Lung cancer biomarker

Abstract

The present invention addresses the problem of providing a lung cancer biomarker and a method for using the same. The aforementioned problem is solved by a method for testing for lung cancer, the test method including (1) a step for detecting at least one protein selected from the group consisting of a protein group (A), a protein group (B), a protein group (C), a protein group (D), and a protein group (E) in a bodily fluid or extracellular vesicles of a bodily fluid collected from a subject.

Description

Lung cancer biomarkers

[0001] The present invention relates to lung cancer biomarkers and the like.

[0002] Lung cancer is a disease with an extremely high mortality rate, and the number of patients in Japan is steadily increasing, placing a social and economic burden. The pathology of lung cancer is thought to be primarily cellular dysplasia caused by genetic abnormalities, but the pathology that causes these genetic abnormalities has not yet been fully elucidated, and the underlying mechanism has not yet been fully elucidated. Among lung cancers, small cell lung cancer (SCLC) is highly invasive and metastatic, and is a fatal disease that progresses even in the early stages. Reasons for the poor prognosis of SCLC include the difficulty of early detection and the lack of effective treatments.

[0003] In recent years, cancer research has explored BM at the genetic level using genetic approaches such as genome and transcriptome analysis. Although deletion of TP53 and RB1 alleles has been reported in SCLC (Non-Patent Document 1), no new clinical treatments have been developed. In 2021, Gay et al. classified SCLC according to the immune-indicating genes ASCL1, NEUROD1, and POUF2F3, demonstrating the possibility of enabling treatment selection (Non-Patent Document 2). The full picture of SCLC has yet to be elucidated, and further research is essential.

[0004] Nature. 2015 Aug 6;524(7563):47-53.Cancer Cell. 2021 Mar 8;39(3):346-360.e7.

[0005] An objective of the present invention is to provide a lung cancer biomarker and a method for using the same.

[0006] As a result of intensive research, the present inventors have found that extracellular vesicles in body fluids collected from subjects or specific protein groups in body fluids are useful as biomarkers for lung cancer. Further research based on this finding led to the completion of the present invention. Specifically, the present invention encompasses the following aspects.

[0007] Item 1. A method for testing for lung cancer, comprising: (1) detecting proteins of group (A), group (B), group (C), group (D), and group (E) in extracellular vesicles or body fluid collected from a subject: (A) Succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional glutamate / proline--tRNA ligase, Pyridoxal-dependent decarboxylase domain-containing protein 1, Platelet-derived growth factor C, Biglycan, Eukaryotic translation initiation factor 3 subunit L, Emerin, Serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, General vesicular transport factor p115, Copine-1, WASH complex subunit5, and a group of proteins consisting of Protein transport protein Sec23A, (B) Immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Gla protein, Ena / VASP-like protein, Beta-hexosaminidase subunit alpha, CD2-associated protein, Oncoprotein-induced transcript 3 protein, Glycosylated lysosomal membrane protein, Transmembrane 7 superfamily member 3, CD81 antigen, EGF-like repeat and discoidin I-like domain-containing protein 3, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1, Transforming growth factor beta activator LRRC32, Claudin-5, Proteasome subunit beta type-1, F-box only protein 50, Ragulator complex protein LAMTOR1, Trypsin-1, Beta-hexosaminidase subunit beta, Monocarboxylate transporter 2, Dyslexia-associated protein KIAA0319-like protein, DnaJ homolog subfamily B member 1, Membrane cofactor protein, Desmoglein-1, B-cell antigen receptor complex-associated protein betaA group of proteins consisting of chain, Raftlin, Synaptic vesicle glycoprotein 2A, Cadherin-1, V-type proton ATPase subunit H, Angiogenin, Ras-related protein Rab-5A, Casein kinase I isoform alpha, Proteasome subunit alpha type-7, Complement receptor type 1, Protein FAM234A, Toll-interacting protein, SID1 transmembrane family member 1, Sushi domain-containing protein 1, Phospholipid-transporting ATPase IA, Charged multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, (C) Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factorreceptor superfamily member 10C、C1GALT1-specific chaperone 1、Cytochrome b5、Beta-enolase、Sorting nexin-1、Catechol O-methyltransferase、Tapasin、Cytochrome b-245 light chain、Protein YIPF5、Neutrophil defensin 3、Armadillo repeat-containing protein 8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrier family 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZdomain-containing protein 8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasminogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、Secretory carrier-associated membrane protein 2、Serine incorporator 3、Calcium-transporting ATPase type 2C member 1、Low affinity immunoglobulin gamma Fc region receptor II-c、AP-3 complex subunit mu-1、Activator of 90 kDa heat shock protein ATPase homolog 1、Brain acid soluble protein 1、Protein transport protein Sec31A、Neutral cholesterol esterA protein group consisting of hydrolase 1, Serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, Ragulator complex protein LAMTOR2, and Tetraspanin-14, (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factor beta-1-induced transcript 1 protein, Volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WD repeat-containing(E) a group of proteins consisting of constitutive coactivator of PPAR-gamma-like protein 1, inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and grancalcin.

[0008] Item 2. The method of detecting a disease according to Item 1, wherein the disease to be detected is small cell lung cancer or squamous cell lung cancer.

[0009] Item 3. The testing method according to Item 1 or 2, wherein the disease to be tested is small cell lung cancer and the protein to be detected comprises at least one selected from the group consisting of protein group (A) and protein group (B), the disease to be tested is squamous cell lung cancer and the protein to be detected comprises at least one selected from the group consisting of protein group (C) and protein group (D), or the disease to be tested is small cell lung cancer and / or squamous cell lung cancer and the protein to be detected comprises at least one selected from the group consisting of protein group (E).

[0010] Item 4. (2A) A step of determining that the subject is suffering from small cell lung cancer when the detected protein contains at least one protein (protein A') selected from the group consisting of protein group (A) and the amount or concentration of protein A' detected in step (1) is equal to or greater than a cutoff value, (2B) A step of determining that the subject is suffering from small cell lung cancer when the detected protein contains at least one protein (protein B') selected from the group consisting of protein group (B) and the amount or concentration of protein B' detected in step (1) is equal to or less than a cutoff value, (2C) A step of determining that the subject is suffering from squamous cell lung cancer when the detected protein contains at least one protein (protein C') selected from the group consisting of protein group (C) and the amount or concentration of protein C' detected in step (1) is equal to or greater than a cutoff value, (2D) determining that the subject is suffering from squamous cell lung cancer if the detected proteins include at least one protein (protein D') selected from the group consisting of protein group (D), and if the amount or concentration of protein D' detected in step (1) is equal to or lower than a cutoff value; and (2E) determining that the subject is suffering from small cell lung cancer and / or squamous cell lung cancer if the detected proteins include at least one protein (protein E') selected from the group consisting of protein group (E), and if the amount or concentration of protein E' detected in step (1) is equal to or higher than a cutoff value.

[0011] Item 5. The testing method according to any one of Items 1 to 4, wherein the protein to be detected includes succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial.

[0012] Item 6. The testing method according to any one of Items 1 to 5, wherein the subject is a human.

[0013] Section 7. Protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E): (A) Succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional A group of proteins consisting of glutamate / proline-tRNA ligase, pyridoxal-dependent decarboxylase domain-containing protein 1, platelet-derived growth factor C, biglycan, eukaryotic translation initiation factor 3 subunit L, emerin, serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, general vesicular transport factor p115, copine-1, WASH complex subunit 5, and protein transport protein Sec23A.(B)Immunoglobulin lambda variable 8-61、HLA class II histocompatibility antigen, DR alpha chain、Protein S100-A16、Suprabasin、High mobility group protein B2、Myelin protein zero-like protein 1、Matrix Gla protein、Ena / VASP-like protein、Beta-hexosaminidase subunit alpha、CD2-associated protein、Oncoprotein-induced transcript 3 protein、Glycosylated lysosomal membrane protein、Transmembrane 7 superfamily member 3、CD81 antigen、EGF-like repeat and discoidin I-like domain-containing protein 3、1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1、Transforming growth factor beta activator LRRC32、Claudin-5、Proteasome subunit beta type-1、F-box only protein 50、Ragulator complex protein LAMTOR1、Trypsin-1、Beta-hexosaminidase subunit beta、Monocarboxylate transporter 2、Dyslexia-associated protein KIAA0319-like protein、DnaJ homolog subfamily B member 1、Membrane cofactor protein、Desmoglein-1、B-cell antigen receptor complex-associated protein beta chain、Raftlin、Synaptic vesicle glycoproteinGroup of proteins consisting of 2A, Cadherin-1, V-type proton ATPase subunit H, Angiogenin, Ras-related protein Rab-5A, Casein kinase I isoform alpha, Proteasome subunit alpha type-7, Complement receptor type 1, Protein FAM234A, Toll-interacting protein, SID1 transmembrane family member 1, Sushi domain-containing protein 1, Phospholipid-transporting ATPase IA, Charged multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, (C) Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factor receptor superfamily member10C、C1GALT1-specific chaperone 1、Cytochrome b5、Beta-enolase、Sorting nexin-1、Catechol O-methyltransferase、Tapasin、Cytochrome b-245 light chain、Protein YIPF5、Neutrophil defensin 3、Armadillo repeat-containing protein 8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrier family 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZ domain-containing protein8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasminogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、Secretory carrier-associated membrane protein 2、Serine incorporator 3、Calcium-transporting ATPase type 2C member 1、Low affinity immunoglobulin gamma Fc region receptor II-c、AP-3 complex subunit mu-1、Activator of 90 kDa heat shock protein ATPase homolog 1、Brain acid soluble protein 1、Protein transport protein Sec31A、Neutral cholesterol ester hydrolase1. A protein group consisting of Serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, Ragulator complex protein LAMTOR2, and Tetraspanin-14, (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factor beta-1-induced transcript 1 protein, Volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WD repeat-containing protein37, Angiopoietin-related protein 3, DnaJ homolog subfamily B member 1, Dedicator of cytokinesis protein 11, Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, Ras-related protein Rab-20, Early activation antigen CD69, TBC1 domain family member 10A, and Receptor-type tyrosine-protein phosphatase epsilon, and (E) a protein group consisting of Constitutive coactivator of PPAR-gamma-like protein 1, Inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and Grancalcin.

[0014] Item 7A. Use of a detector for at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E) for the manufacture of a diagnostic agent for lung cancer.

[0015] Item 7B. A detection agent for detecting at least one protein selected from the group consisting of the above protein group (A), the above protein group (B), the above protein group (C), the above protein group (D), and the above protein group (E), for use as a diagnostic agent for lung cancer.

[0016] Item 7C. Use of a detection agent for at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E) as a diagnostic agent for lung cancer.

[0017] Section 8. Protein group (A), protein group (C), and protein group (E): (A) Succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional glutamate / proline-tRNA ligase, pyridoxal-dependent decarboxylase domain-containing protein 1, platelet-derived growth factor C, biglycan, eukaryotic translation initiation factor 3 subunit L, emerin, serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, general vesicular transport factor p115, copine-1, WASH complex subunit 5, and protein transport protein Sec23A. (C) Transmembrane emp24domain-containing protein 3、Prolow-density lipoprotein receptor-related protein 1、SUN domain-containing protein 2、Follistatin-related protein 1、Leukocyte immunoglobulin-like receptor subfamily A member 6、Acyl-CoA:lysophosphatidylglycerol acyltransferase 1、Tumor necrosis factor receptor superfamily member 10C、C1GALT1-specific chaperone 1、Cytochrome b5、Beta-enolase、Sorting nexin-1、Catechol O-methyltransferase、Tapasin、Cytochrome b-245 light chain、Protein YIPF5、Neutrophil defensin 3、Armadillo repeat-containing protein 8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrierfamily 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZ domain-containing protein 8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasminogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、Secretory carrier-associated membrane protein 2、Serineand (D) a protein group consisting of: (A) a protein group consisting of: ATP-dependent activator 3, calcium-transporting ATPase type 2C member 1, low-affinity immunoglobulin gamma Fc region receptor II-c, AP-3 complex subunit mu-1, activator of 90 kDa heat shock protein ATPase homolog 1, brain acid-soluble protein 1, protein transport protein Sec31A, neutral cholesterol ester hydrolase 1, serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, regulator complex protein LAMTOR2, and tetraspanin-14; and (E) a protein group consisting of: constitutive coactivator of PPAR-gamma-like protein 1, inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and grancalcin; and lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Gla protein, Ena / VASP-like protein, Beta-hexosaminidase subunit alpha, CD2-associated protein, Oncoprotein-induced transcript 3protein、Glycosylated lysosomal membrane protein、Transmembrane 7 superfamily member 3、CD81 antigen、EGF-like repeat and discoidin I-like domain-containing protein 3、1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1、Transforming growth factor beta activator LRRC32、Claudin-5、Proteasome subunit beta type-1、F-box only protein 50、Ragulator complex protein LAMTOR1、Trypsin-1、Beta-hexosaminidase subunit beta、Monocarboxylate transporter 2、Dyslexia-associated protein KIAA0319-like protein、DnaJ homolog subfamily B member 1、Membrane cofactor protein、Desmoglein-1、B-cell antigen receptor complex-associated protein beta chain、Raftlin、Synaptic vesicle glycoprotein 2A、Cadherin-1、V-type proton ATPase subunit H、Angiogenin、Ras-related protein Rab-5A、Casein kinase I isoform alpha、Proteasome subunit alpha type-7、Complement receptor type 1、Protein FAM234A、Toll-interacting protein、SID1 transmembrane family member 1、Sushi domain-containing protein 1、Phospholipid-transporting ATPase IA、ChargedA group of proteins consisting of multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, and (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factorA preventive or therapeutic agent for lung cancer, comprising at least one agent selected from the group consisting of: an enhancer of at least one protein selected from the group consisting of: a protein group consisting of beta-1-induced transcript 1 protein, volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WD repeat-containing protein 37, Angiopoietin-related protein 3, DnaJ homolog subfamily B member 1, Dedicator of cytokinesis protein 11, carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, Ras-related protein Rab-20, early activation antigen CD69, TBC1 domain family member 10A, and receptor-type tyrosine-protein phosphatase epsilon.

