WO2022231191A1 - Composition for cancer diagnosis - Google Patents

Abstract

The present invention provides a composition for cancer diagnosis, comprising a preparation for measuring the expression level of at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1, or a gene encoding same.

Description

composition for diagnosis of cancer

The present invention relates to a composition for diagnosis of cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide, from developing countries to developed countries. For example, according to Korean statistics, in 2017 alone, the incidence and mortality rates were 52.7 and 34.8 per 100,000, respectively, making it the highest cause of cancer-related death among solid cancers. As awareness of this deadly disease has risen, a nationwide lung cancer screening program has been launched in 2019 for people at high risk for lung cancer.

Lung ground glass opacity (GGO) nodules are defined as hazy opacity of lung tissue that does not obscure the underlying bronchi and pulmonary vasculature on chest high-resolution computed tomography (HRCT). Low-dose chest CT scans are widely applied with increasing interest in health screening, increasing the detection rate of GGO and early lung cancer diagnosis rates. As GGO is a radiologically defined lesion, it includes benign lesions with inflammation and parenchymal hemorrhage. However, persistent GGO that does not change for more than 3 months is considered a potential malignancy and can show pathologically various lesions ranging from atypical adenomatous proliferation to invasive lung adenocarcinoma. As early lung adenocarcinoma takes the form of GGO, attention has been focused on the occurrence of each cancer in lung adenocarcinoma. These lesions grow slowly, gradually increase in size, and develop into a solid part over time, leading to invasive cancer. However, although the natural processes of GGO have been studied from a radiological perspective, investigations into molecular and biological aspects are very limited. Therefore, lung cancer progressing in GGO nodule is different from general lung cancer and requires a differentiated diagnosis at an early stage. The present inventor devised a composition for diagnosis of lung cancer to solve the above problems.

An object of the present invention is to provide a composition capable of predicting the possibility of developing cancer early and diagnosing the stage of cancer with high accuracy. Another object of the present invention is to provide a kit capable of predicting the possibility of developing cancer early and diagnosing the stage of cancer with high accuracy. In addition, another object of the present invention is to provide a method of providing information in order to predict the possibility of developing cancer early and to diagnose the stage of cancer with high accuracy. Another object of the present invention is to provide a method for screening a drug for treating the cancer.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present invention, "SPINK1 (Serine Peptidase Inhibitor Kazal Type 1)" is also known as a tumor-associated trypsin inhibitor (TATI) or a pancreatic secreted trypsin inhibitor (PSTI), and it is believed that the enzyme causes cell damage to organs by cleaving the prematurely activated trypsin. It is a gene that encodes a protein that prevents Without its function, the pancreas is repeatedly damaged.

In the present invention, "LPCAT1 (Lysophosphatidylcholine acyltransferase 1)" is a gene that plays an important role in phospholipid metabolism, particularly in the synthesis of pulmonary surfactant and platelet activating factor (PAF), by converting lysophosphatidylcholine to phosphatidylcholine in the presence of acyl-CoA.

In the present invention, "NEAT1 (Nuclear Paraspeckle Assembly Transcript 1)" generates a long non-coding RNA (lncRNA; long non-coding RNA) transcribed at multiple endocrine oncogenes. It is retained in the nucleus and forms a key structural component of the paraspeckles subcellular organelles.

In the present invention, "CEACAM5 (Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5)" encodes a cell surface glycoprotein belonging to the cancer embryonic antigen (CEA) family. The encoded protein is used as a clinical biomarker for gastrointestinal cancer and may promote tumorigenesis through its role as a cell adhesion molecule.

In the present invention, "CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6)" encodes a protein belonging to the cancer embryonic antigen (CEA) family, which is a glycosyl phosphatidylinositol (GPI) anchored cell surface glycoprotein. This family plays a role in cell adhesion and is widely used as tumor markers in serum immunoassay measurements of carcinomas.

In the present invention, "RNASE1 (Ribonuclease A Family Member 1)" encodes a protein belonging to the pancreatic family of secretory ribonucleases, which is a subset of the ribonuclease A superfamily.

In the present invention, "TFF3 (Trefoil Factor 3)" is a member of the trefoil family, which is characterized by having at least one copy of the trefoil motif, a 40 amino acid domain containing three conserved disulfides. These are stable secreted proteins expressed in the gastrointestinal mucosa.

