Exosome-based biomarkers for the diagnosis or prognosis of squamous cell lung cancer and their uses

Abstract

The present invention relates to a diagnostic biomarker composition for squamous cell lung cancer, which comprises PBLD-overexpressing exosomes, and the present invention enables non-invasive and accurate diagnosis of squamous cell lung cancer subtypes.

Description

[Technical Field]

[0001] The present invention relates to an exosome-based biomarker for diagnosing or predicting the prognosis of squamous cell lung cancer, and uses thereof. [Background technology]

[0002] Squamous cell lung cancer is a type of lung cancer that originates from squamous cells, which make up the bronchial mucosa. Squamous epithelium is a flat-shaped epithelium, which refers to the cell layer on the surface of the body and the cell layer that lines the inner surface of body cavities (the space between the body wall and internal organs) and the gastrointestinal tract. Squamous cell carcinoma is primarily found in the center of the lung, is more common in men, is highly associated with smoking, and has been reported to cause symptoms such as coughing, hemoptysis, and wheezing due to partial bronchial obstruction.

[0003] The standard method for diagnosing cancer is tissue biopsy. If an abnormal lesion is identified through low-dose chest CT, tissue for testing is obtained using a needle or other tool under thoracoscope or PET / CT. However, such invasive tissue testing causes pain to the patient and can lead to problems such as bleeding during the biopsy process. There are also concerns about false positives with low-dose chest CT. The false positive rate reported in the National Lung Screen Trial (NSLT) in the US was 26.8%, while that reported in Korea was approximately 14.8%, so a considerable number of false positives have been reported.

[0004] In addition, due to issues such as the high cost of testing and radiation exposure, repeated follow-up tests are difficult, and there are cases where tissue biopsies cannot be performed depending on the patient's condition. Another problem with tissue testing is the heterogeneity of tumor tissues, which means that the biological properties differ between and within tumor tissues, and the information obtained from some tissues may be insufficient to make appropriate treatment and surgical plans.

[0005] The need for early diagnosis of various diseases, including cancer, is becoming more and more pressing. Research is shifting from traditional tissue collection-based tissue biopsy and imaging-based diagnostic methods such as tissue staining to liquid biopsy using biomarkers. As a result of this trend, research into biomarkers, which are molecular biological markers derived from DNA, RNA, metabolites, proteins, and protein fragments, is becoming increasingly active.

[0006] However, biomarkers currently used in lung cancer diagnosis have not been reported to be particularly effective. Although several cancer markers derived from other carcinomas (CEA, SCC-Ag, etc.) that have been reported to have increased diagnostic significance in lung cancer have been applied to lung cancer, their diagnostic sensitivity is extremely low and they are not widely used in clinical practice.

[0007] Therefore, the present inventors aim to complete and provide an invention of a novel biomarker that can diagnose cancer early, non-invasively, with high sensitivity and specificity, and is specific to the squamous cell lung cancer subtype of lung cancer. Summary of the Invention [Problem to be solved by the invention]

[0008] The technical problem to be solved by the present invention is to provide a biomarker composition for diagnosing or predicting the prognosis of squamous cell lung cancer, which comprises exosomes that overexpress PBLD (Phenazine biosynthesis-like domain-containing protein, UniProt accession No. P30039) protein or the gene encoding it.

[0009] Another technical problem that the present invention aims to achieve is to provide a composition for diagnosing or predicting the prognosis of squamous cell lung cancer, comprising a preparation capable of measuring the expression level of PBLD protein in exosomes or the gene encoding it.

[0010] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0011] To achieve the above object, the present inventors provide a biomarker composition for diagnosing or predicting the prognosis of squamous cell lung cancer, which comprises exosomes that overexpress PBLD (Phenazine biosynthesis-like domain-containing protein, UniProt accession No. P30039) protein or the gene encoding it.

[0012] According to another embodiment of the present invention, there is provided a composition for diagnosing or predicting the prognosis of squamous cell lung cancer, comprising a preparation capable of measuring the expression level of PBLD protein in exosomes or the gene encoding it.

[0013] On the one hand, the agent capable of measuring the expression level of the protein may be an antibody or an antigen-binding fragment thereof that specifically binds to the protein; or an aptamer that specifically binds to the protein.

