set to 2;

6. the minimum length of the peptide segment is set to be 7 amino acid residues;

7. the maximum modification number of the peptide fragment is set to be 5;

8. the First-level parent ion mass error tolerance of the First search and the Main search is respectively set to be 20ppm and 5ppm, and the mass error tolerance of the second-level fragment ions is 0.02 Da;

9. alkylation of cysteine is set as fixed modification, variable modification is oxidation of methionine, acetylation of protein N-terminal, deamidation (NQ);

10. the quantitative method was set to TMT-6 plex;

11. the FDR of protein identification and PSM identification is set to be 1%.

Serum and tissue sample collection

Patient serum samples were collected according to standard procedures, briefly, 2ml of peripheral blood was collected from each subject in a blood collection tube, allowed to clot for 30 minutes, the supernatant serum was separated, centrifuged at 1,000 g/min for 15 minutes, collected using an EP tube, and stored in a freezer at-80 ℃ until needed. Serum collection was performed once per treatment cycle for patients with regular follow-up, before each treatment, until the study was complete or a clinical endpoint appeared.

Tissue specimens are obtained during surgical resection or needle biopsy, and the selection of a sample requires a sufficient number of tumor cells and avoidance of necrotic tissue. Fixing with anhydrous formaldehyde, embedding in paraffin, separating tissue with 3um standard in microtome, fixing on anti-dropping glass slide, and drying at 60 deg.C in constant-temperature air-blast drying oven for 2-3 hr.

Enzyme-linked immunosorbent assay (ELISA)

The serum concentration of the candidate protein was measured using human CTSF, FBLN1, AKR1B10, CCL20, SAA1, CXCL1, CXCL3, AXL, AKR1C3, CPNE3 quantitative ELISA kits purchased from oman. And taking out the serum of the patient placed at-80 ℃ for slow thawing, fully shaking and uniformly mixing after thawing, and taking out the ELISA kit from a refrigerator at-4 ℃ for use at room temperature.

50 μ L/100 μ L of standard and patient serum collected according to standard method are added to the microtiter plate wells pre-coated with corresponding antibodies, each sample is repeated 3 times to ensure the accuracy of the experimental results. Blank wells were set, no sample and no ELISA reagents were added, and the rest of the procedure was the same.

After incubation for 45 min in a constant temperature shaker at 37 ℃ the liquid was discarded and the plate was washed repeatedly 4 times for 30 sec each time. Add 50 μ L of biotinylated antibody IgG working solution to the wells (no additional biotin antibody was added for the standard, which had previously incorporated biotin antibody), incubate in a constant temperature shaker at 37 ℃ for 30 minutes, discard the liquid, and wash the plate 4 times for 30 seconds each. Add 50. mu.L of horseradish peroxidase-labeled streptavidin to all wells, incubate in 37 ℃ thermostat water bath for 15 minutes, discard the liquid, repeat the washing of the plate 4 times, each for 30 seconds. To all the wells, 50. mu.L of TMB developing solution A and TMB developing solution B were added, incubated at 37 ℃ to sufficiently react with horseradish peroxidase, and after 15 minutes, 50. mu.L of stop solution was added immediately to terminate the reaction and a color change was observed. The Optical Density (OD) was measured at 450nm in the microplate reader within 15 minutes after the addition of the stop solution, and the OD value of the test sample was determined after setting the blank well OD value to zero. The OD value is proportional to the protein concentration and the protein concentration in the sample is calculated by a fitted public of a standard curve.

Immunohistochemistry (IHC)

