Application of transporter-derived annexin A6 as breast cancer diagnosis marker

Abstract

The invention discloses an application of a transporter-derived annexin A6 as a breast cancer diagnostic marker, which comprises the following steps: screening annexin A6 (ANXA6) as a candidate marker from transporters derived from different molecular typing breast cancer cells through protein spectrum analysis, and separating the transporter from serum through density gradient centrifugation. And detecting the content of the migration body ANXA6 in the serum sample by adopting an ELISA method. ROC curve analysis shows that the AUC value of the serum transporter ANXA6 for diagnosing breast cancer is 0.959, the sensitivity is 93.8%, and the specificity is 85.3%. Compared with the prior art, the serum migration body serves as a novel detection carrier of ANXA6 for the first time, and the problems that an existing detection mode is high in tissue sample invasiveness, dynamic monitoring cannot be achieved, exosome heterogeneity is high, and the signal-to-noise ratio is low are solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of migratory isotopic annexin A6 as a diagnostic biomarker for breast cancer. Background Technology

[0002] Breast cancer (BC) remains the most common malignant tumor among women worldwide and the leading cause of cancer-related death in women. While tissue biopsy remains the gold standard for breast cancer diagnosis, its inherent invasiveness significantly limits its clinical applicability for repeated disease monitoring. Against this backdrop, liquid biopsy, with its advantages of being non-invasive, convenient, and repeatable, has gradually become an important technique for early screening, prognostic assessment, and dynamic monitoring of breast cancer progression. However, current clinical applications of liquid biopsy biomarkers, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomes, all have limitations: these biomarkers are present in extremely low concentrations in complex body fluid environments, exhibit high heterogeneity, and lack sufficient temporal resolution for real-time monitoring of disease progression, thus compromising their reliability and ultimately limiting their further expansion in clinical application.

[0003] Annexin A6 (Gene ID: 309) is one of the larger molecular weight members of the annexin family, involved in regulating multiple processes associated with cancer progression, including cell migration, adhesion, proliferation, and membrane transport. In breast cancer, changes in ANXA6 expression are closely related to tumor invasiveness. Current research on ANXA6 as a biomarker mainly focuses on tissue samples and exosomes (exo). Both of these carriers have inherent limitations, restricting their clinical application value in liquid biopsy, as detailed below: 1. Limitations of ANXA6 in tissue samples Tissue samples must be obtained through puncture or surgical biopsy, which is traumatic for patients and makes repeated sampling during treatment difficult, failing to meet the clinical needs of dynamic monitoring. Furthermore, tissue biopsies only obtain samples from a localized area of ​​the tumor, failing to comprehensively reflect the molecular heterogeneity between the primary and metastatic lesions, or between different metastatic lesions. For proteins like ANXA6, whose expression patterns are complex across different breast cancer subtypes, single-point tissue biopsies are highly prone to misdiagnosis. More importantly, tissue biopsies can only be performed after suspicious lesions are detected by imaging, making them unsuitable as an early screening tool for asymptomatic individuals.

[0004] 2. Limitations of exosome-derived ANXA6 (ANXA6-exo) Exosomes, as liquid biopsy carriers, have the following technical limitations: First, exosomes are vesicles secreted by almost all cell types (including normal cells, immune cells, and tumor cells), and the origin of serum exosome ANXA6 is extremely complex. This high heterogeneity in origin leads to severe dilution of tumor-specific signals, making it difficult to guarantee detection reliability. Second, exosome production is a ubiquitous physiological process in cells and is not directly related to specific cellular behaviors (such as migration and invasion). Therefore, detecting exosome ANXA6 only indicates the possible presence of tumor cells and cannot reflect the crucial clinical information of whether tumor cells are invading and metastasizing. Furthermore, due to the aforementioned heterogeneity and nonspecificity, exosome-based biomarker detection faces the challenge of low signal-to-noise ratio, making it difficult to achieve ideal diagnostic efficacy.

