Diagnostic agent, pancreatic cancer diagnosis kit, detection method, marker, Anti-pancreatic cancer agent, and composition for treating pancreatic cancer

Abstract

A diagnostic agent for pancreatic cancer, the diagnostic agent comprising: a primer set for amplifying SLC12A7 DNA or mRNA in a sample derived from a subject; and / or a probe that binds to the SLC12A7 DNA, mRNA or an amplification product thereof, or an antibody that binds to SLC12A7 protein. An anti-pancreatic cancer agent comprising an SLC12A7 inhibitor as an active ingredient.

Description

Diagnostic agent, pancreatic cancer diagnostic kit, detection method, marker, anti-pancreatic cancer agent, and composition for treating pancreatic cancer

[0001] The present invention relates to a diagnostic agent, a pancreatic cancer diagnostic kit, a detection method, a marker, an anti-pancreatic cancer agent, and a composition for treating pancreatic cancer.

[0002] Pancreatic cancer is an intractable cancer with a poor prognosis, with approximately 60% of cases discovered at an advanced stage. The anaplastic nature of pancreatic cancer and its hypovascular nature make it difficult to identify using diagnostic imaging. Pancreatic enzymes (e.g., AMY, lipase) and tumor markers (e.g., CEA, CA19-9, Span-1, Dupan-2) have been proposed as screening targets (e.g., Non-Patent Documents 1 and 2).

[0003] K Satake, YS Chung, H Yokomatsu, B Nakata, H Tanaka, T Sawada, H Nishiwaki, K Umeyama. A clinical evaluation of various tumor markers for the diagnosis of pancreatic cancer. Int J Pancreatol. 1990 Aug-Nov;7(1-3):25-36.O Nazli, AD Bozdag, T Tansug, R Kir, E Kaymak. The diagnostic importance of CEA and CA 19-9 for the early diagnosis of pancreatic carcinoma. Hepatogastroenterology. 2000 Nov-Dec;47(36):1750-2.

[0004] Currently available tumor markers are useful for diagnosing advanced cancer and monitoring disease progression, but lack sensitivity for diagnosing early-stage cancer, and the development of biomarkers for early diagnosis is an urgent issue for improving prognosis. Therefore, an object of the present invention is to provide a diagnostic agent, a pancreatic cancer diagnostic kit, a detection method, a marker, an anti-pancreatic cancer agent, and a composition for treating pancreatic cancer, which are capable of detecting pancreatic cancer early.

[0005] The present invention includes the following aspects: [1] A diagnostic agent for pancreatic cancer, comprising a primer set for amplifying SLC12A7 DNA or mRNA in a sample derived from a subject, and / or a probe that binds to the SLC12A7 DNA, mRNA, or an amplification product thereof, or an antibody that binds to SLC12A7 protein. [2] The diagnostic agent according to [1], wherein the sample is extracellular vesicles derived from blood. [3] A diagnostic kit for pancreatic cancer, comprising the diagnostic agent according to [1] or [2]. [4] A method for detecting a diagnostic marker for pancreatic cancer, comprising detecting the amount of SLC12A7 DNA, mRNA, or SLC12A7 protein present in a sample derived from a subject. [5] The detection method according to [4], wherein the sample is extracellular vesicles derived from blood. [6] A diagnostic marker for pancreatic cancer, comprising SLC12A7 DNA, mRNA, or SLC12A7 protein isolated from a sample derived from a subject. [7] The marker according to [6], wherein the sample is extracellular vesicles derived from blood. [8] An anti-pancreatic cancer agent comprising an SLC12A7 inhibitor as an active ingredient. [9] A composition for treating pancreatic cancer comprising the anti-pancreatic cancer agent according to [8] and a pharmaceutically acceptable carrier.

[0006] The present invention contributes to the diagnosis and treatment of early-stage pancreatic cancer.