In the present invention, the "ground glass opacity (GGO) nodule" or GGO of the lung is classified as a very slow-growing inactive lung cancer, but is a radiologically defined cancer for the early form of lung cancer, and the management of GGO is well Although not established, changes in radiological findings, such as an increase in size or development of a solid part, lead to surgical treatment. Although the natural processes of GGO have been studied from a radiological perspective, investigations into molecular and biological aspects have been very limited. For the present inventors, GGO is a perfect model for studying the onset of lung cancer, ie, early lung cancer.

In the present invention, "measurement of the expression level of protein" refers to the presence and level of expression of proteins of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1, or TFF3, which are diagnostic markers for cancer in a biological sample in order to diagnose cancer. means process. The expression level measurement or comparative analysis method of the diagnostic marker for cancer includes protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, and SELDI-TOF (Sulface). Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Oakteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2D electrophoresis analysis, liquid chromatography -Mass spectrometry (liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry / Mass Spectrometry), Western blotting, and ELISA (enzyme linked immunosorbent assay), but are not limited thereto .

In the present invention, the "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1, or TFF3. Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies and recombinant antibodies. The antibody can be readily prepared using techniques well known in the art. For example, the polyclonal antibody can be produced by a method well known in the art, including the step of injecting the ADAM8 or the antigen of the cancer embryo antigen into an animal and collecting blood from the animal to obtain a serum containing the antibody. . Such polyclonal antibodies can be prepared from any animal such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, and the like. In addition, monoclonal antibodies can be prepared using hybridoma methods well known in the art, or phage antibody library techniques. The antibody prepared by the above method may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv.

In the present invention, "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized, DNA or RNA-like polymer, and was first introduced by Professors Nielsen, Egholm, Berg and Buchardt of the University of Copenhagen, Denmark in 1991. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases the binding strength and stability to DNA or RNA, resulting in molecular biology , diagnostic assays and antisense therapy.

In the present invention, the "aptamer" is an oligonucleic acid or a peptide molecule, and refers to a ligand-specific DNA or RNA molecule having a high affinity for a protein. Aptamer refers to single-stranded DNA or RNA having a specific binding ability to a specific substance, and has its own unique tertiary structure. It can be mass-produced in a short time and at low cost by using the chemical synthesis technique, and there is little variation between batches, so it has excellent advantages in terms of productivity. In addition, due to its high stability against changes in the surrounding environment, such as pH or temperature, the potential for application in various fields such as the detection of target substances and the development of disease diagnosis sensors is highly evaluated.

In the composition for diagnosis of cancer according to the present invention, the agent for measuring the expression level of a gene encoding at least one selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 is specific to the gene encoding the above. It may include one or more selected from the group consisting of primers, probes and antisense nucleotides that bind positively. Since the information of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 according to the present invention is known, those skilled in the art can easily design primers, probes or antisense nucleotides that specifically bind to the gene encoding the protein based on this. will be able

In the present invention, the "measurement of the expression level of the gene" refers to the presence or absence of a gene encoding at least one selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 to diagnose cancer. It means measuring the amount of a gene as a process. Analytical methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection) assay), Northern blotting, and a DNA chip, but is not limited thereto.

In the present invention, the "primer" is a fragment recognizing a target gene sequence, including a pair of forward and reverse primers, but preferably a primer pair that provides analysis results having specificity and sensitivity. When the nucleic acid sequence of the primer is a sequence that is inconsistent with a non-target sequence present in the sample, and thus amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification, high specificity can be conferred. .

In the present invention, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding. The type of probe is not limited as a material commonly used in the art, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably For example, it may be PNA. More specifically, the probe is a biomaterial derived from or similar thereto, or manufactured in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.

In the present invention, the "diagnosis" refers to determining the susceptibility of a subject to a specific disease or disorder, determining whether the subject currently has a specific disease or disorder, or having a specific disease or disorder Determining a subject's prognosis (e.g., identification of a pre-metastatic or metastatic cancer state, staging the cancer, or determining the responsiveness of a cancer to treatment), or therametrics (e.g., for treatment efficacy); monitoring the state of an object to provide information). For the purpose of the present invention, the diagnosis is to determine whether or not the disease occurs or the likelihood (risk) of the disease, the stage of the cancer, or the survival rate or treatment responsiveness of the cancer patient.

