Biomarker for diagnosing lung cancer and uses thereof

Abstract

The present invention relates to a biomarker for diagnosing non-small cell lung cancer and uses thereof. Specifically, it has been confirmed that lung cancer can be diagnosed with high efficiency by measuring levels of cancer-associated fibroblasts exhibiting high expression of GREM-1 and GAS6 in a biological sample from a patient. Accordingly, the biomarkers may be utilized for the prevention, amelioration, and / or treatment of lung cancer by measuring the levels thereof.

Description

Biomarkers for lung cancer diagnosis and their uses

[0001] An example of the present invention relates to a biomarker for diagnosing lung cancer and its use.

[0002] Cancer-associated fibroblasts (CAFs), also known as peritumoral fibroblasts, are a key component of the tumor stroma. CAFs play a crucial role in promoting tumor growth and progression through various mechanisms, including the secretion of growth factors, chemokines, and extracellular vesicles, and their involvement in extracellular matrix (ECM) remodeling. These modulations effectively regulate key aspects of tumor behavior, including tumor proliferation, metastatic potential, chemotherapy resistance, immune evasion, and reactivation from a dormant state.

[0003] Since the initial identification of CAFs, advances in immunohistochemical methods and single-cell RNA sequencing have led to the redefinition of established CAF markers, including platelet-derived growth factor receptor-α / β, α-smooth muscle actin (αSMA), fibroblast activation protein (FAP), and fibroblast-specific protein-1. However, these proteins are also found in various immune cells and some cancer cells. A technical challenge in isolating CAFs is the lack of specific markers, as most known markers are shared with other cell types. For example, podoplanin is expressed in lymphatic endothelial cells, FAP is expressed in macrophages, and αSMA is primarily expressed in smooth muscle cells.

[0004] These aspects highlight the need for a subtype-specific set of markers that distinguish CAFs from normal fibroblasts (NFs) to accurately identify various CAF populations. Furthermore, while CAF markers have been studied in many cancer types, studies focusing on non-small cell lung cancer (NSCLC) are rare, and information on NSCLC-specific CAF markers is limited. Furthermore, the relationship and function of CAFs at metastatic and primary sites remain unclear.

[0005] Cancer-associated fibroblasts (CAFs), also known as peritumoral fibroblasts, are a key component of the tumor stroma. CAFs play a crucial role in promoting tumor growth and progression through various mechanisms, including the secretion of growth factors, chemokines, and extracellular vesicles, and their involvement in extracellular matrix (ECM) remodeling. These modulations effectively regulate key aspects of tumor behavior, including tumor proliferation, metastatic potential, chemotherapy resistance, immune evasion, and reactivation from a dormant state.

[0006] Since the initial identification of CAFs, advances in immunohistochemical methods and single-cell RNA sequencing have led to the redefinition of established CAF markers, including platelet-derived growth factor receptor-α / β, α-smooth muscle actin (αSMA), fibroblast activation protein (FAP), and fibroblast-specific protein-1. However, these proteins are also found in various immune cells and some cancer cells. A technical challenge in isolating CAFs is the lack of specific markers, as most known markers are shared with other cell types. For example, podoplanin is expressed in lymphatic endothelial cells, FAP is expressed in macrophages, and αSMA is primarily expressed in smooth muscle cells.

[0007] These aspects highlight the need for a subtype-specific set of markers that distinguish CAFs from normal fibroblasts (NFs) to accurately identify various CAF populations. Furthermore, while CAF markers have been studied in many cancer types, studies focusing on non-small cell lung cancer (NSCLC) are rare, and information on NSCLC-specific CAF markers is limited. Furthermore, the relationship and function of CAFs at metastatic and primary sites remain unclear.

[0008] We performed analyses using RNA sequencing to identify distinct markers representing CAF compared to NF in the context of non-small cell lung cancer under various conditions.