[0007] This figure shows an overview of the comprehensive analysis method for exosomal proteins in the serum of pancreatic cancer patients. This figure shows an overview of the method for narrowing down the candidate exosomal proteins obtained. This figure shows a table listing the 11 extracted candidate exosomal proteins. For the candidate proteins, the results of a two-group comparison (Student's t-test) between pancreatic cancer (Stage I, II, III, IV) and non-cancer (benign pancreatic disease + healthy subjects) are shown. Multiple comparisons (anova, Bonferroni correction) between pancreatic cancer and benign pancreatic disease and pancreatic cancer and healthy subjects are also shown, along with published TCGA data. For the candidate proteins and CA19-9, the results of a two-group comparison (Student's t-test) between pancreatic cancer (Stage I, II, III, IV) and non-cancer (benign pancreatic disease + healthy subjects) are also shown. Multiple comparisons (anova, Bonferroni correction) between pancreatic cancer and benign pancreatic disease and pancreatic cancer and healthy subjects are also shown, along with published TCGA data. This figure shows ROC curves for the 11 candidate proteins and CA19-9. (A) Results showing the levels of SLC12A7 RNA expression, intracellular protein expression, and extracellular vesicle protein expression in pancreatic cancer cell lines (Panc-1, Aspc-1, Bxpc-3, Capan-1) and normal pancreatic duct epithelial cells (HPNE). (B) Results showing the levels of SLC12A7 mRNA expression in pancreatic cancer cell lines (Panc-1, Bxpc-3) and normal pancreatic duct epithelial cells (HPNE) under an acidic environment. In the legend, "N" indicates a neutral environment, "6.8" indicates a pH 6.8 environment, and "6.6" indicates a pH 6.6 environment. (A) Results showing the levels of SLC12A7 protein expression in pancreatic cancer cell lines (Panc-1, Bxpc-3) and normal pancreatic duct epithelial cells (HPNE) under an acidic environment. (B) Results of quantification of (A). In the legend, "N" indicates a neutral environment, "6.8" indicates a pH 6.8 environment, and "6.6" indicates a pH 6.6 environment. The results show the protein expression levels of SLC12A7 in an acidic environment in exosomes derived from pancreatic cancer cell lines (Panc-1, Bxpc-3) and normal pancreatic duct epithelial cells (HPNE). (A) and (B) show the quantification of protein expression levels following SLC12A7 knockdown.In the graph, "siSLC12A7 #1" indicates "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21063)," "siSLC12A7 #2" indicates "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21062)," and "siNC" indicates the negative control (Silencer® Select Negative Control siRNA #1: catalog number 4390843). (B) Western blotting results for each cell line in which SLC12A7 was knocked down. CCK-8 assay results using pancreatic cancer cell lines (Panc-1, Bxpc-3) and normal pancreatic ductal epithelial cells (HPNE). In the legend, "normal" indicates a neutral environment, "6.8" indicates a pH 6.8 environment, and "6.6" indicates a pH 6.6 environment. "siSLC12A7 #1" refers to "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21063)," "siSLC12A7 #2" refers to "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21062)," and "siSLC12A7 #NC" refers to the negative control (Silencer® Select Negative Control siRNA #1: catalog number 4390843). These are the results of a scratch assay using pancreatic cancer cell lines (Panc-1, Bxpc-3). "siSLC12A7" refers to "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21063)," and "siNC" refers to the negative control (Silencer® Select Negative Control siRNA #1: catalog number 4390843). This shows the results of a scratch assay using normal pancreatic ductal epithelial cells (HPNE)."siSLC12A7" refers to "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21063)," and "siNC" refers to the negative control (Silencer® Select Negative Control siRNA #1: catalog number 4390843). Figures 13 and 14 show quantitative results of the scratch assay. In the legend, "normal" indicates a neutral environment, "6.8" indicates a pH 6.8 environment, and "6.6" indicates a pH 6.6 environment. "siSLC12A7" refers to "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21063)," and "siNC" refers to the negative control (Silencer® Select Negative Control siRNA #1: catalog number 4390843). (A) Results of a Transwell assay using a pancreatic cancer cell line (Panc-1). (B) Quantification results of (A). In the graph, "normal" indicates a neutral environment, "pH 6.8" indicates a pH 6.8 environment, and "pH 6.6" indicates a pH 6.6 environment. "siSLC12A7" indicates "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21063)," and "siNC" indicates the negative control (Silencer® Select Negative Control siRNA #1: catalog number 4390843). (A) Transwell assay results using a pancreatic cancer cell line (Bxpc-3). (B) Quantification results of (A). In the graph, "normal" indicates a neutral environment, "pH 6.8" indicates a pH 6.8 environment, and "pH 6.6" indicates a pH 6.6 environment."siSLC12A7" indicates "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21063)," and "siNC" indicates the negative control (Silencer® Select Negative Control siRNA #1: catalog number 4390843). (A) Results of a Transwell assay using normal pancreatic duct epithelial cells (HPNE). (B) Results of quantification of (A). In the graph, "normal" indicates a neutral environment, "pH 6.8" indicates a pH 6.8 environment, and "pH 6.6" indicates a pH 6.6 environment. "siSLC12A7" indicates "Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog number s21063)," and "siNC" indicates the negative control (Silencer® Select Negative Control siRNA #1: catalog number 4390843). Pancreatic cancer cell lines (Panc-1, Bxpc-3) were cultured for 24 hours under conditions of pH 7.4 or an acidic environment, and the protein expression levels of ER stress-related molecules were evaluated by Western blotting.