[0014] On the one hand, the preparation capable of measuring the expression level of the gene may be a primer or a probe that specifically binds to the nucleic acid molecule of the gene.

[0015] According to a further embodiment of the present invention,

[0016] a) extracting exosomes from a biological sample isolated from a subject;

[0017] b) measuring the expression level of PBLD protein or the gene encoding it in the extracted exosomes.

[0018] According to one side,

[0019] c) extracting exosomes from a biological sample isolated from a control group;

[0020] d) measuring the expression level of PBLD protein or the gene encoding it in the exosomes;

[0021] e) comparing the expression levels of the protein or the gene encoding it between the subject and the control group.

[0022] On the one hand, if the expression level of the PBLD protein or the gene encoding it in the subject is higher than that in the control group, the subject can be determined to have developed squamous cell lung cancer or can be predicted to be at high risk of developing it.

[0023] On the one hand, if the expression level of the PBLD protein or the gene encoding it in the subject is higher than that in the control group, the subject can be predicted to have a poor prognosis.

[0024] According to another aspect of the present invention, there is provided a kit for diagnosing squamous cell lung cancer, comprising any one of the above diagnostic compositions.

[0025] According to another embodiment of the present invention,

[0026] a) treating a candidate substance with a biological sample isolated from a subject, a cell line, or a non-human animal model;

[0027] b) extracting exosomes from the candidate substance-treated biological sample, cell line, or non-human animal model;

[0028] c) measuring the expression level of PBLD protein or the gene encoding it in the extracted exosomes.

[0029] According to one aspect, the screening method includes, after the above step:

[0030] d) A step of selecting candidate substances that have a reduced expression level of the PBLD protein or the gene encoding it compared to a control sample that has not been treated with the candidate substance may be further included. [Effects of the Invention]

[0031] According to the present invention, early cancer diagnosis and prognosis prediction can be made specifically for the squamous cell lung cancer subtype of lung cancer in a non-invasive manner with high sensitivity and specificity.

[0032] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. [Brief explanation of the drawings]

[0033] [Figure 1] 1 shows a workflow for protein analysis in the present invention. [Figure 2] The difference in protein expression levels between the control group and squamous cell lung cancer group is shown in a volcano plot. [Figure 3] These are the results of PBLD ELISA assays performed on exosomes extracted from the blood of normal and squamous cell patients. [Figure 4] The results of ROC analysis using exosome PBLD are shown. [Figure 5] These are the results of exosome PBLD ELISA performed on blood samples from patients with lung adenocarcinoma and lung squamous cell carcinoma. [Figure 6] 1 shows the results of ROC and AUC analysis for lung adenocarcinoma and lung squamous cell carcinoma. [Figure 7] Exosomes were extracted from the blood of patients with squamous cell lung cancer, and the recurrence-free survival curve was confirmed. DETAILED DESCRIPTION OF THE INVENTION

[0034] The inventors have collected exosomes from a group of squamous cell lung cancer patients, analyzed the proteins therein, and identified PBLD protein, which is expressed at significantly higher levels in the squamous cell lung cancer patient group compared to the control group. They intend to complete and provide this as an invention of a biomarker and a composition for diagnosing or predicting the prognosis of squamous cell lung cancer.

[0035] The present inventors provide a biomarker composition for diagnosing or predicting the prognosis of squamous cell lung cancer, which comprises exosomes that overexpress PBLD (Phenazine biosynthesis-like domain-containing protein, UniProt accession No. P30039) protein or the gene encoding it.

[0036] As used herein, the term "exosomes overexpressing PBLD protein or the gene encoding it" refers to exosomes that express a high level of PBLD protein or the gene encoding it, compared to the PBLD expression level in exosomes isolated from a normal individual or a predetermined cutoff value.

[0037] Exosomes are small nano-sized (30-150 nm) endoplasmic reticulum secreted by many cells. The interior of exosomes and their phospholipid bilayer membranes are known to contain various cell-derived proteins, genetic material (DNA, mRNA, miRNA), lipids, and other substances. Furthermore, tissue-derived exosomes have been reported to reflect the condition of the tissue that secreted them, making them useful for disease diagnosis.