[0005] In recent years, studies have reported on the expression and function of ANXA6 in breast cancer cells (Bouvet et al., Scientific Reports, 2020, 10:21821, doi: 10.1038 / s41598-020-77902-5). Using MDA-MB-231 breast cancer cells as a model, it was found in a cell culture system that intracellular ANXA6 participates in the cell membrane repair process when these cells migrate on type I collagen. Furthermore, knocking down ANXA6 expression using shRNA technology leads to the death of migrating cancer cells. However, this study was limited to the in vitro cellular level, and the ANXA6 detected was an intracellular protein, without addressing the origin of the migratory body in body fluids. Summary of the Invention

[0006] The purpose of this invention is to provide the application of migratory somatic annexin A6 as a diagnostic biomarker for breast cancer, in order to overcome the shortcomings of existing technologies such as high invasiveness of tissue samples, inability to dynamically monitor, high heterogeneity of exosomes, and low signal-to-noise ratio.

[0007] The present invention relates to the application of migratory annexin A6 as a biomarker in the preparation of a kit for diagnosing breast cancer. The kit diagnoses breast cancer by detecting the content of migratory annexin A6 in serum samples, wherein the migratory vesicles are extracellular vesicles obtained from serum by density gradient centrifugation.

[0008] Furthermore, the reagent used to detect the content of migratory annexin A6 is an anti-annexin A6 antibody.

[0009] Furthermore, the kit also includes reagents for isolating migrants from serum.

[0010] Furthermore, the diagnostic threshold for the migratory somatic annexin A6 is 0.5–2.0 ng / mL.

[0011] Furthermore, the migratory myeloprotein A6 showed an AUC value of 0.959, a sensitivity of 93.8%, and a specificity of 85.3% in distinguishing between breast cancer and healthy controls.

[0012] Furthermore, the diagnostic threshold for the migratory myeloprotein A6 is 0.778 ng / mL.

[0013] Furthermore, the serum sample was derived from a human.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This is the first time that a migratory organism has been used as a novel detection carrier for ANXA6, overcoming the inherent limitations of tissue samples and exosomes. Existing technologies for studying ANXA6 as a biomarker are limited to tissue samples and exosomes. This invention extends the detection vector to migration bodies for the first time. This shift is not a simple vector replacement, but rather an application of the unique biological functions of migration bodies. Its advantages are: (1) Overcoming the invasiveness of tissue samples: Migration bodies exist in serum and can be obtained non-invasively, enabling repeated sampling and dynamic monitoring during treatment, solving the clinical pain point that tissue biopsy cannot be used for early screening and dynamic tracking; (2) Overcoming the heterogeneity of exosomes: The formation of migration bodies is closely coupled with cell migration behavior and only forms when cells migrate. Detecting ANXA6 in migration bodies means that these cells are actively migrating and invading, which is a dynamic functional perspective that exosome detection cannot achieve; (3) Improving the signal-to-noise ratio by utilizing the active loading mechanism: Migration bodies have a unique "active loading" mechanism during their formation, which can selectively enrich specific proteins. This invention utilizes the selective enrichment of ANXA6 in migration bodies, which, compared to detecting ANXA6 in serum exosomes, effectively reduces background interference from normal cell sources and significantly improves the accuracy and reliability of detection.

[0015] 2. Unexpected technical effects were achieved. Existing research at the in vitro cellular level has found that double knockdown of ANXA5 / ANXA6 leads to the death of a large number of migrating cancer cells. In contrast to this technical teaching, this invention is the first to propose the use of human serum-derived migrating ANXA6 for breast cancer diagnosis, achieving an unexpectedly significant improvement in diagnostic efficacy. Through large-scale clinical sample validation (n=129), the following specific data on the diagnosis of breast cancer using migrating migrating ANXA6 are provided for the first time:

[0016] An AUC > 0.9 indicates excellent diagnostic efficacy, with a sensitivity of 93.8%, demonstrating the marker's superior discriminative ability. A specificity of 85.3% means that high sensitivity can detect the vast majority of breast cancer patients, resulting in a low rate of missed diagnoses and making it suitable as an early screening indicator.

[0017] Existing technologies do not suggest that migrated somatic ANXA6 can be used for breast cancer diagnosis. This invention is the first to demonstrate that serum migrated somatic ANXA6 can be used as a diagnostic biomarker for breast cancer, and achieves excellent diagnostic efficacy with an AUC as high as 0.959, achieving unexpected technical results.