[0008] <Pancreatic Cancer Diagnostic Marker> This embodiment provides a pancreatic cancer diagnostic marker, which comprises SLC12A7 DNA, mRNA, or SLC12A7 protein isolated from a sample derived from a subject. As described below in the Examples, the present inventors have discovered that SLC12A7 protein is an exosomal protein that is more highly expressed in pancreatic cancer patients than in patients with benign pancreatic diseases and healthy individuals.

[0009] The SLC family (Solute carrier family) is a large family of proteins specialized to cross cell membranes. 378 SLC transporter genes and 51 families have been reported. SLC12A (Solute carrier family 12) transports Na + , K. + , Cl- Solute carrier family 12 member 7 (SLC12A7) is a family of transporters specialized in the transport of K + -Cl - It belongs to the symporter family.

[0010] Examples of the amino acid sequence of SLC12A7 include the sequence registered under Refseq accession number NP_006589 for human SLC12A7, and the gene sequence of SLC12A7 include the sequence registered under Refseq accession number NC_000005 for human SLC12A7.

[0011] In this embodiment, examples of samples include blood, urine, saliva, sweat, tissue exudate, etc., with blood being preferred. Among blood samples, examples include serum and plasma, with serum being preferred, and extracellular vesicles in serum being preferred. In this embodiment, extracellular vesicles (hereinafter also referred to as EVs) refer to vesicles secreted from cells. EVs also include vesicles such as exosomes, microvesicles, and apoptotic bodies. The surface of EVs contains lipids and proteins derived from the cell membrane, and the interior contains intracellular substances such as nucleic acids and proteins.

[0012] Molecules to be detected by diagnostic markers include nucleic acids such as DNA and RNA, proteins, etc. Examples of DNA include genomic DNA and cDNA, and examples of RNA include mRNA and miRNA.

[0013] In this embodiment, it is preferable to use human SLC12A7 mRNA or human SLC12A7 protein in extracellular vesicles purified from serum derived from a subject as a diagnostic marker for pancreatic cancer.

[0014] <<Pancreatic Cancer Diagnostic Agent>> This embodiment provides a pancreatic cancer diagnostic agent comprising a primer set for amplifying SLC12A7 DNA or mRNA in a sample derived from a subject, and / or a probe that binds to the SLC12A7 DNA, mRNA, or an amplification product thereof, or an antibody that binds to SLC12A7 protein.

[0015] The diagnostic agent of this embodiment preferably comprises a primer set for amplifying SLC12A7 mRNA in extracellular vesicles purified from serum derived from a subject, and / or a probe that binds to the mRNA or its amplification product, or an antibody that binds to SLC12A7 protein.

[0016] <Pancreatic Cancer Diagnostic Kit> This embodiment provides a pancreatic cancer diagnostic kit comprising the diagnostic agent of the present invention. The kit of this embodiment preferably comprises a kit for extracting SLC12A7 mRNA (e.g., total RNA) from body fluids, cells, tissues, etc., a fluorescent labeling substance, a nucleic acid amplification reagent, etc. The kit may also comprise an antibody that binds to SLC12A7 protein. Furthermore, in addition to the antibody, the kit may also comprise an ELISA kit for detecting SLC12A7 protein. Pancreatic cancer can be detected early by assessing the expression level of SLC12A7 molecules in a sample derived from a subject.

[0017] <Pancreatic Cancer Diagnostic Device> This embodiment provides a pancreatic cancer diagnostic device comprising a solid phase and a nucleic acid capable of hybridizing with the SLC12A7 gene bound to the solid phase, or a substance having affinity for the SLC12A7 protein.

