A tumor cell intracellular bacterial immunohistochemical detection kit and its use method and application

Abstract

The application discloses a tumor cell intracellular bacterial immunohistochemical detection kit and a use method and application thereof, and belongs to the technical field of tumor detection. The application takes lipoteichoic acid (LTA) and bacterial lipopolysaccharide (LPS) as a breakthrough point, combines immunohistochemical detection, and uses LTA and LPS antibodies to detect the expression of gram-positive bacteria and gram-negative bacteria in tumor cells respectively. It is first confirmed that the expression levels of LTA and LPS in breast cancer tissues increase with the increase of the malignant degree, the stage and the metastasis potential of tumors. The potential risk of lymph node metastasis of tumor tissues with high expression of LTA and LPS increases, and the expression of LTA / LPS in positive metastatic lymph nodes is higher than that in negative lymph nodes. More importantly, the expression of LTA and LPS in breast cancer tumor tissues is related to the overall survival (OS) and recurrence-free survival (RFS) of patients, can be used as a marker for evaluating the malignancy of breast cancer and predicting tumor metastasis, and is helpful for stratifying high-risk and poor-prognosis patients.

Description

Technical Field

[0001] This invention belongs to the field of tumor detection technology, specifically relating to a tumor cell intracellular bacterial immunohistochemical detection kit and its usage and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Breast cancer is one of the most common malignant tumors in women, with complex and heterogeneous pathological types. Some subtypes are highly aggressive and prone to metastasis and recurrence. Although the widespread use of early screening methods has improved overall prognosis, the survival rate of patients with advanced-stage breast cancer remains unsatisfactory. According to the latest report from the American Cancer Society (ACS), breast cancer has ranked first in incidence among female malignant tumors for many years, and second in mortality among cancer-related deaths in women. With population aging and changes in lifestyle, it is projected that by 2040, the number of new breast cancer cases will increase by 40% annually, continuing to pose a significant threat to women's health worldwide. While significant progress has been made in the diagnosis and treatment of breast cancer, effective combined testing is still needed in clinical practice to improve overall treatment outcomes.

[0004] Immunohistochemistry (IHC) utilizes the principle of specific antigen-antibody binding. Through a chemical reaction, a chromogenic agent (fluorescein, enzyme, metal ions, isotopes) labeled with an antibody is colored to identify antigens (peptides and proteins) within tissues or cells, allowing for their localization, qualitative analysis, and relative quantification. For example, enzymes can be covalently linked to antibodies to create enzyme-labeled antibodies. The enzyme's specific catalytic action on the substrate then generates colored, insoluble products or particles with a specific electron density, which can be used to localize various antigenic components on the cell surface or within cells under a conventional microscope or electron microscope. Currently, indirect staining using immunohistochemistry is commonly used. This technique allows for the detection of various antigenic substances, such as proteins, peptides, enzymes, hormones, pathogens, and receptors, at the tissue or cellular level. Due to its high specificity, sensitivity, and accurate localization, and its ability to organically combine morphological and functional studies, this new technology has been widely applied in many fields of biological and medical research. In pathological research, the role and significance of immunohistochemistry are even more important.

[0005] Specific intracellular bacteria exist within tumors and the tumor microenvironment, influencing overall susceptibility to tumor development and progression, concurrent infections, and response to anticancer therapy. Lipoteichoic acid (LTA) is a key component of the cell wall of Gram-positive bacteria, while bacterial lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. The inventors have found that there are currently no reported methods for using LTA and LPS immunohistochemical detection of Gram-negative and Gram-positive bacteria within tumor cells to assess the malignancy, metastatic potential, and patient prognosis of breast cancer. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an immunohistochemical detection kit for bacteria in tumor cells, along with its usage and applications. This invention focuses on lipoteichoic acid (LTA) and bacterial lipopolysaccharide (LPS), combining immunohistochemical detection with anti-LTA and anti-LPS antibodies to detect the expression of Gram-positive and Gram-negative bacteria in tumor cells, respectively. It is the first to demonstrate that the expression levels of LTA and LPS in breast cancer tissues increase with increasing tumor malignancy, stage, and metastatic potential. High expression of LTA and LPS in tumor tissues increases the potential risk of lymph node metastasis, and LTA / LPS expression is higher in positive metastatic lymph nodes than in negative lymph nodes. More importantly, LTA and LPS expression in breast cancer tumor tissues is associated with overall survival (OS) and recurrence-free survival (RFS), serving as biomarkers for assessing breast cancer malignancy and predicting tumor metastasis, and aiding in the stratification of high-risk and poorly prognostic patients.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides the application of LTA / LPS as a diagnostic and malignancy assessment marker and / or prognostic marker in the preparation of breast cancer-related detection products.