In the present invention, the "stage" refers to the extent to which cancer cells have spread and the stage of cancer progression, and the international classification according to the progress of cancer generally follows the TNM stage 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 it has metastasized to other organs. corresponds to

In order to achieve the above object, at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1, or an agent for measuring the expression level of a gene encoding the same, for diagnosis of cancer A composition is provided.

In one embodiment of the present invention, the agent for measuring the expression level of at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 is SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, And it includes at least one selected from the group consisting of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) and aptamer (aptamer) that specifically binds to at least one polypeptide selected from the group consisting of RNASE1. In another embodiment of the present invention, the agent for measuring the expression level of a gene encoding at least one selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 is SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6 and at least one selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to a gene encoding at least one selected from the group consisting of RNASE1. In another embodiment of the present invention, the cancer is breast cancer, ovarian cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-hospital cancer Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer , Small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma. In another embodiment of the present invention, the composition for diagnosing cancer is for diagnosing whether or not developing early stage cancer or predicting the possibility of onset. In another embodiment of the present invention, said early stage cancer is one that progresses in a nodule. In another embodiment of the present invention, the composition for diagnosis of cancer further comprises an agent for measuring the level of TFF3 or the expression level of a gene encoding the same.

In order to achieve the above object, there is provided a kit for diagnosis of cancer comprising the composition for diagnosis of cancer.

In order to achieve the above object, at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 in a biological sample isolated from a subject of interest, or measuring the expression level of a gene encoding the same It provides a method of providing information on the diagnosis of cancer or the stage of cancer, comprising the step of:

In one embodiment of the present invention, the biological sample is whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, 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, synovial fluid, joint aspirate, organ secretions, cells, cells extract (cell extract) or cerebrospinal fluid. In another embodiment of the present invention, at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 measured in a biological sample of the subject of interest, or expression of a gene encoding the same The method further includes comparing the level with a level measured in a normal control group to predict the likelihood of developing cancer or a stage of cancer. In another embodiment of the invention, the cancer is an early stage cancer. In another embodiment of the present invention, said early stage cancer is one that progresses in a nodule. In another embodiment of the present invention, the method for diagnosing cancer or providing information on the stage of cancer further comprises measuring the level of TFF3 or the expression level of a gene encoding the same. In another embodiment of the present invention, the cancer is breast cancer, ovarian cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-hospital cancer Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer , Small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma.

In order to achieve the above object, at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 in the isolated biological sample, or measuring the expression level of a gene encoding the same; contacting a test material with the biological sample; and measuring the expression level of the protein or the gene in the biological sample after contact with the test substance.

In order to achieve the above object, treating the isolated biological sample with a candidate substance expected to induce cancer; And at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 in a biological sample treated with the candidate substance, or measuring the expression level of a gene encoding the same, A method for screening a drug that induces cancer is provided.

According to another aspect of the present invention, the present invention comprises the step of measuring the expression level of at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1, or a gene encoding the same. A method for diagnosing cancer is provided.

According to a specific embodiment of the present invention, the method diagnoses the onset of early stage cancer or predicts the likelihood of developing it.

According to a specific embodiment of the present invention, the cancer in the early stage progresses in the nodule.

According to a specific embodiment of the present invention, the method for diagnosing cancer further comprises measuring the level of TFF3 or the expression level of a gene encoding the same.

In the present invention, by measuring the expression level of at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1, or a gene encoding the same for a liquid biopsy of a subject of interest, cancer is detected at an early stage It has the advantage of being able to diagnose and predict the stage of cancer.

1 shows a heatmap showing the top three marker genes representing each major cell cluster when clustering cells from scRNA-seq and non-malignant adjacent lung tissue and early lung adenocarcinoma.

2 shows a heatmap showing representative genes of each lung epithelial subclusters.

3 shows CAPS in alveoli (i), bronchial epithelium (ii-iii), lung cancer cells with lepidic elements (iv), lung cancer cells with solid elements, and placenta (vi) used as a positive control. It is a staining photograph showing expression.

4 is a horizontal bar graph of 15 differentially expressed genes among early lung cancer cells and remaining lung epithelial cell populations that progress in nodules.