Deparaffinizing the tissue sections in xylene repeated 3 times in a fume hood, hydrating in graded ethanol, rinsing with running water for 10 minutes; dripping 3% endogenous peroxidase blocking agent onto the slices, incubating in a water bath at 37 deg.C for 15 min, removing liquid, soaking the slices in PBS solutionSoaking and cleaning, repeating 5 times for 5 minutes each time; heating sodium citrate antigen retrieval solution (10mmol/L, pH6) to boiling in advance by using a microwave oven, soaking the slices in the antigen retrieval solution after boiling, continuing heating for 20 minutes to retrieve the antigen, and slowly cooling to room temperature; dropwise adding goat serum to seal non-specific binding sites, incubating in a water bath at 37 ℃ for 10 minutes, and discarding liquid, wherein the coverage of all tissues is taken as the standard; the optimal antibody concentrations (CTSF 1: 100; FBLN 11: 300; AKR1B 101: 500) were individually purchased drop-wise from R as previously explored&D systems, Santa Cruz Biotechnology, Abcam recombinant antibodies, in the slide box at 4 degrees C were incubated overnight; taking out the slices, slowing to room temperature, removing the primary antibody, soaking and cleaning the slices in a PBS solution, and repeating for 5 times and 5 minutes each time; adding a biotin-labeled goat anti-mouse/rabbit IgG polymer dropwise, incubating in a water bath kettle at 37 ℃ for 30 minutes, discarding liquid, soaking and cleaning the slices in a PBS solution, and repeating for 5 times and 5 minutes each time; dripping horseradish enzyme labeled streptavidin working solution, incubating in a water bath kettle at 37 ℃ for 30 minutes, discarding the liquid, soaking and cleaning the slices in PBS solution, and repeating for 5 times and 5 minutes each time; dropwise adding Diaminobenzidine (DAB) to fully react under the catalysis of horseradish peroxidase, then discarding the liquid, and washing for 10 minutes in running water; dropwise adding hematoxylin staining solution to mark cell nucleus staining, and washing for 10 minutes with running water; dehydrating with gradient ethanol and xylene for 3 times in a fume hood, and sealing with neutral gum; and observing the staining condition of the section under an upright microscope after the gum is air-dried. Tissues with different antigen expression levels were included in each immunohistochemical experiment to reduce bias due to manipulation errors. The staining was assessed by two independent pathologists, giving a total consideration of staining intensity and staining range: negative is 0 point; weak positive is 1 point; the positive is 2 points; the strong positive is 3 points. Score 2 or more was defined as high expression,<score 2 defines low expression. The positive control ishttp://www.proteinatlas.orgThe method of (1).

Statistical analysis

Statistical analysis of the ELISA data was performed using SPSS 23.0 software and RStudio 1.3.1093 software (R language 4.0.3), with GraphPad Prism 8, and P values less than 0.05 considered statistically significant.

Measuring the difference in serum expression of the candidate protein between the experimental group and each control group using ANOVA (Analysis of Variance); t-test or analysis of variance is used to assess the relationship between serum CTSF, FBLN1 levels and patient clinical variables; the Pearson chi-square test or Fisher's exact test is used to assess the correlation between tissue CTSF, FBLN1 expression and patient clinical variables; and (3) performing tendency score matching by using an R language nonrandom software package, and constructing a random Forest model by using a random Forest software package. Evaluating the predictive value of CTSF, FBLN1 for non-small cell lung cancer brain metastasis patients by sensitivity and specificity, and determining a diagnosis threshold value by ROC curve analysis, wherein the optimal threshold value is determined by the farthest point from the diagonal of the ROC curve and the maximum sum of sensitivity and specificity; logistic regression analysis was used to construct diagnostic predictive models based on the biomarker panels.

Example 1 screening of molecular markers

The PC9 human lung adenocarcinoma cell line used in the invention is purchased from a cell bank of Shanghai biological institute of Chinese academy of sciences, and the PC9-BrM3 lung cancer high brain metastasis cell line is obtained by separating and culturing brain metastasis tumor in a constructed lung cancer high brain metastasis animal model, and the construction method is as follows:

constructing a lung cancer high brain transfer cell line based on an animal model: culturing a parent lung cancer cell strain PC9 stably expressing GFP-luciferase fusion protein (GFP-luciferase), anesthetizing female immunodeficiency BALB-c-nu mice by using ketamine (ketamine) and xylazine (xylazine), and injecting transfected lung cancer PC9 cells into the left ventricle of the mice to enable the nude mice to form intracerebral metastatic tumor focus. Luciferase substrate D-Luciferin is injected into the abdominal cavity, and the condition of the intracerebral metastases is observed through imaging of an animal living body imager.

