Application of urine protein marker SPP1 in preparation of kit for diagnosing esophageal squamous carcinoma

Abstract

The invention provides application of a urine protein marker in preparation of a kit for diagnosing esophageal squamous carcinoma. Wherein the urine protein marker is urine Sected photein 1 (SPP1) protein, and researches prove that the SPP1 is highly expressed in urine of esophageal squamous cell carcinoma patients and can be used for detection and auxiliary diagnosis of esophageal squamous cell carcinoma compared with normal control and esophageal squamous cell carcinoma patient groups. According to the invention, the advantages of noninvasive acquisition, large-scale repeated sampling and convenient preservation of the urine sample are exerted, and SPP1 is detected by using the urine sample.

Description

Technical Field

[0001] This invention relates to the field of esophageal malignant tumors, and more specifically, to the application of a urinary protein marker in the preparation of a preparation for the detection and auxiliary diagnosis of esophageal squamous cell carcinoma. Background Technology

[0002] Esophageal cancer (EC) is one of the most common malignant tumors of the digestive tract, ranking as the 11th most common cause of cancer worldwide (473,000 cases) and the 6th most common cause of cancer-related death (436,000 deaths). Based on its histological classification, esophageal cancer is mainly divided into two types: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), with ESCC being the most common. Esophageal malignancies are a serious threat to human health, with high incidence and poor prognosis, placing a heavy medical burden on society. my country is a high-incidence country for esophageal malignancies, with statistics showing over 246,000 new cases and 188,000 deaths annually. Interestingly, although the overall 5-year survival rate for ECSS is only 20%, the survival rate for early-stage patients is as high as 80%. Therefore, early detection, diagnosis, and treatment of tumors are of great significance for improving the overall prognosis of esophageal cancer patients.

[0003] Endoscopy is currently the most important method for detecting and diagnosing esophageal cancer. During endoscopy, tumor tissue can be obtained and subjected to pathological examination, which is the gold standard for ESCC diagnosis. However, in countries with high ESCC incidence, such as China and Iran, there are objective challenges: a large population and a heavy overall cancer burden; and the fact that endoscopy, as an invasive procedure, carries high risks and relatively high economic and time costs. These factors limit the implementation of large-scale endoscopic screening for high-risk populations, limiting it to targeted screening in key areas with high esophageal cancer incidence. Therefore, it is necessary to identify non-invasive biomarkers suitable for large-scale screening to improve ESCC detection and further enhance its prognosis.

[0004] Currently used pan-tumor serum biomarkers, including SCC, CEA, CA19-9, CA72-4, and CYFRA21-1, lack good sensitivity and specificity for ESCC. Previous literature has identified some serum biomarkers for ESCC diagnosis, but none have explored their application in early lesions in depth. Therefore, while developing clinically applicable biomarkers, it is essential to improve the effectiveness of early ESCC screening.

[0005] Appropriate non-invasive molecular biomarkers are of great significance for the early diagnosis and screening of esophageal cancer. Proteomics has emerged to address this need and holds the promise of making groundbreaking contributions to early esophageal cancer diagnosis and screening. Proteins are fundamental to human life activities, and proteomics is a research field that identifies, analyzes, and studies all proteins within various biological samples. Mass spectrometry, the underlying technology for proteomics, produces high-throughput protein-level data that overcomes the limitations of studying single genes or proteins, providing a holistic view of the protein expression profile of a sample. First proposed by Marc Wilkins in 1996, proteomics stands for "protein complement of the genome," representing the characterization of the proteome, including protein expression, structure, function, interactions, and modifications. Proteomics can also reflect real-time changes in proteins within cells, providing high-throughput, high-sensitivity technical strategies for disease etiology, and is essential for early disease diagnosis, prognosis, and monitoring. Currently, supported by high-throughput technologies, proteomics has become one of the most important methods for understanding complex biochemical processes and exploring gene function.

[0006] Urine has gradually become one of the most promising biological fluids for protein research in clinical samples in recent years. Compared with blood, urine has the following advantages: (1) Urine collection involves a non-invasive procedure that can be repeated to obtain a sufficient sample volume. (2) Compared with blood, urine is easier to analyze and provides a stable and "inert" matrix. Currently, urine has been used to detect more than 2,300 different proteins. Analysis of urinary proteins has revealed a wealth of information about the kidneys, brain, and cardiovascular system and their related diseases, as well as specific types of cancer. Urine has the advantages of being non-invasive, allowing for large-scale repeated sampling, and being easy to preserve. Through non-invasive research on urinary biomarkers, clarifying the molecular mechanisms associated with the occurrence of esophageal cancer has important clinical significance for the early diagnosis, treatment, and prognosis of esophageal cancer.