[0009] Among them, the breast cancer-related testing products include the LTA / LPS immunohistochemical detection kit, which can be used to determine the malignancy of breast cancer, the potential for tumor metastasis, and the patient's prognosis.

[0010] In a second aspect, the present invention provides a kit for the diagnosis and / or prognostic assessment of breast cancer, the kit comprising reagents for detecting LTA / LPS expression;

[0011] Preferably, the kit is an LTA / LPS immunohistochemical detection kit.

[0012] Thirdly, the present invention provides an LTA / LPS immunohistochemical detection kit, the LTA / LPS immunohistochemical detection kit comprising at least the following components: primary antibody, secondary antibody, reagent for inactivating peroxidase and biotin in tissue sections, chromogenic reagent for reacting with horseradish peroxidase and developing color, and reagent for specifically staining cell nuclei in tissues;

[0013] Specifically, the primary antibody refers to anti-LTA and anti-LPS specific antibodies prepared against human LTA and LPS antigens, such as rabbit anti-human LTA / LPS polyclonal antibodies, etc., without being specifically limited here.

[0014] Correspondingly, the secondary antibody only needs to match the species, category, or subclass of the primary antibody. For example, when the primary antibody is a rabbit anti-human LTA / LPS polyclonal antibody, the secondary antibody can be goat anti-rabbit IgG (goat anti-rabbit IgG-HRP).

[0015] Fourthly, the present invention provides a method for using the above-mentioned LTA / LPS immunohistochemical detection kit for immunohistochemical staining diagnosis of tissue sections, the method of use comprising:

[0016] S1. Take the tissue sample from the subject, fix it, and prepare a paraffin slide.

[0017] S2. The treated paraffin tablets reacted with primary and secondary antibodies in sequence.

[0018] S3. After the reaction is complete, the slides are washed, stained with DAB, counterstained with hematoxylin, dehydrated, cleared, and mounted for microscopic observation.

[0019] Fifthly, the present invention provides the application of the above-described LTA / LPS immunohistochemical detection kit and / or method in tumor detection.

[0020] The tumor in question is breast cancer;

[0021] The tumor detection specifically includes: determining the malignancy of breast cancer, assisting in staging, tumor metastasis potential, and patient prognosis.

[0022] One or more of the above technical solutions have the following advantages or beneficial effects:

[0023] The above technical solution is based on the principle that higher LTA / LPS immunohistochemical staining intensity in breast cancer tissue indicates higher malignancy and metastatic potential, a greater likelihood of lymph node metastasis, and lower overall survival (OS) and recurrence-free survival (RFS); conversely, lower intensity LTA / LPS staining indicates lower malignancy and metastatic potential, and higher OS and RFS. Therefore, an LTA / LPS immunohistochemical detection kit was developed. This kit can effectively distinguish breast cancer tissue from normal tissue, assess tumor malignancy, metastatic potential, and patient prognosis, and provide new diagnostic and classification criteria for individualized treatment of breast cancer.

[0024] The above-mentioned kits can effectively predict the malignancy and lymph node metastasis potential of breast cancer and assess the risk of recurrence and metastasis. They can also assess the patient's prognosis, thereby helping to improve the survival rate of breast cancer patients and providing important scientific basis for the prevention and treatment of breast cancer in clinical practice. Therefore, they have good clinical application value and social benefits. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 These are the survival curves of patients with high LTA expression (red) and low LTA expression (blue) in this embodiment of the invention;

[0027] Figure 2 These are the survival curves of patients with high LPS expression (red) and low LPS expression (blue) in this embodiment of the invention;

[0028] Figure 3 These are the relapse-free survival curves for patients with high LTA expression (red) and low LTA expression (blue) in this embodiment of the invention;

[0029] Figure 4 These are the relapse-free survival curves for patients with high LPS expression (red) and low LPS expression (blue) in this embodiment of the invention.