[0018] 3. The detection method is simple and easy to perform, and has high clinical application value. On the one hand, serum can be obtained through routine intravenous puncture, which is non-invasive to patients and facilitates large-scale screening and repeated sampling. On the other hand, the migrations in serum have good stability under appropriate storage conditions, which facilitates sample collection, transportation and centralized testing, making it suitable for multi-center clinical research and application in primary hospitals. The conventional ELISA method is used to detect the ANXA6 content in serum migrations, which is simple to operate, cost-controllable, and easy to promote and apply in hospitals at all levels.

[0019] 4. Providing new tools for precision medicine The migratory somatic ANXA6 biomarker provided by this invention can reflect the migration and invasion behavior of tumor cells, providing a new molecular tool for monitoring the invasion and metastasis of breast cancer and helping to achieve personalized diagnosis and treatment in the context of precision medicine. Attached Figure Description

[0020] Figure 1 Characterization diagrams of migratory bodies in normal breast epithelial cells and breast cancer cells, where: Figure 1 A is a confocal image (green) of WGA-labeled cell migratory bodies, with a magnified view framed in white below; Figure 1 B is a scanning electron microscope image of cell migratory bodies, with the enlarged view shown in red below; Figure 1 C is a transmission electron microscope image of the purified migratory cells after negative staining; Figure 1 D is a Western blot analysis of migration body-specific proteins in cell lysates; Figure 1 E represents the Western blot analysis of the purified migration body-specific proteins. Figure 2 Principal component analysis diagram of protein profiles of normal mammary epithelial cells and breast cancer cells migratory bodies; Figure 3 Correlation heatmap analysis of protein profiles of normal breast epithelial cells and breast cancer cells; Figure 4Cluster heatmap analysis of protein profiles of migratory cells from normal breast epithelial cells and breast cancer cells; Figure 5 Venn diagram analysis of protein profiles of normal mammary epithelial cells and breast cancer cells (common differentially expressed proteins). Figure 6 Venn diagram of protein profile analysis of normal mammary epithelial cells and breast cancer cells (co-upregulated proteins). Figure 7 Venn diagram of protein profile analysis of normal mammary epithelial cells and breast cancer cells (common downregulated proteins). Figure 8 Volcano plot analysis of differential expression in migratory bodies of normal breast epithelial cells and breast cancer cells, including: Figure 8 A is a differential volcano plot of the Hs578T migratory body versus the MCF-10A migratory body; Figure 8 B is a differential volcano plot of the SK-BR-3 migratory body versus the MCF-10A migratory body; Figure 8 C is a differential volcano plot of the MCF-7 migratory body versus the MCF-10A migratory body; Figure 8 D is a volcano plot showing the differences between the BT474 migratory body and the MCF-10A migratory body; Figure 9 The diagram shows the characterization of the migrants in the clinical sample, where: Figure 9 A is a transmission electron microscope (TEM) image of serum migratory bodies; Figure 9 B represents the Western blot analysis of migration-specific marker proteins and ANXA6 in serum migration bodies; Figure 10 The chart shows the differential analysis of ANXA6, NLR, LMR, and PLR in BC, HC, and BBD, where: Figure 10 A represents a comparison of the expression levels of ANXA6 among the three groups: breast cancer group (BC), healthy control group (HC), and benign breast disease group (BBD). Figure 10 B is a comparison of the expression levels of NLR, LMR, and PLR among the three groups: breast cancer group (BC), healthy control group (HC), and benign breast disease group (BBD). Figure 10 C represents the difference in ANXA6 levels between the breast cancer group and the healthy control group plus benign breast disease group (BC vs HC + BBD); Figure 10 D represents the differences in NLR, LMR, and PLR between the breast cancer group and the healthy control group plus benign breast disease group (BC vs HC + BBD). Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Experimental methods in the embodiments that do not specify specific conditions are generally performed according to conventional conditions or conditions recommended by the manufacturer; reagents or instruments used that do not specify the manufacturer are all conventional products that can be purchased commercially.

[0022] 1. Extraction and purification of cell migration bodies (1) Cell culture The following cell lines were used in this experiment: normal breast epithelial cells MCF-10A; triple-negative breast cancer cells Hs578T; HER2-overexpressing breast cancer cells SK-BR-3; luminal type A breast cancer cells MCF-7; and luminal type B breast cancer cells BT-474.

[0023] The cells were cultured at 37°C and 5% CO2. MCF-10A cells were cultured in a dedicated medium; SK-BR-3, Hs578T, and MCF-7 cells were cultured in DMEM complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics; and BT-474 cells were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics.

