Biomarker for judging cancer metastasis or cancer recurrence and application thereof

Abstract

The invention provides a biomarker for judging cancer metastasis or cancer recurrence and application thereof, and relates to the technical field of molecular biology. The biomarker based on STEAP4 protein and / or IGFBP6 protein provided by the invention has the advantages of convenience, rapidness and the like in detection, can provide dynamic monitoring of diseases for patients, and achieves the technical effect of timely discovering cancer metastasis signs or cancer recurrence signs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to biomarkers for judging cancer metastasis or cancer recurrence and applications thereof. Background Art

[0002] The progression of a tumor from the primary stage to the metastatic stage is a complex process, accompanied by significant changes in the tumor microenvironment and tumor characteristics. During the metastatic stage, some specific molecules are highly expressed in tumor tissues, such as the c-MAF gene.

[0003] In the prior art, JP6946385B2 discloses a method for diagnosing, prognosing, and treating breast cancer metastasis. This method predicts and intervenes in breast cancer metastasis by detecting the expression levels of specific molecules in tumor tissue. However, this method has certain limitations. In the early stages of the disease, tumor tissue is often difficult to obtain. By the time metastasis is discovered, the disease has typically progressed to a late stage, making intervention extremely challenging. Therefore, tissue-based detection methods suffer from delayed monitoring, making it difficult to promptly reflect disease changes, and their scope of application is limited. Furthermore, US20080113350A1 discloses a blood test using immunomagnetic enrichment and fluorescence in situ hybridization (FISH) to monitor genetic changes in progressive cancers. While this method generally achieves the goal of predicting cancer progression and metastasis by detecting circulating tumor cells (CTCs) in the blood, it still suffers from limitations due to the nature of the tumor cells. This method is only applicable to a small number of tumor cells with known mutational signatures within a specific cancer type, such as the HER2 subtype of breast cancer. Therefore, detecting circulating tumor cells (CTCs) is not effective for predicting cancer progression in patients with more complex tumors and diverse cancer types. Summary of the Invention

[0004] In view of the above problems, the present invention provides a biomarker, especially a biomarker for judging cancer metastasis or cancer recurrence and its application, so as to predict the probability of cancer metastasis or cancer recurrence.

[0005] To achieve the above objectives, in a first aspect, the present invention protects a biomarker for determining whether cancer metastasis or cancer recurrence exists, characterized in that the biomarker is STEAP4 protein and / or IGFBP6 protein.

[0006] In a second aspect, the present invention protects the use of a biomaterial related to a STEAP4 protein or an IGFBP6 protein for determining whether cancer metastasis or cancer recurrence exists.

[0007] Furthermore, preferably, the biological material includes a nucleic acid molecule encoding the STEAP4 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

[0008] Further, preferably, obtaining a sample to be tested;

[0009] The target protein expression level of the biomarker described above in the sample to be detected is determined by flow cytometry or immunofluorescence staining.

[0010] Furthermore, preferably, the sample to be detected is a biological fluid or tissue, wherein the object to be detected is neutrophils or other myeloid cells in the biological fluid or tissue, and the biological fluid includes but is not limited to serum, blood, or fluids in which cancer metastasis may occur.

[0011] In a third aspect, the present invention protects a biomarker detection product, which includes a substance for detecting the expression level of the biomarker as described above; the function of the product is to identify or assist in identifying the level of cancer metastasis or cancer recurrence.

[0012] Furthermore, preferably, the cancer is selected from one or more of leukemia, brain cancer, prostate cancer, liver cancer, ovarian cancer, gastric cancer, colorectal cancer, pharyngeal cancer, breast cancer, skin cancer, melanoma, lung cancer, sarcoma, cervical cancer, testicular cancer, bladder cancer, endocrine system cancer, endometrial cancer, esophageal cancer, glioma, lymphoma, neuroblastoma, osteosarcoma, pancreatic cancer, pituitary tumor, and kidney cancer.

[0013] In a fourth aspect, the present invention protects a system for predicting cancer metastasis or cancer recurrence, comprising:

[0014] processor;

[0015] and a storage medium containing a program executed by a processor for determining a cancer metastasis level or a cancer recurrence level, wherein the program for determining cancer metastasis or cancer recurrence causes the processor to perform the following steps:

[0016] Obtaining the target protein expression level of the biomarker described above in the sample to be tested;

[0017] Comparing the obtained target protein expression level of the biomarker with a preset expression level threshold;

[0018] The cancer metastasis probability or cancer recurrence probability of the subject to be detected is determined according to the comparison result.