[0038] Here, the present inventors have confirmed that when PBLD protein specifically expressed in exosomes derived from squamous cell lung cancer or the gene encoding it is used, cancer can be accurately and quickly diagnosed and the prognosis can be predicted, thereby completing the present invention.

[0039] As used herein, "diagnosis" refers to predicting the occurrence of a disease, determining the risk or susceptibility of developing the disease, or confirming the existence or characteristics of a pathological condition.

[0040] For the purposes of this invention, the diagnosis refers to a diagnosis of squamous cell lung cancer. In this invention, "squamous cell lung cancer" refers to lung cancer derived from squamous cells, a type of non-small cell cancer derived from squamous epithelial cells that make up the bronchial mucosa.

[0041] The term "diagnostic (bio)marker, (bio)marker for diagnosing, or diagnostic marker" as used herein refers to a marker or a composition contained therein that can distinguish squamous cell lung cancer from a normal control group and diagnose the same, and can distinguish the levels of organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, and sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that are increased or decreased in cells with squamous cell lung cancer compared to normal cells, or can measure such levels.

[0042] In the present invention, the term "prognosis" refers to determining whether or not an individual has recurrence, metastasis, drug responsiveness, or resistance after treatment for squamous cell lung cancer. This includes the concept of predicting not only whether or not an individual has developed squamous cell lung cancer, but also whether or not the individual's future survival prognosis is favorable, by measuring the expression level of PBLD in exosomes isolated from an individual's sample.

[0043] According to a further embodiment of the present invention, there is provided a composition for diagnosing or predicting the prognosis of squamous cell lung cancer, comprising a preparation capable of measuring the expression level of PBLD protein in exosomes or the gene encoding it.

[0044] The term "measuring the expression level" as used herein means measuring the presence or absence, expression, or degree of expression of a specific protein (peptide) or a gene encoding the protein, and specifically, the expression level of the PBLD protein or the mRNA or gene encoding it can be measured.

[0045] On the one hand, the agent capable of measuring the expression level of the protein may be an antibody or an antigen-binding fragment thereof that specifically binds to the protein, or an aptamer that specifically binds to the protein.

[0046] The term "antibody" as used herein refers to a specific immunoglobulin directed against an antigenic site, as that term is known in the art. For example, the antibody may specifically bind to the PBLD protein or a fragment thereof. The fragment refers to a protein fragment having one or more epitopes recognized by an antibody against the protein, and may be, for example, an immunogenic fragment. The antibody form includes polyclonal antibodies, monoclonal antibodies, or recombinant antibodies, and includes all immunoglobulin antibodies. The antibody also includes specialized antibodies such as humanized antibodies.

[0047] Using such antibodies, it is possible to confirm whether the protein is expressed in a biological sample by methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting on polyacrylic gel, or immunoblotting.

[0048] As used herein, the term "antibody fragment" refers to a polypeptide that does not have the structure of an intact antibody, peptide, or protein, but has a specific antigen-binding site or binding domain directed against an antigenic site. The fragment includes functional fragments of an antibody molecule that are not an intact antibody having two light chains and two heavy chains. A functional fragment of an antibody molecule refers to a fragment that retains at least the antigen-binding function, and may be Fab, F(ab'), F(ab')2, or Fv. The binding fragment may contain at least seven or more amino acids, for example, nine or more amino acids, or twelve or more amino acids.

[0049] Analytical methods for detecting the protein include, but are not limited to, Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoassay, immunochemistry assay, immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), and protein chip.

[0050] On the other hand, the preparation capable of measuring the expression level of the gene may be a primer or a probe that specifically binds to the nucleic acid molecule of the gene. The analytical method may be, for example, one or more methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Realtime RT-PCR), RNase protection assay (RPA), Northern blotting, and DNA chip.