[0018] Item 8A. Use of at least one agent selected from the group consisting of: an inhibitor of at least one protein selected from the group consisting of protein group (A), protein group (C), and protein group (E), and an enhancer of at least one protein selected from the group consisting of protein group (B) and protein group (D), for the manufacture of an agent for the prevention or treatment of lung cancer.

[0019] Item 8B. At least one agent selected from the group consisting of an inhibitor of at least one protein selected from the group consisting of protein group (A), protein group (C), and protein group (E), and an enhancer of at least one protein selected from the group consisting of protein group (B) and protein group (D), for use as a preventive or therapeutic agent for lung cancer.

[0020] Item 8C. Use of at least one agent selected from the group consisting of an inhibitor of at least one protein selected from the group consisting of protein group (A), protein group (C), and protein group (E), and an enhancer of at least one protein selected from the group consisting of protein group (B) and protein group (D), as an agent for preventing or treating lung cancer.

[0021] Item 8D. A method for preventing or treating lung cancer, comprising administering to a subject (e.g., a subject in need of lung cancer prevention or treatment, or a subject with lung cancer) at least one agent selected from the group consisting of: an inhibitor of at least one protein selected from the group consisting of protein group (A), protein group (C), and protein group (E), and an enhancer of at least one protein selected from the group consisting of protein group (B) and protein group (D).

[0022] Item 9. Proteins (A), (B), (C), (D), and (E) in extracellular vesicles or body fluids collected from animals treated with the test substance: (A) Succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional glutamate / proline--tRNA ligase, Pyridoxal-dependent decarboxylase domain-containing protein 1, Platelet-derived growth factor C, Biglycan, Eukaryotic translation initiation factor 3 subunit L, Emerin, Serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, general vesicular transport factor p115, Copine-1, WASH complex subunit 5, and proteinA group of proteins consisting of the transport protein Sec23A, (B) Immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Gla protein, Ena / VASP-like protein, Beta-hexosaminidase subunit alpha, CD2-associated protein, Oncoprotein-induced transcript 3 protein, Glycosylated lysosomal membrane protein, Transmembrane 7 superfamily member 3, CD81 antigen, EGF-like repeat and discoidin I-like domain-containing protein 3, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1, Transforming growth factor beta activator LRRC32, Claudin-5, Proteasome subunit beta type-1, F-box only protein 50, Ragulator complex protein LAMTOR1, Trypsin-1, Beta-hexosaminidase subunit beta, Monocarboxylate transporter 2, Dyslexia-associated protein KIAA0319-like protein, DnaJ homolog subfamily B member 1, Membrane cofactor protein, Desmoglein-1, B-cell antigen receptor complex-associated protein betaA group of proteins consisting of chain, Raftlin, Synaptic vesicle glycoprotein 2A, Cadherin-1, V-type proton ATPase subunit H, Angiogenin, Ras-related protein Rab-5A, Casein kinase I isoform alpha, Proteasome subunit alpha type-7, Complement receptor type 1, Protein FAM234A, Toll-interacting protein, SID1 transmembrane family member 1, Sushi domain-containing protein 1, Phospholipid-transporting ATPase IA, Charged multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, (C) Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factorreceptor superfamily member 10C、C1GALT1-specific chaperone 1、Cytochrome b5、Beta-enolase、Sorting nexin-1、Catechol O-methyltransferase、Tapasin、Cytochrome b-245 light chain、Protein YIPF5、Neutrophil defensin 3、Armadillo repeat-containing protein 8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrier family 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZdomain-containing protein 8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasmin ogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、Secretory carrier-associated membrane protein 2、Serine incorporator 3、Calcium-transporting ATPase type 2C member 1、Low affinity immunoglobulin gamma Fc region receptor II-c、AP-3 complex subunit mu-1、Activator of 90 kDa heat shock protein ATPase homolog 1、Brain acid soluble protein 1、Protein transport protein Sec31A、Neutral cholesterolA protein group consisting of ester hydrolase 1, Serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, Ragulator complex protein LAMTOR2, and Tetraspanin-14, (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factor beta-1-induced transcript 1 protein, Volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WDA method for screening for an active ingredient of a preventive or therapeutic agent for lung cancer, using as an index the amount or concentration of at least one protein selected from the group consisting of: (A) a group of proteins consisting of repeat-containing protein 37, Angiopoietin-related protein 3, DnaJ homolog subfamily B member 1, Dedicator of cytokinesis protein 11, Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, Ras-related protein Rab-20, Early activation antigen CD69, TBC1 domain family member 10A, and Receptor-type tyrosine-protein phosphatase epsilon; and (B) a group of proteins consisting of Constitutive coactivator of PPAR-gamma-like protein 1, Inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and Grancalcin.

[0023] Item 10. Proteins (A), (B), (C), (D), and (E) in extracellular vesicles or body fluids collected from animals treated with the test substance: (A) Succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional glutamate / proline--tRNA ligase, Pyridoxal-dependent decarboxylase domain-containing protein 1, Platelet-derived growth factor C, Biglycan, Eukaryotic translation initiation factor 3 subunit L, Emerin, Serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, general vesicular transport factor p115, Copine-1, WASH complex subunit 5, and proteinA group of proteins consisting of the transport protein Sec23A, (B) Immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Gla protein, Ena / VASP-like protein, Beta-hexosaminidase subunit alpha, CD2-associated protein, Oncoprotein-induced transcript 3 protein, Glycosylated lysosomal membrane protein, Transmembrane 7 superfamily member 3, CD81 antigen, EGF-like repeat and discoidin I-like domain-containing protein 3, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1, Transforming growth factor beta activator LRRC32, Claudin-5, Proteasome subunit beta type-1, F-box only protein 50, Ragulator complex protein LAMTOR1, Trypsin-1, Beta-hexosaminidase subunit beta, Monocarboxylate transporter 2, Dyslexia-associated protein KIAA0319-like protein, DnaJ homolog subfamily B member 1, Membrane cofactor protein, Desmoglein-1, B-cell antigen receptor complex-associated protein betaA protein group consisting of chain, Raftlin, Synaptic vesicle glycoprotein 2A, Cadherin-1, V-type proton ATPase subunit H, Angiogenin, Ras-related protein Rab-5A, Casein kinase I isoform alpha, Proteasome subunit alpha type-7, Complement receptor type 1, Protein FAM234A, Toll-interacting protein, SID1 transmembrane family member 1, Sushi domain-containing protein 1, Phospholipid-transporting ATPase IA, Charged multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, (C) Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factorreceptor superfamily member 10C、C1GALT1-specific chaperone 1、Cytochrome b5、Beta-enolase、Sorting nexin-1、Catechol O-methyltransferase、Tapasin、Cytochrome b-245 light chain、Protein YIPF5、Neutrophil defensin 3、Armadillo repeat-containing protein 8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrier family 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZdomain-containing protein 8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasminogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、Secretory carrier-associated membrane protein 2、Serine incorporator 3、Calcium-transporting ATPase type 2C member 1、Low affinity immunoglobulin gamma Fc region receptor II-c、AP-3 complex subunit mu-1、Activator of 90 kDa heat shock protein ATPase homolog 1、Brain acid soluble protein 1、Protein transport protein Sec31A、Neutral cholesterol esterA group of proteins consisting of hydrolase 1, Serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, Ragulator complex protein LAMTOR2, and Tetraspanin-14, (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factor beta-1-induced transcript 1 protein, Volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WD repeat-containingA method for evaluating the inducibility or exacerbation of lung cancer, using as an index the amount or concentration of at least one protein selected from the group consisting of: (A) a group of proteins consisting of ribosomal protein 37, Angiopoietin-related protein 3, DnaJ homolog subfamily B member 1, Dedicator of cytokinesis protein 11, Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, Ras-related protein Rab-20, Early activation antigen CD69, TBC1 domain family member 10A, and Receptor-type tyrosine-protein phosphatase epsilon; and (B) a group of proteins consisting of Constitutive coactivator of PPAR-gamma-like protein 1, Inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and Grancalcin.

[0024] The present invention provides a lung cancer biomarker. Use of the biomarker may enable testing for lung cancer, preventing or treating lung cancer, screening for active ingredients of lung cancer preventive or therapeutic agents, and evaluating the inducibility or exacerbation of lung cancer.

[0025] Quantitative results of proteomic analysis of eight SCLC BM candidate proteins in serum exosomes are shown. HC: healthy control, SCC: squamous cell lung cancer, SCLC: small cell lung cancer, Therapy- / +: before / after treatment, *: p<0.05. Quantitative results of immunoblot analysis of SUCLA2 in serum exosomes are shown. HC: healthy control, SCC: squamous cell lung cancer, SCLC: small cell lung cancer, *: p<0.05. Quantitative results of immunoblot analysis of SUCLA2 in serum exosomes are shown. LD: limited-stage SCLC, ED: extensive-stage SCLC, *: p<0.05. SUCLA2 staining images of lung tissue are shown. SCC: squamous cell lung cancer, SCLC: small cell lung cancer. IHC scores based on SUCLA2 staining images of lung tissue are shown. SCC: squamous cell lung cancer, SCLC: small cell lung cancer, *: p<0.05. ROC curves for serum SUCLA2 or ProGRP, and serum SUCLA2 and ProGRP for SCLC diagnosis are shown. The results of immunoblotting with SUCLA2 antibody for whole cell extracts of each cell line (SBC-3, OS2RA) are shown. siRNA- / +: No / with treatment with siRNA against SUCLA2. Cell proliferation curves for each cell line are shown. The vertical axis shows quantified values ​​obtained by MTT assay. The horizontal axis shows the time elapsed since cell seeding. Si- / +: No / with treatment with siRNA against SUCLA2, *: p<0.05.

[0026] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0027] 1. Lung Cancer Testing Method In one aspect, the present invention relates to a method for testing for lung cancer, which comprises (1) detecting at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E) in extracellular vesicles or body fluid collected from a subject (also referred to herein as the "testing method of the present invention"). This is described below.

[0028] 1-1. Step (1) The type of "lung cancer" to be tested is not particularly limited. Examples of lung cancer include small cell lung cancer, squamous cell carcinoma, adenocarcinoma, and large cell carcinoma, with small cell lung cancer and squamous cell carcinoma being preferred, and small cell lung cancer being particularly preferred. Lung cancer of all classes, grades, and stages according to various classification criteria for the progression of lung cancer can be tested.

[0029] The subject is a living organism that is the target of the testing method of the present invention, and the species of the subject is not particularly limited. Examples of the subject species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, and preferably humans.

[0030] The condition of the subject is not particularly limited, and examples of the subject include a subject whose presence or absence of lung cancer is unknown, a subject whose presence or absence has already been determined by another method to be associated with lung cancer, a subject whose presence or absence has already been determined by another method to be not associated with lung cancer, a subject undergoing treatment for lung cancer, a subject whose presence or absence has been treated for lung cancer, and the like.

[0031] The detection sample in step (1) is preferably extracellular vesicles from a body fluid.

[0032] The body fluid is not particularly limited. Examples of body fluids include whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, exhaled breath, and exhaled breath condensate. Preferred examples include whole blood, serum, plasma, and cerebrospinal fluid, and more preferred examples include whole blood, serum, and plasma. One type of body fluid may be used alone, or two or more types may be used in combination.

[0033] Body fluids can be collected from a subject by methods known to those skilled in the art. For example, whole blood can be collected by drawing blood using a syringe or the like. Serum is a portion of whole blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as the supernatant after clotting of whole blood. Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as the supernatant when whole blood is centrifuged under conditions that do not cause clotting.

[0034] In this specification, blood itself or a sample derived from blood, such as whole blood, serum, or plasma, is referred to as a "blood sample."

[0035] Extracellular vesicles are not particularly limited as long as they are membrane vesicles secreted or released from cells. Extracellular vesicles are generally defined as membrane vesicles that transport intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of cells, thereby facilitating local or systemic intercellular signaling. Examples of extracellular vesicles include exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, and secreted microvesicles.

[0036] Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or in accordance with known methods. Methods for purifying, separating, concentrating, etc. extracellular vesicles include, for example, ultracentrifugation (e.g., pellet-down method, sucrose cushion method, density gradient centrifugation, etc.), methods using immunoaffinity carriers, gel filtration, field-flow fractionation, FACS, etc. Purification, separation, concentration, etc. of extracellular vesicles can also be performed using commercially available kits. These methods may be used alone or in combination of two or more.

[0037] The detection target in step (1) is at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E) (these may be collectively referred to as the "target protein" in this specification).

[0038] Protein group (A) is a protein group consisting of (A) Succinate--CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional glutamate / proline--tRNA ligase, Pyridoxal-dependent decarboxylase domain-containing protein 1, Platelet-derived growth factor C, Biglycan, Eukaryotic translation initiation factor 3 subunit L, Emerin, Serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, General vesicular transport factor p115, Copine-1, WASH complex subunit 5, and Protein transport protein Sec23A.