[0007] Urine is the most commonly used bodily fluid sample in clinical testing, second only to blood. It is a truly non-invasive sample, and mass spectrometry can detect hundreds or even thousands of trace proteins within it. The human urinary proteome contains a wealth of information about intra- and inter-individual differences, as well as physiological and pathological states. Scientists worldwide have been using proteomics to find new protein biomarkers from urine for disease diagnosis, prognostic analysis, and efficacy monitoring. The urinary proteome can reflect not only physiological information such as sex, age, and ethnicity, but also pathological states. Researchers have already identified disease biomarkers using urinary proteomics, including those for chronic kidney disease, lung cancer, liver cancer, and neurological disorders.

[0008] If esophageal cancer biomarkers with good specificity and sensitivity are discovered based on proteomics, it will facilitate large-scale screening of esophageal cancer, enabling early detection and treatment of ESCC patients, which will greatly reduce the mortality rate of the disease. Its significance is profound. Summary of the Invention

[0009] The main objective of this invention is to provide an application of urinary protein markers in the preparation of a reagent kit for diagnosing esophageal squamous cell carcinoma, thereby providing a new non-invasive clinical diagnostic testing method.

[0010] To achieve the above objectives, according to the present invention, an application of a urinary protein marker in the preparation of a kit for diagnosing esophageal squamous cell carcinoma is provided, wherein the urinary protein marker is urinary protein SPP1.

[0011] To achieve the above objectives, according to the intended use of the present invention, a kit for diagnosing esophageal squamous cell carcinoma is provided, the kit comprising a urine protein marker antibody, wherein the urine protein marker antibody is an antibody against SPP1.

[0012] Furthermore, the kit includes a housing and solid-state compounds and reagents disposed within the housing.

[0013] Preferably, the solid support is selected from microplates and coated with anti-SPP1 protein and its polypeptide sheet antibodies.

[0014] Preferably, the reagents include SPP1 standard solution, enzyme-labeled primary and secondary antibodies, chromogenic solution, stop solution, diluent and washing solution.

[0015] Preferably, the kit is an ELISA kit.

[0016] The inventors first collected random urine samples from esophageal squamous cell carcinoma patients and normal controls, and collected the supernatant after centrifugation. Samples and standards of different concentrations were quantitatively added to the corresponding wells, while a universal diluent was added to the blank wells. Samples were then added sequentially, and the optical density (OD) value of the wells was measured at 450 nm. The average OD value of the standard and sample replicates was calculated, and the OD value of the blank wells was subtracted as a correction value. A standard curve of a four-parameter logarithmic function was plotted on logarithmic graph paper with concentration on the x-axis and OD value on the y-axis. The SPP1 content in the samples was calculated. The results showed that SPP1 protein was highly expressed in the urine of esophageal squamous cell carcinoma patients compared to the normal control group.

[0017] The inventors performed immunohistochemical staining on tumor tissue specimens from patients with esophageal squamous cell carcinoma and on normal esophageal tissue specimens. The results showed that SPP1 protein was highly expressed in esophageal squamous cell carcinoma tissue compared to normal esophageal tissue specimens. This indicates a correlation between elevated SPP1 expression in urinary protein and tumor tissue.

[0018] This invention demonstrates through research that, compared with the normal control group, SPP1 protein is highly expressed in the urine of patients with esophageal squamous cell carcinoma, thus proposing that the detection of urinary SPP1 protein can be used for the diagnosis of esophageal squamous cell carcinoma.

[0019] This invention leverages the advantages of urine sample acquisition being non-invasive, allowing for large-scale repeated sampling, and being easy to preserve, to detect SPP1 protein using urine samples.

[0020] To make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings.

[0021] Attached image description.

[0022] Figure 1 This is a flowchart for a study of the biomarker spp1 in urine of patients with esophageal squamous cell carcinoma.

[0023] Figure 2 The ROC curves of SPP1 protein expression in urine in the esophageal squamous cell carcinoma group were compared with those of traditional serum tumor markers.

[0024] Figure 3 This is a box plot showing the expression of SPP1 protein in urine samples from the esophageal squamous cell carcinoma group and the normal control group.

[0025] Figure 4 The immunohistochemical staining analysis of esophageal squamous cell carcinoma tissue and normal esophageal tissue compared the expression of SPP1. Detailed Implementation

[0026] To more clearly express the purpose, technical features, and advantages of this application, the specific implementation process of the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Of course, the embodiments described below are only a part of the invention, not all of it. Therefore, the detailed description of the embodiments of the present invention presented in the accompanying drawings is not intended to limit the scope of the claimed invention, but only to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the protection scope of the present invention.