[0009] The present inventors performed analyses using RNA sequencing to distinguish distinct markers representing CAFs compared to NFs in the context of non-small cell lung cancer under various conditions.

[0010] The present invention provides a composition for diagnosing lung cancer, comprising a preparation for measuring the level of fibroblasts highly expressing GREM1 (Gremlin 1) and GAS6 (Growth Arrest Specific 6).

[0011] In one embodiment of the present invention, the fibroblasts may be cancer-associated fibroblasts.

[0012] In one embodiment of the present invention, the agent for measuring the level of fibroblasts may be at least one selected from the group consisting of antibodies, oligopeptides, ligands, peptide nucleic acids (PNAs), and aptamers that specifically bind to GREM-1 and GAS6 high-expressing fibroblasts.

[0013] In one embodiment of the present invention, the lung cancer may be non-small cell lung cancer.

[0014] In addition, the present invention provides a kit for diagnosing lung cancer, comprising the composition.

[0015] In addition, the present invention provides a method for providing information regarding the diagnosis of lung cancer, comprising the step of measuring the level of GREM-1 and GAS6 high-expressing fibroblasts in a biological sample isolated from an individual.

[0016] In one embodiment of the present invention, the method may further include a step of determining that partial non-small cell lung cancer has developed or is likely to develop when the level of GREM-1 and GAS6 high-expressing fibroblasts in a biological sample isolated from the subject is increased compared to a normal control sample.

[0017] In one embodiment of the present invention, the biological sample is whole blood, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, bronchial washing fluid, bronchoalveolar lavage fluid, synovial fluid, joint aspirate, trachea. It may be organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0018] In addition, the present invention comprises the steps of: measuring the level of GREM-1 and GAS6 high-expressing fibroblasts in a separated biological sample; contacting the biological sample with a test substance;

[0019] A method for screening a drug for treating lung cancer is provided, comprising: a step of measuring the level of GREM-1 and GAS6 high-expressing fibroblasts in the biological sample after contact with the test substance; and a step of determining the test substance as a drug for treating lung cancer when the level of GREM-1 and GAS6 high-expressing fibroblasts in the biological sample decreases.

[0020] A composition for diagnosing lung cancer, comprising a formulation for measuring the level of GREM-1 and GAS6 high-expressing fibroblasts according to one aspect, has been confirmed to be effective as a biomarker for pathological diagnosis of non-small cell lung cancer in non-small cell lung cancer, and can be used to effectively diagnose lung cancer, and further, can effectively treat lung cancer by suppressing GREM-1 and GAS6 high-expressing fibroblasts according to the diagnosis.

[0021] Figures 1A to 1C illustrate differentially expressed genes (DEGs) between CAF and NF. Figure 1A shows a heatmap of 74 DEGs. Figure 1B shows a volcano diagram. Figure 1C shows a heatmap of previously known fibroblast marker genes.

[0022] Figures 2A to 2D show DEGs that show differences in expression levels between CAF and NF. Figure 2A shows a heatmap of selected DEGs comparing CAF and NF. Figure 2B shows a boxplot of DEGs comparing CAF and NF. The adjusted p-value (FDR, false discovery rate) considering multiple comparisons in a generalized linear model is used. COL11A1 and TNC are known fibroblast markers, GREM1 belongs to the BMP pathway, CD36 belongs to the antigen processing machinery pathway, CXCL2 belongs to the chemokine pathway, and GAS6 belongs to the hypoxia pathway. Figure 2C shows a boxplot of DEGs comparing lymph node CAF (LN-CAF), lung CAF (Lung-CAF), and NF. Figure 2D shows Western blot analysis of GREM-1 and GAS6 in LN-CAF, Lung-CAF, and NF derived from squamous cell carcinoma, adenocarcinoma, and non-neoplastic lung.