[0018] The device of this embodiment has the nucleic acid or affinity substance bound to a solid phase. Examples of the solid phase include a glass substrate, a silicon substrate, a plastic substrate, and a metal substrate. Examples of the nucleic acid or affinity substance bound to a solid phase include a nucleic acid array such as a DNA array or an RNA array, or an antibody array. Furthermore, examples of substances having affinity for the SLC12A7 protein include antibodies that bind to the SLC12A7 protein.

[0019] <<Method for Detecting a Diagnostic Marker for Pancreatic Cancer>> This embodiment provides a method for detecting a diagnostic marker for pancreatic cancer, which involves detecting the amount of SLC12A7 DNA, mRNA, or SLC12A7 protein present in a sample derived from a subject. Specifically, the method includes measuring the expression level of SLC12A7 DNA, mRNA, or SLC12A7 protein in a sample derived from a subject in vitro and assessing the subject's incidence of early-stage cancer using the expression level of the DNA, mRNA, or protein. For example, the expression level of SLC12A7 mRNA or SLC12A7 protein in a sample derived from a subject is compared with the control expression level in a subject known to have pancreatic cancer. If the expression level in the sample derived from the subject is no different from the control expression level in the subject with pancreatic cancer, the subject can be diagnosed as having pancreatic cancer. For example, the expression level of SLC12A7 mRNA or SLC12A7 protein in a sample derived from a subject is compared with the control expression level in a healthy control, and if the expression level in the sample derived from the subject is higher than the control expression level in a control suffering from pancreatic cancer, the subject can be diagnosed as suffering from pancreatic cancer.

[0020] In this embodiment, examples of the sample include blood, urine, saliva, sweat, tissue exudate, etc., with blood being preferred. Among blood samples, examples include serum and plasma, with serum being preferred, and extracellular vesicles in serum being preferred.

[0021] Methods for detecting SLC12A7 DNA or mRNA in a sample derived from a subject may include amplifying SLC12A7 DNA or mRNA fragments by PCR using primers and analyzing the amplified products, or analyzing them by a hybridization method using a probe complementary to SLC12A7 DNA or mRNA. From the viewpoint of quantitative analysis, it is preferable to amplify SLC12A7 DNA or mRNA fragments by PCR and analyze the amplified products. Specific quantitative methods include next-generation sequencing (NGS) and real-time PCR (RT-PCR).

[0022] In next-generation sequencers, analyzed DNA fragments are called reads, and the output data is the product of the number of reads and the number of bases determined per read (read length). In this embodiment, it is preferable to amplify the sample SLC12A7 DNA or mRNA using singleplex PCR or multiplex PCR, analyze the base sequence of the amplified product using a next-generation sequencer (NGS), and quantify the amount by counting the number of reads in the relevant region. Multiplex PCR is a method for simultaneously amplifying multiple gene regions by simultaneously using multiple primer pairs in a single PCR reaction system. At least some of the combinations of primers that anneal to specific mRNAs described above may be used in a single PCR reaction system.

[0023] In real-time PCR, the amount of amplified product can be monitored by detecting fluorescence intensity using an intercalator method, a probe method, a cycling probe method, or the like. In this embodiment, SLC12A7 DNA or mRNA is preferably quantified by singleplex or multiplex real-time PCR. In multiplex PCR, at least some of the combinations of various primers that anneal to specific mRNAs described above may be used in a single PCR reaction system.

[0024] In real-time PCR, it is preferable to use a fluorescent dye-labeled probe complementary to SLC12A7 DNA or mRNA. Quantitation methods include absolute quantitation, which determines the actual copy number of the target, and comparative quantitation, which determines the relative value between samples. These methods are used depending on the quality of data to be obtained.

[0025] <Anti-pancreatic cancer agent> This embodiment provides an anti-pancreatic cancer agent containing an SLC12A7 inhibitor as an active ingredient. The SLC12A7 inhibitor is not limited as long as it inhibits the function of SLC12A7, and examples thereof include nucleic acids, proteins, and low-molecular-weight compounds. Examples of substances that directly act on SLC12A7 include low-molecular-weight compounds, proteins, antibodies, and aptamers. Examples of substances that suppress the expression of the gene encoding SLC12A7 include siRNA and antisense oligonucleotides.

[0026] As the anti-pancreatic cancer agent of this embodiment, the SLC12A7 inhibitor may be used in the form of a free form or a pharmaceutically acceptable salt, or may be used in the form of a solvate of the free form or a solvate of the salt.