[0027] This invention mainly includes two stages: 1) Preliminary monitoring stage: Sample preparation. Enzyme-linked immunosorbent assay (ELISA) is a commonly used verification method. ELISA was used to verify the expression of SPP1 in the urine of patients with esophageal squamous cell carcinoma and healthy individuals. Data analysis confirmed that SPP1 was highly expressed in the urine samples of patients with esophageal squamous cell carcinoma; 2) Verification stage: Immunohistochemical detection was performed on tumor tissues and normal esophageal tissues of esophageal cancer patients. It was found that the expression level of SPP1 in esophageal cancer tissues was significantly higher than that in normal esophageal tissues.

[0028] This embodiment collects urine samples from patients with esophageal squamous cell carcinoma and healthy individuals, and uses urine proteomics technology to analyze the differences in urine proteomics between patients with esophageal squamous cell carcinoma and healthy individuals, thereby identifying urine biomarkers related to the diagnosis of esophageal squamous cell carcinoma.

[0029] Research subjects This study included 30 patients who were hospitalized at Weifang People's Hospital from January 2023 to December 2023 and met the diagnostic criteria for esophageal squamous cell carcinoma; 30 healthy controls were also included through a health checkup center during the same period.

[0030] 1. Inclusion and Exclusion Criteria 1.1 Inclusion criteria for patients with esophageal squamous cell carcinoma (1) The patient underwent an upper gastrointestinal endoscopy (gastroscopy / esophagoscopy), and was diagnosed with ESCC by biopsy and pathological examination; (2) The patient had not received any treatment before the urine sample was collected for enrollment.

[0031] 1.2 Exclusion criteria for patients with esophageal squamous cell carcinoma (1) A urinary system disease was found within the past month or there were obvious abnormalities in preoperative related examinations such as blood urea nitrogen, serum creatinine, uric acid, etc.; (2) History of other tumors or hereditary diseases; (3) Patients with severe cachexia and poor appetite; (4) Has received an organ transplant, stem cell transplant, bone marrow transplant, or blood transfusion within the past month; (5) Women during menstruation and pregnant women; (6) Currently participating in other clinical trials that require medication within the past 60 days; (7) Severe cardiovascular disease, uncontrollable infection, or other uncontrollable comorbidities; (8) The patient or their family cannot understand the conditions and objectives of this study; (9) Patients who are mistakenly included but do not meet the inclusion criteria after entering the clinical trial, and patients who refuse to accept biological specimen collection after being included but meet the inclusion criteria, will be removed.

[0032] 1.3 Inclusion criteria for healthy individuals After a comprehensive health check, the medical report did not indicate any major health problems.

[0033] 1.4 Exclusion criteria for healthy individuals The exclusion criteria are basically the same as those for ESCC patients.

[0034] 2. Collection and preservation of urine specimens Urine samples were collected from 30 patients with esophageal squamous cell carcinoma (thoracic surgery ward, Weifang People's Hospital) and 30 healthy controls (health checkup center, Weifang People's Hospital). Sampling was conducted between 6 and 8 AM on the same day, collecting midstream urine from the first urination of the morning. After collection, the urine was transferred to centrifuge tubes, with a collection volume of approximately 10 ml. Urine samples were aliquoted within 8 hours of collection and frozen at -80°C. Mass spectrometry sample preparation and analysis were performed in batches within 3 months of collection.

[0035] 3. Baseline data of subjects included in urinary proteinomics Baseline data of the experimental and control groups were analyzed by urinary proteomics and SPSS statistical analysis.

[0036] Among the patients with esophageal squamous cell carcinoma, there were 23 male patients and 7 female patients, with a mean age of (61.93±8.24) years. 12 patients (40.00%) were smokers, and 14 patients (46.67%) were drinkers. Body Mass Index (BMI): low (<18.5 kg / m²), 1 patient (3.33%); normal (<18.5 kg / m²), 18 patients (60.00%); overweight (<18.5 kg / m²), 6 patients (20.00%); obese (<18.5 kg / m²), 5 patients (6.67%).

[0037] In the normal control group, there were 22 males and 8 females, with a mean age of (60.03±11.21) years. Eight individuals (26.67%) were smokers, and 11 individuals (36.67%) were drinkers. Body Mass Index (BMI): low (<18.5 kg / m²), 0 cases (0); normal (<18.5 kg / m²), 14 cases (46.67%); overweight (<18.5 kg / m²), 12 cases (40.00%); obese (<18.5 kg / m²), 4 cases (13.33%).

