Use of biomarker in metastasis risk diagnosis and treatment of tumor lymph node

Abstract

The present invention relates to a use of a biomarker in the metastasis risk diagnosis and treatment of a tumor lymph node, and specifically relates to a use of specific recognition of a biomarker SPP1+TAM in combination with an existing N0-N3 lymph node metastasis classification system in the clinical metastasis risk diagnosis and treatment of a tumor lymph node. By means of systematical analysis on metastatic lymph nodes and non-metastatic lymph nodes of a variety of tumor patients, it is discovered that SPP1+TAM is associated with a N0-N3 staging system for lymph node metastasis in the tumor lymph node metastasis risk diagnosis. Provided is a clinical application of SPP1+TAM in combination with an N0-N3 staging system in the assisted or alternative metastasis risk diagnosis and guided treatment of a tumor lymph node. Also provided is a corresponding kit for the tumor lymph node metastasis risk diagnosis.

Description

Application of a biomarker in the diagnosis and treatment of tumor lymph node metastasis risk Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomarker SPP1. + Application of TAM specific identification combined with the N0-N3 tumor lymph node metastasis staging system in clinical tumor lymph node metastasis risk diagnosis and treatment. Background Technology

[0002] Lymph node metastasis is the initial step in distant metastasis and a leading cause of death in patients with malignant tumors. Lymph node metastasis is crucial for determining the clinical stage and predicting prognosis of various malignancies. A large number of cancer patients present with lymph node metastasis at their initial consultation. The 5-year survival rate of cancer patients with lymph node metastasis is significantly lower than that of cancer patients without metastasis (Rebecca L Siegel, et al. Cancer statistics, 2022. CA Cancer J Clin. 2022 Jan; 72(1):7-33).

[0003] The distant metastasis of solid tumors is a complex and inefficient process involving multiple consecutive stages, including primary tumor invasion, infiltration, tumor cell survival in the bloodstream, extravasation, and colonization of distant organs. The success of metastasis depends significantly on the microenvironment of the metastatic site. Tumor cells have developed various strategies to evade immune surveillance at the metastatic site. (AW Lambert, et al. Emerging Biological Principles of Metastasis. Cell. 2017 Feb 9; 168(4):670–691.)

[0004] Pathological confirmation after lymph node dissection is the primary method for diagnosing lymph node metastasis. However, this method has limitations, with a false negative rate as high as 20% due to the presence of micrometastases (C Urso, et al. Sentinel lymph nodes in cutaneous melanoma: the experience in the Florence area. Pathologica. 2003 Jun; 95(3):133-9.). Furthermore, current antitumor drugs primarily focus on controlling and shrinking the size of the primary tumor, without addressing distant metastases. Therefore, there is an urgent need to further investigate the underlying mechanisms of tumor lymph node metastasis and explore effective diagnostic techniques and potential treatment strategies.

[0005] Secreted phosphoprotein 1 (SPP1), also known as osteopontin (OPN), is an extracellular phosphoprotein and a multifunctional glycoprotein that plays a crucial role in various tumors, participating in the proliferation and invasion of malignant tumors and regulating the immune system. Some studies have revealed a correlation between SPP1 and N0-N3 stages. For example, SPP1 expression is significantly increased in non-small cell lung cancer (NSCLC) tissues compared to normal tissues. SPP1 was significantly correlated with N stage (p = 0.002) and primary efficacy assessment (p = 0.025), but not with other clinicopathological factors, suggesting that SPP1 expression differs among patients with different N stages (N0, N1, N2, N3) of lung adenocarcinoma (LUAD). Furthermore, SPP1 overexpression is closely associated with clinicopathological features and poor survival outcomes in patients with lung adenocarcinoma LUAD, while this association has not been observed in lung squamous cell carcinoma (Binbin Li, Xue Li, et al. Overexpression of SPP1 is a prognostic indicator of immune infiltration in lung adenocarcinoma. Aging. 2024 Feb 7;16(3):2953–2977).

[0006] Tumor-associated macrophages (TAMs) reside in the tumor microenvironment (TME) and play a crucial role in the survival and progression of tumor cells. TAMs participate in the production of immunosuppressive TMEs within tumors by generating inflammatory mediators, growth factors, cytokines, and chemokines. TAMs can influence angiogenesis and metastatic behavior of tumor cells (TCs) and lead to multidrug resistance. TAMs within the TME can enhance tumor cell metastasis and are located in the stroma and perivascular space (Siva Dallavalasa, et al. The Role of Tumor Associated Macrophages (TAMs) in Cancer Progression, Chemoresistance, Angiogenesis and Metastasis-Current Status. Curr Med Chem. 2021; 28(39):8203-8236.). Some literature has studied the association between TAM and N0-N3 stages, but no correlation has been found between high TAM infiltration and lymph node status (N0, N1-N3) (Xixi Zhao, et al. Prognostic significance of tumor-associated macrophages in breast cancer: a meta-analysis of the literature. Oncotarget. 2017 Feb 25; 8(18):30576–30586).

[0007] SPP1-positive macrophages (SPP1 + Tumor-associated macrophages (TAMs) are a specific subset of TAMs that play a crucial role in promoting tumor metastasis. These macrophages are characterized by high expression of SPP1, which enhances the migration, invasion, and colonization of tumor cells in various cancers. They interact with other cell types in the tumor microenvironment (TME) and secrete various factors that promote the metastatic process (Liang Dong, et al. SPP1+ TAM Regulates the Metastatic Colonization of CXCR4). + Metastasis-Associated Tumor Cells by Remodeling the Lymph Node Microenvironment. Adv Sci. 2024 Nov; 11(44)).

[0008] SPP1 +TAM can promote tumor cell metastasis through the following mechanism (Yan Du, et al. Potential crosstalk between SPP1). + TAMs and CD8 + exhausted T cells promotes an immunosuppressive environment in gastric metastatic cancer.J Transl Med.2024 Feb 16;22(1):158.):

[0009] 1) Intercellular communication: SPP1 + TAM interacts in complex ways with other cells in the TME (including endothelial cells, fibroblasts, and tumor cells), thereby promoting a metastatic environment.

[0010] 2) Extracellular matrix remodeling: SPP1 + TAM can remodel the ECM by secreting factors that break down extracellular matrix (ECM) components, thereby promoting the migration and invasion of tumor cells.

[0011] 3) Angiogenesis: These macrophages can promote angiogenesis, that is, the formation of new blood vessels, which is crucial for tumor metastasis because it can provide oxygen and nutrients to growing tumors and promote the spread of tumor cells.

[0012] 4) EMT induction: SPP1 + TAM can induce epithelial-mesenchymal transition (EMT) in tumor cells. During this process, tumor cells lose their epithelial properties and acquire mesenchymal properties, thereby enhancing their migration and invasion capabilities.

[0013] 5) Lymph node metastasis: SPP1 + TAM can remodel the lymph node microenvironment, creating a favorable microenvironment for tumor cell colonization and metastasis.

[0014] 6) Interaction with tumor cells: SPP1 + TAM can interact directly with tumor cells, enhancing their survival, proliferation, and metastatic potential.

[0015] Current literature only reveals a certain association between SPP1 and the N0-N3 tumor staging system, and the relationship between TAM and N0-N3 is uncertain, but no literature has found any association between SPP1. + There is a correlation between TAM and N0-N3, especially since SPP1 was not found. +There is no correlation between TAM and the current clinical N0-N3 staging system for tumor lymph node metastasis in the diagnosis of tumor lymph node metastasis risk.

[0016] Therefore, although the biomarker SPP1 + TAM has been revealed to have multiple functions in various tumors, such as promoting tumor metastasis and creating a microenvironment conducive to tumor growth. However, there is currently no literature that reveals or guides how to specifically apply it to the clinical diagnosis and treatment of tumor lymph node metastasis, especially how to combine it with the N0-N3 staging system for the clinical diagnosis and treatment of tumor lymph node metastasis.

[0017] This invention, through systematic research and analysis of metastatic and non-metastatic lymph nodes in various cancer patients, discovered the biomarker SPP1. + The association between TAM and the current clinical tumor lymph node metastasis N0-N3 staging system in the diagnosis of tumor lymph node metastasis risk led to the invention and provision of SPP1. + The innovative clinical application of TAM combined with the tumor N0-N3 staging system to assist or replace the diagnosis of tumor lymph node metastasis risk and guide treatment.

[0018] The embodiments of this invention have verified the method proposed in this invention for assisting or replacing the diagnosis of tumor lymph node metastasis risk in gastric cancer, colon cancer, rectal cancer, breast cancer, and ovarian cancer, respectively. It was found that its effect is consistent in multiple cancers. It can further diagnose and guide the treatment of tumor lymph node metastasis risk at an earlier stage based on the existing N0-N3 classification. It can compensate for or even replace the shortcomings of existing pathology in the classification and diagnosis of tumor lymph node metastasis. After clinical application, it is expected to improve the survival rate of patients and reduce medical costs.

[0019] The following section uses gastric cancer as an example to provide a more detailed explanation of the background technology.