[0039] Protein group (A) is a protein whose expression level is higher in small cell lung cancer subjects than in healthy subjects.

[0040] Among proteins in group (A), from the viewpoint that the difference in expression level between healthy subjects and small cell lung cancer subjects is larger and that they can be suitably used for detection and testing, preferred are: Succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial; Immunoglobulin kappa variable 1-39; Phosphoenolpyruvate carboxykinase [GTP], mitochondrial; Eukaryotic translation initiation factor 3 subunit J; AP-1 complex subunit gamma-like 2; Protein-tyrosine sulfotransferase 2; Fibrinogen-like protein 1; Small proline-rich protein 2G; Reelin; E3 ubiquitin-protein ligase TRIM21; CAAX prenyl protease 1 homolog; Alkyldihydroxyacetonephosphate synthase, peroxisomal; SEC23-interacting protein; 2-oxoglutarate dehydrogenase; mitochondrial, Bifunctional glutamate / proline--tRNA ligase, Pyridoxal-dependent decarboxylase domain-containing protein 1, Platelet-derived growth factor C, Biglycan, Eukaryotic translation initiation factor 3 subunit L, Emerin, Serine / threonine-protein kinase A-Raf, 3'(2'),Examples of such proteins include 5'-bisphosphate nucleotidase 1, general vesicular transport factor p115, and Copine-1, and more preferably, succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial, immunoglobulin kappa variable 1-39, phosphoenolpyruvate carboxykinase [GTP], mitochondrial, eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, protein-tyrosine sulfotransferase 2, fibrinogen-like protein 1, small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, and CAAX prenyl protease 1 homolog.

[0041] Protein group (B) consists of (B) Immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Gla protein, Ena / VASP-like protein, Beta-hexosaminidase subunit alpha, CD2-associated protein, Oncoprotein-induced transcript 3 protein, Glycosylated lysosomal membrane protein, Transmembrane 7 superfamily member 3, CD81 antigen, EGF-like repeat and discoidin I-like domain-containing protein 3, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1, Transforming growth factor beta activator LRRC32, Claudin-5, Proteasome subunit beta type-1, F-box only protein 50, Ragulator complex protein LAMTOR1, Trypsin-1, Beta-hexosaminidase subunit beta, Monocarboxylate transporter 2, Dyslexia-associated protein KIAA0319-like protein, DnaJ homolog subfamily B member 1, Membrane cofactor protein, Desmoglein-1, B-cell antigen receptor complex-associated protein beta chain, Raftlin, Synaptic vesicleglycoprotein 2A, Cadherin-1, V-type proton ATPase subunit H, Angiogenin, Ras-related protein Rab-5A, Casein kinase I isoform alpha, Proteasome subunit alpha type-7, Complement receptor type 1, Protein FAM234A, Toll-interacting protein, SID1 transmembrane family member 1, Sushi domain-containing protein 1, Phospholipid-transporting ATPase IA, Charged multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small A group of proteins consisting of phosphatase 1.

[0042] Protein group (B) is a protein whose expression level in small cell lung cancer subjects is lower than that in healthy subjects.

[0043] Among proteins in group (B), from the viewpoint that the difference in expression level between healthy subjects and small cell lung cancer subjects is larger and that they can be suitably used for detection and testing, preferred are immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Gla protein, Ena / VASP-like protein, Beta-hexosaminidase subunit alpha, CD2-associated protein, Oncoprotein-induced transcript 3 protein, Glycosylated lysosomal membrane protein, Transmembrane 7 superfamily member 3, CD81 antigen, EGF-like repeat and discoidin I-like domain-containing protein 3, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1, and Transforming growth factor beta activator LRRC32, Claudin-5, Proteasome subunit beta type-1, F-box only protein 50, Ragulator complex protein LAMTOR1, Trypsin-1, Beta-hexosaminidase subunit beta, Monocarboxylate transporter 2 are preferred, and immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen,These include DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, and Myelin protein zero-like protein 1.

[0044] Protein group (C) includes (C) Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factor receptor superfamily member 10C, C1GALT1-specific chaperone 1, Cytochrome b5, Beta-enolase, Sorting nexin-1, Catechol O-methyltransferase, Tapasin, Cytochrome b-245 light chain, Protein YIPF5, Neutrophil defensin 3, Armadillo repeat-containing protein 8, Estradiol 17-beta-dehydrogenase 11, Peflin, Sorcin, Transmembrane protein 165, Cytochrome P450 4F3, Aldehyde dehydrogenase family 3 member B1, Methyltransferase-like protein 7A, Mucolipin-1, Hydroxyacylglutathione hydrolase, mitochondrial, Myeloid cell nuclear differentiation antigen, Annexin A11, Intercellular adhesion molecule 4, Carcinoembryonic antigen-related cell adhesion molecule 8, Dehydrogenase / reductase SDR family member 7, Transmembraneprotein 87A、Annexin A7、Solute carrier family 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZ domain-containing protein 8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasminogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、SecretoryThis is a group of proteins consisting of carrier-associated membrane protein 2, serine incorporator 3, calcium-transporting ATPase type 2C member 1, low-affinity immunoglobulin gamma Fc region receptor II-c, AP-3 complex subunit mu-1, activator of 90 kDa heat shock protein ATPase homolog 1, brain acid-soluble protein 1, protein transport protein Sec31A, neutral cholesterol ester hydrolase 1, serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, ragulator complex protein LAMTOR2, and tetraspanin-14.

[0045] Protein group (C) is a protein whose expression level is higher in squamous cell lung cancer subjects than in healthy subjects.

[0046] Among proteins in group (C), from the viewpoint that the difference in expression level between healthy subjects and squamous cell lung cancer subjects is larger and that they can be suitably used for detection and testing, preferred are Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factor receptor superfamily member 10C, C1GALT1-specific chaperone 1, Cytochrome b5, Beta-enolase, Sorting nexin-1, Catechol O-methyltransferase, Tapasin, Cytochrome b-245 light chain, Protein YIPF5, Neutrophil defensin 3, and Armadillo repeat-containing protein 8, Estradiol 17-beta-dehydrogenase 11, Peflin, Sorcin, Transmembrane protein 165, Cytochrome P450 4F3, Aldehyde dehydrogenase family 3 member B1, Methyltransferase-like protein 7A, Mucolipin-1, Hydroxyacylglutathione hydrolase,mitochondrial, Myeloid cell nuclear differentiation antigen, Annexin A11, Intercellular adhesion molecule 4, Carcinoembryonic antigen-related cell adhesion molecule 8, Dehydrogenase / reductase SDR family member 7, Transmembrane protein 87A, Annexin A7, Solute carrier family 2, facilitated glucose transporter member 1, Adenosine 3'-phospho 5'-phosphosulfate transporter 1, Interferon-induced 35 kDa protein, Erythrocyte membrane protein band 4.2, Protein argonaute-2, Annexin A5, Cullin-2, Glycophorin-A, Integrin alpha-M, Porphobilinogen deaminase, PDZ domain-containing protein 8, Reticulon-3, Metalloreductase STEAP4, Transmembrane emp24 domain-containing protein 9 are mentioned, and more preferably Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-Co-A:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factor receptor superfamily member 10C are mentioned.,

[0047] Protein group (D) consists of (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factor beta-1-induced transcript 1 protein, Volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WD repeat-containing protein 37, Angiopoietin-related protein 3, DnaJ homolog subfamily B member 1, Dedicator of cytokinesis protein 11, Carboxy-terminal domain RNAThis is a group of proteins consisting of polymerase II polypeptide A small phosphatase 1, Ras-related protein Rab-20, early activation antigen CD69, TBC1 domain family member 10A, and receptor-type tyrosine-protein phosphatase epsilon.

[0048] Protein group (D) is a protein whose expression level in squamous cell lung cancer subjects is lower than that in healthy subjects.

[0049] Among proteins in group (D), from the viewpoint of having a larger difference in expression level between healthy subjects and squamous cell lung cancer subjects and being suitable for use in detection and testing, preferred are Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein CI, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, and RAS protein activator. Examples of such proteins include Ras-like-3, Semaphorin-4B, and Ras-related protein R-Ras2, and more preferably, Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, and Low affinity immunoglobulin gamma Fc region receptor II-b.

[0050] Protein group (E) is a group of proteins consisting of (E) Constitutive coactivator of PPAR-gamma-like protein 1, Inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and Grancalcin.

[0051] Protein group (E) is a group of proteins whose expression levels are higher in small cell lung cancer subjects and squamous cell lung cancer subjects than in healthy subjects.

[0052] Among the protein groups (A) to (E), in one embodiment, the protein groups (A), (C), and (E) are preferred from the viewpoint that they are proteins whose expression level is increased compared to that of healthy subjects and are suitable for use in detection and testing.

[0053] Among the proteins in groups (A) to (E), in one embodiment, succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial, phosphoenolpyruvate carboxykinase [GTP], mitochondrial, constitutive coactivator of PPAR-gamma-like protein 1, AP-1 complex subunit gamma-like 2, CAAX prenyl protease 1 homolog, inter-alpha-trypsin inhibitor heavy chain H4, fibrinogen-like protein 1, and E3 ubiquitin-protein ligase TRIM21 are particularly preferred, as they are also expressed in lung cancer tissues and are more closely associated with pathology. Among these, succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial is particularly preferred, as it can more specifically detect small cell lung cancer, better reflects treatment status, and better reflects pathology.

[0054] The proteins in protein groups (A) to (E) are, in the case of humans, proteins identified by the UniProtKB accession numbers shown in Tables 1 to 8 in the Examples described below. In the case of other biological species, they are orthologs of proteins identified by the UniProtKB accession numbers shown in Tables 1 to 8.

[0055] The number of target proteins in step (1) may be one or a combination of two or more. Combining a larger number of target proteins (e.g., 2, 5, 10, 20, 30, 40, 50 or more) enables more accurate testing for lung cancer, etc.

[0056] Detection is usually performed by measuring the amount or concentration of the target protein. The "concentration" is not limited to absolute concentration, but may also be relative concentration, weight per unit volume, raw data measured to determine absolute concentration, etc.

[0057] The method for detecting a target protein is not particularly limited as long as it can specifically detect part or all of the target protein. Specific examples of detection methods include mass spectrometry, which detects peptides that constitute the target protein, and immunoassays using antibodies that specifically recognize the target protein. The amino acid sequence information of the target protein can be obtained by searching the EBI database (http: / / www.ebi.ac.uk / IPI / IPIhelp.html) using the UniProtKB accession number.

[0058] Suitable examples of immunological assays include immunohistochemical staining, ELISA, EIA, RIA, and Western blotting.

[0059] Mass spectrometry is a method of measuring peptide samples using a mass spectrometer. The peptide samples are converted into gaseous ions (ionization) using an ion source, and then moved in a vacuum in the analytical section, where they are separated and detected according to their mass-to-charge ratio using electromagnetic force or time-of-flight (TOF) differential ionization. Ionization methods using the ion source can be selected from EI, CI, FD, FAB, MALDI, and ESI. Separation methods for the ionized peptide samples in the analytical section can be selected from magnetic deflection, quadrupole, ion trap, time-of-flight (TOF), and Fourier transform ion cyclotron resonance. Tandem mass spectrometry (MS / MS) and triple quadrupole mass spectrometry, which combine two or more mass spectrometry techniques, can also be used. For samples containing phosphorylated peptides, the sample can be enriched using iron ion-immobilized affinity chromatography (Fe-IMAC) before introduction into the mass spectrometer. Alternatively, peptides constituting the target protein can be separated and purified using liquid chromatography (LC) or HPLC to prepare samples. The detector and data processing method can also be selected appropriately. When detecting and quantifying peptides constituting the target protein using mass spectrometry, a peptide with the same amino acid sequence as the peptide and labeled with a stable isotope of known concentration can be used as an internal standard. Such stable isotope-labeled peptides can be appropriately selected from the type, position, number, and other characteristics of amino acids, as long as one or more of the amino acids in the peptide constituting the target protein to be detected contains one or more of 15N, 13C, 18O, and 2H. Such stable isotope-labeled peptides can be chemically synthesized by an appropriate method, such as the F-moc method (Amblard., et al. Methods Mol Biol. 298:3-24 (2005)), using amino acids labeled with stable isotopes. However, they can also be prepared using labeling reagents such as iTRAQ (registered trademark) reagents, ICAT (registered trademark) reagents, ICPL (registered trademark) reagents, and NBS (registered trademark) reagents.

[0060] According to the testing method of the present invention including step (1), the amount and / or concentration of a target protein, which is an indicator for detecting lung cancer, can be provided, thereby assisting in the detection of lung cancer, evaluation of the severity, etc.

[0061] The test results obtained by the test method of the present invention, including step (1), can be used as a therapeutic marker for lung cancer therapeutic drugs, to clarify the pathology of lung cancer, to predict the prognosis of lung cancer, to stratify subjects, to select a treatment method (personalized medicine, treatment responsiveness), to differentiate between intractable lung cancers, and to differentiate the histological type and phenotype of lung cancer.