Figure 5 shows bronchi (i), bronchi (ii), low magnification lung cancer (iii), lung cancer with acinar component (iv), lung cancer with lepidic component (v) and lung cancer with solid component (vi). It is a photograph observing the expression of CEACMA6 in

6 is a graph showing an unsupervised trajectory plot of lung cancer cells and epithelial cell subclusters divided by cell type.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental subject and sample preparation

Samples were collected for histological confirmation and treatment of persistent pure or partial solid ground glass opacity (GGO)-type lung nodules from patients who visited Yonsei University Hospital in Seoul from 2019 to 2020. The following detailed inclusion criteria were: (1) nodules in partial solid GGO or pure GGO status in a chest CT lung setting, (2) no metastatic lesions on PET and brain MRI, which are additional staging tests, and (3) cancer (4) non-smokers or non-smokers for more than 20 years, and (5) consenting to provide samples of residual tumor and adjacent normal lung tissue. Cases receiving adjuvant chemotherapy or neoadjuvant chemotherapy were excluded.

Paired samples of tumor tissue and adjacent normal lung tissue were obtained from 5 patients, lung tumor tissue was obtained from 1 patient, and a total of 11 samples were collected for the study. The specimens collected in the operating room were transferred to the pathology laboratory, and translucent nodules found throughout were examined with frozen sections to confirm lung cancer.

Gel Bead in Emulsion (GEM), library construction, sequencing, and analysis

GEMS and libraries were created by barcoding up to 10,000 cells from each sample using the Chromium Single Cell 3' Protocol. Briefly, single cells, reagents and single gel beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized GEMs using GemCode technology. Transcripts of each cell were individually indexed using GemCode Technology. After capturing polyadenylated mRNA using poly(dT) primers, barcoded full-length cDNA was generated through reverse transcription and paired-end sequencing was performed with an Illumina sequencing system. Data obtained in the laboratory were analyzed using Seurat R package version 3.2.2. Data from independent experiments were first integrated to minimize batch effects and then clustered into individual lung cell types via principal component analysis. Each cell cluster was isolated and analyzed independently for further subclustering.

Cancer cell identification

After obtaining the barcode of the epithelial cells in the lung tumor tissue, the barcode of the cell with disrupted chromosomal gene expression was obtained through infercnv to distinguish it from the non-malignant epithelial cells that may be mixed in the tumor tissue. The epithelial cells found in the tumor tissue were assumed to be lung cancer cells, and cells obtained from adjacent normal-looking lung tissue from the same individual and epithelial cells obtained from the tumor tissue were designated as references. Then the infercnv:run() function was run with the options analysis_mode = "subclusters", cluster_by_groups = F, k_obs_groups = 3 After removal of the underexpressed gene, the reading counts per cell were adjusted to the sum of the median total reading counts for the cells. Each value of the matrix was converted to log(x+1), and a log-fold change value obtained by subtracting the corresponding gene expression level from the average value of the corresponding gene obtained from normal cells was obtained. A value with an absolute value of log-fold-change of 3 or more was obtained, genes were arranged according to chromosomal order, and expression intensity was smoothed using a weighted running average. Under the assumption that most cells were free of CNV, the median expression intensity of each gene in individual cells was concentrated at zero, and the expression values of tumor cells were once again subtracted from the mean values of normal cells. These values were placed symmetrically around the mean to facilitate visual identification of amplification or deletion.

Immunohistochemical research

For validation of the gene of interest, immunohistochemical staining was performed using the antibodies and conditions indicated in Table 1. Formalin-fixed paraffin-embedded tissue blocks of GGO-type lung cancer were obtained from organ tissue archives and made into 4 μum sections. After deparaffinization and rehydration with xylene, endogenous peroxidase activity was quenched by treatment with serially diluted ethanol, PBS, and 0.3% hydrogen peroxide for 10 minutes each. Then, antigen retrieval was performed by immersion in preheated 0.1M citrate buffer (pH 6.0) and boiling three times for 5 minutes. After blocking with a blocking solution for 30 minutes, the primary antibody and the secondary antibody were sequentially treated. For visualization, slides were treated with Envision + System-HRP-labeled polymer anti-rabbit/mouse solution (K5007, Dako, Carpinteria, CA, USA) and DAB chromogen (Novocastra™). Finally, slides were counter-stained with Mayer's hematoxylin solution for 1 min, washed, dehydrated and mounted with coverslips.