[0020] Gastric cancer is the fifth most common malignant tumor worldwide and the third leading cause of cancer-related deaths, with over one million new cases annually (Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024; 74(3):229-263.). Gastric cancer is a malignant tumor originating from the gastric mucosal epithelium, and its incidence is closely related to multiple factors, including changes in dietary structure, increased psychological stress, Helicobacter pylori infection, and the influence of environmental and genetic factors. These factors collectively promote the occurrence and development of gastric cancer, leading to a significant trend of younger onset (Sano T, Coit DG, Kim HH, et al. Proposal of a new stage grouping of gastric cancer for TNM classification: International Gastric Cancer Association staging project. Gastric Cancer. 2017 Mar; 20(2):217-225.). Based on the depth of tumor invasion, gastric cancer can be divided into early gastric cancer and advanced gastric cancer. Early gastric cancer (EGC) refers to tumors confined to the gastric mucosa or submucosa, regardless of whether lymph node metastasis has occurred. Due to the insidious development of gastric cancer, early clinical symptoms are often atypical and easily overlooked or misdiagnosed (Ikenoyama Y, Hirasawa T, Ishioka M, et al. Detecting early gastric cancer: Comparison between the diagnostic ability of convolutional neural networks and endoscopists. Dig Endosc. 2021 Jan; 33(1):141-150.). Advanced gastric cancer (AGC) refers to tumors that have broken through the submucosa, infiltrated deeper, or even metastasized to distant sites.Although the more obvious clinical symptoms of patients with advanced gastric cancer improve the tumor detection rate, the 5-year survival rate after surgery is less than 30%, and the prognosis is poor (Ogata Y, Hatta W, Ohara Y, et al. Predictors of early and late mortality after the treatment for early gastric cancers. Dig Endosc. 2022 May; 34(4):816-825.).

[0021] Lymph node metastasis is the most common route of metastasis for gastric cancer, accounting for approximately 70% of gastric cancer metastases. Related clinical studies have shown that the probability of lymph node metastasis in early gastric cancer ranges from 9.8% to 16.7%. Lymph node metastasis is not only closely related to patient prognosis but is also a crucial factor influencing treatment options. Clinical studies have confirmed that lymph node metastasis is both the most important risk factor for recurrence and a core factor leading to poor prognosis (Lai JF, Kim S, Kim K, et al. Prediction of recurrence of early gastric cancer after curative resection. Ann Surg Oncol. 2009 Jul; 16(7):1896-902.). Therefore, accurate assessment of lymph node metastasis status is of great significance for optimizing individualized treatment strategies, improving poor prognosis, and enhancing patients' quality of life. Furthermore, accurate determination of lymph node staging is crucial for clinical decision-making.

[0022] According to the 8th edition of the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) guidelines, the lymph node staging criteria in the TNM staging system are as follows: N0 indicates no regional lymph node metastasis; N1 indicates 1-2 regional lymph node metastases; N2 indicates 3-6 regional lymph node metastases; and N3 indicates 7 or more regional lymph node metastases. These staging criteria provide important information for assessing the extent of metastasis and prognosis in cancer patients. Some specific tumors use specific staging criteria. For example, ovarian cancer is a female-specific tumor, and it often uses its unique FIGO (International Federation of Gynecology and Obstetrics) staging system. The FIGO staging of ovarian cancer (I-IV) is classified according to the degree of cancer spread; the lower the number, the earlier the stage and the better the prognosis. Stage I indicates that the cancer cells are confined to the ovary or fallopian tube. Stage II indicates that the cancer cells have spread to the pelvis. Stage III indicates that the cancer cells have spread to the lymph nodes or peritoneum. Stage IV indicates that the cancer cells have metastasized to distant sites.

[0023] Unlike the remodeling effect of tumor cells on lymph node structure and microenvironment in metastatic lymph nodes, recent studies have found that before tumor colonization in lymph nodes, tumor cells can induce significant changes in the functional structure and immune status of some draining lymph nodes by secreting various active substances such as cytokines and extracellular vesicles. This is accompanied by a shift in the immune microenvironment towards an immune-tolerant phenotype, gradually forming a pre-metastatic niche. Current research suggests that the pre-metastatic niche provides an ideal "culture dish" for tumor cells and is a necessary condition for tumor metastasis (Peinado H, Zhang H, Matei IR, et al. Pre-metastatic niches: organ-specific homes for metastases. Nat Rev Cancer. 2017 May; 17(5):302-317.). Furthermore, the formation of a pre-metastatic niche indicates that the lymph node is at high risk of metastasis. In this process, complex changes in the vascular system are the core feature of its structural remodeling. Several studies have elucidated the unique pathological features of the pre-metastatic niche, including lymphangiogenesis, high endothelial microvenous remodeling, myeloid cell recruitment, and phenomena such as decreased number and / or weakened function of effector lymphocytes and increased regulatory T cells (Pereira ER, Jones D, Jung K, et al. The lymph node microenvironment and its role in the progression of metastatic cancer. Semin Cell Dev Biol. 2015; 38:98-105.). It is noteworthy that while these microenvironmental changes promote tumor metastasis, they may also reduce the sensitivity and accuracy of existing clinical imaging and pathological methods in identifying metastatic lymph nodes, increasing the risk of preoperative and intraoperative missed diagnoses and misjudgments.

[0024] Currently, the diagnosis of lymph node metastasis in gastric cancer mainly relies on pathology, imaging, and laboratory tests. Among these, abdominal CT has become an important means of preoperative clinical assessment due to its convenient operation and high sensitivity. However, there is still a risk of missed diagnosis in more than 30% of patients (Li Shi, Zhou Jie, Zhao Ping, et al. Relationship between preoperative neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, and C-reactive protein-albumin ratio and postoperative lymph node metastasis in patients with cN0 stage gastric cancer [J]. Chinese Journal of Clinical Oncology, 2021, 48(5):230-234; Sun Z, Hu S, Li J, et al. An application study of CT perfusion imaging in assessing metastatic involvement of perigastric lymph nodes in patients with T1 gastric cancer. Br J Radiol. 2020 Feb 1; 93(1106):20190790.).

[0025] Given that tumor markers are abnormally expressed in tumor cells and often appear earlier than clinical symptoms, their application value in predicting tumor metastasis has gradually attracted attention in recent years. C. Predictive biomarkers in gastric cancer. J Cancer Res Clin Oncol. 2023; 149(1):467-481.). Numerous studies and patents both domestically and internationally have successively developed products and models for the detection and prediction of lymph node metastasis in gastric cancer and other malignant tumors. For example, patent CN202410931935.5 describes the preparation of early gastric cancer lymph node metastasis detection products and the construction of prediction models based on more than five methylation markers. Patent CN202410696460 describes the design of corresponding detection products and computer-aided diagnostic systems based on biomarkers such as SDS, TISMEN, NEB, and GRB14. In addition, patents CN202410114828.3, CN202311702205, and CN202310800086 have developed related products for predicting and predicting lymph node metastasis in lung adenocarcinoma, cervical squamous cell carcinoma, and bile duct carcinoma based on tRF-Tyr-GTA-006, Porphyromonas gingivalis abundance, and RACK1 protein expression levels, respectively. However, these patents and products are still in the theoretical stage and have not yet been approved and implemented in clinical practice.

[0026] The 5-year survival rate for gastric cancer patients varies depending on the degree of lymph node metastasis (N stage). Generally, the survival rate decreases significantly as the stage progresses from N0 (no lymph node metastasis) to N3 (multiple lymph node metastases).

[0027] Survival rate for N stage (lymph node metastasis):

[0028] N0 (no lymph node metastasis): The 5-year survival rate for patients with N0 stage gastric cancer is relatively high, usually exceeding 90%.

[0029] N1 (1-2 lymph node metastases): The survival rate is lower than that of N0, but still relatively high, at approximately 80-85%.

[0030] N2 (3-6 lymph node metastases): Survival rate further declines, typically between 60-70%.

[0031] N3 (tumor involving 7 or more lymph nodes): Survival rate is significantly lower, typically between 20% and 40%. (Hengyi Zhang, et al. Long-term relative survival of patients with gastric cancer from a large-scale cohort: a period-analysis. BMC Cancer. 2024 Nov 18; 24:1420)

[0032] The 5-year survival rates of gastric cancer patients at different N stages show that survival rates gradually decrease and mortality rates increase significantly as N0 increases to N3. This indicates that the higher the N stage, the higher the false negative rate of the pathological or imaging diagnostic methods used to diagnose it. Otherwise, the gradual decrease in survival rate and the significant increase in mortality rate with increasing N stage would not occur. This suggests that the traditional pathological or imaging diagnostic methods used for N staging are clearly inaccurate, leading to inadequate staging, false negatives, and insufficient treatment.

[0033] In addition, most existing technologies focus on the diagnosis of metastatic lymph nodes, but there is a lack of research on the identification of lymph nodes at high risk of metastasis.

[0034] Therefore, there is an urgent clinical need for diagnostic methods with higher accuracy and sensitivity for lymph node metastasis in cancer patients. These methods can guide timely clinical intervention, optimize the scope of surgical clearance, compensate for treatment deficiencies caused by inadequate staging, and thus effectively reduce the overall medical costs for patients and improve the efficiency of medical resource utilization. Summary of the Invention

[0035] Lymph node metastasis is the initial step in distant metastasis and a leading cause of death in many cancer patients. It is crucial for determining the clinical stage and predicting prognosis of various malignancies. SPP1 +TAM has been revealed to have multiple functions, such as promoting tumor metastasis and creating a microenvironment conducive to tumor growth. However, there is currently no literature or technology that reveals or guides how to specifically apply it to the clinical diagnosis and treatment of tumor lymph node metastasis risk.