[0030] Figure 5 These are IHC staining images and statistical diagrams showing the expression of LTA in normal tissues, tumor tissues, and metastatic tumor tissues, respectively, in embodiments of the present invention.

[0031] Figure 6 These are IHC staining images and statistical diagrams of LPS expression in normal tissues, tumor tissues, and metastatic tumor tissues, respectively, in embodiments of the present invention.

[0032] Figure 7 These are IHC staining images and statistical graphs of LTA expression in negative and positive lymph nodes, respectively, in embodiments of the present invention.

[0033] Figure 8 These are IHC staining images and statistical diagrams of LPS expression in negative and positive lymph nodes, respectively, in embodiments of the present invention.

[0034] Figure 9 This is the ROC curve for predicting lymph node metastasis using LTA in an embodiment of the present invention;

[0035] Figure 10 This is the ROC curve for predicting lymph node metastasis using LPS in an embodiment of the present invention. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0038] As mentioned earlier, there are currently no reported methods using immunohistochemistry to assess the malignancy, metastatic potential, and prognosis of breast cancer by detecting the expression of Gram-positive and Gram-negative bacteria in tumor cells using antibodies against lipoteichoic acid (LTA) and bacterial lipopolysaccharide (LPS), respectively. The inventors, through immunohistochemical detection of LTA / LPS in breast cancer tissue, found that LTA / LPS protein expression in breast cancer tissue was significantly higher than in surrounding non-cancerous tissue, and its expression increased significantly with increasing tumor malignancy, stage, and metastasis. This invention is the first to demonstrate that LTA / LPS expression is higher in positive lymph nodes than in negative lymph nodes, suggesting that tumor cells with high LTA / LPS expression have stronger invasive and metastatic capabilities; ROC curves further confirm that both LTA / LPS can effectively predict lymph node metastasis. Furthermore, the overall survival and recurrence-free survival of breast cancer patients with high LTA / LPS expression are lower than those of breast cancer patients with low LTA / LPS expression. Therefore, the expression level of LTA / LPS is closely related to patient survival, tumor malignancy, lymph node metastasis, and TNM stage. Higher LTA / LPS immunohistochemical staining intensity in breast cancer tissue indicates higher malignancy and lymph node metastasis potential; conversely, lower LTA / LPS expression indicates lower malignancy and metastasis potential. High LPS expression is an independent adverse prognostic factor for breast cancer patients. Based on these findings, this invention is proposed.

[0039] In one specific embodiment of the present invention, an application of LTA / LPS as a diagnostic and malignancy assessment marker and / or prognostic marker for predicting tumor metastasis is provided in the preparation of breast cancer-related detection products.

[0040] Among them, the breast cancer-related testing products include the LTA / LPS immunohistochemical detection kit, which can be used to determine the malignancy of breast cancer, the potential for tumor metastasis, and the patient's prognosis.

[0041] In another specific embodiment of the present invention, a kit for the diagnosis and / or prognostic assessment of breast cancer is provided, the kit comprising reagents for detecting LTA / LPS expression;

[0042] Preferably, the kit is an LTA / LPS immunohistochemical detection kit. In another specific embodiment of the present invention, an LTA / LPS immunohistochemical detection kit is provided, the LTA / LPS immunohistochemical detection kit comprising at least the following components: primary antibody, secondary antibody, reagents for inactivating peroxidase and biotin in tissue sections, a chromogenic reagent that reacts with horseradish peroxidase and develops color, and a reagent specifically for staining cell nuclei in tissues;

[0043] The primary antibody specifically refers to LPS and LTA-specific antibodies prepared against human LPS and LTA antigens, such as rabbit anti-human LTA / LPS polyclonal antibodies, etc., without being specifically limited here.

[0044] Correspondingly, the secondary antibody only needs to match the species, category, or subclass of the primary antibody. For example, when the primary antibody is a rabbit anti-human LTA / LPS polyclonal antibody, the secondary antibody can be goat anti-rabbit IgG (goat anti-rabbit IgG-HRP).