Gene Name	Antibody Name	Vendor	Catalog #	Not #	Dilution
CAPS	calcyphosine mouse Ab	Novous Ab	NBP1-91746	000007612	1:1,000
CEACAM6	CEACAM6 mouse Ab	Origene	TA809254	W001	1:200

Single-cell sequencing of early lung adenocarcinoma

Single cell sequencing was performed using 11 samples from a total of 6 subjects. Filtering was performed using mitochondrial gene percentage (<20%), gene count (200 to 10,000) and UMI number (100 to 150,000) to remove noise from empty droplets, debris, doublets and triplets. After this filtering process, the number of cells obtained from normal lung tissue was 53,705 (55.0%) and the number of cells obtained from tumor tissue was 42,371 (44.1%). A total of 96,076 cells were initially divided into 7 major cell groups through dimensionality reduction and sorting, and each cluster was compared with a marker representing each cluster obtained from Seurat's FindConservedMarkers function with a standard marker of known lung cells. After obtaining the barcode of lung cancer cells in the obtained epithelial cell cluster using the infercnv R package, the remaining epithelial cell groups were classified into sub-clusters (see FIG. 1 ). For the purification of lung cancer cells, clusters with epithelial characteristics were obtained from each tumor tissue, and cells with distinct genetic abnormalities were defined as lung cancer cells by inferCNV using cells obtained from normal-looking lung tissues. Among the obtained cell clusters, T cells and related cell types accounted for the largest proportion as in other studies, followed by epithelial cells, including cancer cells and bone marrow cells.

Using CAPS as a ciliary bronchial epithelial cell marker

After removal of cancer cells from the total epithelial cells of the merged normal adjacent lung tissue and tumor tissue, the lung epithelial cells were further characterized after subclustering. The predominant cell type among epithelial cells is characterized by overexpression of CAPS, C20orf85 and C9orf24, followed by secretory club cells characterized by SCGB1A1 and SCGB3A1, small secretoglobin, and genes encoding proteins belonging to BPIFB1 to protect against bacterial exposure. involved in the innate immune response. The respiratory bronchi and alveoli, which are functional units of gas exchange, were mainly composed of terminally differentiated cell populations, type I alveolar cells, and type II alveolar cells. Type I alveolar cells, which form a large surface area air-blood barrier together with pulmonary capillary endothelial cells, showed overexpression of unique genes such as AGER, CAV1, and RTKN2. On the other hand, type II alveolar cells, which produce surfactant from water to reduce alveolar surface tension and thereby prevent lung collapse, were observed in a cell fraction similar to type I alveolar cells and showed overexpression of the surfactant protein family. Several cells with neuroendocrine function overexpressing intrinsic genes such as GRP, CALCA and CPE were observed (see Fig. 2) and could be presumed to be lung neuroendocrine cells.

CAPS is a calcium binding protein involved in cellular signaling and is known to be overexpressed in fluid transport cells lining the epithelium of the fluid-filled cavities of the central nervous system. In the lung, CAPS is known to be partially expressed in bronchial epithelial cells, but observations in lung tissue and lung cancer have been very limited. Therefore, to characterize these cell groups, immunohistochemical (IHC) staining for CAPS was performed with formalin-fixed, paraffin-embedded (FFPE) tissue from normal-looking lung tissue and early lung adenocarcinoma using fallopian tube tissue as a reference sample. CAPS expression was lacking in normal alveolar type I and II cells (refer to i in Fig. 3), whereas it was strongly expressed in ciliary cells of bronchi and bronchi (see ii and iii in Fig. 3). Lung nonmalignant cells with high CAPS expression were enriched with gene set-related cilia and microtubule formation and movement, confirming that CAPS plays an important role in the morphology and function of lung ciliary cells. It was expressed variously in lung cancer specimens (refer to vi and v in FIG. 3). 8 cases of 31 lung cancer tissues showed CAPS positivity of 5% or more, 4 cases showed moderate to strong expression in 50% or more cancer cells, and 4 cases showed 10-30% expression with weak or moderate intensity. . There was no significant correlation between the composition of lung cancer tissue and CAPS expression. Since we only targeted early lung cancer, different CAPS expression in formalin-fixed, paraffin-embedded tissues could hypothesize that CAPS levels are decreased in various developmental states of lung cancer or that the cancer cell origin may be different for each lung cancer.