[0023] Figures 3A to 3C show pathways (gene sets) that show differences in expression between CAF and NF. Figure 3A calculates the GSVA (gene set variation analysis) score for each pathway in well-known databases (e.g., HALLMARK, BIOCARTA, KEGG, REACTOME, WIKI pathway) and selects pathways with significant differences in GSVA scores between CAF and NF and displays them as a heatmap. Figure 3B shows a box plot of GSVA scores for the selected pathways comparing CAF and NF. Figure 3C shows a box plot of GSVA scores for the selected pathways comparing LN-CAF, Lung-CAF, and NF.

[0024] Figures 4A to 4C illustrate CAF subpopulation predictions. Figure 4A shows a heatmap of CAF subpopulations based on GSVA scores. Figure 4B shows a boxplot of GSVA scores for CAF subpopulations comparing CAF and NF. Figure 4C shows a boxplot of GSVA scores for CAF subpopulations comparing LN-CAF, Lung-CAF, and NF.

[0025] Figures 5A and 5B demonstrate the M2 polarization of macrophages by CAFs and the reversal effect through GAS6 or GREM1 inhibition. Figure 5A shows a schematic diagram of an experiment in which PMA-induced M0 macrophages were co-cultured with CAFs or siRNA-treated CAFs at a 5:1 ratio in a trans-well system. Figure 5B shows the results of RT-qPCR analysis for the expression of M1 (CD80, IL-6) and M2 (CD206, IL-10) markers in M0 macrophages after 3 days of co-culture with CAFs derived from primary lung tumors or metastatic lymph nodes.

[0026] The present invention provides a composition for diagnosing or predicting the prognosis of lung cancer, comprising a preparation for measuring the level of GREM-1 and GAS6 high-expressing fibroblasts.

[0027] In the present invention, "GREM-1" is a protein known as an antagonist of bone morphogenetic proteins (BMPs), the expression of which can be increased in cancer-related fibroblasts.

[0028] In the present invention, "GAS6" is a protein secreted from cancer-related fibroblasts, and its expression may increase in cancer-related fibroblasts.

[0029] In the present invention, "high expression" means an increase in the expression of a biomarker compared to a normal control group. For example, it may mean a case where the expression of a biomarker in a sample increases by 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.8 times, 2.0 times, 2.5 times, 3.0 times, 5.0 times, or 10.0 times or more.

[0030] In the present invention, "fibroblasts" are fibroblasts that are mainly located in the dermal layer of the skin and produce collagen, hyaluronic acid, elastin, etc. The present invention provides a composition for diagnosing lung cancer, including a preparation for measuring the level of fibroblasts that highly express GREM-1 and GAS6.

[0031] In the present invention, "GREM-1 (Gremlin 1)" is a protein known as an antagonist of bone morphogenetic proteins (BMPs), the expression of which can be increased in cancer-related fibroblasts.

[0032] In the present invention, "GAS6 (Growth Arrest Specific 6)" is a protein secreted from cancer-related fibroblasts, and its expression may increase in cancer-related fibroblasts.

[0033] In the present invention, "high expression" means an increase in the expression of a biomarker compared to a normal control group. For example, it may mean a case where the expression of a biomarker in a sample increases by 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.8 times, 2.0 times, 2.5 times, 3.0 times, 5.0 times, or 10.0 times or more.

[0034] In the present invention, "fibroblasts" are primarily located in the dermal layer of the skin and play a crucial role in maintaining skin health and elasticity by producing collagen, hyaluronic acid, and elastin. Unlike cancer-associated fibroblasts, normal fibroblasts do not promote tumors and do not accelerate cancer progression. Furthermore, normal fibroblasts do not suppress the immune response, but rather support normal immune function.

[0035] In the present invention, “cancer-associated fibroblasts” refer to cells that play an important role in the tumor microenvironment, exist within cancer tissue, and have a significant influence on the evolution of cancer.

[0036] In one embodiment of the present invention, the method for measuring or comparing the level of the fibroblasts includes, but is not limited to, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, aukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), Western blotting, and ELISA (enzyme linked immunosorbent assay).