[0036] This invention, through systematic analysis of metastatic and non-metastatic lymph nodes from various cancer patients, discovered a biomarker, SPP1-positive tumor-associated macrophages (SPP1). + The association between TAM and the current clinical N0-N3 staging system for tumor lymph node metastasis in the diagnosis of tumor lymph node metastasis risk, thereby providing a SPP1 + This invention represents an innovative clinical application of TAM (Tumor Amplification and Metastasis) in conjunction with the N0-N3 staging system for the auxiliary or alternative diagnosis of tumor lymph node metastasis risk, which can guide treatment. A corresponding diagnostic kit for tumor lymph node metastasis risk is also provided. Based on this, the invention was completed.

[0037] In a first aspect, the present invention provides a method for specifically identifying the biomarker SPP1. + The application of TAM reagents in the auxiliary or alternative diagnosis of tumor lymph node metastasis risk and in the preparation of diagnostic kits for tumor lymph node metastasis risk, wherein the reagents are used to detect SPP1 in lymph node samples from cancer patients. + TAM; when SPP1 is detected in lymph node tissue samples from patients with N0 tumors or those without lymph node metastasis. + TAM indicates that the lymph node has formed a pre-metastatic niche, warning of a high risk of lymph node metastasis. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low; if SPP1 is detected in lymph node tissue samples from N1-N3 tumor patients or tumor patients with confirmed lymph node metastasis... + TAM indicates that the lymph node has formed a pre-metastatic niche and warns of a high risk of lymph node metastasis, or it indicates that lymph node metastasis has already occurred. If SPP1 is not detected... + TAM indicates that the lymph node has not yet formed a pre-metastatic niche, and the risk of lymph node metastasis is low. This application can provide early warning of the risk of tumor lymph node metastasis, assist in diagnosis, and compensate for the inadequacies in treatment caused by the current N0-N3 staging system for tumors based on pathological diagnosis.

[0038] Furthermore, the tumor is one or more of the following: gastric cancer, colorectal cancer, oral squamous cell carcinoma, esophageal cancer, lung cancer, liver cancer, bladder cancer, glioma, ovarian cancer, and / or breast cancer.

[0039] Preferably, the tumor is gastric cancer, colorectal cancer, breast cancer, or ovarian cancer.

[0040] Furthermore, the lymph node tissue samples include non-metastatic and metastatic lymph node tissue samples from cancer patients.

[0041] Furthermore, SPP1 was detected in lymph node tissue samples from N0 tumor patients. + TAM indicates that the lymph node has formed a pre-metastatic niche, warning of a high risk of lymph node metastasis. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

[0042] Furthermore, given that N0 tumor patients are those without regional lymph node metastasis, for tumor patients without lymph node metastasis, such as Nx tumor patients, if SPP1 is detected in lymph node tissue samples from these patients... + TAM indicates that the lymph node has formed a pre-metastatic niche, warning of a high risk of lymph node metastasis. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

[0043] Furthermore, if SPP1 is detected in lymph node tissue samples from N1-N3 gastric cancer patients... + TAM indicates that the lymph node has formed a pre-metastatic niche and warns of a high risk of lymph node metastasis, or indicates that lymph node metastasis has already occurred; if SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

[0044] Furthermore, given that N1 tumor patients have 1-2 regional lymph node metastases, N2 tumor patients have 3-6 regional lymph node metastases, and N3 tumor patients have 7 or more regional lymph node metastases, and considering that data from this patent's embodiments indicate that as long as there is at least one regional lymph node metastasis, essentially 100% of tumor patients will have SPP1 regional lymph node metastases. + A positive TAM result indicates the presence of SPP1 in lymph node tissue samples, regardless of whether the lymph nodes have metastasized or not. Therefore, for cancer patients with confirmed lymph node metastasis, regardless of N staging, the presence of SPP1 in these samples indicates a positive TAM result. + TAM indicates that the lymph node has formed a pre-metastatic niche, warning of a high risk of lymph node metastasis. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

[0045] Secondly, the present invention provides a kit for diagnosing the risk of lymph node metastasis in cancer patients, the kit comprising SPP1 that specifically identifies lymph node tissue samples from cancer patients. +TAM's reagent is used to detect the presence of SPP1 in lymph node tissue samples from cancer patients. + TAM, the diagnosis includes using the kit to detect SPP1 in lymph node tissue samples from tumor patients. + TAM, if SPP1 is detected in lymph node tissue samples from N0 patients or patients with tumors that have not yet been found to have lymph node metastasis. + TAM indicates that the lymph node has formed a pre-metastatic niche, warning of a high risk of lymph node metastasis. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low; if SPP1 is detected in lymph node tissue samples from N1-N3 tumor patients or tumor patients with confirmed lymph node metastasis... + TAM indicates that the lymph node has formed a pre-metastatic niche and warns of a high risk of lymph node metastasis, or it indicates that lymph node metastasis has already occurred. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

[0046] Furthermore, SPP1 was specifically identified in lymph node tissue samples from tumor patients. + TAM requires localization detection, including various immunohistochemical (IHC) localization detection methods, all of which are known and routine methods. Classified by label, these include immunofluorescence, immunoenzyme-linked immunosorbent assay (IEISA), metal ion (immunogold immunoassay, immunoferritin immunoassay), and radioisotope (radioimmunoassay). Immunofluorescence uses fluorescent dyes to label antibodies, and the fluorescence signal is observed under a fluorescence microscope to reveal the antigen. Multiple staining can be performed, allowing simultaneous detection of multiple antigens. Immunoenzyme immunoassay uses enzymes (common enzymes include horseradish peroxidase, alkaline phosphatase, or glucose oxidase) to label antibodies. The antigen is revealed by the colored precipitate produced by the reaction of the enzyme with the substrate. This is one of the most commonly used immunohistochemical methods; DAB (diaminobenzidine) staining is a classic example. Immunogold immunoassay uses colloidal gold particles as labels. Colloidal gold refers to a gold hydrosol, which can rapidly and stably adsorb proteins without significantly affecting their biological activity. Therefore, using colloidal gold-labeled primary or secondary antibodies allows for qualitative, localization, and even quantitative studies of antigens within tissues or cells. Immunoferritin assays utilize the specific binding of antibodies to ferritin, revealing its presence and distribution through a colorimetric reaction. Radioimmunoassay uses radiolabeled antibodies, detected via X-ray imaging. This method offers high sensitivity but carries the risk of radioactive contamination.

[0047] Furthermore, the immunohistochemical (IHC) localization detection method used in this invention can be categorized by staining steps, including direct and indirect methods. The direct method uses a primary antibody labeled with a chromogenic agent (such as an enzyme or fluorescein) to directly bind to the antigen in the tissue, and then the location of the antigen is revealed by color development or fluorescence. Its advantage is its simplicity and speed, but its sensitivity is relatively low. The indirect method first allows the primary antibody to bind to the antigen in the tissue, and then a secondary antibody labeled with a chromogenic agent (an antibody that recognizes the primary antibody) binds to the primary antibody, thereby amplifying the signal. Its advantages are higher sensitivity and wider application.

[0048] Furthermore, the primary antibodies in the direct and indirect methods include antibodies against human tumor-associated macrophage surface markers and Anti-Osteopontin antibodies against the SPP1 protein, or Anti-F4 / 80 antibodies and Anti-Osteopontin antibodies against mouse lymph node tissue.

[0049] Furthermore, the primary antibodies targeting human tumor-associated macrophage surface markers include Anti-CD68, Anti-CD11b, Anti-CD163, Anti-CD206, Anti-CD204, or Anti-Stabilin-1 antibodies. Human macrophage surface markers are mainly divided into universal markers and polarization state markers. Universal markers include CD68 and CD11b, which are expressed in all macrophages. Polarization state markers can distinguish different macrophage subtypes, such as M1 and M2. In this invention, the M2 type is primarily responsible for promoting tumor cell metastasis in lymph node tissues of cancer patients. M2 macrophage surface markers mainly include CD163, CD206, CD204, Stabilin-1, TREM2 (Triggering Receptor Expressed on Myeloid cells 2), PD-L1 (Programmed Death-Ligand 1), B7-H4, MerTK (Mer Tyrosine Kinase), and CCR2 (CC Chemokine Receptor Type 2). Antibodies against these macrophage surface markers can be used in this invention.

[0050] Preferably, the antibody targeting the surface markers of human tumor-associated macrophages is an Anti-CD68 antibody or an Anti-CD163 antibody.

[0051] Furthermore, the kit includes, but is not limited to, dewaxing and dehydration reagents (such as xylene, anhydrous ethanol and graded ethanol), washing buffers (such as distilled water, PBS buffer, PBST buffer), antigen retrieval reagents (such as citrate buffer), and 3% hydrogen peroxide solution.

[0052] Furthermore, the kit contains secondary antibodies labeled with different markers, including secondary antibodies corresponding to primary antibodies of different species (such as mouse and rabbit), such as Alexa Fluor 488-labeled secondary antibody used for rabbit primary antibodies in immunofluorescence assays, and Alexa Fluor 594-labeled secondary antibody used for mouse primary antibodies, to achieve multiplex labeling detection.