[0024] (2) Extraction of migration bodies The prepared cells were seeded into 150 mm cell culture dishes pre-coated with 1 µg / mL fibronectin and cultured for 24-48 h according to the cell proliferation rate. Once the cells reached a suitable density, the culture medium was discarded, and the cells were gently washed once with pre-cooled PBS. Trypsin-EDTA digestion solution was then added to prepare a single-cell suspension. All centrifugation steps were performed at 4°C. (1) Centrifuge at 600g for 10 min and collect the supernatant; (2) Centrifuge at 2000g for 20 min to remove cell debris and other impurities; (3) Centrifuge at 20,000g for 30 min to obtain crude extract of the migrating body.

[0025] The crude migratings were resuspended in extraction buffer and thoroughly mixed with 10% OptiPrep™ separation buffer at a 1:1 volume ratio, for a total volume of 2 mL. This mixture was gently spread onto a pre-prepared iodixanol density gradient, with gradients from bottom to top of 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, and 5%, each gradient volume being 1 mL. The mixture was then ultracentrifuged at 150,000 g for 4 h. After centrifugation, each gradient fraction was slowly collected from top to bottom, 1 mL per tube. Fractions from layers 4-6 were collected, mixed with 1 mL of PBS, and centrifuged at 18,000 g for 30 min. The resulting precipitate was resuspended in PBS, washed, and then centrifuged again at 20,000 g for 30 min. The final purified migrating precipitate was used directly for subsequent experiments.

[0026] 2. Identification and characterization of migrants (1) Observation by confocal laser scanning microscope Cells were pre-coated with 10 µg / mL fibronectin solution in 35 mm confocal microscopy dishes at 37°C for at least 30 min. Cells were then seeded into the treated dishes and cultured at 37°C with 5% CO2 for 16–20 h. Cells were fixed with 4% paraformaldehyde at room temperature for 10 min, washed 2–3 times with PBS, and incubated with 1 µg / mL WGA-Alexa Fluor™ 488 staining solution for 10 min in the dark. After washing with PBS, images were acquired using a high-resolution laser scanning confocal microscope. (See also...) Figure 1 A. Migratory bodies are visible at the ends of contractile fibers at the posterior margin of the cell, and are marked with green fluorescence.

[0027] (2) Observation by scanning electron microscope Place 25 mm cell-specific glass slides in a six-well plate and coat the slides with 10 µg / mL fibronectin at 37°C for at least 30 min. Seed the cells onto the slides and culture for 16–20 h as usual. Add 1 mL of 2.5% glutaraldehyde fixative and fix at room temperature for 2 h. Discard the fixative and wash three times with PBS for 15 min each time. Then fix with 1% osmium tetroxide in the dark for 1–2 h. After fixation, discard the osmium tetroxide and wash three more times with PBS for 15 min each time.

[0028] The samples were dehydrated using a gradient of ethanol: 30%, 50%, 70%, 80%, 90%, and 95% ethanol were applied sequentially for 15 minutes each, followed by 100% ethanol for 20 minutes. Fresh 100% ethanol was then used to continue dehydration. After dehydration, the samples were dried using a critical point dryer. The dried samples were then fixed to a carbon support film stage with conductive adhesive and ion sputtered with platinum for approximately 120 seconds. (See [link to relevant documentation]). Figure 1 B. Images were acquired using a Hitachi Regulus 8100 scanning electron microscope.

[0029] (3) Observation by transmission electron microscopy Add 10 μL of the purified migrating suspension to a copper grid and allow it to stand for 1 min to adsorb. Carefully blot away excess liquid with filter paper, add 10 μL of uranium acetate staining solution, and negatively stain at room temperature for 1 min. Blot away the staining solution again with filter paper, allow to air dry at room temperature for several minutes, and then observe and acquire images using a Hitachi HT7800 transmission electron microscope at an accelerating voltage of 80-120 kV. See [link to documentation] Figure 1 C. The results showed that the extracted vesicles had typical migratory body morphology and structure, with a diameter of approximately 0.5-3 μm.

[0030] (4) Western blot verification of migration body-specific proteins Equal amounts of cells and migratory proteins were taken and subjected to SDS-PAGE electrophoresis. After transfer to a membrane, the cells were incubated overnight at 4°C with primary antibodies against ITGB1, EOGT, PIGK, GM130, and β-actin, respectively. After washing, the cells were incubated with secondary antibodies and then subjected to ECL staining.