[0062] 1-2. Step (2) In one embodiment, the testing method of the present invention includes the following steps: (2A) determining that the subject is suffering from small cell lung cancer when the detected protein contains at least one protein (protein A') selected from the group consisting of protein group (A) and the amount or concentration of protein A' detected in step (1) is equal to or greater than a cutoff value; (2B) determining that the subject is suffering from small cell lung cancer when the detected protein contains at least one protein (protein B') selected from the group consisting of protein group (B) and the amount or concentration of protein B' detected in step (1) is equal to or less than a cutoff value; (2C) determining that the subject is suffering from squamous cell lung cancer when the detected protein contains at least one protein (protein C') selected from the group consisting of protein group (C) and the amount or concentration of protein C' detected in step (1) is equal to or greater than a cutoff value; (2D) determining that the subject suffers from squamous cell lung cancer if the detected protein contains at least one protein (protein D') selected from the group consisting of protein group (D) and if the amount or concentration of protein D' detected in step (1) is equal to or lower than a cutoff value, and (2E) determining that the subject suffers from small cell lung cancer and / or squamous cell lung cancer if the detected protein contains at least one protein (protein E') selected from the group consisting of protein group (E) and if the amount or concentration of protein E' detected in step (1) is equal to or higher than a cutoff value. The testing method of the present invention, which includes these steps, makes it possible to diagnose lung cancer.

[0063] In one embodiment of the present invention, in the above steps, instead of determining the presence or absence of small cell lung cancer, the stage of progression can be determined. For example, the testing method of the present invention can further include a step of determining that the subject has small cell lung cancer or that the stage of small cell lung cancer in the subject is high if the detected protein includes succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial and if the amount or concentration of succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial detected in step (1) is equal to or greater than a cutoff value.

[0064] The cutoff value can be appropriately set by one skilled in the art from the perspectives of sensitivity, specificity, positive predictive value, negative predictive value, etc., and can be a value determined on a case-by-case basis or a predetermined value based on the amount and / or concentration of the target protein in extracellular vesicles or body fluid collected from a control subject (e.g., a subject not affected or affected by lung cancer, a subject with a low or high index value for the severity / progression of lung cancer, etc.). The cutoff value can be, for example, 0.7 to 1.5 times the amount and / or concentration of the target protein in extracellular vesicles or body fluid collected from a reference subject or subject group (e.g., the mean, median, etc., if there are multiple subjects). The cutoff value can also be set by performing statistical analysis based on, for example, receiver operating characteristic (ROC) curve analysis (more specifically, a method using the Youden index is exemplified).

[0065] In a preferred embodiment of step (2), when the subject is a specimen during or after treatment for lung cancer, the therapeutic effect can be assessed by setting the cutoff value based on, for example, the amount and / or concentration of the target protein in a previous sample from the same specimen.

[0066] 2. Diagnosis of lung cancer with higher accuracy When a subject is determined to have lung cancer by the testing method of the present invention including step (2), lung cancer can be diagnosed with higher accuracy by combining the testing method of the present invention with a step of having a lung cancer doctor diagnose the subject. Furthermore, since the testing method of the present invention can detect lung cancer more accurately, combining the above steps with the testing method of the present invention can more efficiently and accurately diagnose "the subject having lung cancer."

[0067] 3. Treatment of Lung Cancer If a subject is determined to have lung cancer by the testing method of the present invention including step (2), the testing method of the present invention can be further combined with the step of administering a diagnosis by a physician if the subject has been diagnosed with lung cancer as described above in "2. Diagnosis of Lung Cancer with Higher Accuracy," and the test method of the present invention can be further combined with the step of administering a diagnosis by a physician (3) to treat the subject for the disease, thereby enabling the subject to be treated for the disease. Furthermore, since the testing method of the present invention can detect lung cancer more accurately, combining step 3 with the testing method of the present invention or the combination of the testing method of the present invention and the step of administering a diagnosis by a physician can enable more efficient and reliable treatment of subjects with lung cancer.

[0068] The treatment method for lung cancer is not particularly limited, but a representative example is drug therapy. The pharmaceuticals used in drug therapy are not particularly limited, but examples include various anticancer drugs. Examples of anticancer drugs include alkylating agents, antimetabolites, microtubule inhibitors, antibiotic anticancer drugs, topoisomerase inhibitors, platinum preparations, molecular targeted drugs, hormone drugs, and biological preparations, and preferred examples include antimetabolites, antibiotic anticancer drugs, and platinum preparations. Examples of alkylating agents include cyclophosphamide, ifosfamide, nitrosoureas, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine. Examples of antimetabolites include enocitabine, carmofur, capecitabine, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, gemcitabine, cytarabine, cytarabine ocfosfate, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, cladribine, doxifluridine, hydroxycarbamide, mercaptopurine, etc. Examples of microtubule inhibitors include alkaloid anticancer drugs such as vincristine, and taxane anticancer drugs such as docetaxel and paclitaxel. Antibiotic anticancer drugs include, for example, mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, aclarubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, zinostatin stimalamer, etc. Topoisomerase inhibitors include CPT-11, irinotecan, nogitecan, which have topoisomerase I inhibitory activity, and etoposide and sobuzoxane, which have topoisomerase II inhibitory activity. Platinum drugs include, for example, cisplatin, nedaplatin, oxaliplatin, carboplatin, etc.Examples of hormone drugs include dexamethasone, finasteride, tamoxifen, astrozole, exemestane, ethinylestradiol, chlormadinone, goserelin, bicalutamide, flutamide, prednisolone, leuprorelin, letrozole, estramustine, toremifene, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, progesterone, mepitiostane, etc. Examples of biological drugs include interferon α, β, and γ, interleukin 2, ubenimex, and dried BCG. Examples of molecularly targeted drugs include rituximab, alemtuzumab, trastuzumab, cetuximab, panitumumab, imatinib, dasatinib, nilotinib, gefitinib, erlotinib, temsirolimus, bevacizumab, VEGF trap, sunitinib, sorafenib, tosituzumab, bortezomib, gemtuzumab ozogamicin, ibritumomab ozogamicin, ibritumomab tiuxetan, tamibarotene, and tretinoin. In addition to the molecular targeted drugs specified herein, other molecular targeted drugs include inhibitors that target angiogenesis, such as human epidermal growth factor receptor 2 inhibitors, epidermal growth factor receptor inhibitors, Bcr-Abl tyrosine kinase inhibitors, epidermal growth factor tyrosine kinase inhibitors, mTOR inhibitors, and vascular endothelial growth factor receptor 2 inhibitors (α-VEGFR-2 antibodies), various tyrosine kinase inhibitors such as MAP kinase inhibitors, inhibitors that target cytokines, proteasome inhibitors, and antibody-anticancer drug combinations. These inhibitors also include antibodies. Pharmaceuticals can be used in combination of one, two, or three or more types.

[0069] 4. Lung Cancer Test Agent and Test Kit In one aspect, the present invention relates to a lung cancer test agent (sometimes referred to herein as the "test agent of the present invention") that includes a detection agent for at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E). This will be described below.

[0070] Protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), lung cancer, etc. are defined in the same way as in "1. Lung cancer testing methods" above.

[0071] The detection agent is not particularly limited as long as it can specifically detect the target protein, and examples of the detection agent include antibodies against the target protein.

[0072] The detection agent may be modified as long as its function is not significantly impaired. Examples of modifications include the addition or introduction of labels such as fluorescent dyes, luminescent substances, dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, gold colloids, biotin, etc.

[0073] The detection agent can also be used by immobilizing it on any solid phase, and therefore the test agent of the present invention can be provided in the form of a substrate on which the detection agent is immobilized (for example, a microarray chip on which a probe is immobilized, or another example, an ELISA plate on which an antibody is immobilized).

[0074] The solid phase used for immobilization is not particularly limited as long as it can immobilize antibodies, etc., and examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. There are no particular limitations on the immobilization of the detection agent to the solid phase.

[0075] The antibody is not particularly limited as long as it selectively (specifically) recognizes the target protein. Here, "selectively (specifically) recognizes" means that the target protein can be specifically detected, for example, in Western blotting or ELISA, but is not limited thereto, and may be any antibody that allows a person skilled in the art to determine that the detected product is derived from the target protein.

[0076] The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, and portions of the above antibodies that have antigen-binding ability, such as Fab fragments and fragments produced by an Fab expression library. The antibodies of the present invention also include antibodies that have antigen-binding ability to a polypeptide consisting of at least 8 consecutive amino acids, preferably 15 amino acids, and more preferably 20 amino acids, from the amino acid sequence of the target protein.

[0077] Methods for producing these antibodies are well known, and the antibodies of the present invention can be produced according to these conventional methods (Current Protocols in Molecular Biology, Chapters 11.12-11.13 (2000)). Specifically, when the antibodies of the present invention are polyclonal, they can be obtained from the serum of a non-human animal such as a rabbit by conventional methods using a target protein expressed and purified in E. coli or the like, or an oligopeptide having a partial amino acid sequence of the target protein, synthesized according to conventional methods. On the other hand, when the antibodies of the present invention are monoclonal, they can be obtained from hybridoma cells prepared by immunizing a non-human animal such as a mouse with a target protein expressed and purified in E. coli or the like, or an oligopeptide having a partial amino acid sequence of the target protein, according to conventional methods (Current Protocols in Molecular Biology, edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.4-11.11).

[0078] The target protein used as an immunizing antigen for antibody production can be obtained by DNA cloning, constructing each plasmid, transfecting the host, culturing the transformant, and recovering the protein from the culture, based on known gene sequence information. These procedures can be performed according to methods known to those skilled in the art or described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, D.M. Glover, IRL PRESS (1985)).

[0079] Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding a target protein in a desired host cell is prepared, which is then introduced into the host cell to transform it, the transformant is cultured, and the target protein is recovered from the resulting culture, thereby obtaining a protein as an immunogen for producing the antibody of the present invention. Alternatively, a partial peptide of the target protein can be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.

[0080] The antibodies of the present invention may also be prepared using an oligopeptide having a partial amino acid sequence of the target protein. The oligo(poly)peptide used to produce such antibodies does not need to have functional biological activity, but it is desirable that it has immunogenic properties similar to those of the target protein. Preferred examples include oligo(poly)peptides that have these immunogenic properties and consist of at least 8, preferably 15, and more preferably 20 consecutive amino acids in the amino acid sequence of the target protein.

[0081] The production of antibodies against such oligo(poly)peptides can be carried out by using various adjuvants depending on the host to enhance immunological responses, including, but not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, surfactants such as keyhole limpet hemocyanin and dinitrophenol, and human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0082] The test agent of the present invention may be in the form of a composition. The composition may contain other components as needed. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0083] The test agent of the present invention may be in the form of a kit. In addition to the detection agent or the composition containing the same, the kit may also contain other components that can be used to detect target proteins in extracellular vesicles in the body fluids of a subject or in a blood sample. Specific examples of such components include various reagents (e.g., secondary antibodies, buffer solutions, etc.), instruments (e.g., instruments for purifying, separating, and concentrating extracellular vesicles (e.g., columns, etc.)), and the like.

[0084] 5. Preventive or Therapeutic Agent for Lung Cancer In one aspect, the present invention relates to a preventive or therapeutic agent for lung cancer (sometimes referred to herein as the "agent of the present invention") comprising at least one agent selected from the group consisting of an inhibitor of at least one protein selected from the group consisting of protein group (A), protein group (C), and protein group (E), and an enhancer of at least one protein selected from the group consisting of protein group (B) and protein group (D). This will be explained below.

[0085] Protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), lung cancer, etc. are defined in the same way as in "1. Lung cancer testing methods" above.

[0086] Examples of inhibitors include antibodies against the target protein. The antibodies may be the same as those described above in "4. Lung cancer test agents and test kits."

[0087] Another example of an inhibitor is an inhibitor of the expression of a protein of interest.

[0088] The expression inhibitor of a target protein is not particularly limited as long as it can suppress the expression level of the target protein, its mRNA, its precursor, etc., and examples include small interfering RNA (siRNA) specific to the gene of the target protein, microRNA (miRNA) specific to the gene of the target protein, antisense nucleic acid specific to the gene of the target protein, and expression vectors thereof; ribozymes specific to the gene of the target protein; and gene editing agents for the gene of the target protein using the CRISPR / Cas system.

[0089] In addition, expression inhibition means suppressing the expression level of a target protein, its mRNA, etc. to, for example, 1 / 2, 1 / 3, 1 / 5, 1 / 10, 1 / 20, 1 / 30, 1 / 50, 1 / 100, 1 / 200, 1 / 300, 1 / 500, 1 / 1000, or 1 / 10,000 or less, and also includes reducing these expression levels to zero.

[0090] Examples of enhancers include those that enhance the expression of a target protein.

[0091] The expression enhancer for a target protein is not particularly limited as long as it can enhance the expression level of the target protein, its mRNA, its precursor, etc., and examples thereof include an expression vector for the target protein.

[0092] In addition, enhanced expression means increasing the expression level of a target protein, its mRNA, etc., by, for example, 2-, 3-, 5-, 10-, 20-, 30-, 50-, 100-, 200-, 300-, 500-, 1000-, or 10,000-fold.

[0093] The siRNA for the gene encoding the target protein is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target protein. In one embodiment, the siRNA is preferably, for example, 18 or more bases, 19 or more bases, 20 or more bases, or 21 or more bases in length. Furthermore, the siRNA is preferably, for example, 25 or less bases, 24 or less bases, 23 or less bases, or 22 or less bases in length. The upper and lower limits of the siRNA length described herein may be arbitrarily combined. For example, the following combinations are conceivable: a lower limit of 18 bases and an upper limit of 25, 24, 23, or 22 bases; a lower limit of 19 bases and an upper limit of 25, 24, 23, or 22 bases; a lower limit of 20 bases and an upper limit of 25, 24, 23, or 22 bases; or a lower limit of 21 bases and an upper limit of 25, 24, 23, or 22 bases.