[0053] Furthermore, it also includes auxiliary reagents such as blocking solutions, DAPI staining agents, or DAPI-containing anti-fluorescence quenching mounting tablets.

[0054] Furthermore, the SPP1 + TAM can be detected by methods such as immunohistochemistry, immunofluorescence, or multicolor immunohistochemistry. The various reagents in the kit can be flexibly selected and combined according to actual detection needs to achieve the detection of SPP1 in biological samples. + The sensitive and specific detection of TAM enables early warning of the risk of tumor lymph node metastasis and auxiliary diagnosis of metastatic lymph nodes.

[0055] Furthermore, the tumor is one or more of the following: gastric cancer, colorectal cancer, esophageal cancer, lung cancer, liver cancer, bladder cancer, glioma, ovarian cancer, and / or breast cancer.

[0056] Preferably, the tumor is gastric cancer.

[0057] Furthermore, the biological sample includes a patient's lymph node tissue sample.

[0058] Furthermore, the lymph node tissue samples include non-metastatic lymph node tissue samples and metastatic lymph node tissue samples.

[0059] Furthermore, the specific types of the kits include, but are not limited to, immunohistochemistry kits and / or immunofluorescence kits.

[0060] Furthermore, the immunohistochemistry kit typically includes dewaxing and hydration reagents, antigen retrieval solution, primary antibodies against SPP1 protein and macrophages (such as human macrophage surface marker CD68 or mouse macrophage surface marker F4 / 80), horseradish peroxidase secondary antibody, DAB chromogenic solution or green, black, blue, purple, and yellow chromogenic reagents for horseradish peroxidase, blocking solution, washing solution, mounting medium, and detailed operating instructions. Through a standardized staining procedure, the target cells in the sample can be located and detected.

[0061] Furthermore, the immunofluorescence kit typically includes primary antibodies against SPP1 protein and macrophages (such as human macrophage surface marker CD68 or mouse macrophage surface marker F4 / 80), fluorescently labeled secondary antibodies (such as Alexa Fluor 488, Alexa Fluor 594, etc.), DAPI staining agent, mounting medium, various buffers, and standardized operating instructions. Through multiplex immunofluorescence labeling, simultaneous detection and high-resolution visualization of target molecules can be achieved.

[0062] Furthermore, the diagnostic methods described in the kits include one or more direct and indirect methods, which can be selected according to different experimental and clinical needs. The reagents in the above-mentioned kits can be flexibly combined and are suitable for detecting SPP1 in biological samples. + The sensitive and specific detection of TAM enables early warning of the risk of tumor lymph node metastasis and auxiliary diagnosis of metastatic lymph nodes.

[0063] Thirdly, the present invention further provides a clinical application that guides precision cancer treatment by assisting or replacing the diagnosis of lymph node metastasis risk in cancer patients.

[0064] Furthermore, when SPP1 is detected in lymph node tissue samples from patients with N0 tumors or those without lymph node metastasis... + TAM indicates that the lymph node has formed a pre-metastatic niche, warning of a high risk of lymph node metastasis. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

[0065] Furthermore, if SPP1 is detected in lymph node tissue samples from N1-N3 tumor patients or those with confirmed lymph node metastases... + TAM indicates that the lymph node has formed a pre-metastatic niche and warns of a high risk of lymph node metastasis, or it indicates that lymph node metastasis has already occurred. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

[0066] Furthermore, when SPP1 was detected in lymph node tissue samples from cancer patients... + During TAM (Transient Ambient Hyperplasia), if the lymph node is diagnosed as having formed a pre-metastatic niche or has already metastasized, surgical dissection or other methods can be used to remove lymph nodes at high risk of metastasis or those that have already metastasized. Combined with test results, this allows for stratified management and individualized treatment of patients, compensating for treatment deficiencies caused by insufficient staging in traditional pathological or imaging diagnoses. Therefore, through lymph node metastasis risk early warning and auxiliary diagnosis, this invention can guide timely clinical intervention, optimize the scope of surgical dissection, and compensate for treatment deficiencies caused by inadequate staging. Beneficial effects

[0067] 1. In terms of auxiliary diagnosis, this invention detects SPP1 in lymph node tissue samples from tumor patients. + TAM can not only identify high-risk lymph nodes at an early stage, but also identify metastatic lymph nodes. Based on the fact that pathological diagnosis can only identify metastatic lymph nodes, it realizes dual auxiliary diagnosis for identifying high-risk lymph nodes and metastatic lymph nodes. This can make up for the shortcomings in treatment caused by the insufficient staging and accuracy of the current tumor N0-N3 staging system based on pathological diagnosis.

[0068] 2. Regarding diagnostic accuracy and sensitivity, this invention targets SPP1. + TAM, a specific biomarker, provides more precise molecular-level diagnostic evidence through immunohistochemical and immunofluorescence localization detection methods, significantly improving diagnostic sensitivity and specificity compared to pathological and imaging diagnoses. Furthermore, this invention employs standardized laboratory testing procedures, objectively quantifying detection indicators with clear interpretation criteria, allowing for seamless integration with existing pathology platforms without requiring additional specialized equipment, thus ensuring the accuracy and reproducibility of test results. Because this molecular diagnostic method is more sensitive and accurate than pathological and imaging diagnostic methods, with a higher positive detection rate, it has the potential to replace pathological or imaging diagnostic methods to a certain extent or in certain scenarios after sufficient clinical data accumulation.

[0069] 3. In terms of cost-effectiveness, the detection method of this invention has been optimized and standardized, making it suitable for use in routine pathology laboratories. It only requires the equipment of routine pathology testing, and has high economic efficiency and applicability.

[0070] 4. Through early warning and auxiliary diagnosis of lymph node metastasis risk, this invention can guide timely clinical intervention, optimize the scope of surgical clearance, and compensate for treatment deficiencies caused by inadequate staging, thereby effectively reducing the overall medical costs for patients and improving the efficiency of medical resource utilization. Therefore, this invention has significant technical advantages and clinical translational value in the field of risk assessment and auxiliary diagnosis of tumor lymph node metastasis. Attached Figure Description

[0071] Figure 1 shows the SPP1 cells screened out by spatial transcriptome analysis. + TAM. Note: A. UMAP plot after dimensionality reduction and clustering; B. Heatmap of cell types enriched in the regions of each subpopulation; C. Distribution map of the proportion of different cell types in each tissue; D. Cell annotation of each spatial transcriptome sequencing point using classic cell marker genes of gastric cancer and lymph nodes; E. SPP1 + TAM is enriched in metastatic lymph nodes; F.SPP1 + TAM tissue mapping in lymph nodes.

[0072] Figure 2 shows the immunofluorescence detection of SPP1 in gastric cancer lymph node tissue. + The presence of TAM. Note: A. Immunofluorescence assay for SPP1 in metastatic lymph nodes of gastric cancer patients. + The presence of TAM; BC immunofluorescence assay for SPP1 in lymph nodes of N0 gastric cancer patients. + The existence of TAM.

[0073] Figure 3 shows the immunofluorescence detection of SPP1 in breast cancer lymph node tissue. + The presence of TAM. Note: A. Immunofluorescence assay for SPP1 in metastatic lymph nodes of breast cancer patients. + The presence of TAM; BC immunofluorescence assay for SPP1 in lymph nodes of N0 breast cancer patients. + The existence of TAM.

[0074] Figure 4 shows the immunofluorescence detection of SPP1 in colon cancer lymph node tissue. + The presence of TAM. Note: A. Immunofluorescence assay for SPP1 in metastatic lymph nodes of colorectal cancer patients. + The presence of TAM; BC immunofluorescence assay for SPP1 in lymph nodes of N0 colon cancer patients. + The existence of TAM.

[0075] Figure 5 shows the immunofluorescence detection of SPP1 in rectal cancer lymph node tissue. + The presence of TAM. Note: A. Immunofluorescence assay for SPP1 in metastatic lymph nodes of rectal cancer patients. + The presence of TAM; BC immunofluorescence assay for SPP1 in lymph nodes of N0 rectal cancer patients. + The existence of TAM.

[0076] Figure 6 shows the immunofluorescence detection of SPP1 in ovarian cancer lymph node tissue. + The presence of TAM. Note: A. Immunofluorescence assay for SPP1 in metastatic lymph nodes of ovarian cancer patients. + The presence of TAM; BC. Immunofluorescence assay for SPP1 in lymph nodes of ovarian cancer patients without lymph node metastasis. + The existence of TAM.

[0077] Figure 7 shows the detection of SPP1 in the lymph nodes of a mouse popliteal lymph node model. + TAM. Note: A. In vivo imaging and HE staining were used to verify the formation of lymph node metastasis in a mouse popliteal lymph node metastasis model; B. Immunofluorescence assays were used to detect SPP1 in metastatic and non-metastatic lymph nodes of mice. + The existence of TAM.

[0078] Figure 8 shows the detection and verification of lymph node metastasis-related indicators. Note: A. HE staining verifies the formation of lymph node metastasis in the mouse popliteal lymph node metastasis model; B. Immunofluorescence assay detects SPP1 in metastatic and non-metastatic lymph nodes of mice. + The existence of TAM.