[0019] The beneficial effects of the present invention are as follows:

[0020] The biomarker detection based on STEAP4 protein and / or IGFBP6 protein provided by the present invention has multiple advantages such as convenience and rapidity, and can provide patients with dynamic monitoring of the disease, achieving the technical effect of timely detecting signs of cancer metastasis or cancer recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a correlation analysis diagram of the expression levels and expression percentages of STEAP4 protein and IGFBP6 protein in neutrophils derived from lung tissue of a mouse model in an embodiment of the present invention;

[0022] Figure 2 This is an analysis diagram showing the relationship between the expression of STEAP4 protein and IGFBP6 protein in lung tissue-derived neutrophils and metastasis based on a mouse model in an embodiment of the present invention;

[0023] Figure 3 This is a graph showing the expression of STEAP4 protein and IGFBP6 protein in blood neutrophils in a mouse model according to an embodiment of the present invention;

[0024] Figure 4 This is an expression analysis diagram showing the expression ratio and degree of STEAP4 protein and IGFBP6 protein in blood neutrophils in a mouse model according to an embodiment of the present invention;

[0025] Figure 5 : is a ROC curve diagram of the predictive ability of STEAP4 protein and IGFBP6 protein for metastasis in the embodiment of the present invention;

[0026] Figure 6 This is a graph showing the expression ratio of STEAP4 protein in the blood neutrophils of pan-cancer patients;

[0027] Figure 7 Violin plots comparing STEAP4 and IGFBP6 at various tumor stages based on mouse models;

[0028] Figure 8 A representative graph showing the expression ratio of STEAP4 protein in neutrophils in the blood of breast cancer metastasis patients and healthy subjects; the control group is a blood sample not stained with STEAP4 (FMO), and the experimental group is a sample stained with STEAP4.

[0029] Figure 9 This is a statistical graph of flow cytometry experimental results. DETAILED DESCRIPTION

[0030] The present invention will be described in more detail with reference to the following examples. However, the scope of protection of the present invention is not limited to the following examples.

[0031] In order to clearly illustrate the embodiments of the present invention, some technical terms involved in the present invention are explained below.

[0032] "Subject" refers to any individual to whom drug administration is directed. A subject can be a vertebrate, such as a mammal. Thus, a subject can be a human. The term does not denote a particular age or sex. Thus, it is intended to encompass both adult and newborn subjects, whether male or female. A patient is a subject suffering from a disease or condition. The term "patient" includes both human and animal subjects.

[0033] "Inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% decrease in the activity, response, condition, or disease compared to a natural or control level. Thus, a decrease can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to a natural or control level.

[0034] The term "prognosis" includes a prediction about the likely course of a disease or disease progression, particularly concerning the likelihood of disease remission, disease relapse, and death.

[0035] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. The term includes active treatment, which is treatment specifically directed at ameliorating the disease, pathological condition, or condition, as well as causal treatment, which is treatment directed at eliminating the cause of the disease, pathological condition, or condition in question. The term also includes palliative treatment, which is treatment intended to relieve symptoms rather than cure the disease, pathological condition, or disorder; preventive treatment, which is treatment intended to minimize or partially or completely inhibit the progression of the disease, pathological condition, or condition in question; and supportive treatment, which is treatment used to supplement another specific treatment directed at ameliorating the disease, pathological condition, or condition in question.

[0036] The term "tumor" refers to a new growth formed by the proliferation of localized tissue cells in response to various tumorigenic factors. Because such a new growth often presents as a space-occupying, mass-like protrusion, it is also called a neoplasm. The term "tumor" as used herein encompasses both benign and malignant tumors. However, benign tumors generally do not metastasize and generally have a good prognosis. Therefore, the present invention is more applicable to the detection of malignant tumor cells.

[0037] The term "malignant tumor," also known as "cancer" in Traditional Chinese Medicine (TCM), refers to abnormal cell proliferation that can potentially invade other parts of the body. It is a disease caused by a malfunction in the mechanisms controlling cell division and proliferation. In addition to uncontrolled cell division, cancer cells can also invade surrounding normal tissues and even metastasize to other parts of the body through the circulatory or lymphatic systems.