[0051] As used herein, the term "primer" refers to a nucleic acid sequence having a free 3' hydroxyl group that can form base pairs with a template complementary to a specific base sequence and serve as a starting point for copying the template strand. A primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature. For example, PCR amplification can be performed using sense and antisense primers having a sequence of 7 to 50 nucleotides specific to the gene or mRNA encoding the PBLD protein, and the presence or absence of squamous cell lung cancer in an individual can be diagnosed by measuring the amount of the desired product. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected using techniques well known in the art. The primers can have a length of 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80, 20 to 50, or 20 to 30 nt.

[0052] As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, that can specifically bind to a target nucleic acid, e.g., mRNA, and may be labeled to determine the presence, content, and expression level of a specific mRNA. Probes may be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, etc. For example, hybridization may be performed using a probe having a nucleic acid sequence complementary to the gene or mRNA encoding the PBLD protein, and the mRNA expression level may be measured based on the degree of hybridization to diagnose whether or not an individual has developed squamous cell lung cancer. Selection of an appropriate probe and hybridization conditions may be appropriately determined using techniques known in the art. The probe may have 10 to 100, 15 to 100, 10 to 80, 10 to 50, 10 to 30, 10 to 20, 15 to 80, 15 to 50, 15 to 30, 15 to 20, 20 to 100, 20 to 80, 20 to 50, or 20 to 30 nt.

[0053] The primers or probes may be chemically synthesized using phosphoramidite solid support synthesis or a wide variety of other well-known methods. These nucleic acid sequences may also be modified by a variety of methods known in the art. Examples of such modifications include methylation, capping, substitution of one or more natural nucleotides with their analogs, or internucleotide modifications, such as uncharged linkers (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkers (e.g., phosphorothioates, phosphorodithioates, etc.). The primers or probes may also be modified with a label capable of directly or indirectly providing a detectable signal. Examples of such labels include radioisotopes, fluorescent molecules, or biotin.

[0054] According to a further embodiment of the present invention, there is provided a diagnostic kit for squamous cell lung cancer, comprising a preparation capable of measuring the expression level of a PBLD protein or the gene encoding it.

[0055] For example, a biological sample may be obtained from a specimen containing tissue suspected or suspected to be a site of squamous cell lung cancer, exosomes may be extracted from the sample, and a composition containing a PBLD protein detection agent may be used to confirm the expression level of the corresponding protein in the exosomes, thereby providing information for diagnosing squamous cell lung cancer. The kit may be an immunoassay kit.

[0056] According to another embodiment of the present invention, a diagnostic kit for squamous cell lung cancer includes a preparation for detecting the gene encoding the PBLD protein and provides information useful for diagnosing squamous cell lung cancer. For example, a biological sample is obtained from a specimen containing tissue suspected or suspected to be at risk of squamous cell lung cancer, and exosomes are extracted from the sample to extract nucleic acids encoding the PBLD protein. Using a composition containing a preparation for detecting the nucleic acids, the level of expression of the nucleic acids encoding the corresponding proteins in the sample can be determined (in the case of a reverse-transcribed mRNA sample, cDNA and a preparation for detecting the cDNA can be applied), providing information for diagnosing squamous cell lung cancer. The kit may also be a DNA chip.

[0057] According to a further embodiment of the present invention, the method comprises the steps of: a) extracting exosomes from a biological sample isolated from a subject;

[0058] b) A method for providing information for diagnosing or predicting the prognosis of squamous cell lung cancer is provided, which comprises a step of measuring the expression level of PBLD protein or the gene encoding it in the extracted exosomes.

[0059] As used herein, the term "subject" refers to any organism in which squamous cell lung cancer develops or may develop, and specific examples may include, but are not limited to, mammals such as dogs, cats, mice, rats, monkeys, cows, pigs, minipigs, livestock, and humans.

[0060] The term "sample" as used herein refers to a substance derived from the individual, and may specifically include tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, etc., and preferably may be any one or more selected from the group consisting of blood, plasma, lung tissue biopsy, nasopharyngeal swab, sputum, and saliva, but is not limited to these.

[0061] According to one side,

[0062] c) extracting exosomes from a biological sample isolated from a control group;

[0063] d) measuring the expression level of PBLD protein or the gene encoding it in the exosomes;

[0064] e) comparing the expression levels of the protein or the gene encoding it between the subject and the control group.