[0079] Figure 9 shows the detection of niche-related indicators before lymph node metastasis. Note: A. Immunofluorescence assay to detect SPP1 in lymph node tissue of N0 patients. + A. The presence of TAM was detected, and the tissue was stained with HE; B. Immunohistochemical experiments were performed to detect the expression of CD163, CD31-positive blood vessels, MECA-79-positive high endothelial veins, and PDPN-positive lymphatic vessels in the lymph node tissue of N0 patients, and immunofluorescence experiments were performed to detect Treg; C. The corresponding statistical results in Figure B.

[0080] Figure 10 shows the ROC curves for detecting niche-related indicators before lymph node metastasis.

[0081] Figure 11 shows the verification of niche-related indicators before lymph node metastasis. Note: Immunohistochemical experiments were used to detect the expression of CD163, CD31-positive blood vessels, MECA-79-positive high endothelial veins, and PDPN-positive lymphatic vessels in the lymph node tissue of N0 patients. Immunofluorescence experiments were used to detect Treg.

[0082] Figure 12 shows the ROC curves for detecting and validating niche-related indicators before lymph node metastasis. Detailed Implementation

[0083] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0084] Terminology Explanation:

[0085] Lymph node metastasis (N) staging is based on the 8th edition of the American Joint Committee on Cancer (AJCC) gastric cancer staging system and the guidelines of the Union for International Cancer Control (UICC), namely N0: no regional lymph node metastasis, N1: 1-2 regional lymph node metastases, N2: 3-6 regional lymph node metastases, and N3: 7 or more regional lymph node metastases.

[0086] Tumor-draining lymph nodes (TDLNs) are the first lymph nodes to receive lymphatic drainage from the primary lesion and serve as the initiation site for anti-tumor immunity. Antigen-presenting cells (APCs), including dendritic cells (DCs) and tumor-associated macrophages (TAMs), migrate to the TDLN carrying tumor antigens under the influence of chemokines. They then cross-present these antigens to naive T cells, thereby inducing CD8+ immunization. + T cells proliferate and differentiate, then gradually infiltrate the tumor site via peripheral circulation to exert their effector killing function, continuously supplying "troops" throughout the entire immune response.

[0087] D2 lymph node dissection is a standard surgical procedure for gastric cancer. Its core principle is to thoroughly remove the perigastric lymph nodes to reduce tumor recurrence and improve survival rates. D2 dissection requires removal of at least the second-station lymph nodes, which are located relatively far from the primary tumor. In addition, both visible and invisible lymph nodes and adipose tissue must be removed.

[0088] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0089] Example 1: Spatial transcriptomics was used to identify the characteristic presence of SPP1-positive tumor-associated macrophages (SPP1) in metastatic lymph nodes of gastric cancer. + TAM)

[0090] 1.1 Sample Selection and Processing

[0091] Lymph node samples were selected from four patients with gastric cancer, including lymph node tissues from two patients with stage N0 and two patients with stage N3. Sample information is shown in Table 1.

[0092] Table 1. Information on lymph node samples from patients.

[0093] 1.2 Cell type identification and distribution analysis

[0094] (1) Using the classic cell type marker genes of gastric cancer and lymph nodes, the cell type of each spatial transcriptome sequencing spot was defined by the AddModule scoring method.

[0095] (2) Statistical analysis of the proportion of different cell types in each tissue.

[0096] 1.3 Macrophage Subpopulation Analysis

[0097] (1) Based on the macrophage subpopulation marker gene sets reported in previous studies (PMID:35303421 and PMID:32302573), the gene set characteristics of different macrophage subpopulations were scored using AddModule.

[0098] (2) Based on the median UMI of the SPP1 gene in macrophage spots, macrophages were divided into SPP1 and SPP1 groups. + TAM and SPP1 - TAM (two groups)

[0099] (3) SPP1 + TAM and SPP1 - The two groups of macrophages from TAM were projected back onto tissue slices from spatial transcriptome sequencing to visualize their spatial distribution.

[0100] 1.4 Results

[0101] Spatial transcriptome sequencing of the primary tumor lesions and their paired lymph nodes in four patients revealed that two patients had no lymph node metastasis (N0 group), while two patients had lymph node metastasis (N3 group). Analysis identified 11 major cell types, including B cells, vascular endothelial cells, dendritic cells (DCs), epithelial cells, fibroblasts, lymphatic endothelial cells, macrophages, NK cells, plasma cells, smooth muscle cells, and T cells. UMAP dimensionality reduction analysis showed that these cell types formed unique clustering patterns (Figure 1A). Further analysis of the correspondence between cell clusters and cell types (Figure 1B) revealed that certain cell types were significantly enriched in specific clusters. Comparison of different experimental groups showed that the proportion of macrophages in the N3-LN group was significantly higher than that in the N0-LN group (Figures 1C, 1D). Further scoring of the gene sets of different macrophage types revealed SPP1... + TAM was enriched in metastatic lymph nodes (Figure 1E). Finally, after projecting this type of macrophage back into lymph node tissue sections, SPP1 was found. + TAM is specifically present in metastatic lymph nodes but absent in non-metastatic normal lymph nodes (Figure F). These results indicate that SPP1 + TAM may play an important role in lymph node metastasis, providing important clues for further research on its function in the tumor microenvironment.

[0102] Example 2: Establishing a clinical cohort of gastric cancer patients to validate SPP1 + Characteristic presence of TAM in metastatic lymph nodes of gastric cancer

[0103] This study included paraffin-embedded lymph node tissue from 115 patients who underwent radical gastrectomy for N0 and 77 patients who underwent N1-N3 stage gastric cancer, as well as plasma samples from some patients. All samples were obtained from the First Affiliated Hospital of China Medical University.

[0104] Patients included in the study were those who underwent radical gastrectomy for total / distal / proximal gastric cancer combined with D2 lymph node dissection at the Department of Gastrointestinal Oncology Surgery, First Affiliated Hospital of China Medical University. All patients had complete postoperative pathology reports and lymph node tissue specimens.

[0105] All procedures and operations in this study complied with the principles of the Declaration of Helsinki, and written informed consent was obtained from all patients for all clinical samples. This study was approved by our hospital's Institutional Review Committee.

[0106] 2.1 Immunofluorescence detection of SPP1 + Presence of TAM in lymph node tissue of gastric cancer patients

[0107] In conducting immunofluorescence detection of SPP1 + Before the TAM experiment, the following reagents and materials need to be prepared: primary antibodies for detecting the target molecule, including Anti-CD68 antibody (mouse monoclonal antibody, 1:100) and Anti-Osteopontin antibody (rabbit monoclonal antibody, 1:50) for human lymph node tissue; fluorescently labeled secondary antibodies, including Alexa Fluor 488 (anti-mouse, 1:500) and Alexa Fluor 594 (anti-rabbit, 1:500); BSA blocking solution for blocking non-specific binding; dewaxing and dehydration reagents (such as xylene, anhydrous ethanol, graded ethanol), slide washing buffers (distilled water, PBS buffer, PBST buffer), antigen retrieval reagent (citrate buffer), 3% hydrogen peroxide solution prepared by mixing 30% hydrogen peroxide solution and methanol solution in a 1:9 ratio, and mounting medium containing DAPI to prevent fluorescence quenching. In addition, a microscope slide scanning system and an inverted fluorescence microscope are required. These reagents and equipment together constitute a complete immunofluorescence detection system for detecting SPP1 in lymph node tissues of human and mouse gastric cancer lymph node metastasis models. + The existence of TAM is determined. The specific steps are as follows:

[0108] (1) Place the tissue sections in an oven and bake them;

[0109] (2) After baking, remove the slices and cool them to room temperature. Then, perform gradient dewaxing and dehydration on the slices: soak them in xylene I, II and III at each stage, then soak them in anhydrous ethanol I and II respectively. Remove the slices and soak them in 90%, 80% and 70% ethanol in sequence. Remove the slices.

[0110] (3) Wash the slides with distilled water at room temperature;

[0111] (4) Prepare an appropriate amount of hydrogen peroxide solution by mixing 30% hydrogen peroxide solution and methanol solution in a 1:9 ratio, and incubate;

[0112] (5) Wash the slides with distilled water at room temperature;

[0113] (6) Place the tissue sections in citrate buffer for antigen retrieval;

[0114] (7) Remove the container from the equilibrium temperature and bring it to room temperature;

[0115] (8) Wash the slides with PBS buffer at room temperature;

[0116] (9) Wipe the moisture around the tissue section dry, draw a circle around the tissue with an immunohistochemistry pen, add BSA blocking solution evenly, and place it in a humid chamber to stand at room temperature.

[0117] (10) Prepare an appropriate amount of primary antibody dilution solution according to the experimental design and sample type. The primary antibody information and dilution ratio are shown in Table 2.

[0118] Table 2 Human Lymph Node Tissue SPP1 + Antibody for TAM staining

[0119] (11) Discard the blocking solution, add an appropriate amount of primary antibody diluent to evenly cover the tissue surface, and incubate overnight in the dark.

[0120] (12) The next day, remove the humidification chamber and equilibrate to room temperature, then wash the slides at room temperature with PBST buffer.

[0121] (13) Add an appropriate amount of fluorescent secondary antibody dilution solution in the dark and incubate at room temperature; information on the secondary antibody and dilution ratios are shown in Table 3.