[0037] In one embodiment of the present invention, the fibroblasts may be cancer-associated fibroblasts.

[0038] In one embodiment of the present invention, the agent for measuring the level of fibroblasts may be at least one selected from the group consisting of antibodies, oligopeptides, ligands, peptide nucleic acids (PNAs), and aptamers that specifically bind to GREM-1 and GAS6 high-expressing fibroblasts.

[0039] As used herein, "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, it may be an antibody that binds to IL-1ß high-expressing macrophages. The antibody of the present invention includes all polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The antibody can be easily produced using techniques well known in the art. In addition, the antibody of the present invention includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of an antibody molecule. A functional fragment of an antibody molecule means a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.

[0040] In this specification, the above "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized polymer similar to DNA or RNA, and was first introduced in 1991 by Professors Nielsen, Egholm, Berg, and Buchardt of the University of Copenhagen, Denmark. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases its binding affinity and stability to DNA or RNA, and is therefore used in molecular biology, diagnostic analysis, and antisense therapy.

[0041] As used herein, the term "aptamer" refers to a ligand-specific DNA or RNA molecule, which is an oligonucleotide or peptide molecule and has high affinity for a protein. An aptamer is a single-stranded DNA or RNA molecule that has a specific binding ability to a specific substance and has its own unique 3D structure. It can be mass-produced in a short time and at low cost using chemical synthesis techniques, and has excellent productivity as there is almost no variation between batches. In addition, it has high stability against changes in the surrounding environment, such as pH or temperature, and its potential for use in various fields, such as the development of sensors for target substance detection and disease diagnosis, is highly evaluated.

[0042] In one embodiment of the present invention, the lung cancer may be non-small cell lung cancer. In this case, "non-small cell lung cancer" (NSCLC) refers to all epithelial lung cancers except small cell lung cancer, accounting for approximately 85% of all lung cancers and including major subtypes such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

[0043] In the present invention, the term "diagnosis" includes determining a subject's susceptibility to a specific disease or condition, determining whether a subject currently has a specific disease or condition, determining a prognosis of a subject with a specific disease or condition (e.g., identifying a pre-metastatic or metastatic cancer state, determining the stage of cancer, or determining the responsiveness of cancer to treatment), or therametrics (e.g., monitoring the condition of a subject to provide information on the efficacy of treatment). For the purposes of the present invention, the diagnosis is to determine whether or not the subject has developed the disease or the likelihood of developing the disease (risk), the stage of the cancer, or the survival rate or responsiveness to treatment of a cancer patient.

[0044] In the present invention, the "stage" refers to the extent to which cancer cells have spread and the stage of cancer progression. The international classification according to the progression of cancer generally follows the TNM staging classification. Here, 'T (Tumor Size)' is a classification according to the size of the primary tumor, 'N (Lymph Node)' is a classification according to the degree of lymph node metastasis, and 'M (Metastasis)' is a classification according to whether or not there is metastasis to other organs.

[0045] In addition, the present invention provides a kit for diagnosing lung cancer, comprising the composition.

[0046]

[0047] In addition, the present invention provides a method for providing information regarding the diagnosis of lung cancer, comprising the step of measuring the level of GREM-1 and GAS6 high-expressing fibroblasts in a biological sample isolated from an individual.

[0048] In this specification, "subject" means any living creature, including humans, rats, mice, and livestock. As a specific example, it may be a mammal, including humans.

[0049] In one embodiment of the present invention, the method may further include a step of determining that non-small cell lung cancer has developed or is likely to develop if the level of GREM-1 and GAS6 high-expressing fibroblasts in a biological sample isolated from the subject is increased compared to a normal control sample.