[0031] See Figure 1 D、 Figure 1 E. The results showed that: ① the migratory bodies expressed specific proteins ITGB1, EOGT, and PIGK, but did not express the cellular protein GM130; ② in the cells, the SK-BR-3 and BT474 cell lines did not express ITGB1 and EOGT; ③ all cells expressed specific proteins GM130 and β-actin. The above results proved that the migratory bodies were successfully purified.

[0032] 3. Protein proteomic analysis of the migration body and screening of candidate biomarkers (1) Protein spectrum analysis Proteomic analysis was performed on migratory bodies derived from breast cancer cell lines (Hs578T, SK-BR-3, MCF-7, BT474) and migratory bodies derived from normal breast epithelial cells (MCF-10A), with three biological replicates for each group.

[0033] See Figure 2 Principal component analysis results show that the contribution rates of principal component 1 and principal component 2 are 31.2% and 21.3%, respectively. See Figure 3 , Figure 4The results showed that, except for the Hs578T cell migration proteomic data, whose intra-group correlation coefficients ranged from 0.695 to 1.000, the intra-group correlations of the other cell migrations were all ≥0.883, indicating good correlation between samples. The inter-group correlation coefficients of all cell migrations were greater than or equal to 0.448, indicating that there was also good correlation between migrations from different cell types.

[0034] (2) Screening of differentially expressed proteins Using MCF-10A cell migrations derived from normal breast epithelial cells as a control, and with a |log2 fold change| ≥ 1 and P ≤ 0.05 as the screening threshold for differentially expressed proteins, differential expression analysis was performed on the proteomic profiles of four breast cancer cell lines and MCF-10A cell migrations.

[0035] See Figure 5 , Figure 6 , Figure 7 , Figure 8 The results showed that: ① Hs578T-derived migrations upregulated 133 proteins, downregulated 621 proteins, and had no significant difference in 2512 proteins; ② SK-BR-3-derived migrations upregulated 449 proteins, downregulated 1179 proteins, and had no significant difference in 1638 proteins; ③ MCF-7-derived migrations upregulated 750 proteins, downregulated 662 proteins, and had no significant difference in 1854 proteins; ④ BT474-derived migrations upregulated 341 proteins, downregulated 803 proteins, and had no significant difference in 2122 proteins.

[0036] See Figure 5 , Figure 6 , Figure 7 A total of 156 commonly expressed differentially expressed proteins were screened from four types of breast cancer cell migrations, of which 9 were commonly upregulated and 141 were commonly downregulated. For differential expression data, please refer to [link to differential expression data]. Figure 8 .

[0037] (3) Candidate biomarker determination Based on the comprehensive proteomic results, annexin A6 (ANXA6) was selected from nine commonly upregulated proteins as a candidate diagnostic biomarker for further clinical validation.

[0038] 4. Isolation of serum migratory bodies and detection of ANXA6 levels (clinical validation) (1) Serum sample collection Serum samples in this embodiment were all collected from the First Affiliated Hospital of Army Medical University. All cases were pathologically confirmed, and all samples were collected at the initial diagnosis, before any treatment was received. The specific definitions are as follows: ① Benign breast disease (BBD), including but not limited to the following pathological types: fibroadenoma of the breast, intraductal papilloma of the breast, cystic hyperplasia of the breast, inflammatory lesions of the breast, and adenosis of the breast.

[0039] ② Breast Cancer (BC): Primary breast cancer confirmed by pathological histology, including the following molecular subtypes: triple-negative breast cancer (TNBC), HER2 overexpression type, luminal A type, and luminal B type.

[0040] A total of 129 serum samples were included in this study, comprising a healthy control group (HC, n=30), a benign breast disease group (BBD, n=34), and a breast cancer group (BC, n=65). Peripheral blood inflammatory and immune markers NLR (neutrophil-to-lymphocyte ratio), LMR (lymphocyte-to-monocyte ratio), and PLR (platelet-to-lymphocyte ratio) were also collected for comparative analysis.