[0027] The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include hydrochloride, sulfate, hydrobromide, hydroiodide, phosphate, nitrate, benzoate, methanesulfonate, 2-hydroxyethanesulfonate, p-toluenesulfonate, acetate, propanoate, oxalate, malonate, succinate, glutarate, adipate, tartrate, maleate, fumarate, malate, mandelate, etc. The solvate is not particularly limited as long as it is a pharmaceutically acceptable solvate, and examples thereof include hydrates, organic solvents, etc.

[0028] Pancreatic cancer is an hypovascular tumor, and its malignant progression is triggered by tissue acidification in a hypoxic environment. As will be described later in the Examples, since SLC12A7 inhibitors exert their anticancer effects in an acidic environment, the antipancreatic cancer agent of this embodiment is preferably applied to patients whose pancreatic cancer tissues are in an acidic environment.

[0029] <Composition for Treating Pancreatic Cancer> In one embodiment, the present invention provides a composition for treating pancreatic cancer, comprising the anti-pancreatic cancer agent and a pharmaceutically acceptable carrier.

[0030] The composition for treating pancreatic cancer of this embodiment can be administered orally in the form of, for example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., or parenterally in the form of inhalants, injections, suppositories, topical skin preparations, etc.

[0031] As the pharmaceutically acceptable carrier, those usually used in the preparation of pharmaceutical compositions can be used without any particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, etc.; excipients such as starch, crystalline cellulose, etc.; swelling agents such as alginic acid, etc.; solvents for injections such as water, ethanol, glycerin, etc.; adhesives such as rubber-based adhesives, silicone-based adhesives, etc. The pharmaceutically acceptable carriers can be used alone or in combination of two or more.

[0032] The composition for treating pancreatic cancer of this embodiment may further contain additives. Examples of additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavorings such as peppermint and rhododendron oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives, etc. The additives can be used alone or in combination of two or more.

[0033] (Administration Method) The administration method of the anti-pancreatic cancer agent or the composition for treating pancreatic cancer is not particularly limited and may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc. For example, tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc. are administered orally. Injections are administered intravenously alone or mixed with common replacement fluids such as glucose or amino acids, and may also be administered intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally as needed. Suppositories are administered rectally. Topical skin preparations are applied, pasted, or sprayed onto the affected area. Inhalants are administered using devices such as nebulizers, metered-dose inhalers, and dry powder inhalers.

[0034] (Dosage) The dosage of an anti-pancreatic cancer agent or a composition for treating pancreatic cancer varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined in general. However, in the case of oral administration, for example, 1 μg to 10 g of active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. In the case of an injection, for example, 0.1 μg to 1 g of active ingredient may be administered per day, for example, 0.001 to 200 mg per day. In the case of a suppository, for example, 1 μg to 10 g of active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. In the case of a topical skin preparation, for example, 1 μg to 10 g of active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. In the case of an inhalant, for example, 1 μg to 10 g of active ingredient may be administered per single inhalation, for example, 0.01 to 2000 mg per single inhalation.

[0035] In one embodiment, the present invention provides an SLC12A7 inhibitor, a pharmaceutically acceptable salt thereof, or a solvate thereof for the treatment of pancreatic cancer.

[0036] In one embodiment, the present invention provides a method for treating pancreatic carcinoma, comprising administering to a patient in need thereof an effective amount of an SLC12A7 inhibitor, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0037] In one embodiment, the present invention provides use of an SLC12A7 inhibitor, a pharmaceutically acceptable salt thereof, or a solvate thereof for producing an anti-pancreatic cancer agent or a composition for treating pancreatic cancer.

[0038] Other Embodiments [Pancreatic Cancer Diagnostic Agent] In one embodiment, the present invention provides a pancreatic cancer diagnostic agent comprising a primer set for amplifying at least one molecule selected from the group consisting of RNMT, PLOD3, SF3B1, PTAFR, PLOD1, SSRP1, KCNH5, STRBP, NEU1, PSAT1, and SLC12A7 in a sample derived from a subject, and / or a probe that binds to the molecule or its amplification product, or an antibody that binds to the molecule.