[0038] "Tumor cell metastasis," as used in this application, refers to the process by which tumor cells migrate from their original site of origin to other parts of the body by invading the circulatory system and continuing to grow. Benign tumors generally do not metastasize to other locations, but patients who do experience metastasis have a very poor prognosis. Because cancer cells metastasize to various parts of the body, cancer treatment becomes more difficult.

[0039] As used herein, the term "tumor microenvironment" refers to the microenvironment surrounding tumor cells, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules, and the extracellular matrix. Tumors and their surroundings are closely intertwined and constantly interact. Tumors can influence their microenvironment by releasing cell-signaling molecules, promoting angiogenesis and inducing immune tolerance. Immune cells in the microenvironment can influence the growth and development of cancer cells.

[0040] Examples of protein detection assays include, but are not limited to, immunoassays, such as enzyme-linked immunosorbent assay (ELISA), Western blot, dot blot, radioimmunoassay (RIA), fluorescent immunoassay (FIA), immunoprecipitation, and the like.

[0041] Examples of nucleic acid detection include, but are not limited to, PCR, reverse transcription PCR, real-time quantitative PCR, and RNA-seq sequencing such as single-cell RNA-seq sequencing.

[0042] STEAP4, also known as STAMP2 and TNFAIP9, is a metalloreductase encoded by the STEAP4 gene and belongs to the STEAP family. Members of this family typically have six transmembrane spans. It is present on the cell membrane and in the Golgi apparatus and is closely associated with tumors [STEAP: Aprostate-specific cell-surface antigen highly expressed in human prostate tumors, Proc Natl Acad Sci USA, 1999; Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction, Nat. Commun., 2018; STEAP proteins: from structure to applications in cancer therapy, Mol. Cancer Res, 2012].

[0043] The gene and protein sequences of STEAP are available at NCBI Gene: https: / / www.ncbi.nlm.nih.gov / gene?Db=gene&Cmd=DetailsSearch&Term=79689 and Protein Atlas: https: / / www.proteinatlas.org / ENSG00000127954-STEAP4.

[0044] IGFBP6 protein, insulin-like growth factor binding protein 6 (IGFBP-6) is a protein encoded by the IGFBP6 gene. IGFBP6 is the sixth member of the IGFBP family and is more likely to bind to IGF-II in a specific manner than IGFBP1 [Bach LA, et al. J Biol Chem 1993; 268(13): 9246-9254]. In animal models and human subjects, four IGFBPs (IGFBP-1, IGFBP2, IGFBP3, and IGFBP5) have been associated with T1D, prevention of obesity, and induction of insulin resistance [Mohamed-Ali V, et al. Clin Endocrinol (Oxf) 1999; 50(2): 221-228; Feldman B et al.].

[0045] IGFBP-6 binds to IGF-II with a higher affinity than IGF-I and is a specific inhibitor of IGF-II. IGFBP-6 plays a complex role in tumor development, progression, and metastasis. On the one hand, it can inhibit the proliferation and invasion of certain tumor cells; on the other hand, within specific tumor microenvironments, it may also promote tumor growth and metastasis. (IGFBP-6: At the Crossroads of Immunity, Tissue Repair and Fibrosis, Int J Mol Sci., 2022).

[0046] The expression levels of STEAP4 and IGFBP6 are determined or measured in a sample from a subject. The sample can be from a tissue sample or biological fluid where metastasis may occur. Sample processing first extracts neutrophils as the test subject; in a specific implementation process, monocytes can also be used as the test subject. Because IGFBP6 is a secretory protein, it can be secreted by cells into the microenvironment and may be higher in the tissues or serum of metastatic individuals. Specifically, the sample can be a biological fluid or a cell sample. Biological fluids include but are not limited to serum, blood, or fluids where cancer metastasis may occur, such as other body fluids related to cancer metastasis, such as pleural effusion, ascites, etc. Cell samples are only on neutrophils.

[0047] The terms "and / or", "or / and", and "and / or" used in the present invention include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0048] Example 1

[0049] 1. Materials and Methods

[0050] 1.1 Experimental Materials

[0051] 1.1.1 Antibodies used: IGFBP6 ELISA: Human IGFBP6 ELISA Kit (EHIGFBP6, Invitrogen, USA); STEAP4 flow cytometry: CD15 antibody: BD Horizon TM BV421 Mouse Anti-Human CD15 (567008, BD); CD16 antibody: BD Pharmingen TM PE Mouse Anti-Human CD16 (561313, BD); STEAP4 Antibody: Human STAMP2 / STEAP4 Alexa 647-conjugatedAntibody (FAB4626R, R&D).