[0041] (2) Isolation of serum migrants Blood was collected using a yellow-topped centrifuge tube and centrifuged at 1000g for 10 minutes at 4°C to remove blood cells. The serum was then transferred to a 2 mL low-absorption centrifuge tube and centrifuged at 2000g for 10 minutes at 4°C to further remove cell debris and residual blood cells. The supernatant was collected into a 1.5 mL low-absorption centrifuge tube. The tube was then centrifuged at 20,000g for 60–90 minutes at 4°C, and 90% of the supernatant was aspirated. 900 µL of PBS was added, and the tube was centrifuged again at 20,000g for 10 minutes at 4°C, and the remaining supernatant was aspirated. The resulting precipitate is the serum mitozoite, used for subsequent experiments.

[0042] (3) Identification of serum migrants See Figure 9 A. Transmission electron microscopy imaging showed that the isolated serum migratory bodies exhibited typical migratory body structures. See also Figure 9 B. Western blot analysis confirmed the presence of migration-specific marker proteins ITGB1, PIGK, and target protein ANXA6 in serum migrations.

[0043] (4) Preparation of migratory body lysis buffer Collect 1 mL of serum migratory body precipitate and add RIPA lysis buffer (high potency, main components: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and multiple inhibitors such as sodium orthovanadate, sodium fluoride, EDTA, and leupeptin). Incubate on ice for 20 min, gently shaking every 10 min to ensure complete lysis of the migratory bodies. Centrifuge the lysed migratory body suspension at 1000 g for 10 min at pre-cooled 4°C. Carefully aspirate the supernatant and transfer it to a new sterile centrifuge tube, avoiding aspiration of the bottom precipitate. This is the migratory body lysis buffer.

[0044] (5) ELISA detection of ANXA6 content ① Reagent preparation Remove the kit and allow it to equilibrate at room temperature for 30 minutes. Prepare the washing buffer, standard dilution buffer, and other reagents according to the instructions.

[0045] ② Standard product preparation Take out the ANXA6 standard and perform serial dilutions with the standard diluent provided with the kit to prepare a series of standard curve working solutions (0, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 ng / mL). Prepare and use immediately.

[0046] ③Sampling and Capture Add the lysis buffer of the standard and clinical sample migrations to each well of the ELISA plate, with 3 replicates per group and a sample volume of 100 μL / well. After sample addition, seal the ELISA plate with sealing film and incubate at 37°C for 60 min to allow ANXA6 in the sample to fully bind to the anti-ANXA6 capture antibody pre-coated on the ELISA plate. After incubation, discard the liquid in the wells, shake off excess water, add 330 μL of washing buffer to each well, let stand for 30 s, then discard. Repeat the washing process 3 times, patting dry on absorbent paper after each wash.

[0047] ④ Add enzyme-labeled detection antibodies Add 100 μL of horseradish peroxidase (HRP)-labeled anti-ANXA6 detection antibody working solution to each well. Seal the ELISA plate with sealing film and incubate at 37°C for 60 min to allow the detection antibody to specifically bind to ANXA6 bound to the capture antibody. After incubation, discard the liquid in the wells, shake off excess water, add 330 μL of washing buffer to each well, let stand for 30 s, then discard. Repeat the washing process 5 times, patting dry on absorbent paper after each wash to remove unbound enzyme-labeled antibody.

[0048] ⑤ Color development Add 90 μL of TMB substrate solution to each well, gently shake the plate to distribute the substrate solution evenly, and incubate at 37°C in the dark for 10-20 min. During this period, closely observe the color change of the standard wells. When a clear gradient of blue appears in the standard wells, stop the reaction immediately.

[0049] ⑥ Termination and Measurement Add 50 μL of stop solution (2 M sulfuric acid) to each well, gently shake to mix, and terminate the colorimetric reaction. At this point, the solution will rapidly change from blue to yellow. Ensure that there are no water droplets at the bottom of the ELISA plate and no air bubbles in the wells. Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0050] ⑦ Result Calculation A standard curve was plotted with the concentration of the standard on the x-axis and the OD value on the y-axis, and the regression equation was calculated. The OD value of the test sample was then substituted into the regression equation to calculate the actual expression concentration of ANXA6 in the migratory lysate.