[0045] The reagent used to inactivate peroxidase and biotin in the tissue sections can be a 0.3% H2O2 solution.

[0046] The colorimetric reagent that reacts with horseradish peroxidase and produces a color is a DBA solution.

[0047] The reagent specifically used for staining cell nuclei in tissues can be hematoxylin.

[0048] In another specific embodiment of the present invention, the LTA / LPS immunohistochemical detection kit may further include a fixative, a dewaxing and hydration agent, a serum blocking agent, a buffer solution, an antibody diluent, a dehydrating and clearing agent, etc.

[0049] The fixative can be a neutral buffered 4% paraformaldehyde solution, Bouin's solution, or mDF solution.

[0050] The serum blocking agent is generally an antibody from the animal's own serum, which is from the same source as the secondary antibody, and can bind to sites in the tissue that can bind to the secondary antibody beforehand; calf serum, BSA, sheep serum, etc. can also be used, but the source cannot be the same as the primary antibody.

[0051] The buffer solution can be PBS buffer.

[0052] PBS can be used as antibody diluent, or a special antibody diluent containing sodium azide preservative, BSA stabilizer, etc., which facilitates multiple recycling of antibodies, can be used.

[0053] The dewaxing hydration agent and the dehydrating and clarifying agent are both xylene and ethanol (including anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol), but they are used in completely opposite order.

[0054] In another specific embodiment of the present invention, a method for using the above-mentioned LTA / LPS immunohistochemical detection kit for immunohistochemical staining diagnosis of tissue sections is provided, the method comprising:

[0055] S1. Take the tissue sample from the subject, fix it, and prepare a paraffin slide.

[0056] S2. The treated paraffin tablets reacted with primary and secondary antibodies in sequence.

[0057] S3. After the reaction is complete, the slides are washed, stained with DAB, counterstained with hematoxylin, dehydrated, cleared, and mounted for microscopic observation.

[0058] In step S1, the tissue to be tested can be breast tissue (such as breast cancer tissue).

[0059] In step S2, the paraffin sheet processing procedure includes: dewaxing and hydrating the prepared paraffin sheet, performing antigen repair, then inactivating peroxidase and biotin and performing serum blocking.

[0060] In step S3, the microscopic observation includes: randomly selecting three fields of view for each sample, with approximately 500 cells per group; semi-quantitative scoring based on hematoxylin staining intensity (0, negative; 1, +; 2, ++; 3, +++); and classifying positive cells into four levels based on the percentage (<1%, 0 points; 1-25%, 1 point; 26-50%, 2 points; 60-75%, 3 points; 76-100%, 4 points); combining the two scores for each sample to obtain the final LTA / LPS expression score (IHC sum score). The cutoff points for expression levels are defined as: 0-4 points, low expression; 5-8 points, high expression.

[0061] In another specific embodiment of the present invention, the above-described LTA / LPS immunohistochemical detection kit and / or the above-described method of use are provided for application in tumor detection.

[0062] The tumor in question is breast cancer.

[0063] The tumor detection specifically includes: determining the malignancy of breast cancer, assisting in staging, tumor metastasis potential, and patient prognosis.

[0064] Preferably, the tumor metastatic potential includes lymph node metastatic potential.

[0065] Preferably, the patient prognosis includes overall survival and recurrence-free survival for breast cancer patients.

[0066] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0067] Example 1

[0068] The method for assessing the malignancy, metastatic potential, and prognosis of breast cancer using an LTA / LPS immunohistochemical detection kit includes three parts: sample collection and processing, immunohistochemical experiments and result evaluation, and statistical analysis of the obtained data. Specifically, it is divided into the following steps:

[0069] 1. Sample Collection and Processing

[0070] A total of 261 breast cancer tumor tissues, related adjacent normal tissues, and some lymph node tissues were collected from the Affiliated Hospital of Qingdao University. Detailed clinical and pathological data were collected, and follow-up data were recorded, including overall survival (OS) and recurrence-free survival (RFS) at the last visit to the hospital. Tumor staging and histological classification were determined according to the American Joint Committee on Cancer (AJCC) 8th edition. This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (QYFY WZLL 28840), and informed consent was obtained from all participants.