Lung cancer cells located closer to terminally differentiated lung epithelial cell clusters

Lung cancer cells were located closer to the terminally differentiated lung epithelial cell clusters. The lung cancer cells in the sample originate at the bronchi-alveolar conduit junction. In UMAP, it was observed that lung cancer cells were clustered in the centers of ciliary cells, club cells, alveolar type I and alveolar type II. The top 15 highly differentially expressed genes between lung cancer cells and the rest of the lung epithelial cells are shown in Figure 4 and Table 2. These differentially expressed genes were actively expressed in secretory vesicles and surfactant pathways, confirming that the origin of early lung carcinogenesis was closely related to type II alveolar cells. It was confirmed that lung cancer cells had different markers according to the order of type II alveolar cells and type I and ciliary bronchial cells. They confirmed that they overexpress CEACAM6 and CEACAM5, which are surface glycoproteins that play a role in intercellular adhesion in a calcium- and fibronectin-independent manner. It was confirmed that CEACAM6 was not stained at all in the components of normal lung tissue, and it was strongly expressed in lung cancer cells regardless of histological subtype and could be involved in early tumor formation (see FIG. 5 ).

Gene name	avg_logFC	pct.1	pct.2
SPINK1	2.119588	0.47	0.056
NAPSA	1.891179	0.951	0.199
HOPX	1.580921	0.928	0.371
LPCAT1	1.528898	0.844	0.186
AREG	1.478092	0.844	0.451
LAPTM4B	1.430558	0.805	0.214
NEAT1	1.384919	0.949	0.884
SFTPB	1.372976	0.976	0.398
CEACAM6	1.36579	0.852	0.414
CTSH	1.34259	0.941	0.745
DRAM1	1.314869	0.819	0.18
TRAM1	1.303142	0.871	0.604
LY6E	1.300678	0.912	0.716
RNASE1	1.293394	0.905	0.394
CEACAM5	1.287858	0.536	0.033
PIFO	-1.6252	0.245	0.611
RSPH1	-1.69681	0.249	0.634
SNTN	-1.69975	0.058	0.579
C2orf40	-1.77478	0.031	0.54
SLPI	-1.77846	0.782	0.913
TFF3	-1.84996	0.265	0.689
C9orf24	-1.89412	0.239	0.654
C11orf88	-1.95616	0.079	0.613
C1orf194	-1.97282	0.131	0.622
GSTA1	-2.06876	0.248	0.739
C20orf85	-2.27844	0.088	0.64
CAPS	-2.56457	0.49	0.81
BPIFB1	-2.5811	0.088	0.41
SCGB1A1	-3.45752	0.844	0.947

As shown in Table 2, it was observed that SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 expression increased in GGO-type lung cancer cells. Through this, it was possible to diagnose this by observing increased expression of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 or decreased expression of TFF3 in early lung cancer nodules that could only be diagnosed radiologically among early lung cancers.

As a result of performing a trajectory analysis on the lung epithelial cell group in which the ciliated cells were set as the root state, differentiation into type I and type II alveolar cells was observed in ciliated cells according to a similar time. Indeed, when lung cancer cells were projected onto the trajectory of a group of normal-looking lung epithelial cells, they followed either type I alveolar cells or type II alveolar cells (see Fig. 6). Similar to the previous findings, club cells were observed in all processes according to the similar time of differentiation between bronchial cells and alveolar cells, whereas neuroendocrine cells were observed in the middle of the ciliary to alveolar cell differentiation time point. When representative genes of major lung epithelial cell clusters were aligned over time, synchrony between genes related to surfactant homeostasis and cancer-specific genes such as CEACAM6 was observed, confirming that surfactant-associated pathways are strongly involved in early lung cancer development. .

Correlation between lung cancer stage and genetic markers

As a result of performing trajectory analysis of ciliated cells on the lung epithelial cell population, the differentiation of ciliated cells into type I and type II alveolar cells was observed. When representative genes of major lung epithelial cell clusters were sorted over time, synchrony was confirmed between genes associated with surfactant homeostasis and cancer-specific genes such as CEACAM6. This confirmed that the surfactant-related pathway is strongly involved in the development of early lung cancer.