[0051] 5. Application of migratory ANXA6 in the diagnosis of breast cancer (1) Analysis of differences in expression levels The concentrations of ANXA6 in serum migratory cells from the HC, BBD, and BC groups were measured using ELISA. Simultaneously, peripheral blood inflammatory immune markers NLR, LMR, and PLR were also measured. The results showed: ①See Figure 10 As shown in Figures A and C, there were significant differences in ANXA6 levels among BC, BBD, and HC (P<0.005). ②NLR and PLR differ only between BC and HC; ③See Figure 10 As shown in Figure B, LMR differs between BC and HC, and between BC and BBD; ④See Figure 10 As shown in Figure D, the differences in ANXA6, NLR, LMR, and PLR between BC and HC+BBD were statistically significant, with P values ​​of <0.0001, 0.0039, <0.0001, and 0.0134, respectively.

[0052] (2) ROC curve analysis The diagnostic efficacy of ANXA6, NLR, LMR, and PLR was evaluated using ROC curve analysis. Table 1 shows the ROC curve analysis between breast cancer patients and healthy individuals.

[0053] Table 2 shows the ROC curve analysis of breast cancer vs. benign breast diseases:

[0054] Table 3 shows the ROC curve analysis of breast cancer vs. healthy individuals + benign breast diseases:

[0055] Conclusion: 1. As shown in Table 1, when the ROC curve distinguishes between breast cancer (BC) and healthy individuals (HC), the AUC value of ANXA6 is 0.959 (95% CI: 0.922-0.995), with a sensitivity of 93.8%, a specificity of 85.3%, and a cutoff value of 0.778 ng / mL. Its diagnostic efficacy is significantly better than traditional inflammatory and immune markers such as NLR, LMR, and PLR.

[0056] 2. Referring to Table 2, when the ROC curve distinguishes between breast cancer (BC) and benign breast disease (BBD), the AUC value of ANXA6 is 0.714 (95% CI: 0.610-0.818), with a sensitivity of 47.7%, a specificity of 93.3%, and a cutoff value of 1.787 ng / mL; when combined with LMR for diagnosis, the AUC increases to 0.764.

[0057] 3. Referring to Table 3, when the ROC curve distinguishes between breast cancer (BC) and "healthy individuals with benign breast disease" (HC+BBD), the AUC value of ANXA6 is 0.844 (95% CI: 0.779-0.909), with a sensitivity of 81.5%, a specificity of 75.0%, and a cutoff value of 1.130 ng / mL; when combined with NLR, LMR, and PLR for diagnosis, the AUC increases to 0.864.

[0058] In summary, ANXA6 performed best in distinguishing breast cancer patients from healthy individuals (AUC 0.959), and its combined use with inflammatory and immune markers can further improve diagnostic accuracy.

[0059] All data were statistically analyzed using SPSS 26.0 and GraphPad Prism 10 software. Comparisons between two groups were performed using t-tests or Mann-Whitney U tests, and comparisons among multiple groups were performed using one-way ANOVA. ROC curve analysis was used to calculate AUC, sensitivity, specificity, and Youden's index. A p-value < 0.05 was considered statistically significant.

[0060] The above experimental results show that when serum ANXA6 is used to diagnose breast cancer, the area under the curve (AUC) reaches 0.959, the diagnostic sensitivity is 93.8%, and the specificity is 85.3%, indicating that it can be used as a biomarker for breast cancer diagnosis.

[0061] The English abbreviations, full English names, and Chinese translations used in the instruction manual are shown in Table 4:

[0062] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. The application of migratory somatic annexin A6 as a biomarker in the preparation of a kit for diagnosing breast cancer, characterized by: The kit diagnoses breast cancer by detecting the level of migratory annexin A6 in serum samples, wherein the migratory vesicles are extracellular vesicles obtained from serum by density gradient centrifugation.

2. The application according to claim 1, characterized in that, The reagent used to detect the content of migratory annexin A6 is an anti-annexin A6 antibody.

3. The application according to claim 2, characterized in that, The kit also includes reagents for isolating the migrants from serum.

4. The application according to claim 3, characterized in that, The diagnostic threshold for the migratory somatic annexin A6 is 0.5–2.0 ng / mL.

5. The application according to claim 4, characterized in that, The migratory myeloprotein A6 differentiated breast cancer from healthy controls with an AUC of 0.959, a sensitivity of 93.8%, and a specificity of 85.3%.

6. The application according to claim 5, characterized in that, The diagnostic threshold for the migratory myeloprotein A6 was 0.778 ng / mL.

7. The application according to any one of claims 1 to 6, characterized in that, The serum samples were derived from humans.

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