[0071] 2. Perform immunohistochemical experiments and evaluate the results.

[0072] (1) Tissue Fixation: After obtaining breast cancer samples from the patient and adjacent normal tissue samples, fixation is required to maintain the inherent morphology and structure of the cells, preserve the antigenicity of the tissue cells, remove lipids that hinder antigen-antibody binding, and facilitate good staining results for the antigen-antibody conjugates. 4% neutral buffered paraformaldehyde solution is commonly used (Bouin's solution, mDF solution, etc., can also be used). The tissue fixation time is best within 12 hours and should generally not exceed 24 hours. This is because the detection intensity of tissue antigens will gradually decrease with prolonged fixation time.

[0073] (2) Slide Preparation: Paraffin slides can be made. Paraffin slides are easy to preserve, have a beautiful structure, and can be preserved for a long time. First, dehydrate the tissue with a gradient of ethanol (from low to high, usually starting with 50% alcohol, then 70%, 85%, 95% to pure alcohol), then soak the tissue in a clearing agent, then completely soak the tissue in paraffin, and finally section it. Generally, paraffin sections are about 5μm in size. After sectioning, mount (lift) and bake the slides. A thin layer of protein glycerin can be spread on the glass slide, then the paraffin slide can be flattened in warm water (about 45℃), lifted and placed on the glass slide, excess water can be absorbed with filter paper, and the glass slide can be placed in a 45℃ oven to dry.

[0074] (3) Dewaxing and hydration of paraffin slides: In order for antibodies and other reagents to fully bind and react with antigens in the tissue, breast cancer sample slides need to be washed sequentially with xylene and graded ethanol (from high to low). Xylene I 10 min, xylene II 10 min; anhydrous ethanol 5 min, 95% ethanol 5 min, 85% ethanol 5 min, 75% ethanol 5 min; phosphate buffered saline (PBS) wash twice, and place on a shaker.

[0075] (4) Antigen retrieval: During formaldehyde or paraformaldehyde fixation, some antigens in tissues undergo protein cross-linking and aldehyde group blocking, thus losing their antigenicity. Antigen retrieval allows intracellular antigenic determinants to be re-exposed, improving antigen detection rates. Commonly used retrieval methods can be divided into three types from strongest to weakest: high-pressure retrieval, microwave retrieval, and trypsin retrieval. Generally, tissue sections can be placed in a retrieval solution and then microwaved at medium heat for 6 minutes * 4 times. Note that after microwave retrieval, allow it to cool naturally for about 30 minutes.

[0076] (5) Inactivation of peroxidase and biotin: If the chromogenic system is a peroxidase (HRP) system, or in the traditional ABC and SP methods, the immunohistochemical reaction results are easily interfered with by endogenous peroxidase and biotin. To prevent non-specific color development, inactivation with hydrogen peroxide and avidin is necessary. Furthermore, some tissues may have high levels of endogenous peroxidase, requiring inactivation. The sample slide can be incubated in 0.3% H2O2 for 10 min. Then wash twice in PBS solution for 10 min each time.

[0077] (6) Serum blocking: Serum blocking is used to eliminate non-specific binding sites and non-specific staining. The blocking serum is generally the animal's own serum from the same source as the secondary antibody, and it can bind to sites in the tissue that can bind to the secondary antibody beforehand; calf serum, BSA, sheep serum, etc. can also be used, but the source cannot be the same as the primary antibody. It can be blocked at room temperature for 10-30 minutes to prevent over-blocking.

[0078] (7) Primary and secondary antibody treatment:

[0079] There are several incubation temperatures for primary antibodies: 4℃, room temperature, and 37℃, with 4℃ yielding the best results. Incubation time depends on temperature and antibody concentration. It can be incubated at 37℃ for 1-2 hours, while at 4℃ it needs to be incubated overnight, followed by a 45-minute warm-up at 37℃ after removal from the refrigerator.

[0080] Secondary antibodies can be incubated at room temperature or 37°C for 30 minutes to 1 hour. Furthermore, the species, class, or subclass of the secondary antibody must match that of the primary antibody.