[0039] Molecules to be detected by diagnostic agents include nucleic acids such as DNA and RNA, and proteins. Examples of DNA include genomic DNA and cDNA, and examples of RNA include mRNA and miRNA. When the molecules to be detected are nucleic acids, the diagnostic agent preferably contains a primer set for amplifying these molecules and / or a probe that binds to the molecules or their amplification products. When the molecules to be detected are proteins, the diagnostic agent preferably contains an antibody that binds to these molecules.

[0040] In this embodiment, the sample may include cells derived from tissue fragments, blood, urine, saliva, sweat, tissue exudate, etc., with blood being preferred. Among blood samples, serum and plasma may be mentioned, with serum being preferred, and extracellular vesicles in serum being preferred.

[0041] [Method for detecting a diagnostic marker for pancreatic cancer] In one embodiment, the present invention provides a method for detecting a diagnostic marker for pancreatic cancer, which comprises detecting the abundance of at least one molecule selected from the group consisting of RNMT, PLOD3, SF3B1, PTAFR, PLOD1, SSRP1, KCNH5, STRBP, NEU1, PSAT1, and SLC12A7 from a sample derived from a subject.

[0042] This embodiment enables early diagnosis of pancreatic cancer in a minimally invasive manner.

[0043] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0044] [Sample collection] Blood samples were collected from 40 pancreatic cancer patients (Stage I to IV), 10 healthy individuals, and 10 patients with benign pancreatic diseases. The samples were centrifuged at 1650G for 5 minutes to separate the serum, which was then dispensed into tubes and stored at -80°C.

[0045] [Outline of comprehensive analysis of exosomal proteins in serum from pancreatic cancer patients] The collected serum was analyzed as follows (1) to (3). The method is outlined below (see Figure 1). Exosomes were isolated by immunoprecipitation, and proteins encapsulated in exosomes were extracted using S-Trap micro spin columns. Proteins were then analyzed by LC / MS using a Q Exactive and UltiMate 3000 Nano LC system for proteomic analysis.

[0046] (1) Exosome isolation: immunoprecipitation. Using the exosome markers CD9 and CD63 as targets, 500 μl of patient serum was added to beads coated with anti-CD9 and anti-CD63 antibodies, and the beads were incubated on a rotator to isolate exosomes.

[0047] (2) Extraction of exosomal proteins and LC / MS pretreatment. The exosomes were solubilized in TEAB buffer (supplemented with 5% SDS), and exosomal proteins were extracted. The amount of exosomal protein was measured using a Micro BCA Protein Assay Kit, and the digestion efficiency of the solubilized sample was evaluated using 13.8 ng of Pierce Digestion Indicator for Mass Spectrometer. The exosomal proteins were then reduced, alkalized, and ionized. S-Trap protein binding buffer was added to the ionized solution. The sample was then loaded onto S-Trap micro spin columns, washed with 150 μL of S-Trap buffer, and incubated in digestion buffer at 47°C for 2 hours. Unwanted peptides were eluted, then lyophilized, and stored at -80°C.

[0048] (3) Comprehensive Analysis by LC / MS: Peptides were dissolved in 10 μL of 0.1% formic acid solution to confirm their quality. Proteomics analysis was then performed using a Q Exactive equipped with an UltiMate 3000 Nano LC System 1. Specifically, 1 μg of sample was injected onto a column, which was then connected to another column. 0.1% acetonitrile (A) was used as the mobile phase, and 0.1% formic acid (B) was used as the buffer. Data-dependent acquisition (DDA) was performed in positive ion mode.

[0049] [Analysis and refinement of candidate exosome proteins obtained] To refine the 5,197 candidate exosome proteins obtained, we analyzed them in the following order (see Figure 2). st Step 2: A three-group comparison (ANOVA, Bonferroni correction) was performed between pancreatic cancer (Stage I, II, III, IV), benign pancreatic disease, and healthy individuals. This resulted in 24 types of findings. nd step: 1 st For candidate exosomal proteins that showed significant differences in step 1, a two-group comparison (Student's t-test) was performed between pancreatic cancer (Stage I, II, III, IV) and non-cancer (benign pancreatic disease + healthy subjects), narrowing the list to 21 types. Furthermore, multiple comparisons (Anova, Bonferroni correction) were performed between pancreatic cancer and benign pancreatic disease, and pancreatic cancer and healthy subjects for the candidate exosomal proteins that showed significant differences in at least one of the groups, resulting in 17 candidates that showed significant differences. 3 rd Step 4: We performed a logistic regression analysis adjusted for age and sex to examine the influence of these factors on the amount of exosomal protein, and narrowed it down to 14 types. th Step 3: The true positive rate (sensitivity) and false positive rate (1 - specificity) of each candidate exosomal protein were graphed using an ROC curve. Based on the 70% sensitivity of CA19-9, the gold standard for diagnosing pancreatic cancer, for early-stage pancreatic cancer, a true positive rate of 70% or higher was set as the cutoff on the ROC curve. Candidates with a false positive rate of 30% or lower were selected, resulting in a narrowing down to 11 types (see Figure 3).