[0094] The siRNA may be a small hairpin RNA (shRNA). The shRNA can be designed so that a portion thereof forms a stem-loop structure. For example, if the sequence of a certain region is designated sequence a and the complementary strand to sequence a is designated sequence b, the shRNA can be designed so that these sequences are present in a single RNA strand in the order of sequence a, spacer, and sequence b, with a total length of 45 to 60 bases. Sequence a is a partial sequence of the base sequence encoding the target protein. The target region is not particularly limited, and any region can be used as a candidate. The length of sequence a is 19 to 25 bases, preferably 19 to 21 bases.

[0095] The gene-specific siRNA for a target protein may have additional bases at the 5' or 3' end. The length of the additional bases is usually about 2 to 4 bases. The additional bases may be DNA or RNA, but using DNA may improve the stability of the nucleic acid. Examples of such additional base sequences include, but are not limited to, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', and uuuuu-3'.

[0096] siRNA may have a protruding sequence (overhang) at the 3' end, specifically, dTdT (dT stands for deoxythymidine) added. Alternatively, blunt ends without additional ends may be used. The sense and antisense strands of siRNA may have different numbers of bases. For example, "asymmetrical interfering RNA (aiRNA)" may be used, in which the antisense strand has protruding sequences (overhangs) at the 3' and 5' ends. A typical aiRNA has an antisense strand consisting of 21 bases and a sense strand consisting of 15 bases, with a 3-base overhang structure at each end of the antisense strand.

[0097] The location of the target sequence of the gene-specific siRNA for the target protein is not particularly limited, but in one embodiment, it is desirable to select the target sequence from the 5'-UTR and up to about 50 bases from the start codon, as well as from regions other than the 3'-UTR. It is preferable to confirm the specificity of the selected target sequence by examining the selected candidate target sequences for homology to 16-17 consecutive bases in mRNAs other than the target using homology search software such as BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ). For target sequences whose specificity has been confirmed, double-stranded RNA may be designed as an siRNA consisting of a sense strand with a 3'-terminal overhang of TT or UU at 19-21 bases following AA (or NA), and an antisense strand with a sequence complementary to the 19-21 bases and a 3'-terminal overhang of TT or UU. Furthermore, shRNA, which is a precursor of siRNA, can be designed by appropriately selecting any linker sequence (e.g., approximately 5-25 bases) that can form a loop structure and linking the sense strand and antisense strand via the linker sequence.

[0098] siRNA and / or shRNA sequences can be searched for using search software provided free of charge on various websites, including, for example, the siRNA Target Finder provided by Ambion (http: / / www.ambion.com / jp / techlib / misc / siRNA_finder.html), the insert design tool for pSilencer® Expression Vector (http: / / www.ambion.com / jp / techlib / misc / psilencer_converter.html), and GeneSeer provided by RNAi Codex (http: / / codex.cshl.edu / scripts / newsearchhairpin.cgi).

[0099] siRNA can be prepared by synthesizing the sense and antisense strands of a target sequence on mRNA using an automated DNA / RNA synthesizer, denaturing them in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute, and then annealing them at about 30 to about 70°C for about 1 to about 8 hours. Alternatively, siRNA can be prepared by synthesizing shRNA, which serves as a precursor to siRNA, and cleaving it using the RNA-cleaving protein dicer.

[0100] The target protein gene-specific miRNA may be any miRNA as long as it inhibits the translation of the gene encoding the target protein. For example, instead of cleaving the target mRNA like siRNA, the miRNA may inhibit its translation by pairing with the target's 3' untranslated region (UTR). The miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA. The length of the miRNA is not particularly limited; the length of the pri-miRNA is typically several hundred to several thousand bases, the length of the pre-miRNA is typically 50 to 80 bases, and the length of the mature miRNA is typically 18 to 30 bases. In one embodiment, the target protein gene-specific miRNA is preferably pre-miRNA or mature miRNA, and more preferably mature miRNA. Such target protein gene-specific miRNA may be synthesized by known techniques or purchased from a company that provides synthetic RNA.

[0101] A gene-specific antisense nucleic acid for a target protein is a nucleic acid containing a base sequence complementary or substantially complementary to the base sequence of the mRNA of a gene encoding the target protein, or a portion thereof. The nucleic acid functions to inhibit synthesis of the target protein by binding to the mRNA to form a specific and stable duplex. Antisense nucleic acids may be DNA, RNA, or DNA / RNA chimeras. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H), causing selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only a sequence in the mRNA, but also a sequence in an intron region in the initial translation product of the gene for the target protein. Intron sequences can be determined by comparing the genomic sequence with the cDNA base sequence of the gene for the target protein using homology search programs such as BLAST and FASTA.

[0102] The length of the target region of a gene-specific antisense nucleic acid for a target protein is not limited, as long as hybridization of the antisense nucleic acid results in inhibition of translation into the target protein. The gene-specific antisense nucleic acid for a target protein may be the entire sequence or a partial sequence of the mRNA encoding the target protein. Considering ease of synthesis, antigenicity, intracellular internalization, and other issues, oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred, but are not limited to these. More specifically, preferred target regions for antisense nucleic acids include, but are not limited to, the 5'-end hairpin loop, 5'-end untranslated region, translation initiation codon, protein coding region, ORF translation termination codon, 3'-end untranslated region, 3'-end palindrome region, and 3'-end hairpin loop of the gene for the target protein.

[0103] The gene-specific antisense nucleic acid of a target protein may not only hybridize with the mRNA or initial transcription product of the gene of the target protein and inhibit translation into protein, but may also bind to these genes, which are double-stranded DNA, to form a triplex and inhibit transcription into RNA (antigene).

[0104] The nucleotide molecules constituting the above-mentioned target protein gene-specific siRNA, target protein gene-specific miRNA, and target protein gene-specific antisense nucleic acid may contain various chemical modifications to improve stability (chemical and / or enzymatic) and specific activity (affinity with RNA). For example, to prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide constituting the antisense nucleic acid can be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. Furthermore, the hydroxyl group at the 2'-position of the sugar (ribose) of each nucleotide may be substituted with -OR (R = CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl for the carbonyl group at the 2-position. Furthermore, some of the nucleotide molecules constituting siRNA or miRNA may be substituted with natural DNA.

[0105] The gene-specific siRNA for a target protein, the gene-specific miRNA for a target protein, and the gene-specific antisense nucleic acid for a target protein can be prepared by determining the target sequence of the mRNA or initial transcription product based on the cDNA sequence or genomic DNA sequence of the gene for the target protein and synthesizing a complementary sequence thereto using a commercially available automated DNA / RNA synthesizer. Furthermore, the antisense nucleic acids containing the various modifications described above can also be chemically synthesized by known techniques.

[0106] Expression vectors for target protein gene-specific siRNA, target protein gene-specific miRNA, target protein gene-specific antisense nucleic acid, and target protein expression vectors are not particularly limited, as long as they incorporate target protein gene-specific siRNA, target protein gene-specific miRNA, target protein gene-specific antisense nucleic acid, target protein mRNA, etc. in an expressible state. Typically, the expression vector contains a promoter sequence and a polynucleotide containing the target protein gene-specific siRNA, target protein gene-specific miRNA, target protein gene-specific antisense nucleic acid, or target protein coding sequence (and, if necessary, a transcription termination signal sequence), as well as other sequences as necessary. The promoter is not particularly limited, and examples include RNA polymerase II (pol II) promoters such as the CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, and CAG promoter; and RNA polymerase III (pol III) promoters such as mouse and human U6-snRNA promoters, human H1-RNase P RNA promoter, and human valine-tRNA promoter. The other sequences are not particularly limited, and various known sequences that can be contained in expression vectors can be used. Examples of such sequences include a replication origin and a drug resistance gene. The types of drug resistance genes and vectors are exemplified above.

[0107] Another example of a gene expression inhibitor for a target protein is a gene-specific ribozyme for the target protein. In the narrow sense, "ribozyme" refers to RNA with enzymatic activity for cleaving nucleic acids. However, in this application, it also encompasses DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme nucleic acid is the self-splicing RNA found in infectious RNAs such as viroids and virusoids, and is known to have hammerhead and hairpin types. Hammerhead types exert their enzymatic activity with approximately 40 bases. By arranging several bases (totaling approximately 10 bases) adjacent to the hammerhead structure at both ends to be complementary to the desired cleavage site in the mRNA, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the advantage of using only RNA as a substrate and therefore not attacking genomic DNA. When the mRNA of a gene encoding a target protein is double-stranded, the target sequence can be made single-stranded by using a hybrid ribozyme containing an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase [Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)]. Furthermore, when a ribozyme is used in the form of an expression vector containing DNA encoding it, a hybrid ribozyme can be further linked to a modified tRNA sequence to promote translocation of the transcript into the cytoplasm [Nucleic Acids Res., 29(13): 2780-2788 (2001)].

[0108] The target of application of the agent of the present invention is not particularly limited, and examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.

[0109] The form of the drug of the present invention is not particularly limited, and depending on the intended use of the drug of the present invention, it may take a form that is commonly used for each intended use.

[0110] In terms of forms, when the application is a medicine, health enhancer, nutritional supplement (supplement, etc.), etc., examples include formulations suitable for oral ingestion (oral formulations) such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), and jellies; and formulations suitable for parenteral ingestion (parenteral formulations) such as nasal drops, inhalants, rectal suppositories, inserts, enemas, jellies, injections, patches, lotions, and creams.

[0111] When the application is a food composition, the form may be liquid, gel or solid food, such as juice, soft drinks, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake, infant formula, cake mix, powdered or liquid dairy products, bread, cookies, etc.

[0112] When the application is an oral composition, the form may be any form such as liquid (solution, emulsion, suspension, etc.), semi-solid (gel, cream, paste, etc.), solid (tablet, particulate, capsule, film, kneaded material, molten solid, waxy solid, elastic solid, etc.), more specifically, dentifrices (toothpaste, liquid dentifrice, liquid dentifrice, powder dentifrice, etc.), mouthwash, liniment, patch, mouth freshener, food (e.g., chewing gum, tablet candy, candy, gummy, film, troche, etc.), etc.

[0113] The drug of the present invention may further contain other components as needed. The other components are not particularly limited as long as they are components that can be incorporated into, for example, medicines, food compositions, oral compositions, health promotion agents, nutritional supplements (supplements, etc.), and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0114] The total content of the inhibitors and enhancers of the target protein in the drug of the present invention depends on the type, use, mode of use, target, and condition of the target, and is not limited to, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0115] The amount of the composition of the present invention to be applied (e.g., administered, ingested, inoculated, etc.) is not particularly limited as long as it is an effective amount that exhibits medicinal efficacy, and is generally 0.1 to 1,000 mg / kg body weight per day in terms of the weight of the active ingredient. The above dosage is preferably administered once a day or in two or three divided doses, and can be increased or decreased as appropriate depending on the patient's age, condition, and symptoms.

[0116] 6. Screening Method for Active Ingredients of Lung Cancer Prophylactic or Therapeutic Agents In one aspect, the present invention relates to a screening method for active ingredients (or candidate substances thereof) of prophylactic or therapeutic agents for lung cancer, which uses as an index the amount or concentration of at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E) in extracellular vesicles or body fluid collected from an animal treated with a test substance (also referred to herein as the "screening method for active ingredients of the present invention"). This is described below.

[0117] The definitions of body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), lung cancer, and measurement of the amount or concentration of target proteins are the same as those in "1. Lung cancer testing methods" above.

[0118] The animal species is not particularly limited, and examples of the animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.

[0119] A wide range of test substances can be used, regardless of whether they are naturally occurring or artificially produced. Furthermore, not only purified compounds but also compositions containing a variety of compounds and animal and plant extracts can be used. Compounds are not limited to low-molecular-weight compounds, but also include high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.

[0120] More specifically, the method for screening for an active ingredient of the present invention comprises at least one step selected from the group consisting of: a step of selecting the test substance as an active ingredient for a preventive or therapeutic agent for lung cancer when the value of the index for at least one protein selected from the group consisting of protein group (A), protein group (C), and protein group (E) is lower than the amount or concentration of the corresponding protein in extracellular vesicles of body fluid or a blood sample collected from an animal that has not been treated with the test substance; and a step of selecting the test substance as an active ingredient for a preventive or therapeutic agent for lung cancer when the value of the index for at least one protein selected from the group consisting of protein group (B) and protein group (D) is higher than the amount or concentration of the corresponding protein in extracellular vesicles of body fluid or a blood sample collected from an animal that has not been treated with the test substance.

[0121] The corresponding protein means a protein that is the same as the target protein used as an indicator.

[0122] "Low" means, for example, that the index value is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, or 1 / 100 of the control value.

[0123] "High" means, for example, that the index value is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold higher than the control value.

[0124] 7. Method for Evaluating Lung Cancer Induction or Exacerbation In one aspect, the present invention relates to a method for evaluating lung cancer induction or exacerbation using, as an indicator, the amount or concentration of at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E) in extracellular vesicles of body fluids or a blood sample collected from an animal treated with a test substance (also referred to herein as the "toxicity evaluation method of the present invention"). This is described below.

[0125] The definitions of body fluid, extracellular vesicles, protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), lung cancer, measurement of the amount or concentration of the target protein, animal species, test substance, etc. are the same as those in "1. Lung cancer testing method" and "6. Screening method for active ingredients of lung cancer preventive or therapeutic agents" above.