[0065] As used herein, the term "control group" refers to general individuals who have not developed squamous cell lung cancer, a group of non-squamous cell lung cancer patients, a non-patient group, and the like.

[0066] According to one aspect, if the expression level of the protein or the gene encoding it in the subject is higher than that in the control group, the subject can be diagnosed as having developed squamous cell lung cancer or predicted to be at a high risk of developing squamous cell lung cancer. Preferably, if the expression level of the protein is 1.6-fold or more higher than that in the control group, the subject can be diagnosed as having developed squamous cell lung cancer or predicted to be at a high risk of developing squamous cell lung cancer.

[0067] On the one hand, if the expression level of the PBLD protein or the gene encoding it in the subject is higher than that in the control group, the subject is predicted to have a poor prognosis, where the poor prognosis is a relatively short recurrence-free survival period.

[0068] The expression levels of the above genes are compared to the control group, but in another aspect, the exosome pbld concentration can be determined to be 90 pg / ml as the standard, and if it is higher than this, it can be determined to be a high expression level, and if it is lower, it can be determined to be a low expression level.

[0069] According to a further embodiment of the present invention,

[0070] a) treating a candidate substance with a biological sample isolated from a subject, a cell line, or a non-human animal model;

[0071] b) extracting exosomes from the candidate substance-treated biological sample, cell line, or non-human animal model;

[0072] c) measuring the expression level of PBLD protein or the gene encoding it in the extracted exosomes.

[0073] The "subject" has the same meaning as previously described herein. The "sample" is as previously described herein, and may preferably be a tissue biopsy sample.

[0074] The "cell line" may preferably be a squamous cell lung cancer cell line.

[0075] The "animal model" may preferably be a squamous cell lung cancer animal model.

[0076] The expression level of the PBLD protein or the gene encoding it can be measured as described above in this specification.

[0077] According to one aspect, the screening method includes, after the step:

[0078] d) A step of selecting a candidate substance that has a reduced expression level of the PBLD protein or the gene encoding it compared to a control sample that has not been treated with the candidate substance may be further included.

[0079] The terms used in the embodiments are merely used for the purpose of explanation and are not to be construed as being limiting. A singular expression includes a plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprise" or "have" indicate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0080] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention pertains. Commonly used predefined terms should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and should not be interpreted as having an ideal or overly formal meaning unless expressly defined herein.

[0081] The present invention can be modified in various ways and has various embodiments, and specific embodiments will be described in detail below with reference to the drawings and detailed description. However, this is not intended to limit the present invention to the specific embodiments, and it should be understood that the present invention includes all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention. In the description of the present invention, if a detailed description of related publicly known technology is deemed to obscure the gist of the present invention, the detailed description will be omitted.

[0082] Hereinafter, the embodiments will be described in detail with reference to the accompanying drawings. However, various modifications may be made to the embodiments, and the scope of the patent application is not limited or restricted by the embodiments. It should be understood that all modifications, equivalents, or alternatives to the embodiments are included in the scope of the patent.

[0083] In addition, when describing the present embodiment with reference to the drawings, the same reference numerals will be used to refer to the same components regardless of the reference numerals, and redundant descriptions thereof will be omitted. In describing the present embodiment, if a detailed description of related prior art is determined to unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

[0084] Since the present invention can be modified in various ways and can have multiple embodiments, specific embodiments will be illustrated in the drawings and described in detail below. However, this does not limit the present invention to the specific embodiments, and it should be understood that the present invention includes all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention. In describing the present invention, if a detailed description of related publicly known technology is considered to obscure the gist of the present invention, the detailed description will be omitted.

[0085] Embodiment 1. Sample Collection

[0086] Embodiment 1-1. Cell culture medium

[0087] The cell lines used in the present invention were HPAepiC, BEAS-2B, HSAEC1-KT, H1703, H2170, and SW900, each of which was cultured at 2 × 10 cells per 150 mm cell culture dish. 6After dispensing, the cells were grown in a 36°C incubator until they filled approximately 70-80% of the cell culture dish. After 48 hours of culture in cell culture medium containing exosome-removed FBS, the culture medium was collected and centrifuged at 1,500 rpm for 5 minutes to remove cellular debris. After centrifugation at 3,000 rpm for 30 minutes, the supernatant was collected and stored in a -80°C ultra-low temperature freezer until exosome extraction.