Enrichment and culture of lung cancer brain metastatic cells: injecting a lung cancer cell line PC9 into an immunodeficient mouse, forming tumor by intracerebral tumor transfer after 3 weeks, separating the intracerebral tumor transfer, performing primary culture to obtain a lung cancer brain transfer cell line PC9-BrM1, injecting a PC9-BrM1 cell line into the immunodeficient mouse in the manner of intracardial injection, separating the intracerebral tumor transfer and primary culture again, repeating the step for 3 times to obtain the lung cancer high brain transfer cell line so as to enrich the lung cancer cell brain transfer characteristics, and sequentially obtaining the lung cancer brain transfer cell lines PC9-BrM2 and PC9-BrM 3.

A proteomics database was constructed using a general method, and serum and tissue samples were further collected, and the protein expression amount was measured by enzyme linked immunosorbent assay (ELISA) and Immunohistochemistry (IHC).

The experimental results are as follows:

mass spectrometry quality control detection

In the mass precision distribution of the mass spectrometer in FIG. 1A, the first-order mass errors are all within 10ppm, which accords with the precision of the orbitrap mass spectrum, and the qualitative and quantitative analysis of the protein is not influenced by the overlarge mass deviation, wherein the score of the spectrogram matching peptide segment is inversely related to the distribution of the mass deviation. In the number of peptide fragments corresponding to each protein shown in fig. 1B, most proteins correspond to more than two peptide fragments, which is beneficial to increasing the accuracy and reliability of the quantitative result. The RSD (Relative standard deviation) among triplicate samples shown in FIG. 1C was seen to fluctuate slightly and to be highly reproducible.

In conclusion, the mass spectrometric data meet the standards.

Bioinformatics analysis results

Proteomics is used for exploring protein spectrum difference change analysis of diseases, and is widely applied to identification of novel disease biomarkers or treatment targets. In order to screen proteins of lung cancer which are specifically changed in the brain transfer process, the secretion of an animal-derived lung cancer high brain transfer cell line PC9-BrM3 and parent lung adenocarcinoma cell PC9 is collected and subjected to high-throughput quantitative proteomic detection. 3619 proteins are identified in the omics detection result, wherein 3183 proteins can be quantified. Using fold upregulation ratio (BrM3/PC9) >1.3 as a screening criterion to identify differential proteins in brain metastasis subgroup cell lines compared to the parental lung adenocarcinoma PC9 cell line, 773 upregulated proteins and 587 downregulated proteins were identified (table 3, fig. 2A).

TABLE 3 statistics of differentially expressed protein amounts

Among these differential proteins, we initially looked at the following 10 proteins (fig. 2B):

CTSF (cathepsin F),

FBLN1 (senescence Key protein 1),

AKR1B10(Aldo-keto reductase family 1member B10, aldoketoreductase family 1member B10),

CCL20(C-C motif chemokine 20, CC motif chemokine 20),

SAA1(Serum amyloid A-1),

CXCL1(Growth-regulated alpha),

CXCL3(C-X-C motif chemokine 3, CXC motif chemokine 3),

AXL (Tyrosine-kinase receptor UFO), Tyrosine-protein kinase receptor UFO,

AKR1C3(Aldo-keto reductase family 1member C3),

CPNE3(Copine-3, calcium-dependent membrane-bound protein 3).

The serum candidate protein expression of clinical samples was verified in cohort 1 using ELISA, which included 20 LCBM (non-small cell lung cancer brain metastasis) patients, 20 ALC (advanced non-small cell lung cancer metastasis non-small cell lung cancer), 20 ELC (early-stage non-small cell lung cancer) patients, and 20 HG (health group) patients in cohort 1. (according to the TNM stage of lung cancer, the group of surgically treatable stage I-IIIA non-small cell lung cancers is early stage lung cancer)

As can be seen from the results shown in fig. 2C, three candidate proteins, CTSF, FBLN1 and AKR1B10, were significantly up-regulated in the serum of non-small cell lung cancer brain metastasis patients compared to each control group, indicating that CTSF, FBLN1 and AKR1B10 are likely to be potential diagnostic markers for non-small cell lung cancer brain metastasis.