[0050] In one embodiment of the present invention, the biological sample is whole blood, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, bronchial washing fluid, bronchoalveolar lavage fluid, synovial fluid, joint aspirate, trachea. It may be organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0051] In addition, the present invention comprises the steps of: measuring the level of GREM-1 and GAS6 high-expressing fibroblasts in a separated biological sample; contacting the biological sample with a test substance;

[0052] A method for screening a drug for treating lung cancer is provided, comprising: a step of measuring the level of GREM-1 and GAS6 high-expressing fibroblasts in the biological sample after contact with the test substance; and a step of determining the test substance as a drug for treating lung cancer when the level of GREM-1 and GAS6 high-expressing fibroblasts in the biological sample decreases.

[0053] In this specification, “treatment” may mean any action that improves or beneficially changes the symptoms of lung cancer in a subject by administering a pharmaceutical composition according to one aspect.

[0054] In one embodiment of the present invention, the step of measuring the level of fibroblasts may be measuring the level of GREM-1 and GAS6 high-expressing cancer-associated fibroblasts.

[0055]

[0056] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0057]

[0058] Examples and Experimental Examples

[0059] 1. Experimental method

[0060] (1) Isolation and culture of CAF and NF

[0061] Isolation and culture of CAFs and NFs were performed as described in a previous report. Briefly, CAFs were isolated from tumor tissue (primary tumor or metastatic lymph nodes), and NFs were isolated from non-tumor tissues most distant from the tumor in resected lungs of NSCLC patients. Non-tumor and tumor specimens were washed three times with phosphate-buffered saline (PBS; HyClone, Logan, UT, USA). Tissue samples were minced into 1-2 mm pieces and dissociated using 1 mL of 0.25% collagenase type I (STEMCELL Technologies, Vancouver, Canada) in an incubator at 37°C for 30 minutes to 1 hour. Enzymatically dissociated tissue samples were centrifuged at 1300 rpm for 3 minutes. The pellet was resuspended in DMEM / F12 (Gibco, Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (FBS; Gibco), and 1% antibiotic-antimycotic solution (Gibco). The pellet was then cultured in 100 mm culture dishes. Cells were expanded for 7–14 days until 90% confluence was reached. Cells migrating from the seeded tissue fragments were collected after trypsin-EDTA treatment (Gibco). The harvested cells were passed through a 100 μm filter (Falcon, Franklin Lakes, NJ, USA) and seeded into 100 mm culture dishes. The cells were then expanded for three passages and cryopreserved in complete DMEM containing 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) until analysis. CAFs were confirmed by negative expression of EpCAM or E-cadherin by flow cytometry or RT-PCR.

[0062]

[0063] (2) RNA base sequence analysis

[0064] Total RNA was extracted from cultured cell pellets of CAFs or NFs using the RNeasy Mini Kit (Qiagen). Nucleic acid extraction was performed according to the manufacturer's instructions. The quality and quantity of extracted nucleic acids were assessed using a Nanodrop 8000 UV-Vis spectrophotometer (NanoDrop Technologies Inc.), a Qubit 3.0 fluorometer (Life technologies, Inc.), and a 4200 TapeStation (Agilent Technologies, Inc.). Sequencing libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina, Inc.) according to the manufacturer's protocol. Paired-end sequencing of the RNA libraries was performed on a HiSeq 2500 sequencing platform (Illumina, Inc.). After removing low-quality bases from the FASTQ files, sequencing reads were aligned to the human reference genome (hg19) using STAR v2.5.2b, and expected counts were calculated using RSEM v1.3 to estimate gene expression.