[0088] Embodiment 1-2. Blood sample

[0089] Whole blood samples collected in EDTA and SST tubes were centrifuged at 3,000 rpm for 30 minutes at 4°C to recover plasma and serum, which were then stored in an ultra-low temperature freezer at -80°C until exosome extraction.

[0090] Embodiment 2. Extraction of exosomes from a sample

[0091] To extract exosomes from the cell culture medium, 200 ml of culture medium was centrifuged at 5,000 rcf for 15 minutes using a 100 KDa Amicon Ultra 50 ml filter tube, and 0.5 ml of the concentrate was placed in a size exclusion chromatography column, after which PBS was poured in to obtain 6-9 fractions of exosome concentrate (0.5 ml per fraction, 2 ml in total) for use.

[0092] To extract plasma-derived exosomes, plasma stored in a -80°C ultra-low temperature freezer was centrifuged at 10,000 rcf for 30 minutes at 4°C to remove precipitates. 0.5 ml of plasma was then placed in a size exclusion chromatography column, and PBS was added to obtain 11-12 fractions of exosome-enriched solution (0.5 ml per fraction, 2 ml in total) for use.

[0093] Embodiment 3. Protein Analysis

[0094] To identify exosomal proteins derived from squamous cell lung cancer cell lines, exosomes were extracted from three types of normal lung cells and three types of squamous cell lines and analyzed for protein content. The concentrations of exosomal proteins used for protein content analysis are shown in Table 1 below.

[0095] [Table 1]

[0096] Among the exosome proteins for the protein analysis, the peptide concentrations required for LC-MS analysis were measured using a tryptophan-based fluorescence assay method, and the concentrations are shown in Table 2 below.

[0097] [Table 2]

[0098] Protein analysis was performed as follows. A 200µg volume of exosome sample derived from a squamous cell lung cancer cell line was taken, completely dried, and then dissolved in 2% SDS. Protein pretreatment was performed using the FASP method. After protein digestion, each peptide sample was labeled with Tandem Mass Tag (TMT6plex) reagent and combined. The peptides were then separated into 24 peptide fractions, and each fraction was analyzed by NanoLC-MS. A schematic diagram of this process is shown in Figure 1.

[0099] To document the differences in protein expression between the control and squamous cell lung cancer groups, a t-test was performed to identify proteins that met all criteria: p-value < 0.05 and fold change > 1.2. The results were plotted on a volcano plot, shown in Figure 2. As a result, 62 proteins were found to be upregulated in the squamous cell lung cancer group, compared with 34 in the control group. Among these, PBLD (Phenazine biosynthesis-like domain-containing protein, UniProt accession no. P30039) was identified as a squamous cell lung cancer-specific exosome marker.

[0100] Embodiment 4: Evaluation of diagnostic usefulness using PBLD

[0101] To evaluate the diagnostic utility of exosomal PBLD in patients with squamous cell lung cancer, exosomes were extracted from the blood of nine normal subjects and 29 squamous cell lung cancer patients, and a PBLD ELISA assay was performed. The results are shown in Figure 3. ELISA analysis confirmed that PBLD concentrations were 1.6-fold higher (p=0.0013) in the squamous cell lung cancer patient group compared to the normal group, and that concentrations increased with increasing pathological stage.

[0102] The results of the ROC analysis using exosomal PBLD are shown in Figure 4 , and the AUC value was confirmed to be 0.864, with a sensitivity of 93.1% and a specificity of 66.7%.

[0103] Embodiment 5: Evaluation of diagnostic comparison between lung adenocarcinoma and lung squamous cell carcinoma using PBLD

[0104] To evaluate the diagnostic utility of exosomal PBL D for lung adenocarcinoma and squamous cell carcinoma, exosomal PBL D ELISA was performed on the blood of 10 normal subjects, 15 lung adenocarcinoma subjects, and 15 squamous cell carcinoma subjects, as shown in Figure 5. ELISA analysis revealed that the concentration of exosomal PBL D in the lung adenocarcinoma subjects was 1.4-fold higher (p=0.2164) than in the normal subjects, but 2.2-fold higher (p=0.0115) in the squamous cell carcinoma subjects.