[0126] More specifically, the toxicity evaluation method of the present invention includes at least one step selected from the group consisting of: a step of determining that the test substance has lung cancer-inducing or exacerbating properties when the value of the indicator for at least one protein selected from the group consisting of protein group (A), protein group (C), and protein group (E) is higher than the amount or concentration of the corresponding protein in extracellular vesicles of body fluid or a blood sample collected from an animal that has not been treated with the test substance; and a step of determining that the test substance has lung cancer-inducing or exacerbating properties when the value of the indicator for at least one protein selected from the group consisting of protein group (B) and protein group (D) is lower than the amount or concentration of the corresponding protein in extracellular vesicles of body fluid or a blood sample collected from an animal that has not been treated with the test substance.

[0127] The corresponding protein means a protein that is the same as the target protein used as an indicator.

[0128] "High" means, for example, that the index value is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold higher than the control value.

[0129] "Low" means, for example, that the index value is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, or 1 / 100 of the control value.

[0130] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0131] Materials and Methods Study Design: Proteome analysis enrolled three patients with pretreatment small cell lung cancer (SCLC), three patients with posttreatment SCLC, three patients with squamous cell lung cancer (SCC), and three healthy controls. The healthy controls were recruited from Osaka University Hospital. Serum samples were collected and stored at -80°C. The medical records of these patients were retrospectively analyzed. Quantitative proteomics was performed using liquid chromatography-mass spectrometry (LC-MS / MS) on these samples.

[0132] Cell lines and cell culture: SBC-3 and OS2RA cells were cultured in RPMI supplemented with penicillin (100 U / ml), streptomycin (100 mg / ml), and 10% FBS. All cells were cultured on collagen-coated plates, maintained in a humidified incubator containing 5% CO2, and routinely tested at 37°C to ensure the absence of mycoplasma contamination. Cells were cultured for 3–4 days and passaged before reaching confluence.

[0133] Isolation of exosomes (EVs) for proteomics: EVs were isolated using the MagCapture Exosome Isolation Kit PS Ver. 2 (FUJIFILM WAKO, Japan) according to the manufacturer's instructions. EV size distribution and number were measured using NanoSight nanoparticle tracking analysis (Malvern Instruments, Malvern, UK).

[0134] Western blotting. Cultured cells were lysed in RIPA Lysis and Extraction Buffer (No. 89900; Thermo Fisher Scientific) containing Complete Mini Protease Inhibitor Cocktail (Roche), followed by centrifugation to remove pelleted cell / tissue debris. Protein samples were loaded onto NuPAGE 4-12% Bis-Tris gels (Invitrogen). For immunoblot analysis, the gels were electroblotted onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with Blocking One (Nacalai Tesque) and incubated with specific primary antibodies followed by appropriate secondary antibodies. The following primary antibodies were used for immunoblotting: mouse anti-Flotillin-1 antibody (610821; BD Biosciences), anti-TSG101 antibody (ab125011, Abcam), anti-calnexin antibody (ab22595, Abcam), CD9 monoclonal antibody (AHS0902; Thermo Fisher Scientific), and β-Actin (13E5) Rabbit mAb (HRP Conjugate) (51255, Cell Signaling). Immunoreactive signals were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and detected using an ImageQuant LAS500 (GE Healthcare). Bands were quantified using ImageJ. The levels of each protein were expressed as the ratio of the signal intensity of the target protein to that of β-actin.

[0135] <Lung cancer tissue specimens> Tissue array slides (codes LC1241 and LC1201b) of lung cancer tissue specimens, including control non-cancer specimens that were not infected with HCV or HBV, were purchased (US Biomax, Rockville, MD).

[0136] Immunohistochemistry: Two tissue array slides were used for immunostaining. Immunohistochemical staining of these samples and pathologist review were performed by the Institute of Applied Medicine (Osaka, Japan). After deparaffinization, antigen retrieval was performed by autoclaving at 125°C for 15 minutes. To block endogenous peroxidase activity, the samples were incubated with 3% bovine serum albumin at room temperature for 1 hour. They were then incubated with anti-SUCLA2 antibody (ab190845; Abcam) overnight at 4°C. Subsequent secondary antibody reactions were performed at room temperature for 30 minutes using horseradish peroxidase-conjugated anti-rabbit secondary antibody (02-6102; Invitrogen).

[0137] <Histological Analysis> The percentage of SUCLA2-positive cells was assessed using immunohistochemical samples stained with the anti-SUCLA2 antibody described above. Images were processed and reconstructed using BZ-X Analyzer software (Keyence Corp., Osaka, Japan) according to the manufacturer's instructions. For each sample, three randomly selected fields were used to calculate the ratio of the number of DAB-positive cells to the number of cells in a single field, and the average value of the three fields was used as the percentage of SUCLA2-positive cells in that sample. All quantitative measurements were performed under the same light and optical conditions for each field.

[0138] <Fluorescent Immunohistochemistry> Cell lines were cultured on chamber slides for several days and allowed to adhere. (D11134H: Matsunami Glass Industry Co., Ltd.) Cells were fixed in 4% paraformaldehyde (Nacalai Tesque) for 20 minutes. Then, cells were permeabilized for 10 minutes in PBS containing 0.1% Triton X-100. Slides were blocked at room temperature for 30 minutes (Blocking One Histo, 06349-64, Nacalai). Slides were incubated with the following primary antibodies for 60 minutes at room temperature: anti-SUCLA2 antibody (ab190845: Abcam), Cleaved Caspase-3 (Asp175) Antibody #9661 (Cell Signaling), and GAPDH Poluclonal antibody (Cat No. 10494-1-AP: Proteintech). Then, the slides were incubated with the following secondary antibodies for 60 minutes at room temperature: goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor TM 488 (A-11070; Invitrogen) Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor TM 594 (A-11005: Invitrogen). Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (12745-74: Nacalai Tesque) was used for mounting. Photographs were taken using a BZ-X800 fluorescence microscope (KEYENCE).

[0139] Cell viability assay: Cells were seeded in 96-well plates at 1 x 104 cells per well in quintuplicate. After allowing the cells to adhere overnight, they were treated with siRNA for 24 hours. Cell viability was measured by a colorimetric MTT assay using Cell Count Kit-8 (CK04, Dojindo, Kumamoto, Japan). Absorbance was measured at 570 nm. The mean and SD were calculated.

[0140] <Mycoplasma testing> Genomic DNA was obtained from all cell lines using the AllPrep DNA / RNA mini kit (QIAGEN). Mycoplasma detection was performed using the e-Myco Plus Mycoplasma PCR detection kit (iNtRON, Jungwon-gu, Korea). Only cells that were confirmed to be mycoplasma-negative were used in this study.

[0141] <Knockdown experiment> Knockdown of SUCLA2 expression in cultured cells (SBC-3, OS2RA). Cells were cultured for 48 hours before analysis and then transfected with siRNA against SUCLA2 (SiRNA: AM16708, HSS112960, HSS189576 (Thermos Fisher Scientific)). Transfection was performed using Lipofectamine RNAiMAX transfection reagent (Life Technologies) according to the manufacturer's protocol.

[0142] <RNA> Total RNA was purified using the RNeasy mini kit (Kiergen, Hilden, Germany).

[0143] Statistical analysis: All statistical evaluations of experiments using T cell lines were repeated at least three times. The following statistical analyses were performed using JMP Pro 14.0.0 software (SAS Institute Japan, Tokyo, Japan): Welch's t-test, one-way analysis of variance, Spearman's rank correlation coefficient, receiver operating characteristic curve, and logistic regression analysis. A P value of less than 0.05 was considered statistically significant. Volcano plots and principal component analysis were performed in R studio.

[0144] Proteome analysis of serum EVs was performed as previously described (ERJ Open Res. 2021 Mar 22;7(1):00658-2020). The exosome eluate was prepared by adding 10x phase-shifting surfactant (PTS) buffer (120 mM sodium deoxycholate, 500 mM NH4HCO3), 120 mM sodium N-lauroyl sarcosinate, and boiling at 95°C for 5 minutes. 20 mM iodoacetamide was then added and kept at 37°C for 30 minutes in the dark for alkylation. The exosomes were then digested overnight at 37°C with 1 μg trypsin (Wako Chemical, Tokyo, Japan) and 2 mAU LysC (Wako Chemical, Tokyo, Japan). 1% trifluoroacetic acid (TFA) was added to the digestion solution, and the solution was centrifuged at 20,000 g for 10 minutes to precipitate the wash solution. The supernatant containing the fragmented peptides was desalted using a C18-SCX StageTip and further dried using a centrifugal evaporator. An analytical column (75 μm × 20 cm, ReproSil-Pur C18-AQ, 1.9 μm resin, in-house packed; Dr. Maisch, Ammerbuch, Germany) was used. The Orbitrap Fusion Lumos mass spectrometer was operated in 5 GPF (gas-phase fractionation)-DIA mode (fragment resolution 50,000, precursor resolution 120,000). Automatic gain control (AGC) targets were 1e6 and 2e5 for MS1 and MS2, maximum IITs were 250 ms and 86 ms for MS1 and MS2, NCE was 30, and precursor isolation windows were 2 m / z, covering 418–494, 490–566, 562–638, 634–710, and 706–782 m / z (5xGPF). Individual samples were analyzed in DIA mode (fragment resolution 30,000, precursor resolution 120,000, AGC targets 4e5 and 2e5 for MS1 and MS2, maximum IIT 100 ms and 54 ms for MS1 and MS2, NCE 30, precursor isolation window 8 m / z). DIA data were analyzed using DIA-NN (version 1.7.12) with the following default settings: scan window setting and automatic mass accuracy tolerance. Search results were refined and filtered to the 1% precursor level.MS files were searched using UniProt.

[0145] <Bioinformatics analysis of proteome> To analyze and elucidate biologically relevant proteomic pathways and molecular networks, we employed the following tools: Ingenuity Pathways Analysis (IPA, Qiagen, Inc., Redwood City, CA, USA) for upstream analysis and enrichment analysis.

[0146] Molecular Network and Pathway Analysis of Proteome and Transcriptome: KeyMolnet (viewer program version 6.2, content version 9.7.20210930154837, KM Data Inc.) was used to analyze molecular networks and pathways in EV proteomics and lung tissue proteomics. Briefly, KeyMolnet is a commercial knowledge base containing core content derived from a selection of reliable reviews and secondary content derived from important original articles in major journals. This database contains approximately 260,000 relationships between human genes, proteins, microRNAs, and small molecules. Molecular network analysis was performed using a "start points and end points" network search algorithm to evaluate the molecular networks through which differentially expressed molecules affect target molecules (analysis of upstream networks of target molecules) and, conversely, the molecular networks through which target molecules affect these molecules (analysis of downstream networks of target molecules). The pathways associated with the resulting molecular network were analyzed, and the significance of the similarity between the extracted network and the canonical pathway was calculated as an HS score using a formula based on the hypergeometric distribution. An HS score of 20 or higher was considered statistically significant.

[0147] Transmission electron microscopy: EVs were fixed with 2% paraformaldehyde and incubated with the following primary antibodies: anti-CD9 (MM2 / 57; Thermo Fisher Scientific), anti-CD14 [EPR3653] (ab133335; Abcam, Cambridge, UK), and anti-lipopolysaccharide-binding protein (LBP) polyclonal (PA5-21642; Invitrogen, USA). Immunoreactive EVs were visualized using anti-mouse IgG (H+L) (EMGMHL10) and anti-rabbit IgG (H+L) (EMGFAR10; BBI Solutions, UK) antibodies.

[0148] Results: Proteomics Discovery of Novel Biomarkers for SCLC Based on TEM, most vesicles isolated from healthy controls and patients with SCC and SCLC similarly expressed the common EV marker protein CD9 on their surface. Furthermore, isolated EVs were positive for CD9 and TSG 101, but negative for calnexin and haptoglobin. Nanoparticle tracking analysis revealed that serum EVs in both groups were smaller than 200 nm in diameter and indistinguishable in size and number.

[0149] Using untargeted proteomics, we identified 3907 proteins in EVs obtained from <20 μL of serum from healthy controls (HCs) and patients with SCLC and SCC. Notably, 180 proteins were significantly different between HCs and SCLCs (P < 0.05; 118 proteins were upregulated and 62 proteins were downregulated in SCLCs). Further filtering by identified protein peptides > 2 yielded 82 proteins (Tables 1–3). Of these, 28 proteins were selected as more promising candidates after filtering by SCLC / Ctrl fold change > 1.5.

[0150]

[0151]

[0152]

[0153] Similarly, for SCC, 271 proteins were significantly different between HC and SCC (P<0.05, UP142 and Down122 in SCC), and further narrowing down the search by Identified Protein Petide > 2 yielded 119 proteins (Tables 4-7). Of these, 76 proteins were selected as more promising candidates by SCC / Ctrl fold change > 1.5.

[0154]

[0155]

[0156]

[0157]

[0158] Four proteins (FAM120A, GCA, ITIH4, and SCFD1) were commonly elevated in both SCLC and SCC, and these four molecules may be important in the formation of hilar carcinoma.

[0159] PCA analysis clearly separated HC, SCC, and SCLC by their respective BMs. Bioinformatics (Metascape) analysis of specific top pathways in SCLC and SCC revealed that distinct pathways were upregulated in each protein group. In SCLC, COP2-mediated vesicular transport via the TCA cycle was the top pathway, while in SCC, Goldi vesicle transport and neutrophil degranulation were the top pathways.

[0160] Next, to further narrow down the molecules specifically altered in serum proteomics, we performed proteomics analysis on tissues from three SCLC and three SCC patients with no significant differences in patient background. Tissue proteomics analysis yielded 8,839 proteins, of which 320 proteins were significantly different at P < 0.05. Of these, 121 proteins were significantly elevated in SCLC compared with SCC. Of the 28 proteins identified as BM candidates in serum exosomes, we were able to narrow down the list to eight proteins that were also significantly elevated in SCLC in tissue proteomics (Table 8).