[0065]

[0066] (3) Differential expression and gene set enrichment analysis

[0067] To compare gene expression levels between CAF and NF, differential expression (DE) analysis was performed using generalized linear modeling via the glmFit function in the R package egdeR v3.36.1. The batch effect according to tissue origin (lung or lymph node) was adjusted for expression counts using the design matrix in the R package edgeR. Significant DE genes were selected based on the following criteria: FDR-adjusted p-value < 0.05, logCPM > 2, and absolute log fold change (logFC) > 1. K-means-based clustering was performed on the expression profiles of significant DE genes, and heatmaps were generated using the R package ComplexHeatmap v2.10.0. Gene set enrichment analysis (GSVA) ​​was performed on the HALLMARK, C2 (curation), and C6 (oncology) gene set (pathway) databases in the R package MSigDB v7.4.1. GSVA scores were calculated for each gene set (pathway) using the R package GSVA v1.42.0. Gene sets were considered to exhibit significant DE according to the following criteria: absolute value of log2 fold change (logFC) > 0.4 and FDR-adjusted p-value < 0.05 by nonparametric (e.g., Kruskal-Wallis) test. For pairwise comparisons, t-tests were performed and statistical significance was determined by FDR-adjusted p-values.

[0068]

[0069] (4) CAF subgroup prediction

[0070] To decipher CAF into three subgroups (myCAF, iCAF, and apCAF), the GSVA R package (v1.42.0) was used to estimate GSVA scores based on specific gene sets reported in pancreatic cancer. myCAF, annotated with lung cancer, was also estimated with GSVA scores based on highly expressed genes (COL4A1, ACTA2, MEF2C, MYG11, ITGA7). Differences in GSVA scores between CAF subgroups were analyzed using the Kruskal-Wallis test. The threshold for statistical significance was an FDR-adjusted p-value < 0.05.

[0071]

[0072] (5) Western blot

[0073] Cells were harvested and suspended in lysis buffer (150 mM NaCl, 25 mM Tris-Cl, pH 7.4, 1% NP-40, 10 mM EDTA) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). After boiling for 10 min, the cell lysate was centrifuged at 13,000 rpm for 10 min at room temperature (RT). Protein samples were quantified using a modified BCA protein assay reagent (Pierce, cat. no. 23228). A 20 μg volume of protein sample was electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA) using a Trans-Blot electrophoresis cell transfer system (Bio-Rad Laboratories, Hercules, CA, USA). The PVDF membranes onto which the proteins were transferred were incubated for 1 h in 5% BSA in 1X TBST (150 mM NaCl, 50 mM Tris-Cl, pH 7.5, 0.1% Tween 20), washed with 1X TBST, and then incubated overnight at 4°C in primary antibodies diluted 1:500 in TBST containing 5% BSA. Detection was performed with a chemiluminescent substrate (Thermo Fisher) followed by exposure to ECL solution (Thermo Fisher, 34094). Western blots were performed by applying primary antibodies against GAS6 (Abcam, ab264098), GREM1 (R&D Systems, AF956), α-SMA (Abcam, ab7817), and β-actin (Santa Cruz, sc-47778) to the blots at a ratio of 1:500.

[0074]

[0075] 2. Experimental Results

[0076] (1) Patient characteristics

[0077] Twenty-two cancer-associated fibroblast (CAF) samples and 11 normal fibroblast (NF) samples were included in the study from 22 patients with non-small cell lung cancer (NSCLC). The 22 CAF samples consisted of 12 adenocarcinomas and 10 squamous cell carcinomas (SqCCs): 16 were derived from primary tumors and 6 from metastatic lymph nodes. Additionally, 12 samples were derived from stage I or II cancers, and 10 samples were derived from stage III or IV cancers. The 11 NF samples included 8 adenocarcinomas and 3 SqCCs, all from non-neoplastic lungs. Ten pairs of CAF and NF were isolated from the same patients.

[0078]

[0079] (2) Analysis of genes differentially expressed between CAF and NF

[0080] Figure 1 shows the differentially expressed genes (DEGs) between CAF and NF using 22 CAF samples and 11 NF samples. Figure 1A shows a heatmap of 74 DEGs, which clearly distinguished CAF from NF and CAF of lymph node origin (LN-CAF) from CAF of lung origin (Lung-CAF) during unsupervised clustering. The volcano plot showed 66 genes significantly upregulated (e.g., GAS6, GREM1, CD36, COL11A1, IGF2) and 8 genes significantly downregulated (e.g., TNC, CXCL2, MMP1) in CAF (Figure 1B). Figure 1C shows a heatmap of significant DEGs of known fibroblast markers, which also clearly distinguished CAF from NF and LN-CAF from Lung-CAF.