[0122] Table 3. Secondary antibody information and dilution ratio

[0123] (14) After discarding the secondary antibody, wash the slides with PBS-T buffer at room temperature in the dark, and then wash with PBS buffer.

[0124] (15) Add an appropriate amount of DAPI-containing anti-fluorescence quenching mounting medium, cover with a glass slide, and store in the dark after mounting.

[0125] (16) Scan the slide using a microscope slide scanning system, take images using an inverted fluorescence microscope, and save the images.

[0126] 2.2 Results

[0127] As shown in Table 4, the non-metastatic lymph node SPP1 of 115 N0 patients + Immunofluorescence staining with TAM revealed that SPP1 was absent in 67 cases. +TAM, while 48 cases had SPP1 + TAM. SPP1 was performed on metastatic and non-metastatic lymph nodes in 77 patients with lymph node metastasis. + TAM immunofluorescence staining showed that SPP1 was present in all 77 metastatic lymph nodes. + TAM, SPP1 was present in 76 cases of non-metastatic lymph nodes. + TAM. Representative immunofluorescence results are shown in Figure 2. This indicates the presence of SPP1 in patient samples from N1-N3. + During TAM, a pre-metastasis niche has already been formed or lymph node metastasis has occurred, and its sensitivity and specificity are both 100%.

[0128] Table 4. Statistics of staining results

[0129] As shown in Table 5, this study further analyzed the SPP1 of all postoperative lymph node tissues from 6 patients with N0, 4 with N1, 5 with N2, and 3 with N3 gastric cancer. + TAM testing showed that SPP1 was detectable in some lymph nodes of patients in stage N0. + TAM, while SPP1 was present in all lymph node samples from patients in stages N1-N3. + TAM.

[0130] Table 5. SPP1 in lymph nodes of gastric cancer patients at different N stages + Summary of TAM test results

[0131] Example 3: Establishing clinical cohorts of other cancer patients to validate SPP1 + The characteristic presence of TAM in other metastatic lymph nodes of tumors.

[0132] To verify SPP1 +To further validate the presence of TAM in lymph node tissues from patients with breast cancer, colon cancer, rectal cancer, and ovarian cancer, samples different from those in Example 2 were selected. Paraffin-embedded lymph node tissue specimens from 20 patients with breast cancer, 10 with colon cancer, and 9 with rectal cancer who underwent radical surgery were included. All specimens were obtained from the First Affiliated Hospital of China Medical University. All patients had complete postoperative pathology reports and lymph node tissue specimens, and had not received neoadjuvant chemoradiotherapy before surgery. In addition, this study also included lymph node tissues from 12 patients with ovarian cancer at different FIGO stages. All specimens were obtained from Shengjing Hospital of China Medical University. Enrolled patients underwent radical ovarian cancer surgery combined with systematic lymph node dissection at the Department of Gynecology of Shengjing Hospital, and had complete postoperative pathology data and paraffin-embedded lymph node tissue specimens. They had not received neoadjuvant therapy before surgery. SPP1 in lymph nodes from different tumor patients was analyzed according to the method in Example 2. + TAM was tested.

[0133] As shown in Table 7-9, immunofluorescence staining results indicated that SPP1 was detected in only a portion of the lymph nodes in all lymph node tissues from patients with N0 stage breast cancer, colon cancer, and rectal cancer. + The presence of TAM was observed; while SPP1 was detected in all lymph node tissues of N+ (N1-N3 stage) patients. + TAM. Similarly, the outcomes in ovarian cancer patients (see Table 11) also showed the same trend: in all lymph nodes of ovarian cancer patients without lymph node metastasis, only a portion of the lymph nodes had SPP1. + TAM, and SPP1 can be detected in all lymph node tissues of ovarian cancer patients with lymph node metastasis. + TAM. Further analysis revealed that SPP1 was detected in only a portion of lymph node specimens from patients with stage I and II ovarian cancer. + TAM, while SPP1 can be detected in all lymph nodes of patients with stage III and IV ovarian cancer. + TAM. The above results are consistent with the findings of the gastric cancer cohort study, suggesting that SPP1... + TAM is not only specifically present in metastatic lymph nodes of gastric cancer, but also exhibits high specificity in metastatic lymph nodes of other solid tumors such as breast cancer, colon cancer, rectal cancer, and ovarian cancer, making it worthy of wider application. Representative immunofluorescence results are shown in Figure 3-6.

[0134] Table 6. Statistics on the number of patients with breast cancer, colon cancer, and rectal cancer.

[0135] Table 7. SPP1 in lymph nodes of breast cancer patients at different N stages + Summary of TAM test results

[0136] Table 8. SPP1 in lymph nodes of colon cancer patients at different N stages + Summary of TAM test results

[0137] Table 9. SPP1 in lymph nodes of rectal cancer patients at different N stages. + Summary of TAM test results

[0138] Table 10 Statistics on the number of ovarian cancer patients

[0139] Table 11 SPP1 in lymph nodes of ovarian cancer patients at different N stages + Summary of TAM test results

[0140] Example 4: A mouse model of popliteal lymph node metastasis was established, and SPP1 was detected. + Characteristic presence of TAM in metastatic lymph nodes of gastric cancer

[0141] 4.1 Experimental Cells

[0142] The GA0518 cell line was used in cell experiments. The gastric cancer GA0518 cell line was cultured in a cell culture incubator containing 5% CO2 using 1640 medium supplemented with 10% fetal bovine serum. When the cells grew to about 80%, they were passaged into new culture dishes at a ratio of 1:3.

[0143] 4.2 Construction of a stable GA0518-luciferase cell line using a lentiviral transfection system

[0144] (1) 3*10 5 One GA0518 cell was seeded into a well plate and cultured in a 5% CO2 incubator;

[0145] (2) When the cell confluence is 20-30%, remove the cells, wash them with PBS, add medium containing 10% fetal bovine serum, change the medium, add an appropriate amount of lentivirus suspension to each well according to the MOI determined in the pre-experiment; after culturing in a 5% CO2 incubator, remove the virus-containing medium and replace it with complete medium for culturing.

[0146] (3) Since the lentivirus has been transferred with the puromycin resistance gene, the cells are in good condition 3-4 days after transfection. Puromycin is then added for selection. Puromycin is added during cell passage or medium change until almost no cells die, and then the selection is ended.

[0147] 4.3 Establishment and tissue sampling of a mouse popliteal lymph node metastasis model

[0148] BALB / c-nude mice aged 4-5 weeks were used for the experiment and were housed in an SPF-grade environment for modeling. During the housing period, all groups of mice were allowed free access to food and water. The culture temperature was 22℃~24℃, the relative humidity was 45%~65%, and the day and night cycles were maintained.

[0149] All animal experiments in this study were approved by the Ethics Committee of China Medical University, and all procedures complied with animal ethics requirements.

[0150] A popliteal lymph node metastasis model was established in BALB / c-nude mice using the GA0518 cell line. Fourteen metastatic lymph node tissue samples and ten non-metastatic lymph node tissue samples were collected. Details are as follows:

[0151] Establishment of a mouse model of popliteal lymph node metastasis:

[0152] (1) Remove the cultured cells, discard the original culture medium in the dish, rinse twice with PBS, and then add trypsin to digest the cells.

[0153] (2) Discard the trypsin, add PBS to detach the cells, collect them in EP tubes and place them on ice;

[0154] (3) Count the cell concentration and prepare the cells into 2×10⁻⁶ cells. 6 Cells / 25μL of cell suspension;

[0155] (4) Inject 25 μL of cell suspension into the paw pad of each mouse;

[0156] (5) One week after injection, Luciferase substrate solution was injected intraperitoneally, and the tumor formation in mice was detected by a small animal in vivo imaging system.

[0157] (6) After observing for 4-7 weeks, the mice were sacrificed, the popliteal lymph nodes were dissected, and the volume of the popliteal lymph nodes was measured.

[0158] (7) Embed the popliteal lymph nodes to make paraffin tissue sections.

[0159] Mouse lymph node sampling and embedding:

[0160] (1) Mouse tissue embedding: Mouse lymph node tissue was removed and fixed in 4% paraformaldehyde, and then soaked in 75% ethanol.

[0161] (2) Place the tissue into a small embedding box or wrap it with embedding paper before placing it into the embedding box, and soak it in the following order to dehydrate: 80% ethanol → 95% ethanol I → 95% ethanol I → anhydrous ethanol I → anhydrous ethanol II.

[0162] (3) Soak the wax block in the following order to achieve transparency: 1:1 mixture of xylene and ethanol → xylene I → xylene II;

[0163] (4) Melt the soft wax and hard wax in advance, and then infiltrate the tissue in the following order: soft wax → hard wax;

[0164] (5) Place the tissue into a mold, inject paraffin wax, and let it cool;

[0165] (6) Place the embedded tissue block on a microtome, slice it, spread it in a slide bleaching machine, transfer the slice to an adhesive glass slide, label it, bake the slide, and perform subsequent immunofluorescence and immunohistochemical experiments.