[0050] The results of a two-group comparison (Student's t-test) between pancreatic cancer (Stage I, II, III, IV) and non-cancer (benign pancreatic disease + healthy subjects) for the 11 candidate proteins and CA19-9, the results of multiple comparisons (Anova, Bonferroni correction) between pancreatic cancer and benign pancreatic disease, and pancreatic cancer and healthy subjects, and published TCGA data are shown in Figures 4 and 5. ROC curves for the 11 candidate proteins and CA19-9 are also shown in Figure 6.

[0051] Among 11 candidate proteins, we focused on SLC12A7. We examined the RNA expression level, intracellular protein expression level, and protein expression level in EVs of SLC12A7 in pancreatic cancer cell lines (Panc-1, Aspc-1, Bxpc-3, Capan-1) and normal pancreatic duct epithelial cells (HPNE) (see Figure 7). We confirmed that SLC12A7 RNA expression level, intracellular protein expression level, and protein expression level in EVs tended to be higher in pancreatic cancer cell lines compared to HPNE.

[0052] [Cell lines and culture conditions] The following experiments were performed using human pancreatic cancer cell lines Panc-1 and BxPC-3, and a normal pancreatic duct epithelial cell line, hTERT-HPNE. Panc-1 and hTERT-HPNE cells were cultured in high-glucose DMEM medium (Dulbecco's Modified Eagle Medium, Gibco), and BxPC-3 cells in RPMI-1640 medium (Gibco), both supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were cultured under normal conditions (pH 7.4) or acidic conditions (pH 6.8, pH 6.6). Acidic conditions were achieved by adding hydrochloric acid to the medium to adjust the pH.

[0053] [Gene and protein expression analysis under acidic conditions] The cells were cultured under normal or acidic conditions for 24 to 72 hours, after which RNA and protein were extracted. Quantitative PCR (qPCR) was performed using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) to analyze the expression levels of target genes, including SLC12A7. The results are shown in Figure 8. Western blotting was performed for protein expression analysis. The results are shown in Figure 9.

[0054] [SLC12A7 Expression in Normal and Acidic Environments] As shown in Figure 8, pancreatic cancer cell lines showed higher SLC12A7 mRNA expression levels compared to hTERT-HPNE in a normal environment at pH 7.4. However, no significant changes in mRNA expression were observed in either pancreatic cancer cell lines or hTERT-HPNE in an acidic environment. In contrast, as shown in Figure 9, protein expression analysis confirmed that expression increased in pancreatic cancer cell lines in an acidic environment, whereas expression decreased in hTERT-HPNE. Similarly, protein expression analysis was performed on exosomes derived from each cell line. As shown in Figure 10, SLC12A7 protein expression levels on exosomes were also confirmed to increase in pancreatic cancer cell lines in an acidic environment.

[0055] [SLC12A7 knockdown by siRNA] Silencer Select siRNA (Thermo Fisher Scientific, siSLC12A7; catalog numbers s21062 and s21063) was used to silence SLC12A7 gene expression. The siRNA was transfected into cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific). 48 hours later, knockdown efficiency was assessed by qPCR and Western blotting.

[0056] [Cellular Function Analysis] (1) CCK-8 Assay (Evaluation of Cell Proliferation Potential) The CCK-8 assay (Cell Counting Kit-8, Dojindo) was used to evaluate cell proliferation potential. 5,000 cells were seeded into a 96-well plate at 5,000 cells / well, and after a specified time, the medium was replaced with one containing CCK-8 reagent (final concentration 10%). The incubation temperature was 37°C, 5% CO ２After 2 hours of incubation, the absorbance (450 nm) was measured using a microplate reader. Experiments were performed under independent conditions in at least three wells, and the average was calculated.