[0081] Figure 2 shows six genes (GAS6, GREM1, CD36, COL11A1, TNC, and CXCL2) among the DEGs between CAF and NF: COL11A1 and TNC (fibroblast markers), GREM1 (bone morphogenetic protein [BMP] pathway), CD36 (antigen processing machinery pathway), CXCL2 (chemokine pathway), and GAS6 (hypoxia pathway). Figure 2A shows a heatmap of these DEGs, clearly distinguishing CAF from NF and LN-CAF from Lung-CAF. COL11A1, GREM1, CD36, and GAS6 were significantly elevated in CAF, while TNC and CXCL2 showed higher levels in NF (all FDR, p ≤ 0.0001) (Figure 2B). COL11A1 expression was higher in Lung-CAF than in LN-CAF or NF (FDR, p ≤ 0.0001) (Fig. 2C). CD36 expression was highest in LN-CAF and exceeded the levels of Lung-CAF and NF (all FDR, p ≤ 0.0001). CXCL2 expression was higher in LN-CAF than in Lung-CAF, but lower than in NF (all FDR, p ≤ 0.0001). Both GREM1 and GAS6 showed stronger expression in Lung-CAF and LN-CAF compared to NF (all FDR, p ≤ 0.0001) (Fig. 2C). Western blot analysis also showed that the protein expression of GREM1 and GAS6 was higher in both LN-CAF and Lung-CAF compared to NF, regardless of histological subtype (Fig. 2D).

[0082]

[0083] (3) Gene set enrichment analysis between CAF and NF

[0084] Figure 3A shows a heatmap of Gene Set Variation Analysis (GSVA) ​​scores for selected gene sets comparing CAF and NF. The GSVA score clearly distinguished CAF from NF but did not completely distinguish LN-CAF from Lung-CAF. Gene sets related to hypoxia response (Hallmark Hypoxia) and epithelial-to-mesenchymal transition (EMT) (Hallmark EMT) were upregulated in CAF compared to NF (all false discovery rate (FDR), p ≤ 0.0001) (Figure 3B). Conversely, the gene set related to Reactome Mismatch Repair showed a significantly higher GSVA score in NF than in CAF (FDR, p ≤ 0.0001). Additionally, LN-CAF showed higher GSVA scores in gene sets such as Hallmark Hypoxia and Hallmark EMT compared to Lung-CAF (FDR, p ≤ 0.0001) (Fig. 3C).

[0085]

[0086] (4) CAF subgroup analysis

[0087] Figure 4A shows a heatmap of subpopulations of CAF and NF based on previously reported gene sets, which clearly distinguished CAF from NF but did not completely distinguish LN-CAF from Lung-CAF. CAF showed significantly higher GSVA scores for the myofibroblastic CAF (myCAF) subpopulation compared to NF (FDR, p ≤ 0.0001), and NF showed significantly higher GSVA scores for the antigen-presenting CAF (apCAF) subpopulation compared to CAF (FDR, p ≤ 0.01). GSVA scores for the inflammatory CAF (iCAF) subpopulation were similar between CAF and NF (Figure 4B). Both Lung-CAF and LN-CAF showed significantly higher GSVA scores for the myCAF subgroup compared to NF (all FDRs, p ≤ 0.0001), and NF showed significantly higher GSVA scores for the apCAF subgroup compared to Lung-CAF (FDR, p ≤ 0.01) (Fig. 4C). Additionally, both LN-CAF and NF showed significantly higher GSVA scores for the iCAF subgroup compared to Lung-CAF (all FDRs, p ≤ 0.05).