[0052] 1.1 STEAP4 and IGFBP6 are specifically overexpressed in neutrophils of metastatic tissues

[0053] Through single-cell sequencing analysis, the expression characteristics of neutrophil surface proteins associated with tumor metastasis can be discovered, which can be used as potential biomarkers for early diagnosis, prognosis assessment and treatment monitoring of tumors.

[0054] In a mouse model, single-cell sequencing analysis of neutrophils in tumor tissue revealed that STEAP4 and IGFBP6 were specifically expressed in the lung tissue of mice with lung metastasis, but not in neutrophils in normal lung tissue or in the lung tissue of mice with primary lung cancer. Figure 1 As shown, this trend was consistent across five mouse models of metastasis.

[0055] The relationship between the expression level and percentage of STEAP4 and IGFBP6 in lung tissue-derived neutrophils and their metastasis. Figure 1 As can be seen in the left figure, STEAP4 and IGFBP6 are almost not expressed in the healthy and primary lung cancer (KP) mouse models. Both lung metastasis models have high expression (ratio>60%). Figure 1 The right panel shows that, unlike the left panel, the two lung metastasis models (MMTV-PyMT and 4T1), shown in the right panel, show high expression of STEAP4 and IGFBP6 under metastatic conditions, while the three metastasis models (M39M, B16-F10, and AT3) show low expression under normal conditions. These results suggest that elevated expression of STEAP4 and IGFBP6 is highly correlated with cancer metastasis, and that both STEAP4 and IGFBP6 have significant potential for predicting metastasis.

[0056] Neutrophils were analyzed by single-cell transcriptome sequencing in four different lung metastasis models: rhabdomyosarcoma lung metastasis model M39M, melanoma lung metastasis model B16-F10, breast cancer lung metastasis model AT3, and breast cancer lung metastasis model 4T1. Figure 2 As shown, it was found that both STEAP4 and IGFBP6 were highly expressed in neutrophils under metastatic conditions, while the expression levels of both were extremely low in normal mouse lung tissue, indicating that STEAP4 and IGFBP6 each have the ability to detect cancer metastasis as a biomarker.

[0057] 1.2 STEAP4 and IGFBP6 are specifically highly expressed in blood neutrophils

[0058] By observation Figure 3 It can be seen that in the single-cell transcriptome data of blood neutrophils, for normal (Normal) and mice with primary tumors (Primary tumor) and breast cancer lung metastasis model AT3 and breast cancer lung metastasis model 4T1, it can be seen that STEAP4 and IGFBP6 are highly expressed in the blood neutrophils of metastatic (Lung met) mice, but are almost not expressed in the neutrophils of normal and primary tumor-bearing mice, indicating that the two can serve as blood markers for detecting cancer metastasis separately or in combination.

[0059] By observation Figure 4 It can be seen that STEAP4 and IGFBP6 are found specifically in individuals with breast cancer lung metastasis in the single-cell transcriptome data of blood neutrophils, with an expression ratio of up to 80% in neutrophils, while they are almost not expressed in healthy mice or in primary pancreatic cancer mouse models. This phenomenon was found in both types of breast cancer mice, indicating that STEAP4 and IGFBP6 can not only serve as tissue markers for detecting cancer metastasis in a variety of cancers from different sources ( Figure 1-2 ), can also become blood biomarkers for detecting cancer metastasis ( Figure 3-4 ) potential. Among them, blood biomarkers have great application value: compared with tissue testing, they are more convenient and faster, and support the dynamic monitoring of the development of cancer metastasis.

[0060] 1.3 ROC analysis illustrates the predictive ability of STEAP4 for metastasis