Example 2, validation of diagnostic value of 3 potential markers for brain metastasis of non-small cell lung cancer

The study included 379 cases of non-small cell lung cancer, 30 cases of PBT (primary brain tumor), and 50 health examiners (HG) in the serum validation cohort 2 to further validate the diagnostic value of 3 potential markers for brain metastasis of non-small cell lung cancer. 379 patients with non-small cell lung cancer included 204 cases of non-small cell Lung Cancer Brain Metastases (LCBM), 40 cases of LM (liver metastases, single organ liver metastases); 50 cases of BM (bone metastasis, single organ bone metastases); 40 cases of advanced non-small cell lung cancer (ALC) without distant organ metastasis, and 45 cases of Early Lung Cancer (ELC). There were no significant differences between the subgroups in the cohort in terms of age, sex and pathological typing.

As shown in fig. 3, serum CTSF (fig. 3A) and FBLN1 (fig. 3B) levels were significantly elevated in lung cancer brain metastasis patients (both P <0.001) compared to the controls, whereas AKR1B10 (fig. 3C) was elevated in the advanced lung cancer groups (LCBM, LM, BM, and ALC) in each group (comparison alone) and not specifically elevated in the lung cancer brain metastasis group.

When compared between the control groups, it was seen that the serum CTSF level in the early lung cancer patients was significantly increased compared to the healthy group (P0.009), while the serum levels of FBLN1 and AKR1B10 were not significantly different between the early lung cancer patients and the healthy group (P0.293, P0.05). Patients with advanced non-small cell lung cancer (LCBM, LM, BM, ALC) had significantly elevated serum levels of CTSF and FBLN1 (both P <0.001) compared to patients with early stage lung cancer.

Therefore, both CTSF and FBLN1 are specifically increased in serum of a patient with non-small cell lung cancer brain metastasis, and in addition, the CTSF level is correspondingly and gradually increased along with the occurrence and development process of the lung cancer, and different critical values (healthy population vs early lung cancer; early lung cancer vs late lung cancer; brain metastasis vs other late lung cancer) are defined, so that the CTSF can be used for detecting the occurrence of the brain metastasis, and can also be used for screening and identifying the early lung cancer and evaluating the lung cancer progression state.

In contrast, FBLN1 does not reflect the occurrence of early stage lung cancer, but only increases as lung cancer progresses to an advanced stage. When the sample size is expanded, AKR1B10 is found to be not stably and specifically highly expressed in a lung cancer brain transfer patient, but is generally highly expressed in advanced lung cancer, but can reflect the progress degree of the lung cancer to a certain extent.

The result shows the potential role of serum CTSF in early lung cancer diagnosis.

Example 3 validation of CTSF in early diagnosis

Again, the ROC curve was used to verify the diagnostic accuracy of CTSF:

in the ROC curve of 45 cases of Early Lung Cancer (ELC) vs 50 health examiners (HG), AUC:0.709, sensitivity: 53.3%, intentionally: 90%, Cut off value: 58.42. as shown in particular in fig. 4.

45 cases of Early Lung Cancer (ELC) vs late stage (four-stage no distant target organ metastasis ALC, lung cancer brain metastasis LCBM, lung cancer liver metastasis LM, lung cancer bone metastasis BM) all late stages were pooled in a ROC analysis and compared to early lung cancer patients. The AUC values of each marker in the ROC curve are: CTSF 0.836, CEA0.849, CA 1250.412, NSE 0.399, SCC 0.510, CYFRA 21-10.376; table 4 shows cut off, specificity and sensitivity, and FIG. 5 shows the results.

TABLE 4 application of markers in differentiating early and late lung cancer

	cut off	Sensitivity of the device	Degree of specificity
				CTSF	54.535	94.30％	75.0％
CEA	0.835	90.9％	75.0％
				CA125	149.04	14.8％	99.2％
NSE	20.205	20.5％	98.9％
				SCC	0.925	85.9％	50.0％
CYFRA211	17.76	14.8％	99.6％

As shown in fig. 5: other common lung cancer markers can not distinguish early lung cancer from late lung cancer, so the CFSF disclosed by the invention has a wider application range in practice.

The results show that the CFSF has good diagnosis accuracy in screening early-stage lung cancer patients and distinguishing early-stage and late-stage patients, and has good clinical application value.