[0081] In this embodiment, the primary antibody used in immunohistochemistry was a specific monoclonal antibody against human LPS and LTA antigens (Hycult Biotech, LPS: HM6011; LTA: HM5018). The secondary antibody was the corresponding anti-mouse antibody.

[0082] PBS can be used as antibody diluent, or a special antibody diluent containing sodium azide preservative, BSA stabilizer, etc., which facilitates multiple recycling of antibodies, can be used.

[0083] (8) Slide cleaning (immersion, rinsing and washing): In order to prevent non-specific staining caused by the residue of primary and secondary antibodies, it is especially important to strengthen the cleaning (extend the time and increase the number of times). For example, cleaning before primary antibody incubation: 3 min * 3 times, cleaning after primary antibody incubation: 5 min * 5 times.

[0084] (9) DAB staining: Add DBA solution, develop color, observe the brown background under an optical microscope, and rinse with tap water immediately to stop the staining. The DAB staining time is not fixed and is mainly controlled by the microscope. Rinse when strong specific staining and light background staining are observed.

[0085] (10) Counterstaining: Hematoxylin counterstaining (nuclear dye) is commonly used. The purpose is to form cell outlines, thereby better locating the target protein. After counterstaining, the slide is rinsed with running water, then placed in hydrochloric acid alcohol for a few seconds, rinsed with running water again, and finally placed in ammonia water to return to blue.

[0086] (11) Dehydration and Clearing Mounting: The dehydration and clearing process is the reverse of the dewaxing process. Clean the slides sequentially with a gradient of ethanol (from low to high) followed by xylene: 75% ethanol 5 min → 85% ethanol 5 min → 95% ethanol 10 min → anhydrous ethanol 10 min → xylene 10 min. Absorb the liquid and allow to air dry at room temperature. For clearing, xylene II for 10 min and xylene I for 10 min can be used. For long-term preservation, neutral resin is generally used for mounting. A drop of mounting solution can be placed directly on the tissue on the slide, then a coverslip is placed on top, taking care to avoid air bubbles. Allow to air dry for half a day.

[0087] (12) Observe under a microscope and evaluate the results.

[0088] Immunohistochemical staining scoring method: Two experienced pathologists independently performed blind evaluations. When significant differences existed (score difference > 2), a third pathologist was required to analyze the results. Observation was performed under an optical microscope, with three fields of view randomly selected for each sample, approximately 500 cells per group. The staining of the target protein within tumor cells was assessed: a semi-quantitative scoring method was used based on hematoxylin staining intensity (0, negative; 1, +; 2, ++; 3, +++), and the percentage of positive cells was categorized into four levels (<1%, 0 points; 1-25%, 1 point; 26-50%, 2 points; 60-75%, 3 points; 76-100%, 4 points). The two scores for each sample were combined to obtain the final LTA / LPS expression score (IHC sum score). The cutoff points for expression levels were defined (verified by X-tiles): 0-4 points, low expression; 5-8 points, high expression.

[0089] 3. Statistical analysis of the obtained data.

[0090] The correlation between LTA / LPS expression and clinicopathological parameters was analyzed using the chi-square test or Fisher's exact test. Cumulative overall survival and recurrence-free survival were calculated using the Kaplan-Meier method. Statistical differences between subgroups were calculated using the log-rank test. Multivariate analysis using the Cox regression model was used to identify independent prognostic factors.

[0091] Based on the above experiments and data analysis, such as Figure 1 and Figure 2 As shown, clinical data indicate that breast cancer patients with high LTA / LPS expression have lower overall survival (OS) than those with low LTA / LPS expression; Figure 3 and Figure 4 As shown, patients with high LTA and LPS expression tend to have lower RFS (recovery survival). These findings are the first to confirm that Gram-positive / negative bacterial expression within tumor cells is associated with survival in breast cancer patients. LTA / LPS expression is significantly higher than in surrounding non-cancerous tissues, and its expression also increases significantly with increasing tumor malignancy, stage, and metastasis. Figure 5 , Figure 6 The difference in LTA / LPS expression levels between negative and positive lymph nodes suggests that high LTA / LPS expression indicates a greater likelihood of lymph node metastasis in tumor tissue. Figure 7 , Figure 8 ).