[0161]

[0162] Pathway analysis using tissue Metascape analysis also revealed significant changes in key pathways in SCLC, such as methylmalonyl and the TCA cycle, suggesting the possibility that metabolic systems may be involved in tumor pathogenesis. The key pathways in SCC were neutrophil degranulation and angiogenesis. In this study, we compared the analysis of serum exosomes from SCLC with that of lung tissue, and found that changes in the TCA cycle were highly elevated in both. The fact that the same pathways are upstream suggests that serum exosomes may reflect the pathophysiology of tissues, which is an important finding.

[0163] After narrowing down the biomarkers from serum exosomes and tissues, eight molecules remained as promising SCLC BM candidates (Table 8). Of the eight serum exosome mutations, SUCLA2 and PCK2 were selected as final candidate molecules, as they were significantly elevated in SCLC compared with HC and SCC and significantly decreased with therapeutic intervention (Figure 1). Comparing SUCLA2 and PCK2, SUCLA2, which is directly involved in the TCA cycle, was selected as the final candidate molecule. Protein-protein interaction (PPI) maps confirmed that SUCLA2 and the associated TCA cycle molecules SUCLG1 and SDHB were also significantly elevated in SCLC. KeyMolnet confirmed that MAP kinase p38 and the angiotensin receptor-associated protein ATRAP were elevated in the upstream pathway of SUCLA2 (SCS-bataA). KGA (GLS), a binding molecule of SUCLA2, was also elevated.

[0164] <Usefulness of SUCLA2 as a Diagnostic and Staging Marker> We confirmed whether SUCLA2, selected as a novel BM candidate, could actually function as a clinical marker. First, we performed WB analysis of SUCLA2 in exosomes from four HC, four SCC, and four SCLC patients. We found high SUCLA2 expression in SCLC exosomes (Figure 2). Furthermore, Western blotting of exosomes from four SCLC LD (limited-stage) and four ED (extensive-stage) cases confirmed significantly elevated SUCLA2 expression in the ED cases (Figure 3). Next, we quantified SUCLA2 expression using tissue arrays. Immunostaining of SUCLA2 in the tissue arrays demonstrated that SUCLA2 staining was stronger in the cytoplasm of SCLC than in SCC (Figures 4 and 5). Finally, ELISA was performed on serum samples from HC and SCLC patients, confirming that SUCLA2, when used in combination with the existing BM, ProGRP, increased the AUC of the receiver operating characteristic curve. The usefulness of SUCLA2 as a new BM was confirmed (Figure 6).

[0165] <SUCLA2 Depletion Promotes Apoptosis> We analyzed the function of SUCLA2. First, we knocked down SUCLA2 in the cell lines SBC3 and OS2RA. WB confirmed that SUCLA2 was lost (Figure 7). RNA sequencing and IPA analysis were performed on the SUCLA2-knockdown cell lines. Results confirmed that SUCLA2 knockdown altered the endocrine system and tissue proliferation. DAPI staining confirmed that SUCLA2 knockdown reduced SUCLA2 expression. In the cell lines, SUCLA2 knockdown significantly reduced cell proliferation (Figure 8). Cspase 3 / 7 staining confirmed enhanced apoptosis in SUCLA2-knockdown cells. This suggests that apoptosis may be responsible for the cell loss. Furthermore, WB confirmed that SUCLA2 knockdown increased active PARP. SUCLA2 knockdown in SBC3 increased sensitivity to the anticancer drug CDDP. These results confirmed that SUCLA2 is a molecule strongly involved in tumor survival.

Claims

1. A method for testing lung cancer, comprising: (1) detecting proteins (A), (B), (C), (D), and (E) in extracellular vesicles or body fluid collected from a subject; (A) Succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial; Immunoglobulin kappa variable 1-39; Phosphoenolpyruvate carboxykinase [GTP], mitochondrial; Eukaryotic translation initiation factor 3 subunit J; AP-1 complex subunit gamma-like 2; Protein-tyrosine sulfotransferase 2; Fibrinogen-like protein 1; Small proline-rich protein 2G; Reelin; E3 ubiquitin-protein ligase TRIM21; CAAX prenyl protease 1 homolog; Alkyldihydroxyacetonephosphate synthase; peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional glutamate / proline--tRNA ligase, Pyridoxal-dependent decarboxylase domain-containing protein 1, Platelet-derived growth factor C, Biglycan, Eukaryotic translation initiation factor 3 subunit L, Emerin, Serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, General vesicular transport factor p115, Copine-1, WASH complex subunit 5, and ProteinA group of proteins consisting of the transport protein Sec23A, (B) Immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Gla protein, Ena / VASP-like protein, Beta-hexosaminidase subunit alpha, CD2-associated protein, Oncoprotein-induced transcript 3 protein, Glycosylated lysosomal membrane protein, Transmembrane 7 superfamily member 3, CD81 antigen, EGF-like repeat and discoidin I-like domain-containing protein 3, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1, Transforming growth factor beta activator LRRC32, Claudin-5, Proteasome subunit beta type-1, F-box only protein 50, Ragulator complex protein LAMTOR1, Trypsin-1, Beta-hexosaminidase subunit beta, Monocarboxylate transporter 2, Dyslexia-associated protein KIAA0319-like protein, DnaJ homolog subfamily B member 1, Membrane cofactor protein, Desmoglein-1, B-cell antigen receptor complex-associated protein betaA group of proteins consisting of chain, Raftlin, Synaptic vesicle glycoprotein 2A, Cadherin-1, V-type proton ATPase subunit H, Angiogenin, Ras-related protein Rab-5A, Casein kinase I isoform alpha, Proteasome subunit alpha type-7, Complement receptor type 1, Protein FAM234A, Toll-interacting protein, SID1 transmembrane family member 1, Sushi domain-containing protein 1, Phospholipid-transporting ATPase IA, Charged multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, (C) Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factorreceptor superfamily member 10C、C1GALT1-specific chaperone 1、Cytochrome b5、Beta-enolase、Sorting nexin-1、Catechol O-methyltransferase、Tapasin、Cytochrome b-245 light chain、Protein YIPF5、Neutrophil defensin 3、Armadillo repeat-containing protein 8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrier family 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZdomain-containing protein 8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasminogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、Secretory carrier-associated membrane protein 2、Serine incorporator 3、Calcium-transporting ATPase type 2C member 1、Low affinity immunoglobulin gamma Fc region receptor II-c、AP-3 complex subunit mu-1、Activator of 90 kDa heat shock protein ATPase homolog 1、Brain acid soluble protein 1、Protein transport protein Sec31A、Neutral cholesterol esterA protein group consisting of hydrolase 1, Serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, Ragulator complex protein LAMTOR2, and Tetraspanin-14, (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factor beta-1-induced transcript 1 protein, Volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WD repeat-containingand (E) a group of proteins consisting of Constitutive coactivator of PPAR-gamma-like protein 1, Inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and Grancalcin.

2. The method of claim 1, wherein the disease to be examined is small cell lung cancer or squamous cell lung cancer.

3. The method of claim 1, wherein the disease to be examined is small cell lung cancer and the protein to be detected comprises at least one selected from the group consisting of protein group (A) and protein group (B); the disease to be examined is squamous cell lung cancer and the protein to be detected comprises at least one selected from the group consisting of protein group (C) and protein group (D); or the disease to be examined is small cell lung cancer and / or squamous cell lung cancer and the protein to be detected comprises at least one selected from the group consisting of protein group (E).

4. (2A) determining that the subject suffers from small cell lung cancer when the detected protein contains at least one protein (protein A') selected from the group consisting of protein group (A) and the amount or concentration of protein A' detected in step (1) is equal to or higher than a cutoff value; (2B) determining that the subject suffers from small cell lung cancer when the detected protein contains at least one protein (protein B') selected from the group consisting of protein group (B) and the amount or concentration of protein B' detected in step (1) is equal to or lower than a cutoff value; (2C) determining that the subject suffers from squamous cell lung cancer when the detected protein contains at least one protein (protein C') selected from the group consisting of protein group (C) and the amount or concentration of protein C' detected in step (1) is equal to or higher than a cutoff value; (2D) determining that the subject is suffering from squamous cell lung cancer when the detected protein contains at least one protein (protein D') selected from the group consisting of protein group (D) and the amount or concentration of protein D' detected in step (1) is equal to or lower than a cutoff value; and (2E) determining that the subject is suffering from small cell lung cancer and / or squamous cell lung cancer when the detected protein contains at least one protein (protein E') selected from the group consisting of protein group (E) and the amount or concentration of protein E' detected in step (1) is equal to or higher than a cutoff value. The method of claim 1, further comprising at least one step selected from the group consisting of:

5. The method according to any one of claims 1 to 4, wherein the protein to be detected includes Succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial.

6. The testing method according to any one of claims 1 to 4, wherein the subject is a human.

7. Protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E): (A) Succinate--CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, A group of proteins consisting of mitochondrial, bifunctional glutamate / proline-tRNA ligase, pyridoxal-dependent decarboxylase domain-containing protein 1, platelet-derived growth factor C, biglycan, eukaryotic translation initiation factor 3 subunit L, emerin, serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, general vesicular transport factor p115, Copine-1, WASH complex subunit 5, and protein transport protein Sec23A.(B)Immunoglobulin lambda variable 8-61、HLA class II histocompatibility antigen, DR alpha chain、Protein S100-A16、Suprabasin、High mobility group protein B2、Myelin protein zero-like protein 1、Matrix Gla protein、Ena / VASP-like protein、Beta-hexosaminidase subunit alpha、CD2-associated protein、Oncoprotein-induced transcript 3 protein、Glycosylated lysosomal membrane protein、Transmembrane 7 superfamily member 3、CD81 antigen、EGF-like repeat and discoidin I-like domain-containing protein 3、1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1、Transforming growth factor beta activator LRRC32、Claudin-5、Proteasome subunit beta type-1、F-box only protein 50、Ragulator complex protein LAMTOR1、Trypsin-1、Beta-hexosaminidase subunit beta、Monocarboxylate transporter 2、Dyslexia-associated protein KIAA0319-like protein、DnaJ homolog subfamily B member 1、Membrane cofactor protein、Desmoglein-1、B-cell antigen receptor complex-associated protein beta chain、Raftlin、Synaptic vesicle glycoproteinA protein group consisting of 2A, Cadherin-1, V-type proton ATPase subunit H, Angiogenin, Ras-related protein Rab-5A, Casein kinase I isoform alpha, Proteasome subunit alpha type-7, Complement receptor type 1, Protein FAM234A, Toll-interacting protein, SID1 transmembrane family member 1, Sushi domain-containing protein 1, Phospholipid-transporting ATPase IA, Charged multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, (C) Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factor receptor superfamily member10C、C1GALT1-specific chaperone 1、Cytochrome b5、Beta-enolase、Sorting nexin-1、Catechol O-methyltransferase、Tapasin、Cytochrome b-245 light chain、Protein YIPF5、Neutrophil defensin 3、Armadillo repeat-containing protein 8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrier family 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZ domain-containing protein8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasminogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、Secretory carrier-associated membrane protein 2、Serine incorporator 3、Calcium-transporting ATPase type 2C member 1、Low affinity immunoglobulin gamma Fc region receptor II-c、AP-3 complex subunit mu-1、Activator of 90 kDa heat shock protein ATPase homolog 1、Brain acid soluble protein 1、Protein transport protein Sec31A、Neutral cholesterol ester hydrolase1. A group of proteins consisting of Serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, Ragulator complex protein LAMTOR2, and Tetraspanin-14, (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factor beta-1-induced transcript 1 protein, Volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WD repeat-containing proteinand (E) a group of proteins consisting of Constitutive coactivator of PPAR-gamma-like protein 1, Inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and Grancalcin.