[0038] There were no statistically significant differences in baseline information such as gender, age, smoking, alcohol consumption, and body mass index between the two groups (P≥0.05), as shown in Table 1.

[0039] Table 1: Baseline Data

[0040] 4. Preparation of urine protein samples Centrifugation: The urine sample was slowly thawed on ice and centrifuged at 3500g for 10 minutes at 4°C to remove detached cells and larger impurities from the urine.

[0041] 5. Preparation of reagents for enzyme-linked immunosorbent assay (ELISA) of urine proteins Reagent preparation: The reagents used in this experiment should be prepared and used immediately in accordance with the instructions for use.

[0042] ① Sample diluent (ELISA assay / sample diluent buffer): Dilute it to 1× with distilled water; ② Preparation of Standard (Human SSP1 Standard, recombinant human SPP1): Add 400 μL of sample diluent to the SPP1 standard powder and dissolve it thoroughly. The concentration of the solution at this point is 18000 pg / ml. Dilute this standard solution stepwise to seven gradients, including a concentration of 0 pg / ml. Take 200 μL of a standard solution with a concentration of 18000 pg / mL and add it to 400 μL of sample diluent. This solution is the second concentration gradient solution, with an SPP1 concentration of 6000 pg / mL. Then, take another 200 μL from the second concentration gradient solution and add it to 400 μL of sample diluent. This solution is the third concentration gradient solution, with an SPP1 concentration of 2000 pg / mL. Next, take another 200 μL from the third concentration gradient solution and add it to 400 μL of sample diluent. This solution is the fourth concentration gradient solution, with an SPP1 concentration of 666.7 pg / mL. Repeat this process until the sixth concentration gradient is reached, at which point the SPP1 concentration is 74.07 pg / mL. The seventh concentration gradient is the pure sample diluent, with an SPP1 concentration of 0 pg / mL. ③ Human SPP1 Biotin Conjugate: First, centrifuge the human SPP1 biotin conjugate. Then, add 100 μL of 1× diluent to the bottle, transfer and mix well. Then, dilute the above reagent 80 times with 1× assay diluent for the next ELISA step. ④ Streptavidin-HRP solution: Before use, mix the streptavidin-HRP solution with a centrifuge and pipette, and dilute the streptavidin-HRP solution with 1× assay diluent by 500 times (Note: Do not store diluted streptavidin-HRP solution; it must be prepared within 15 minutes before use). ⑤ Prepare 1× Wash buffer: Equilibrate 20× Wash buffer to room temperature and mix well. Add 20 ml of Wash buffer concentrate diluent to 380 ml of distilled water to prepare 1× Wash buffer. Store in the refrigerator and use within 1 month.

[0043] 6. Determination of protein concentration in urine using enzyme-linked immunosorbent assay (ELISA). Ensure that urine samples, reagents, and 96-well plates (HumanSPP1 Antibody Coated Wells, 96-well plate) treated with enzyme-linked immunosorbent assay (ELISA) are brought to normal room temperature (18-25°C) before the experiment begins. ① Sample loading: Add 100 μL of urine sample and standard solutions of different concentration gradients to the corresponding 96-well plate, cover with a sealing film, place on a shaker and gently shake, and incubate at room temperature for 2.5 hours; ② First wash: Remove the protective film, discard the urine sample and standard solution in the well, invert the sample onto clean paper and gently pat dry to ensure all liquid is removed. Then add 300 μL of 1× washing buffer to each well, let stand for 30 seconds, then gently shake and discard all liquid. Invert the sample onto clean paper again and gently pat dry to ensure all liquid is removed. This washing process is repeated a total of 4 times, ensuring that no liquid is visible to the naked eye after each wash. ③ Add human SPP1 biotin conjugate: Add 100 μL of detection antibody solution to each well, attach a new blocking membrane, place on a shaker and gently shake, incubate at room temperature for 1 hour; ④ Second cleaning: Repeat the cleaning steps in ②; ⑤ Add streptavidin solution-HRP solution: Add 100 μL of streptavidin solution to each well, cover with a new sealing membrane, place on a shaker and shake gently, and incubate at room temperature for 45 min; ⑥ Third cleaning: Repeat the cleaning steps in ②; ⑦ Add ELISA colorimetric TMB reagent: Add 100 μL of streptavidin solution to each well, protect from light, attach a new sealing membrane, place on a shaker and gently shake, incubate at room temperature for 30 min; ⑧ Add ELISA stop solution: Add 50 μL of reaction stop solution to each well, shake gently, and immediately measure the absorbance at 450 nm using a microplate reader; ⑨ Plot a standard curve based on the absorbance of gradient concentration standards, and calculate the correlation coefficient R between absorbance and standard concentration. 2 When R 2 A linear fit of >95% indicates a good result, and the data at this level can be analyzed and applied. The concentration of the urine sample is calculated based on the fitting formula used for the experimental standard. ⑩ Box-scatter plots of SPP1 protein levels in the esophageal squamous cell carcinoma (ESCC) group and the healthy controls (CTL) were plotted based on urine sample data. The results showed that the amount of SPP1 protein in the urine of ESCC patients was significantly higher than that in the healthy controls. For detailed results, please see [link to results]. Figure 2 .