[0061] In this example, based on the single-cell RNA-seq data of lung tissue from the preclinical mouse model, the expression percentage of STEAP4 or IGFBP6 in neutrophils was used as a variable to determine whether the tissue source was a lung metastasis sample. Figure 5As can be seen, the proportion of STEAP4 expression in tissue-derived neutrophils can be used to classify metastatic and non-metastatic individuals. The predictive ability of STEAP4 was found to be 0.938, with an optimal threshold of 13.1% STEAP4 expression (above which the threshold predicts metastasis), a specificity of 0.87, and a sensitivity of 0.88. These results demonstrate that STEAP4 has extremely high predictive accuracy in distinguishing individuals with metastasis from those without metastasis, making it a promising biomarker. Similarly, the predictive AUC for IGFBP6 was 0.99, with an optimal threshold of 9.57%, a specificity of 1, and a sensitivity of 0.94, indicating that IGFBP6 has extremely high predictive accuracy in distinguishing individuals with metastasis from those without metastasis, and can also be used as a promising biomarker. Combining the average expression percentages of both factors as a variable for predicting metastasis yielded an AUC of 0.98, an optimal threshold of 14.1%, a specificity of 0.93, and a sensitivity of 0.94. Note: Due to different technical errors among different technologies, the cell positive rates at protein and RNA levels may also vary. Therefore, the thresholds for neutrophil expression percentages of STEAP4 and IGFBP6 for other technologies may be different.

[0062] 1.4 Expression of STEAP4 in neutrophils in pan-cancer patients

[0063] The expression results of STEAP4 in blood neutrophils of various cancer patients as shown in Table 1 were obtained.

[0064] Specifically, the experimental data for this study were derived from single-cell transcriptome data of human blood-derived neutrophils from several published articles [see Table 1]. The types of cancer in the samples in Table 1 were statistically analyzed [see Table 2]. By calculating the expression level and expression ratio of STEAP4 in neutrophils (cells with read counts > 0 were recorded as STEAP4+ cells), the differences in STEAP4 expression ratios among the groups were compared. The results are shown in Table 1. Figure 6 shown.

[0065] Table 1 Statistics of STEAP4 expression in neutrophils of pan-cancer patients

[0066]

[0067]

[0068] Table 2 Statistics of the number of pan-cancer patients

[0069] Tumor type healthy transfer No transfer Cervical squamous cell carcinoma and cervical adenocarcinoma (CESC) 0 0 2 Colorectal cancer (CRC) 0 3 2 Gallbladder cancer (GBC) 0 4 0 Gastrointestinal stromal tumors (GIST) 0 1 0 Hepatocellular carcinoma (HCC) 0 5 3 Healthy control group (healthy) 18 0 0 Cholangiocarcinoma (ICC) 0 6 10 Lung cancer (LC) 0 5 2 Pancreatic adenocarcinoma (PAAD) 0 5 0 Renal cell carcinoma (RCC) 0 1 0 Gastric adenocarcinoma (STAD) 0 0 1 Thymoma (THYM) 0 0 1

[0070] By observation Figure 6 As can be seen in Table 2, since the data come from multiple cancer types including colorectal cancer, gallbladder cancer, and gastric cancer, for patients with metastasis, the expression ratio of STEAP4 in neutrophils is also specifically increased during metastasis, indicating that the STEAP4 protein has the ability to predict metastasis in all cancer types and acts as a biomarker indicating the risk of cancer metastasis in multiple cancer types.

[0071] 1.5 Cancer metastasis progression stage and STEAP4 or IGFBP6 expression

[0072] Single-cell data from neutrophils in lung metastasis tissues of mouse models suggested that STEAP4 or IGFBP6 has the ability to indicate the stage of cancer metastasis progression, and is significantly upregulated in neutrophils in the early stages of metastasis, indicating that both STEAP4 and IGFBP6 are highly valuable biomarkers.

[0073] By observing the single-cell transcriptome data of neutrophils in a mouse model Figure 7 As can be seen, in a primary lung cancer model (KP), a mouse model without lung metastasis, no STEAP or IGFBP6 expression was upregulated in lung neutrophils. In contrast, in a breast cancer lung metastasis model (MMTV-PyMT), STEAP4 and IGFBP6 expression in neutrophils from metastatic tissues increased with cancer progression, with significant upregulation in neutrophils already at the early stages of metastasis. These results suggest that STEAP4 and IGFBP6 expression levels are positively correlated with the risk of cancer metastasis. Low levels of expression reflect earlier or no metastasis, while high levels are strongly associated with later cancer progression and a strong metastatic outbreak. Therefore, STEAP4 and IGFBP6 expression detection can be used for dynamic monitoring of cancer metastasis risk.