[0166] mouse lymph node SPP1 + TAM staining:

[0167] In conducting immunofluorescence detection of SPP1 + Before the TAM experiment, the following reagents and materials need to be prepared: primary antibodies for detecting the target molecule, including Anti-F4 / 80 antibody (mouse monoclonal antibody, 1:200) and Anti-Osteopontin antibody (rabbit monoclonal antibody, 1:300) for mouse lymph node tissue. The primary antibody information is shown in Table 12. The remaining reagents and procedures are the same as those for human lymph node tissue SPP1. + TAM detection.

[0168] Table 12 SPP1 of mouse lymph node tissue + Antibody for TAM staining

[0169] 4.4 Results

[0170] After successfully establishing a gastric cancer popliteal lymph node metastasis model, SPP1 in the lymph nodes of mice was analyzed. + TAM was used for detection. The staining results are shown in Table 8. SPP1 was detected in all 14 metastatic lymph nodes. + The presence of TAM was observed; however, in 8 out of 10 cases without metastatic lymph nodes, SPP1 was absent. + Two cases of TAM were observed. Representative animal experimental results are shown in Figure 7. This further demonstrates that SPP1 is present in metastatic lymph nodes. + The presence of TAM, with a sensitivity and specificity of 100%.

[0171] Table 13 Mouse lymph node SPP1 + TAM staining

[0172] Example 5: A mouse model of popliteal lymph node metastasis was established, and SPP1 was detected. + Characteristic presence of TAM in metastatic lymph nodes of gastric cancer

[0173] To verify SPP1 +The characteristic presence of TAM in metastatic lymph nodes of gastric cancer was further validated using samples different from those in Example 3. Following the method in Example 3, a popliteal lymph node metastasis model was constructed using C57BL / 6 mice, and SPP1 in the mouse lymph nodes was analyzed. + TAM was tested.

[0174] The staining results are shown in Table 14. SPP1 was detected in all 6 cases of metastatic lymph nodes. + The presence of TAM was observed; however, in 2 of the 5 cases without metastatic lymph nodes, SPP1 was absent. + Three cases of TAM were observed. Representative animal experimental results are shown in Figure 8. This further confirms that SPP1 is present in metastatic lymph nodes. + The presence of TAM, with a sensitivity and specificity of 100%.

[0175] Table 14 Mouse lymph node SPP1 + TAM staining

[0176] Example 6: Immunohistochemical staining and immunofluorescence experiments to verify the pre-metastatic niche for biomarker detection.

[0177] To analyze SPP1 + The presence of TAM in metastatic lymph nodes of stage N0 gastric cancer was analyzed, and SPP1 was further selected from 20 cases of stage N0 lymph nodes. + Patients with TAM and 20 patients without SPP1 + Patients with TAM underwent pre-metastatic niche assessment. Specific analytical indicators included the following staining markers: CD163 (M2 macrophage marker), CD31 (vascular endothelial cell marker), MECA-79 (high endothelial venous cell marker), PDPN (lymphatic endothelial cell marker), and Treg cells identified by dual markers of CD3 and FOXP3. The rationale for using CD163-positive cell counts to determine the formation of pre-metastatic niches in gastric cancer lymph node metastasis has been published in the literature (Yukie Go, Hiroaki Tanaka, et al. Tumor-Associated Macrophages Extend Along Lymphatic Flow in the Pre-metastatic Lymph Nodes of Human Gastric Cancer. Ann Surg Oncol. 2016 Feb:23 Suppl 2:S230-5).

[0178] 6.1 SPP1 was not detected in N0 patients. + TAM and SPP1 exist +Changes in various evaluation indicators of TAM's ecological niche before migration

[0179] Before conducting immunohistochemical assays to detect pre-metastatic niche formation in lymph nodes, the following reagents and materials are required: primary antibodies for detecting the target molecules, including Anti-CD163 antibody (rabbit monoclonal antibody, 1:400), Anti-MECA-79 antibody (rat monoclonal antibody, 1:200), and Anti-CD31 antibody (mouse monoclonal antibody, 1:400) for human lymph node tissue; and corresponding secondary antibodies, including Goat Anti-Rabbit IgG Antibody (H+L), Biotinylated (anti-rabbit, 1:200), Goat Anti-Mouse IgG Antibody (H+L) (anti-mouse, 1:200), and Goat Anti-Rat IgG. Antibody (H+L) (anti-rat, 1:200); blocking solution prepared with BSA and 10% donkey serum for blocking nonspecific binding; dewaxing and dehydration reagents (e.g., xylene, anhydrous ethanol, graded ethanol); washing buffer (distilled water, PBS buffer); antigen retrieval reagent (citric acid buffer); 3% hydrogen peroxide solution prepared by mixing 30% hydrogen peroxide solution and methanol solution in a 1:9 ratio; DAB substrate kit for color development; hematoxylin staining solution, hydrochloric acid alcohol differentiation solution, and lithium carbonate blue solution for subsequent staining; and neutral resin for mounting. In addition, an immunohistochemistry kit (e.g., The kit includes the ABC-HRP Standard reagent kit, a microscope slide scanning system, and a microscope. These reagents and equipment together constitute a complete immunohistochemical detection system for detecting SPP1 in human gastric cancer lymph node tissue. + The existence of TAM is determined. The specific steps are as follows:

[0180] (1) Place the tissue sections in an oven and bake them;

[0181] (2) After baking, remove the slices and cool them to room temperature. Then, perform gradient dewaxing and dehydration on the slices: soak them in xylene I, II and III at each stage, then soak them in anhydrous ethanol I and II, remove the slices, and then soak them in 90%, 80% and 70% ethanol in sequence.

[0182] (3) Wash the slides with distilled water at room temperature;

[0183] (4) Prepare an appropriate amount of 3% hydrogen peroxide solution by mixing 30% hydrogen peroxide solution and methanol solution in a 1:9 ratio, and incubate at room temperature in the dark.

[0184] (5) Wash the slides with distilled water at room temperature;

[0185] (6) Place the tissue sections in citrate buffer for antigen retrieval;

[0186] (7) Remove the container from the equilibrium temperature and bring it to room temperature;

[0187] (8) Wash the slides with PBS buffer at room temperature;

[0188] (9) Mark around the tissue, add the blocking solution prepared with 3% BSA and 10% donkey serum evenly, and place it in a humidified box to stand at room temperature;

[0189] (10) Prepare an appropriate amount of primary antibody dilution solution according to the experimental design and sample type. The primary antibody information and dilution ratio are shown in Table 15.

[0190] Table 15. SPP1 in lymph node tissue of N0 patients + Antibody for TAM staining

[0191] (11) Discard the blocking solution, add an appropriate amount of primary antibody diluent, and incubate overnight in the dark;

[0192] (12) The next day, remove the humidification chamber and bring it to room temperature. Wash the slides with PBS buffer at room temperature.

[0193] (13) Add an appropriate amount of secondary antibody dilution solution and incubate at room temperature; information on secondary antibodies and dilution ratios are shown in Table 16.

[0194] Table 16 Secondary Antibody Information and Dilution Ratio

[0195] (3) After discarding the secondary antibody diluent, wash the slides with PBS buffer at room temperature;

[0196] (4) Use Prepare an appropriate amount of ABC-HRP Standard immunohistochemistry kit using working solution A: working solution B: PBS buffer in a ratio of 1:1:100. Add the solution dropwise and react at room temperature.

[0197] (5) Wash slides with PBS buffer at room temperature;

[0198] (6) Using the DAB substrate kit, prepare an appropriate amount of DAB working solution in the ratio of DAB substrate solution: diluent = 1:9 in the dark, and observe the color development under a microscope.

[0199] (7) Stain with hematoxylin, then rinse the stain with running water;

[0200] (8) Hydrochloric acid-alcohol differentiation solution reaction;

[0201] (9) Then place in lithium carbonate blueing solution and wash with water;

[0202] (10) Complete the dehydration in the following soaking order and reaction time: 70%, 80%, 90% alcohol, then soak in anhydrous ethanol I and II; after dehydration, air dry, add neutral resin and then seal.

[0203] (11) Scan the slide using a microscope slide scanning system and save the image.

[0204] 6.2 Immunohistochemical score

[0205] After all tissue sections were stained, they were scanned using a digital slide scanner to generate electronic images, which were then scored using a double-blind method by two independent clinical pathologists.

[0206] (1) The scoring criteria for CD163 immunohistochemical staining are shown in Tables 17 and 18. First, the scores are determined based on the intensity of the immunohistochemical staining and the size of the stained area:

[0207] Table 17 Specific Standards for Staining Intensity Scoring

[0208] Table 18 Specific Standards for Scoring Dyeing Area

[0209] The final staining score is calculated by multiplying the staining intensity score by the staining area score.

[0210] (2) Blood vessel and lymphatic vessel density scoring: The counting was performed according to the Weidner criteria. The slide was observed in a panoramic view to determine the hot spot with the highest blood vessel density. Any single brown-yellow granular endothelial cell or cord-like endothelial cell cluster was counted as one positive blood vessel. The count was then performed under a high-power light microscope at 200x magnification. Five fields of view were counted for each case, and the average value was taken as the blood vessel density or lymphatic vessel density of that case.