[0092] Table 1. Correlation between LTA / LPS expression and clinicopathological features in breast cancer patients

[0093]

[0094] Table 2. Univariate and multivariate Cox proportional hazards analysis of breast cancer patients

[0095]

[0096]

[0097] As shown in Table 1, increased expression of LPS and LTA in tumor cells was associated with N stage (P<0.001) and TNM stage (P<0.001) in breast cancer patients, while their expression was not associated with age (P=0.942 / 0.366), tumor location (p=0.150 / 0.983), T stage (p=0.061 / 0.820), or pathological grade (P=0.641 / 0.438). To determine the role of LPS and LTA in overall survival (OS) of breast cancer patients, univariate analysis was performed using the Kernel-Müller method, as shown in Table 2. The results showed that patients with high LPS (P=0.001) and LTA expression (P=0.002), advanced N stage (P=0.007), and advanced TNM stage (P=0.015) had lower OS. Advanced pathological grade (P=0.063) also tended to indicate lower OS, although this tendency was not statistically significant. Further multivariate analysis revealed that high levels of LPS (P = 0.008) were an independent adverse prognostic factor.

[0098] Example 2: Validation of LTA / LPS Expression

[0099] Tumor tissues and related adjacent normal tissues from 130 breast cancer patients were collected from a hospital in Qingdao. The experimental methods were the same as in Example 1.

[0100] A validation cohort was constructed using 130 breast cancer patients to detect LTA / LPS expression in tumor tissues, and ROC curves were used to calculate the efficacy of LTA / LPS expression in predicting lymph node metastasis.

[0101] ROC curve results indicate that LTA / LPS expression in tumor tissue can effectively predict lymph node metastasis in patients (AUC under the curve were 0.712 and 0.697, respectively, P < 0.001). Figure 9 , Figure 10 ).

[0102] In summary, LTA / LPS expression in breast cancer tissue increases with increasing tumor malignancy, stage, and metastatic potential, with high LTA / LPS expression suggesting a greater likelihood of lymph node metastasis. LPS can serve as a biomarker for assessing breast cancer malignancy and predicting tumor metastasis, aiding in the stratification of high-risk and poorly prognostic patients.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of LTA and LPS as diagnostic and malignancy assessment markers and / or prognostic markers for predicting tumor metastasis in the preparation of breast cancer-related detection products; The breast cancer-related testing products mentioned are LTA and LPS immunohistochemical detection kits that can be used to determine the malignancy of breast cancer, the potential for tumor metastasis, and the prognosis of patients. The tumor metastatic potential includes lymph node metastasis potential; The patient prognosis includes overall survival and recurrence-free survival for breast cancer patients; The method of using the LTA and LPS immunohistochemical detection kit for the diagnosis of tissue sections by immunohistochemical staining includes: S1. Take the tissue sample from the subject, fix it, and prepare a paraffin slide. S2. The treated paraffin tablets reacted with primary and secondary antibodies in sequence. S3. After the reaction is complete, the slides are washed, stained with DAB, counterstained with hematoxylin, dehydrated, cleared, and mounted for observation under a microscope. In step S1, the tissue to be tested is breast tissue; the breast tissue is breast cancer tissue. Alternatively, in step S2, the paraffin sheet processing procedure includes: dewaxing and hydrating the prepared paraffin sheet, performing antigen retrieval, then inactivating peroxidase and biotin and performing serum blocking. Alternatively, in step S3, the microscopic observation includes: Three fields of view were randomly selected for each sample, with approximately 500 cells in each group; Semi-quantitative scoring was performed based on the intensity of hematoxylin staining, where 0 was negative; 1 was positive; 2 was ++; and 3 was +++. The cells are divided into four grades based on the percentage of positive cells: <1%, 0 points; 1-25%, 1 point; 26-50%, 2 points; 60-75%, 3 points; 76-100%, 4 points. The two scores for each sample are combined to obtain the final LTA / LPS expression score; the cutoff points for expression level are defined as: 0-4 points, low expression; 5-8 points, high expression.

Images