[0074] Example 2

[0075] Neutrophil isolation

[0076] Fresh whole blood samples were collected from human donors. Peripheral blood was slowly layered onto Polymorphprep (Polymorphprep, No. 1895), taking care not to mix the blood into the Polymorphprep layer below. Centrifuged at 500g for 35 minutes at 20°C using the lowest acceleration / deceleration setting. Carefully aspirate the neutrophil layer (polymorphonuclear leukocytes) using a pipette to avoid contamination of the upper mononuclear cell layer. Wash the cells with 5mL 50% HBSS (Gibco, 24020117) and resuspend by gently pipetting. Centrifugation was achieved by the following steps: centrifugation at 350g for 10 minutes at 20°C, then removing the supernatant (avoid tilting the tube to prevent cell loss). The cells were resuspended in 10mL HBSS, centrifuged at 300g for 5 minutes at 20°C, and treated with RBC lysis buffer (Invitrogen, 00-4333-57). After lysis, the cells were centrifuged again (300 g, 5 minutes, 20° C.), washed with 10 mL of HBSS, and centrifuged at 300 g for 5 minutes at 20° C. The final pellet contained purified neutrophils.

[0077] Flow cytometry staining

[0078] Resuspend the isolated neutrophils in working buffer (PBS containing 2% FBS). Block Fc receptors for 15 minutes at room temperature using Human BD FcBlock (BD, 564220). Add the antibody staining mixture (BD Horizon TM BV421 mouse anti-human CD15, BD 567008; Alexa 647-labeled anti-human STEAP4, R&D Systems FAB4626R; BD Pharmingen TM PE mouse anti-human CD16, BD 561313) in working buffer (PBS containing 2% FBS) and incubated at 4°C in the dark for 30 minutes. Cells were washed twice with staining buffer and analyzed on a BD FACS Aria SORP. FlowJo TM Data processing was performed using FlowJo software (FlowJo LLC).

[0079] This flow cytometry method is used to analyze neutrophils from fresh blood. In this example, single cells are screened by FSC and SSC to exclude cell debris and aggregates; appropriate gating is set to exclude lymphocytes and retain other white blood cells. All white blood cells are further screened to exclude red blood cells and other non-white blood cell components; live / dead cell dyes such as PerCP-Cy5.5 are used to screen the live cell population. In this example,

[0080] Single cells were screened using FSC and SSC, debris and aggregates were excluded, and a single cell population was obtained. All white blood cells were further screened, lymphocytes were excluded, and a white blood cell population of 83.0% was obtained. Live cells were screened using a live / dead cell dye (PerCP-Cy5.5), and a live cell population of 99.2% was obtained. + 、CD16 + Neutrophils were specifically selected based on the double-positive characteristics of the neutrophils. After the above steps, neutrophils were successfully isolated from fresh blood of healthy donors.

[0081] The analysis results are as follows Figure 8 and Figure 9 The STEAP4-positive cell population was divided using flow cytometry FMO control, and its proportion was calculated. Figure 8 A comparison of breast cancer patients with lung metastasis and healthy controls revealed that healthy controls had very low rates of STEAP4-expressing neutrophils (0.30% and 0.31%), while those with metastasis had significantly higher rates, at 22.7% and 20.9%, respectively. This suggests that STEAP4 is specifically expressed in patients with lung metastasis, while it is virtually absent in healthy controls.

[0082] Healthy people (HD), breast cancer patients without metastasis (No Met), and breast cancer patients with metastasis found that STEAP4 appeared very specifically in patients with metastasis. + STEAP4 is expressed in neutrophils, while healthy individuals and patients without metastasis express almost no STEAP4. The average expression rate in healthy individuals is 0.34%, while the average rate in patients with non-metastatic breast cancer is 1.38%. However, the rate in patients with metastatic breast cancer is 25.0%, a significant difference of 74 times that in healthy individuals and 18 times that in patients with non-metastatic breast cancer. This data demonstrates that the expression rate of STEAP4 on neutrophils is a highly differentiated, high-quality blood marker that can successfully identify patients with cancer metastasis.

[0083] In specific implementations, the threshold for indicating cancer metastasis risk is determined based on the specific detection method used and the type of cancer. For example, in fresh blood from breast cancer patients, neutrophils are sorted using flow cytometry, and an expression ratio of 20% or more indicates a high risk of cancer metastasis.

[0084] By observation Figure 9The boxplot shows that the percentage of STEAP4-positive neutrophils in the lung metastasis group was significantly higher than in the other two groups, indicating that STEAP4 is highly expressed in neutrophils in patients with lung metastasis. The data points in the lung metastasis group were relatively concentrated, with a small standard error range, indicating low data variability and reliable results. The prediction level was AUC 1.0, indicating that the percentage of STEAP4-positive neutrophils has strong predictive power as a biomarker for lung metastasis and can distinguish patients with lung metastasis from other populations. These flow cytometry results demonstrate that the percentage of STEAP4-positive neutrophils is significantly elevated in patients with lung metastasis, and the concentrated distribution of data indicates strong predictive power, suggesting its potential as a lung metastasis biomarker for early diagnosis and prognostic assessment.