A total of 820 B cells composed of two subtypes of B cells, follicular and MALT B cells were observed in this study using GGO-type lung cancer and normal-looking adjacent peripheral lung tissue. Of these B cells, 89.1% were found in tumor tissue, which was the most prominent cell rich in tumor tissue among immune cells constituting lung tissue. Follicular B cells showed a relatively high proportion compared to the MALT B cell subtype.

We present (i) independently clustered cells obtained from individual tumor samples and adjacent normal lung tissue, (ii) individually extracted epithelial cell barcodes obtained from tumor tissue, (iii) cell groups obtained from normal lung tissue and non- Lung cancer cells were defined as epithelial clusters obtained from tumor tissue as reference, (iv) unsupervised clustering method applied in the inferecnv package, and (v) cells with small changes in clear chromosomal gene expression. This was meaningful in improving the accuracy of tumor biomarkers, trajectories, and differentially expressed gene results obtained by accurately designating cancer cells from other non-malignant epithelial components within the tumor tissue. Interestingly, cancer cells from these early nonsmokers not only showed heterogeneity between samples, but also clearly showed heterogeneity of CNVs within individual samples. It was possible to obtain important results by linking the proportions of lepidic, acinar, papillary, micropapillary and solid parts with histological components.

The present inventors were able to provide a new diagnostic method for very early lung cancer development and tumor proliferation by confirming not only intertumoral heterogeneity but also intratumoral heterogeneity in early GGO-type lung cancer.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (17)

1. At least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1, or an agent for measuring the expression level of a gene encoding the same, a composition for diagnosis of cancer.
2. According to claim 1,

   The agent for measuring the expression level of at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 is SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 selected from the group consisting of A composition comprising at least one selected from the group consisting of an antibody that specifically binds to at least one polypeptide, an oligopeptide, a ligand, a peptide nucleic acid (PNA), and an aptamer.
3. According to claim 1,

   The agent for measuring the expression level of a gene encoding at least one selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 is selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 A composition comprising at least one selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to a gene encoding at least one of the
4. According to claim 1,

   The cancer is breast cancer, ovarian cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer , bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervoussystem tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma.
5. According to claim 1,

   The composition for diagnosis of cancer is for diagnosing whether or not the onset of early stage cancer or predicting the possibility of onset.
6. 6. The method of claim 5,

   The composition of claim 1, wherein the early stage cancer progresses in the nodule.
7. The method of claim 1,

   The composition for diagnosis of cancer further comprises an agent for measuring the level of TFF3 or the expression level of a gene encoding the same.
8. A kit for diagnosing cancer comprising the composition for diagnosing cancer according to any one of claims 1 to 7.
9. In a biological sample isolated from a subject of interest, at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1, or a gene encoding the same, cancer comprising the step of measuring the expression level a method of providing information regarding the diagnosis or staging of cancer.
10. 10. The method of claim 9,

    The biological sample includes whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears ( 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, synovial fluid, joint aspirate, organ secretions, cells, cell extract or cerebrospinal fluid ( cerebrospinal fluid), a method of providing information.
11. 10. The method of claim 9,

    At least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 measured with respect to the biological sample of the subject of interest, or the expression level of a gene encoding the same, measured in a normal control group The method of providing information, further comprising the step of predicting the likelihood of developing cancer or determining the stage of the cancer compared to the .
12. 10. The method of claim 9,

    wherein the cancer is an early stage cancer.
13. 13. The method of claim 12,

    The method of providing information, wherein the early stage cancer progresses in the nodule.
14. 10. The method of claim 9,

    The method of providing information about the diagnosis of cancer or the stage of cancer further comprises measuring the level of TFF3 or the expression level of a gene encoding the same.
15. 10. The method of claim 9,

    The cancer is breast cancer, ovarian cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer , bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervoussystem tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma.
16. Measuring the expression level of at least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1, or a gene encoding the same in the isolated biological sample;

    contacting a test substance with the biological sample; and

    A method for screening a drug for treating cancer, comprising measuring the expression level of the protein or the gene in the biological sample after contacting the test substance.
17. treating the isolated biological sample with a candidate substance predicted to induce cancer; and

    At least one polypeptide selected from the group consisting of SPINK1, LPCAT1, NEAT1, CEACAM5, CEACAM6, and RNASE1 in the biological sample treated with the candidate substance, or the expression level of a gene encoding the same. Cancer, comprising: How to screen for drugs that induce

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