8. Protein group (A), protein group (C), and protein group (E): (A) Succinate--CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional A group of proteins consisting of glutamate / proline-tRNA ligase, pyridoxal-dependent decarboxylase domain-containing protein 1, platelet-derived growth factor C, biglycan, eukaryotic translation initiation factor 3 subunit L, emerin, serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, general vesicular transport factor p115, copine-1, WASH complex subunit 5, and protein transport protein Sec23A. (C) Transmembrane emp24domain-containing protein 3、Prolow-density lipoprotein receptor-related protein 1、SUN domain-containing protein 2、Follistatin-related protein 1、Leukocyte immunoglobulin-like receptor subfamily A member 6、Acyl-CoA:lysophosphatidylglycerol acyltransferase 1、Tumor necrosis factor receptor superfamily member 10C、C1GALT1-specific chaperone 1、Cytochrome b5、Beta-enolase、Sorting nexin-1、Catechol O-methyltransferase、Tapasin、Cytochrome b-245 light chain、Protein YIPF5、Neutrophil defensin 3、Armadillo repeat-containing protein 8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrierfamily 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZ domain-containing protein 8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasminogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、Secretory carrier-associated membrane protein 2、Serineand (D) a protein group consisting of: (A) Immunoglobulin gamma Fc region receptor II-c, AP-3 complex subunit mu-1, Activator of 90 kDa heat shock protein ATPase homolog 1, Brain acid soluble protein 1, Protein transport protein Sec31A, Neutral cholesterol ester hydrolase 1, Serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, Ragulator complex protein LAMTOR2, and Tetraspanin-14; and (E) a protein group consisting of Constitutive coactivator of PPAR-gamma-like protein 1, Inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and Grancalcin, as well as a protein group (B) and a protein group (D): (B) Immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Gla protein, Ena / VASP-like protein, Beta-hexosaminidase subunit alpha, CD2-associated protein, Oncoprotein-induced transcript 3protein、Glycosylated lysosomal membrane protein、Transmembrane 7 superfamily member 3、CD81 antigen、EGF-like repeat and discoidin I-like domain-containing protein 3、1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1、Transforming growth factor beta activator LRRC32、Claudin-5、Proteasome subunit beta type-1、F-box only protein 50、Ragulator complex protein LAMTOR1、Trypsin-1、Beta-hexosaminidase subunit beta、Monocarboxylate transporter 2、Dyslexia-associated protein KIAA0319-like protein、DnaJ homolog subfamily B member 1、Membrane cofactor protein、Desmoglein-1、B-cell antigen receptor complex-associated protein beta chain、Raftlin、Synaptic vesicle glycoprotein 2A、Cadherin-1、V-type proton ATPase subunit H、Angiogenin、Ras-related protein Rab-5A、Casein kinase I isoform alpha、Proteasome subunit alpha type-7、Complement receptor type 1、Protein FAM234A、Toll-interacting protein、SID1 transmembrane family member 1、Sushi domain-containing protein 1、Phospholipid-transporting ATPase IA、ChargedA group of proteins consisting of multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, and (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factorA preventive or therapeutic agent for lung cancer, comprising at least one agent selected from the group consisting of: an enhancer of at least one protein selected from the group consisting of a protein group consisting of beta-1-induced transcript 1 protein, Volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WD repeat-containing protein 37, Angiopoietin-related protein 3, DnaJ homolog subfamily B member 1, Dedicator of cytokinesis protein 11, Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, Ras-related protein Rab-20, Early activation antigen CD69, TBC1 domain family member 10A, and Receptor-type tyrosine-protein phosphatase epsilon.

9. Protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E) in extracellular vesicles or body fluids collected from animals treated with the test substance: (A) Succinate--CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional glutamate / proline--tRNA ligase, Pyridoxal-dependent decarboxylase domain-containing protein 1, Platelet-derived growth factor C, Biglycan, Eukaryotic translation initiation factor 3 subunit L, Emerin, Serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, General vesicular transport factor p115, Copine-1, WASH complex subunit 5, and Protein transportA group of proteins consisting of protein Sec23A, (B) Immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Gla protein, Ena / VASP-like protein, Beta-hexosaminidase subunit alpha, CD2-associated protein, Oncoprotein-induced transcript 3 protein, Glycosylated lysosomal membrane protein, Transmembrane 7 superfamily member 3, CD81 antigen, EGF-like repeat and discoidin I-like domain-containing protein 3, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1, Transforming growth factor beta activator LRRC32, Claudin-5, Proteasome subunit beta type-1, F-box only protein 50, Ragulator complex protein LAMTOR1, Trypsin-1, Beta-hexosaminidase subunit beta, Monocarboxylate transporter 2, Dyslexia-associated protein KIAA0319-like protein, DnaJ homolog subfamily B member 1, Membrane cofactor protein, Desmoglein-1, B-cell antigen receptor complex-associated protein beta chain, Raftlin, SynapticA group of proteins consisting of vesicle glycoprotein 2A, Cadherin-1, V-type proton ATPase subunit H, Angiogenin, Ras-related protein Rab-5A, Casein kinase I isoform alpha, Proteasome subunit alpha type-7, Complement receptor type 1, Protein FAM234A, Toll-interacting protein, SID1 transmembrane family member 1, Sushi domain-containing protein 1, Phospholipid-transporting ATPase IA, Charged multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, (C) Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factor receptor superfamilymember 10C、C1GALT1-specific chaperone 1、Cytochrome b5、Beta-enolase、Sorting nexin-1、Catechol O-methyltransferase、Tapasin、Cytochrome b-245 light chain、Protein YIPF5、Neutrophil defensin 3、Armadillo repeat-containing protein 8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrier family 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZ domain-containingprotein 8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein 10、Transmembrane protein with metallophosphoesterase domain、Activated CDC42 kinase 1、Golgi reassembly-stacking protein 2、Ubiquitin-conjugating enzyme E2 variant 1、Calpain-5、DnaJ homolog subfamily C member 13、Golgi integral membrane protein 4、Transmembrane protein 40、Urokinase plasminogen activator surface receptor、WD repeat-containing protein 13、Copine-2、Integrin beta-2、Optineurin、Inactive rhomboid protein 2、Secretory carrier-associated membrane protein 2、Serine incorporator 3、Calcium-transporting ATPase type 2C member 1、Low affinity immunoglobulin gamma Fc region receptor II-c、AP-3 complex subunit mu-1、Activator of 90 kDa heat shock protein ATPase homolog 1、Brain acid soluble protein 1、Protein transport protein Sec31A、Neutral cholesterol ester hydrolase1. A protein group consisting of Serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, Ragulator complex protein LAMTOR2, and Tetraspanin-14, (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoprotein M6-a, Dermcidin, Low affinity immunoglobulin gamma Fc region receptor II-b, Cystatin-A, B-cell antigen receptor complex-associated protein beta chain, Lipolysis-stimulated lipoprotein receptor, Apolipoprotein C-I, Prolactin-inducible protein, Immunoglobulin epsilon heavy chain, Multimerin-2, Anthrax toxin receptor 1, EGF-like repeat and discoidin I-like domain-containing protein 3, P-selectin glycoprotein ligand 1, Putative trypsin-6, Beta-hexosaminidase subunit alpha, Solute carrier family 35 member D3, RAS protein activator like-3, Semaphorin-4B, Ras-related protein R-Ras2, Transforming growth factor beta-1-induced transcript 1 protein, Volume-regulated anion channel subunit LRRC8D, Fermitin family homolog 2, Plexin-D1, Regulator of G-protein signaling 19, WD repeat-containing proteinand (E) a group of proteins consisting of Constitutive coactivator of PPAR-gamma-like protein 1, Inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and Grancalcin.

10. Protein group (A), protein group (B), protein group (C), protein group (D), and protein group (E) in extracellular vesicles or body fluids collected from animals treated with the test substance: (A) Succinate-CoA ligase [ADP-forming] subunit beta, mitochondrial, Immunoglobulin kappa variable 1-39, Phosphoenolpyruvate carboxykinase [GTP], mitochondrial, Eukaryotic translation initiation factor 3 subunit J, AP-1 complex subunit gamma-like 2, Protein-tyrosine sulfotransferase 2, Fibrinogen-like protein 1, Small proline-rich protein 2G, Reelin, E3 ubiquitin-protein ligase TRIM21, CAAX prenyl protease 1 homolog, Alkyldihydroxyacetonephosphate synthase, peroxisomal, SEC23-interacting protein, 2-oxoglutarate dehydrogenase, mitochondrial, Bifunctional glutamate / proline--tRNA ligase, Pyridoxal-dependent decarboxylase domain-containing protein 1, Platelet-derived growth factor C, Biglycan, Eukaryotic translation initiation factor 3 subunit L, Emerin, Serine / threonine-protein kinase A-Raf, 3'(2'),5'-bisphosphate nucleotidase 1, General vesicular transport factor p115, Copine-1, WASH complex subunit 5, and Protein transport protein Sec23A, (B) Immunoglobulin lambda variable 8-61, HLA class II histocompatibility antigen, DR alpha chain, Protein S100-A16, Suprabasin, High mobility group protein B2, Myelin protein zero-like protein 1, Matrix Glaprotein、Ena / VASP-like protein、Beta-hexosaminidase subunit alpha、CD2-associated protein、Oncoprotein-induced transcript 3 protein、Glycosylated lysosomal membrane protein、Transmembrane 7 superfamily member 3、CD81 antigen、EGF-like repeat and discoidin I-like domain-containing protein 3、1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase delta-1、Transforming growth factor beta activator LRRC32、Claudin-5、Proteasome subunit beta type-1、F-box only protein 50、Ragulator complex protein LAMTOR1、Trypsin-1、Beta-hexosaminidase subunit beta、Monocarboxylate transporter 2、Dyslexia-associated protein KIAA0319-like protein、DnaJ homolog subfamily B member 1、Membrane cofactor protein、Desmoglein-1、B-cell antigen receptor complex-associated protein beta chain、Raftlin、Synaptic vesicle glycoprotein 2A、Ca dherin-1、V-type proton ATPase subunit H、Angiogenin、Ras-related protein Rab-5A、Casein kinase I isoform alpha、Proteasome subunit alpha type-7、Complement receptor type 1、Protein FAM234A、Toll-interactingA group of proteins consisting of protein, SID1 transmembrane family member 1, Sushi domain-containing protein 1, Phospholipid-transporting ATPase IA, Charged multivesicular body protein 2a, Anoctamin-6, Dedicator of cytokinesis protein 11, Thromboxane A2 receptor, Tetraspanin-32, Cell cycle control protein 50A, Acidic fibroblast growth factor intracellular-binding protein, Ras-related protein Rab-22A, and Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1, (C) Transmembrane emp24 domain-containing protein 3, Prolow-density lipoprotein receptor-related protein 1, SUN domain-containing protein 2, Follistatin-related protein 1, Leukocyte immunoglobulin-like receptor subfamily A member 6, Acyl-CoA:lysophosphatidylglycerol acyltransferase 1, Tumor necrosis factor receptor superfamily member 10C, C1GALT1-specific chaperone 1, Cytochrome b5, Beta-enolase, Sorting nexin-1, Catechol O-methyltransferase, Tapasin, Cytochrome b-245 light chain, Protein YIPF5, Neutrophil defensin 3, Armadillo repeat-containing protein8、Estradiol 17-beta-dehydrogenase 11、Peflin、Sorcin、Transmembrane protein 165、Cytochrome P450 4F3、Aldehyde dehydrogenase family 3 member B1、Methyltransferase-like protein 7A、Mucolipin-1、Hydroxyacylglutathione hydrolase, mitochondrial、Myeloid cell nuclear differentiation antigen、Annexin A11、Intercellular adhesion molecule 4、Carcinoembryonic antigen-related cell adhesion molecule 8、Dehydrogenase / reductase SDR family member 7、Transmembrane protein 87A、Annexin A7、Solute carrier family 2, facilitated glucose transporter member 1、Adenosine 3'-phospho 5'-phosphosulfate transporter 1、Interferon-induced 35 kDa protein、Erythrocyte membrane protein band 4.2、Protein argonaute-2、Annexin A5、Cullin-2、Glycophorin-A、Integrin alpha-M、Porphobilinogen deaminase、PDZ domain-containing protein 8、Reticulon-3、Metalloreductase STEAP4、Transmembrane emp24 domain-containing protein 9、Transmembrane emp24 domain-containing protein 4、Myeloid-derived growth factor、Ankyrin-1、Transmembrane emp24 domain-containing protein10. Protein group consisting of Transmembrane protein with metallophosphoesterase domain, Activated CDC42 kinase 1, Golgi reassembly-stacking protein 2, Ubiquitin-conjugating enzyme E2 variant 1, Calpain-5, DnaJ homolog subfamily C member 13, Golgi integral membrane protein 4, Transmembrane protein 40, Urokinase plasminogen activator surface receptor, WD repeat-containing protein 13, Copine-2, Integrin beta-2, Optineurin, Inactive rhomboid protein 2, Secretory carrier-associated membrane protein 2, Serine incorporator 3, Calcium-transporting ATPase type 2C member 1, Low affinity immunoglobulin gamma Fc region receptor II-c, AP-3 complex subunit mu-1, Activator of 90 kDa heat shock protein ATPase homolog 1, Brain acid soluble protein 1, Protein transport protein Sec31A, Neutral cholesterol ester hydrolase 1, Serine / threonine-protein kinase OSR1, Golgi phosphoprotein 3, AP-2 complex subunit mu, Ragulator complex protein LAMTOR2, and Tetraspanin-14, (D) Alpha-amylase 1, Nostrin, Matrix Gla protein, Neuronal membrane glycoproteinM6-a、Dermcidin、Low affinity immunoglobulin gamma Fc region receptor II-b、Cystatin-A、B-cell antigen receptor complex-associated protein beta chain、Lipolysis-stimulated lipoprotein receptor、Apolipoprotein C-I、Prolactin-inducible protein、Immunoglobulin epsilon heavy chain、Multimerin-2、Anthrax toxin receptor 1、EGF-like repeat and discoidin I-like domain-containing protein 3、P-selectin glycoprotein ligand 1、Putative trypsin-6、Beta-hexosaminidase subunit alpha、Solute carrier family 35 member D3、RAS protein activator like-3、Semaphorin-4B、Ras-related protein R-Ras2、Transforming growth factor beta-1-induced transcript 1 protein、Volume-regulated anion channel subunit LRRC8D、Fermitin family homolog 2、Plexin-D1、Regulator of G-protein signaling 19、WD repeat-containing protein 37、Angiopoietin-related protein 3、DnaJ homolog subfamily B member 1、Dedicator of cytokinesis protein 11、Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1、Ras-related protein Rab-20、Early activationA method for evaluating the inducibility or aggravation of lung cancer, using as an indicator the amount or concentration of at least one protein selected from the group consisting of: (A) a group of proteins consisting of antigen CD69, TBC1 domain family member 10A, and Receptor-type tyrosine-protein phosphatase epsilon, and (B) a group of proteins consisting of constitutive coactivator of PPAR-gamma-like protein 1, Inter-alpha-trypsin inhibitor heavy chain H4, Sec1 family domain-containing protein 1, and Grancalcin.

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