[0044] Using this method, the inventors detected the SPP1 protein content in the morning urine of 30 patients with esophageal squamous cell carcinoma and plotted ROC curves. Comparison with traditional serum tumor markers CEA (carcinoembryonic antigen), CA199 (carbohydrate antigen 199), and CA724 (carbohydrate antigen 72-4) revealed that this protein exhibited good sensitivity and specificity. For detailed results, please refer to [link to relevant documentation]. Figure 3 .

[0045] 7. Immunohistochemical verification of spp1 protein in esophageal squamous cell carcinoma and normal esophageal control tissues. Paraffin-embedded esophageal squamous cell carcinoma and normal esophageal control tissue samples were cut into 4-mm sections. Slides were dewaxed with xylene for 20 min, followed by a gradient dehydration with 100%, 100%, 95%, and 75% ethanol for 2 min each time. After washing with phosphate-buffered saline (PBS) for 10 min, antigen retrieval was performed, followed by incubation with 3% H₂O₂ for 15 min, PBS washing for 10 min, incubation with anti-human SPP1 monoclonal antibody, washing with PBS, incubation with horseradish peroxidase-labeled secondary antibody at 37°C for 20 min, and washing with PBS for 15 min. Chromogen chromogen was stained with 3,3′-diaminobenzidine solution for 5 min, counterstained with hematoxylin for 2 min, dehydrated with a gradient dehydration of 75%, 95%, 100%, and 100% ethanol, washed with xylene, and mounted with natural resin. Results were observed under a microscope; for detailed results, please refer to [link to relevant documentation]. Figure 4 The figure clearly shows that spp1 protein expression is significantly upregulated in the tissues of patients with esophageal squamous cell carcinoma.

[0046] This indicates that the elevated SPP1 levels in the urine of patients with esophageal squamous cell carcinoma are correlated with the occurrence and development of esophageal squamous cell carcinoma, and we can use this correlation to diagnose esophageal squamous cell carcinoma.

[0047] In this embodiment, the urine protein marker antibody is placed on a solid-phase carrier; preferably, the solid-phase carrier is selected from an ELISA plate, more preferably, the membrane carrier is selected from a nitrocellulose membrane, a glass cellulose membrane, or a nylon membrane; preferably, the urine protein marker antibody is a monoclonal antibody; preferably, the kit is an ELISA kit.

[0048] This embodiment utilizes proteomics technology to study biomarkers in urinary esophageal squamous cell carcinoma (ESCC) patients. Compared with blood-related tumor markers, it not only has higher sensitivity and specificity but is also more easily accepted by patients in terms of collection. This may be of great significance for conducting large-scale screening of esophageal squamous cell carcinoma patients.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications and variations can be made to the invention by those skilled in the art without departing from its scope. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of secreted phosphoprotein 1 (SPP1) in the preparation of agents for the detection and auxiliary diagnosis of esophageal squamous cell carcinoma.

2. A reagent kit for diagnosing esophageal squamous cell carcinoma, characterized in that, The kit includes a urine protein marker antibody, which is Secreted phosphoprotein 1 (SPP1).

3. The reagent kit according to claim 2, characterized in that, The SPP1 protein is derived from urine.

4. The application according to claim 2, characterized in that, The preparation is a urine SPP1 detection kit for patients with esophageal squamous cell carcinoma.

5. The application according to claim 4, characterized in that, The urinary protein marker antibody is mounted on a solid-phase support.

6. The application according to claim 5, characterized in that, The solid-bonded composite is a microporous plate.

7. The application according to claim 6, characterized in that, SPP1 antibody was coated onto microplates.

8. The application according to claim 5, characterized in that, The reagents include SPP1 standard solution, enzyme-labeled secondary antibody, chromogenic solution, stop solution, diluent and washing solution.

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