[0057] (2) Scratch Assay (Evaluation of Cell Migration Ability) A scratch assay was used to evaluate cell migration ability. Cells were seeded in a 6-well plate, and after forming a confluent monolayer, a uniform scratch was made using a 200 μL pipette tip. The cells were washed with PBS and replaced with serum-free medium (or low FBS). The width of the scratch was photographed using an inverted microscope after 0, 12, and 24 hours. The area of ​​cells that had migrated into the scratch was quantified and calculated using ImageJ software.

[0058] (3) Transwell assay (evaluation of cell invasiveness) Cell invasiveness was evaluated using a Transwell assay (Corning, 8 μm pore size). Matrigel (Corning, 1:8 dilution) was applied to the membrane surface of the upper chamber and allowed to gel at 37°C for 30-60 minutes. Cells (5 × 10 cells) suspended in serum-free medium were placed in the upper chamber. ４ Cells were seeded in the upper chamber, and 10% FBS-containing medium was added to the lower chamber as an inducer. After 24 hours, non-invading cells in the upper chamber were removed with a cotton swab, and cells that had invaded the lower chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of invading cells was counted in at least three random fields, and the average was calculated.

[0059] Involvement of SLC12A7 in Pancreatic Cancer Cell Line Phenotypes. To clarify the functional role of SLC12A7, we generated knockdown (KD) lines of pancreatic cancer cells and hTERT-HPNE using siRNA (siSLC12A7). The results of SLC12A7 knockdown in each cell line are shown in Figure 11. The knockdown (KD) lines were compared with wild-type (WT) cells for various functional analyses. As shown in Figure 12, CCK-8 assays confirmed that pancreatic cancer cells had higher proliferation potential than hTERT-HPNE cells. In an acidic environment compared to pH 7.4, proliferation was enhanced in WT cells, but decreased in KD cells. As shown in Figures 13 to 15, scratch assays confirmed that pancreatic cancer cells had higher migration potential than hTERT-HPNE cells. In an acidic environment compared to pH 7.4, migration was enhanced in WT cells, but not in KD cells. As shown in Figures 16 to 18, the Transwell assay showed that the invasive ability of pancreatic cancer cell lines was significantly enhanced in an acidic environment, whereas the KD cell line showed no enhancement of invasive ability. hTERT-HPNE showed almost no invasive ability.

[0060] [Changes in ER stress in acidic environments] Pancreatic cancer cells were seeded into 24-well plates and cultured for 24 hours under either pH 7.4 or acidic conditions. The protein expression levels of SLC12A7 and ER stress-related molecules (PERK, p-PERK, ATF6, ATF4, EIF, and pEIF) were assessed by Western blotting. As shown in Figure 19, the expression levels of pPERK, pEIF, and ATF4, as well as ATF6, increased in acidic conditions. This suggests that the PERK-ATF4 pathway selectively induces transcription of stress response genes, leading to the accumulation of SLC12A7. Furthermore, the increase in some proteins, including SLC12A7, increases the folding and transport load in the ER, and ATF6 acts as a reinforcement mechanism for protein folding.

[0061] The present invention contributes to the diagnosis and treatment of early-stage pancreatic cancer.

Claims

1. A diagnostic agent for pancreatic cancer, comprising a primer set for amplifying SLC12A7 DNA or mRNA in a sample derived from a subject, and / or a probe that binds to the SLC12A7 DNA, mRNA or its amplification product, or an antibody that binds to SLC12A7 protein.

2. The diagnostic agent according to claim 1, wherein the sample is blood-derived extracellular vesicles.

3. A pancreatic cancer diagnostic kit comprising the diagnostic agent according to claim 1 or 2.

4. A method for detecting a diagnostic marker for pancreatic cancer, which comprises detecting the amount of SLC12A7 DNA, mRNA, or SLC12A7 protein present in a sample derived from a subject.

5. The detection method according to claim 4, wherein the sample is blood-derived extracellular vesicles.

6. A diagnostic marker for pancreatic cancer, comprising SLC12A7 DNA or mRNA or SLC12A7 protein isolated from a sample derived from a subject.

7. The marker of claim 6, wherein the sample is blood-derived extracellular vesicles.

8. An anti-pancreatic cancer agent containing an SLC12A7 inhibitor as an active ingredient.

9. A composition for treating pancreatic cancer, comprising the anti-pancreatic cancer agent according to claim 8 and a pharmaceutically acceptable carrier.

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