[0211] Table 19 Staining of lymph nodes in patients

[0212] 6.3 Results

[0213] To further analyze SPP1 in non-metastatic lymph nodes + The pattern of TAM existence is that SPP1 is absent in N0 patients. + TAM and SPP1 exist + The TAM case was analyzed. The results are shown in Figure 9, indicating the presence of SPP1. + TAM's non-metastatic lymph nodes and the absence of SPP1 +Compared to lymph node tissue in TAM, it showed an increased number of M2 macrophages, a relatively increased number of blood vessels with larger luminal areas, thinner walls and larger luminal areas in high endothelial veins, an increased number of lymphatic vessels, and a significantly increased number of Tregs, suggesting the presence of SPP1. + The non-metastatic lymph nodes of TAM have formed a pre-metastatic niche.

[0214] Furthermore, using the CD163 results, ROC curves were plotted using GraphPad Prism 8.0.1 software. The specific method was as follows: based on SPP1... + Different cut-off thresholds were set for TAM expression values, and the sensitivity and specificity at each threshold were calculated, resulting in ROC curves. Subsequently, the Youden index (Sensitivity + Specificity - 1) was used to evaluate each cut-off value, and the cut-off value with the highest Youden index was selected as the optimal threshold for determining pre-metastatic niche formation in lymph node metastasis. Patient samples were then grouped based on this cut-off value to evaluate diagnostic efficacy, and the area under the ROC curve (AUC) was calculated.

[0215] Based on the ROC curves above, SPP1 was found to be... + When TAM was used to determine the pre-lymph node metastasis niche in patients, the AUC was 0.8225 (see Figure 10). This indicates that SPP1 was the most prevalent niche in N0 stage patient samples. + TAM (Transurethral Aminoacrylamide) has high specificity in determining the formation of pre-metastatic niches in lymph nodes. Youden index analysis showed an optimal cut-off value of 5.0; at this threshold, the sensitivity was 65%, the specificity was 100%, and the diagnostic efficacy was optimal. These results indicate that SPP1 is present in lymph node samples from N0 patients. + As a biomarker, TAM can accurately determine the formation of pre-metastatic niches in lymph nodes, providing a strong basis for clinical staging and treatment decisions.

[0216] Example 7: Immunohistochemical staining to verify the pre-metastatic niche for biomarker detection

[0217] To verify the relationship between pre-metastatic niche formation and SPP1 in N0 patients. +To further evaluate the pre-metastatic niche of lymph nodes, 10 patients with SPP1+ macrophages in N0 stage lymph nodes (different from those in Example 5) and 10 patients without SPP1+ macrophages were selected and assessed using immunohistochemistry and immunofluorescence techniques. Specific analytical indicators included the following staining markers: CD163 (M2 macrophage marker), CD31 (vascular endothelial cell marker), MECA-79 (high endothelial venous cell marker), PDPN (lymphatic endothelial cell marker), and Treg cells identified by dual markers of CD3 and FOXP3.

[0218] Table 20 Staining of lymph nodes in patients

[0219] The results are shown in Figure 11, which are consistent with the results in Figure 5, indicating the presence of SPP1. + TAM's non-metastatic lymph nodes and the absence of SPP1 + Compared with TAM lymph node tissue, it has an increased number of M2 macrophages, a relatively increased number of blood vessels with a larger lumen area, thinner walls of high endothelial veins with a larger lumen area, an increased number of lymphatic vessels, and a significantly increased number of Tregs, proving that the non-metastatic lymph nodes with SPP1+TAM have formed a pre-metastatic niche.

[0220] Furthermore, using the CD163 results, ROC curves were plotted using GraphPad Prism 8.0.1 software. The specific method was as follows: based on SPP1... + Different cut-off thresholds were set for TAM expression values, and the sensitivity and specificity at each threshold were calculated, with ROC curves plotted accordingly. Subsequently, the Youden index was used to evaluate each cut-off value, and the cut-off value with the largest Youden index was selected as the optimal threshold for determining pre-metastatic niche formation in lymph node metastasis. Patient samples were then grouped based on this cut-off value to evaluate diagnostic efficacy, and the area under the ROC curve (AUC) was calculated.

[0221] Based on the ROC curves above, SPP1 was found to be... + When TAM was used to determine the pre-lymph node metastasis niche in patients, the AUC was 0.8850 (see Figure 12). This indicates that SPP1 was the most prevalent niche in N0 stage patient samples. + TAM (Transient Acid Analysis) has high specificity in determining the formation of pre-metastatic niches in lymph nodes. Youden index analysis showed an optimal cut-off value of 2.8; at this threshold, the sensitivity was 70%, specificity was 90%, and diagnostic efficacy was optimal. These results indicate that SPP1 is present in lymph node samples from N0 patients. +As a biomarker, TAM can accurately determine the formation of pre-metastatic niches in lymph nodes, providing a strong basis for clinical staging and treatment decisions.

Claims

1. Specific recognition of the biomarker SPP1 + The application of TAM (SPP1-positive tumor-associated macrophages) reagents in the preparation of diagnostic kits for lymph node metastasis risk in cancer patients is characterized by, If SPP1 is detected in lymph node tissue samples from patients with N0 tumors or those without lymph node metastases... + TAM indicates that the lymph node has formed a pre-metastatic niche, warning of a high risk of lymph node metastasis. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low; if SPP1 is detected in lymph node tissue samples from N1-N3 tumor patients or tumor patients with already identified metastatic lymph nodes... + TAM indicates that the lymph node has formed a pre-metastatic niche and warns of a high risk of lymph node metastasis, or it indicates that lymph node metastasis has already occurred. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

2. The application as described in claim 1, characterized in that, The reagent is specifically designed to identify SPP1 in lymph node tissue samples from cancer patients. + TAM antibodies include antibodies against human tumor-associated macrophage surface markers and anti-Osteopontin antibodies against the SPP1 protein.

3. The application as described in claim 2, characterized in that, The antibodies targeting human tumor-associated macrophage surface markers include Anti-CD68 antibody, Anti-CD11b antibody, Anti-CD163 antibody, Anti-CD206 antibody, Anti-CD204 antibody, or Anti-Stabilin-1 antibody.

4. The application as described in any one of claims 1 to 3, characterized in that, The tumors include one or more of the following: gastric cancer, colorectal cancer, oral squamous cell carcinoma, esophageal cancer, lung cancer, liver cancer, bladder cancer, glioma, breast cancer, or ovarian cancer.

5. A kit for diagnosing the risk of lymph node metastasis in cancer patients, said kit comprising SPP1 that specifically identifies lymph node tissue samples from cancer patients. + TAM's reagents, including antibodies against surface markers of human tumor-associated macrophages and an anti-Osteopontin antibody against the SPP1 protein, are characterized by... The diagnosis includes using the kit to detect SPP1 in lymph node tissue samples from cancer patients. + TAM, if SPP1 is detected in lymph node tissue samples from patients with N0 tumors or those without lymph node metastases. + TAM indicates that the lymph node has formed a pre-metastatic niche, warning of a high risk of lymph node metastasis. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low; if SPP1 is detected in lymph node tissue samples from N1-N3 tumor patients or tumor patients with already identified metastatic lymph nodes... + TAM indicates that the lymph node has formed a pre-metastatic niche and warns of a high risk of lymph node metastasis, or it indicates that lymph node metastasis has already occurred. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

6. The reagent kit as described in claim 5, characterized in that, It also includes SPP1 in lymph node tissue samples from cancer patients. + For TAM indirect immunohistochemical localization detection, the corresponding marker-labeled secondary antibody or for direct localization detection, the marker-labeled antibody against human tumor-associated macrophage surface markers and the Anti-Osteopontin antibody against SPP1 protein, wherein the marker is fluorescein, enzyme (including horseradish peroxidase, alkaline phosphatase and / or glucose oxidase), metal ion and / or radioisotope.

7. The kit as described in claim 5 or 6, characterized in that, The antibodies targeting human tumor-associated macrophage surface markers include Anti-CD68 antibody, Anti-CD11b antibody, Anti-CD163 antibody, Anti-CD206 antibody, Anti-CD204 antibody, or Anti-Stabilin-1 antibody.

8. The reagent kit as described in claim 6, characterized in that, It also includes dewaxing and hydration reagents, antigen retrieval solution, chromogenic reagents of different colors for the corresponding markers, blocking solution, washing solution and / or mounting medium required for the indirect or direct immunohistochemical localization detection.

9. The kit according to any one of claims 5 to 8, characterized in that, The tumors include one or more of the following: gastric cancer, colorectal cancer, oral squamous cell carcinoma, esophageal cancer, lung cancer, liver cancer, bladder cancer, glioma, breast cancer, or ovarian cancer.

10. The application as described in claim 1 or the kit as described in claim 5, characterized in that, When the tumor is ovarian cancer, if SPP1 is detected in lymph node tissue samples from patients with FIGO stage I-II or ovarian cancer without lymph node metastasis. + TAM indicates that the lymph node has formed a pre-metastatic niche, warning of a high risk of lymph node metastasis. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low. If SPP1 is detected in lymph node tissue samples from patients with FIGO stage III-IV ovarian cancer or those with confirmed lymph node metastases... + TAM indicates that the lymph node has formed a pre-metastatic niche and warns of a high risk of lymph node metastasis, or it indicates that lymph node metastasis has already occurred. If SPP1 is not detected... + TAM indicates that the lymph node has not formed a pre-metastatic niche and the risk of lymph node metastasis is low.

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