[0085] pass Figure 8 and Figure 9 The results showed that STEAP4 + The expression ratios of these proteins vary by several dozen-fold, making it easy to identify patients with metastasis. Furthermore, data from MMTV-PyMT mice suggest that the proportion of STEAP4- or IGFBP6-positive neutrophils increases with metastasis progression, and detecting their expression levels can indicate the risk of cancer metastasis.

[0086] In addition, in MMTV-PyMT mice, the protein expression of STEAP4 and IGFBP6 on blood neutrophils was examined by immunofluorescence staining. The results showed that STEAP4 was expressed in blood neutrophils of mice with breast cancer lung metastasis. + IGFBP6 + The levels of STEAP4 and IGFBP6 gradually increase in the early, middle, and late stages of cancer progression, and lung metastasis occurs in the middle and late stages of cancer. This data supports the use of STEAP4 and IGFBP6 as blood markers to dynamically monitor the risk of cancer metastasis.

[0087] Example 3

[0088] A biomarker detection product, comprising a substance for detecting the expression level of the biomarkers described above; the function of the product is to identify or assist in identifying the level of cancer metastasis or cancer recurrence. In other words, the product is used to assist in identifying the level of cancer metastasis, and the product may be a kit. The kit contains substances for detecting the expression level of specific biomarker proteins. By detecting the expression level of the target proteins STEAP4 and / or IGFBP6 in the sample to be tested, it helps doctors or researchers assess the possibility of cancer metastasis.

[0089] This kit includes the following components: specific antibodies targeting selected biomarkers associated with cancer metastasis (STEAP4 and / or IGFBP6), which can be used to detect target protein expression levels in a sample. Fluorescent or enzyme markers are used to label the specific antibodies (STEAP4 and / or IGFBP6) for detection of target protein expression using methods such as flow cytometry and enzyme-linked immunosorbent assay (ELISA). Treatment reagents, such as red blood cell lysis buffer, cell fixative, and permeabilization reagents, are used to pre-treat the sample to make it suitable for subsequent assays. The instructions provide detailed instructions on the procedure, reagent preparation, assay conditions, and result interpretation to guide users in the correct use of the kit.

[0090] Collect biological samples from the individual to be tested, such as blood or tissue sections. Pre-treat the sample according to the kit instructions. For example, for blood samples, use an erythrocyte lysis buffer to remove red blood cells (RBCs) to obtain a white blood cell suspension. For tissue sections, fix and permeabilize the sample to allow the antibody to recognize and bind to the target protein within the cells. The treated sample is incubated with the specific antibody provided in the kit to allow the antibody to bind to the target protein. The antibody-bound sample is then tested according to the detection method used (e.g., flow cytometry, ELISA, etc.). For example, in flow cytometry, fluorescently labeled antibodies are used to detect the fluorescence intensity of the target protein (STEAP4 and / or IGFBP6) on the cell surface or within the cell. In ELISA, the concentration of the target protein (STEAP4 and / or IGFBP6) in the sample is determined by the color change produced by the reaction of the enzyme-labeled antibody with the substrate. The target protein expression data obtained by the assay is compared with the standard curve of the calibrator provided in the kit to calculate the specific expression level of the target protein (STEAP4 and / or IGFBP6). The probability of cancer metastasis in the patient is determined based on a preset expression threshold (e.g., 20%). For example, if the expression level of the target protein (STEAP4 and / or IGFBP6) is above the threshold, the risk of cancer metastasis is high; if the expression level is below or equal to the threshold, the risk of cancer metastasis is low.

[0091] Example 4

[0092] The present invention protects a system for predicting cancer metastasis or cancer recurrence, comprising: a processor; and a storage medium containing a program executed by the processor for determining the level of cancer metastasis or cancer recurrence, wherein the program for determining the level of cancer metastasis or cancer recurrence causes the processor to perform the following steps: obtaining the expression level of a target protein of a biomarker as described above in a sample from a subject to be tested; comparing the obtained expression level of the target protein of the biomarker with a preset expression level threshold; and determining the probability of cancer metastasis or cancer recurrence in the subject to be tested based on the comparison result. The system determines the probability of cancer metastasis or cancer recurrence in the subject to be tested by detecting the expression level of the target protein of a specific biomarker (STEAP4 and / or IGFBP6) and comparing it with the preset expression level threshold.