[0088]

[0089] (5) Induction of macrophage polarization by CAF-derived signals

[0090] Figure 5A is a schematic representation of a 3-day co-culture experiment using a trans-well system between CAFs derived from non-small cell lung cancer patients and M0 macrophages induced by phorbol 12-myristate 13-acetate (PMA). CAFs were compared with CAFs transfected with siRNA targeting GREM1 or GAS6. RT-qPCR analysis revealed that the expression of both M1 macrophage markers (CD80 and IL-6) and M2 macrophage markers (CD206 and IL-10) was increased in all CAF samples (Figure 5B). However, when co-cultured with CAFs in which GREM1 or GAS6 was inhibited, the expression of M2 markers (CD206 and IL-10) was significantly reduced, while the decrease in M1 marker expression was inconsistent.

[0091]

[0092] Therefore, through this example, it was confirmed that cancer-associated fibroblasts derived from non-small cell lung cancer patients specifically up-express multiple genes, including GREM1 and GAS6, compared to normal fibroblasts, and the expression of the genes was confirmed to be related to increased activity of a set of genes related to hypoxia response and epithelial-mesenchymal transition. In addition, it was confirmed that GREM1 and GAS6 high-expressing fibroblasts are involved in immune regulation within the tumor microenvironment by inducing M2-like phenotype polarization of macrophages. Accordingly, it was confirmed that by measuring the level of GREM1 and GAS6 high-expressing fibroblasts, a composition and kit that can be utilized for lung cancer diagnosis can be provided, and it was confirmed that it can also be applied as a drug screening method to evaluate the effectiveness of a substance targeting the fibroblasts.

[0093]

[0094] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A composition for diagnosing lung cancer comprising a preparation for measuring levels of GREM-1 (Gremlin 1) and GAS6 (Growth Arrest Specific 6) high-expression fibroblasts.

2. A composition according to claim 1, wherein the fibroblasts are cancer-associated fibroblasts.

3. In claim 1, the agent for measuring the level of fibroblasts is a composition comprising at least one selected from the group consisting of antibodies, oligopeptides, ligands, peptide nucleic acids (PNAs), and aptamers that specifically bind to GREM-1 and GAS6 high-expressing fibroblasts.

4. A composition according to claim 1, wherein the lung cancer is non-small cell lung cancer (NSCLC).

5. A lung cancer diagnostic kit comprising the composition of any one of claims 1 to 4.

6. A method for providing information regarding the diagnosis of lung cancer, comprising the step of measuring the level of fibroblasts highly expressing GREM-1 (Gremlin 1) and GAS6 (Growth Arrest Specific 6) in a biological sample isolated from an individual.

7. A method for providing information, further comprising a step of determining that non-small cell lung cancer has developed or is likely to develop if the level of GREM-1 and GAS6 high-expressing fibroblasts in a biological sample isolated from the subject is increased compared to a normal control sample in claim 6.

8. In claim 6, the biological sample is whole blood, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, bronchial washing fluid, bronchoalveolar lavage fluid, synovial fluid, joint aspirate A method of providing information, which is aspirate), organ secretions, cell, cell extract, or cerebrospinal fluid.

9. A step of measuring the levels of GREM-1 (Gremlin 1) and GAS6 (Growth Arrest Specific 6) high-expression fibroblasts in isolated biological samples; A step of contacting a test substance with the above biological sample; A step of measuring the levels of GREM-1 and GAS6 high-expression fibroblasts in the biological sample after contact with the above test substance; and A method for screening a drug for treating lung cancer, comprising: a step of determining the test substance as a drug for treating lung cancer when the level of GREM-1 and GAS6 high-expressing fibroblasts in the biological sample decreases.

10. A drug screening method according to claim 9, wherein the step of measuring the level of fibroblasts is measuring the level of cancer-associated fibroblasts that highly express GREM-1 and GAS6.

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