[0105] The results of the ROC analysis are shown in Figure 6. The exosome-PBLD AUC for lung adenocarcinoma was 0.65 (p = 0.2020), and the AUC for lung squamous cell carcinoma was 0.80 (p = 0.126), confirming the significance of the exosome-PBLD in diagnosing lung squamous cell carcinoma.

[0106] Embodiment 6: Prognosis evaluation of squamous cell lung cancer using PBLD

[0107] To evaluate the prognosis of squamous cell lung cancer patients using exosomal PBLD, exosomes were extracted from the blood of 18 squamous cell lung cancer patients, and a PBLD ELISA assay was performed. Kaplan-Meier survival analysis was performed to generate recurrence-free survival curves, as shown in Figure 7. The recurrence-free survival analysis revealed that the recurrence-free survival period for patients with high exosomal PBLD concentrations was approximately 10 months, a significant difference (p=0.048) compared to the patient group with low concentrations (approximately 57 months).

[0108] Although the embodiments of the present invention have been described in detail above with reference to the drawings, the present invention is not limited to the above-described embodiments, and a person skilled in the art may apply various technical modifications and variations based on the above. For example, the described techniques may be performed in an order different from that described, and / or the components of the described systems, structures, devices, circuits, etc. may be combined or combined in a form different from that described, or may be substituted or replaced by other components or equivalents, and still achieve appropriate results.

[0109] Accordingly, other implementations, variations, and equivalents of the following claims are within the scope of the following claims.

Claims

1. A biomarker composition for diagnosing or predicting the prognosis of squamous cell lung cancer, comprising exosomes that overexpress PBLD (phenazine biosynthesis-like domain-containing protein, UniProt accession No. 30039) protein or the gene encoding it.

2. A composition for diagnosing or predicting the prognosis of squamous cell lung cancer, comprising a preparation capable of measuring the expression level of PBLD protein in exosomes or the gene encoding it.

3. 3. The composition for diagnosing or predicting the prognosis of squamous cell lung cancer according to claim 2, wherein the preparation capable of measuring the expression level of the protein is an antibody or an antigen-binding fragment thereof that specifically binds to the protein, or an aptamer that specifically binds to the protein.

4. The composition for diagnosing or predicting the prognosis of squamous cell lung cancer according to claim 2, wherein the preparation capable of measuring the expression level of the gene is a primer or probe that specifically binds to the nucleic acid molecule of the gene.

5. a) extracting exosomes from a biological sample isolated from a subject; b) measuring the expression level of PBLD proteins or genes encoding them in the extracted exosomes; A method for providing information for diagnosing or predicting prognosis of squamous cell lung cancer, comprising:

6. The method comprises: c) extracting exosomes from a biological sample isolated from the control group; d) measuring the expression level of PBLD proteins or genes encoding them in the exosomes; e) comparing the expression levels of said protein or the gene encoding it in said subject and said control group; The information providing method according to claim 5, further comprising:

7. The information providing method of claim 6, wherein if the expression level of the PBLD protein or the gene encoding it in the subject is higher than that in the control group, the subject is determined to have developed squamous cell lung cancer or is predicted to be at high risk of developing it.

8. The method for providing information according to claim 6 , wherein if the expression level of the PBLD protein or the gene encoding it in the subject is higher than that in the control group, the subject is predicted to have a poor prognosis.

9. A diagnostic kit for squamous cell lung cancer, comprising the composition according to any one of claims 2 to 4.

10. a) treating a biological sample isolated from a subject, a cell line, or a non-human animal model with a candidate substance; b) extracting exosomes from the candidate substance-treated biological sample, cell line, or non-human animal model; c) measuring the expression level of PBLD proteins or genes encoding them in the extracted exosomes; A method for screening a therapeutic agent for squamous cell lung cancer, comprising:

11. The screening method of claim 10, further comprising, after the step d) selecting candidate substances that have a reduced expression level of PBLD protein or the gene encoding it compared to a control sample in which the candidate substance has not been treated.

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