[0093] Among them, the processor is responsible for executing the program stored in the storage medium to perform data processing and analysis; the storage medium is used to store the program executed by the processor for judging the level of cancer metastasis or cancer recurrence, as well as related data and thresholds. The detection equipment is used to detect the target protein expression level of the biomarker in the sample of the person to be tested. The detection equipment can be a flow cytometer, an enzyme-linked immunosorbent assay (ELISA) device, an immunofluorescence microscope, etc. The specific equipment is selected according to the biomarker and sample type to be detected. The data input / output interface is used to input information and test data of the person to be tested, and to output judgment results and reports.

[0094] Collect biological samples from the person to be tested, such as blood, tissue sections, etc. The samples are pretreated, such as red blood cell lysis of blood samples, fixation and permeabilization of tissue sections, etc., to make the samples suitable for subsequent detection steps. The treated samples are incubated with a substance for detecting protein expression levels (such as a specific antibody) to allow the antibody to bind to the target protein. According to the detection equipment and method used, the sample bound to the antibody is detected to obtain the expression data of the target protein. For example, a flow cytometer is used to detect the fluorescence intensity of the target protein on the cell surface or inside, or an ELISA is used to determine the concentration of the target protein in the sample. The target protein expression data obtained by the detection is transmitted to the processor in the system through the data input interface. The processor executes the program in the storage medium to process and analyze the data. The processor compares the target protein expression level of the acquired biomarker with the preset expression level threshold. Based on the comparison result, the probability of cancer metastasis or cancer recurrence of the person to be tested is determined. The determination result is output through the data output interface, and a report is generated for reference by doctors or researchers. A cancer metastasis and recurrence prediction system based on biomarker protein expression levels accurately determines the probability of cancer metastasis or recurrence by measuring the expression of target proteins in samples from patients and comparing them with preset thresholds. This system offers advantages such as ease of use, high accuracy, and applicability to a variety of cancer types, providing a powerful tool for early cancer diagnosis, treatment monitoring, and prognostic assessment.

[0095] The various experimental supplies involved in this article are all conventional experimental supplies, which can be easily obtained through various means (such as purchase, self-preparation, etc.) before the date of this application.

[0096] Although the present invention has been described in detail above using general descriptions, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A biomarker for determining cancer metastasis or cancer recurrence, characterized in that: The biomarkers are STEAP4 protein and / or IGFBP6 protein.

2. Application of biomaterials related to STEAP4 protein or IGFBP6 protein in determining cancer metastasis or cancer recurrence.

3. The use according to claim 2, characterized in that The biological material includes a nucleic acid molecule encoding a STEAP4 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

4. The use according to claim 2, characterized in that Obtaining samples to be tested; Flow cytometry or immunofluorescence staining is used to determine the target protein expression level of the biomarker according to claim 1 in the sample to be tested.

5. The use according to claim 4, characterized in that The sample to be detected is a biological fluid or tissue.

6. A biomarker detection product, comprising a substance for detecting the expression level of the biomarker according to claim 1; the function of the product is to identify or assist in identifying the level of cancer metastasis or cancer recurrence.

7. The biomarker detection product according to claim 6, characterized in that: The cancer is selected from one or more of leukemia, brain cancer, prostate cancer, liver cancer, ovarian cancer, gastric cancer, colorectal cancer, pharyngeal cancer, breast cancer, skin cancer, melanoma, lung cancer, sarcoma, cervical cancer, testicular cancer, bladder cancer, endocrine system cancer, endometrial cancer, esophageal cancer, glioma, lymphoma, neuroblastoma, osteosarcoma, pancreatic cancer, pituitary tumor, and kidney cancer.

8. A system for determining cancer metastasis or cancer recurrence, characterized in that: include: processor; and a storage medium containing a program executed by a processor for determining cancer metastasis or cancer recurrence, wherein the program for determining the level of cancer metastasis or cancer recurrence causes the processor to perform the following steps: Obtaining the target protein expression level of the biomarker according to claim 1 in the sample to be tested; Comparing the obtained target protein expression level of the biomarker with a preset expression level threshold; The cancer metastasis probability or cancer recurrence probability is determined according to the comparison result.

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