Application of FABP5 / S100A9 protein complex in preparation of reagent kit for rapid detection of breast cancer metastasis

Abstract

The application provides application of an FABP5 / S100A9 protein complex in preparation of a kit for rapidly detecting breast cancer metastasis. The application firstly finds that a protein complex (FABP5 / S100A9) in human serum exosomes is related to breast cancer metastasis, the complex protein significantly increases in the body of a breast cancer metastasis patient, and promotes breast cancer metastasis, which indicates the value of the complex protein in breast cancer metastasis detection. A kit based on detection of the binding condition of the FABP5 / S100A9 complex in patient serum and the protein expression level in the patient body is provided. The application is used for determining metastatic breast cancer according to the expression level and co-precipitation condition of the complex protein, and provides a basis for formulating a clinical precise treatment scheme and prognosis prediction of a patient, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of testing and diagnostic technology, specifically relating to the application of a FABP5 / S100A9 protein complex in the preparation of a rapid detection kit for breast cancer metastasis. Background Technology

[0002] Breast cancer (BC) is the most common malignant tumor among women worldwide. It is a highly heterogeneous disease characterized by differences in molecular features. Clinically, it is classified into three main subtypes based on hormone receptor (ER and PR) and HER2 (ERBB2) status: luminal ER-positive and PR-positive, further subdivided into luminal A and B, HER2-positive, and triple-negative breast cancer (TNBC). Metastatic breast cancer affects approximately 30% of breast cancer patients and accounts for about 90% of breast cancer deaths. Common distant metastases of breast cancer are bone (~70%), liver (~30%), brain (10%-30%), lung, and lymph nodes. Like other cancers, the pathogenesis of metastatic breast cancer requires the activation of important pathways to maintain proliferation, resistance to apoptosis, angiogenesis, immune resistance, and the migration and invasion capabilities required for disseminated tumor cells (DTCs) to reach distant metastatic sites.

[0003] Clinically, the main treatment methods for breast cancer include surgical resection, radiotherapy, chemotherapy, and targeted therapy. Surgical treatment is primarily for patients with tumors within a certain size range, with breast-conserving surgery being the most common approach (accounting for 75%-85%). However, for multicentric invasive carcinomas and extensive ductal carcinoma in situ, total mastectomy is necessary. Some patients have achieved similar remission and survival rates to those who underwent total mastectomy through a combination of breast-conserving surgery and radiotherapy. Currently, recent advances in molecular biology and immunology have enabled the development of highly targeted therapies for various forms of breast cancer. The main goal of targeted therapy is to inhibit specific targets / molecules that support tumor progression.

[0004] In multiple studies targeting different cancer types, FABP5 (Fatty acid-binding protein 5) has been shown to inhibit apoptosis and promote cancer cell proliferation, colony formation, epithelial-mesenchymal transition (EMT), migration, cell invasion, and in vitro angiogenesis. In vivo, this translates into increased tumor growth, metastasis, and reduced survival. The mechanism underlying this primarily involves FABP5-mediated activation of various TFs. This leads to increased expression of key proteins involved in cell survival, prevention of apoptosis, cell proliferation, migration and invasion, lipid storage, and angiogenesis.

[0005] S100A9 is upregulated in multiple cancer types, including breast cancer (invasive ductal carcinoma), colitis-associated colon cancer, hepatocellular carcinoma, gastric cancer, lung adenocarcinoma, colorectal cancer, apocrine breast cancer, non-small cell lung cancer, and squamous cervical cancer. S100A9 levels are also elevated in nasopharyngeal carcinoma stromal and bladder transitional cell carcinoma. S100A9 may have both tumor-promoting and tumor-suppressing effects in breast cancer. In invasive ductal carcinoma of the breast, S100A9 immunopositivity is associated with mitotic activity, MIB-1 proliferation index (recombinant Ki-67 monoclonal antibody), HER2 overexpression, poor tumor differentiation, vascular invasion, lymph node metastasis, and poor pathological stage. S100A9 is associated with high-grade, negative ER and PR status, high Ki67 and p53 expression, and ERBB2 and EGFR expression. S100A9 expression was also closely associated with 10 basic protein characteristics: CK5 / 6, CD10, EGFR, CAV1, CD44, ETS1, MET, Moesin, GATA3, and CK19.

[0006] Currently, there is no research on the role of the FABP5 / S100A9 protein complex in breast cancer metastasis. In this patent, a series of experiments demonstrate that the FABP5 / S100A9 complex is upregulated in patients with breast cancer metastasis and that the two proteins co-precipitate in the patients' bodies. Summary of the Invention

[0007] The purpose of this invention is to solve the diagnostic problem of metastatic breast cancer and to provide an application of the FABP5 / S100A9 protein complex in the preparation of a rapid detection kit for breast cancer metastasis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides the application of the FABP5 / S100A9 protein complex in the preparation of a rapid detection kit for breast cancer metastasis.

[0010] The rapid detection kit for breast cancer metastasis uses an immunoprecipitation method to detect the expression level and co-precipitation of the FABP5 / S100A9 protein complex in the serum of patients with breast cancer metastasis.

[0011] The rapid detection kit for breast cancer metastasis includes rabbit-derived FABP5 polyclonal antibody, rabbit-derived S100A9 polyclonal antibody, and immunoprecipitation detection reagent.

[0012] The immunoprecipitation assay reagents include Protein A / G magnetic beads, IP Lysis / Wash Buffer, PBS (pH=7.2-7.4), and 5× SDS-PAGE protein loading buffer.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] By jointly detecting the protein expression levels of FABP5 and S100A9, the binding of FABP5 and S100A9 in the serum of breast cancer patients can be analyzed to screen for metastatic breast cancer, providing an important basis for the precise diagnosis and treatment of tumors and the prediction of prognosis. Attached Figure Description

[0015] Figure 1 This is a mass spectrum of the ELGVGIALR peptide fragment of FABP5 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0016] Figure 2 This is the FEETTADGR peptide mass spectrum of FABP5 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0017] Figure 3 This is a mass spectrum of the FEETTADGRK peptide fragment of FABP5 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0018] Figure 4 This is a mass spectrum of the DLQNFLK peptide fragment of S100A9 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0019] Figure 5 This is a mass spectrum of the LGHPDTLNQGEFK peptide fragment of S100A9 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0020] Figure 6 This is a mass spectrum of the LTWASHEK peptide fragment of S100A9 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0021] Figure 7 The mass spectrum of the MHEGDEGPGHHHKPGLGEGTP peptide fragment of S100A9 in the protein complex (FABP5 / S100A9) in serum exosomes.

[0022] Figure 8 Western blot validation of FABP5 / S100A9 protein expression levels in serum exosomes of healthy individuals, patients with non-metastatic breast cancer, and patients with metastatic breast cancer; n=10, **** indicates P<0.0001.

[0023] Figure 9 The binding and expression of the FABP5 / S100A9 complex in patients with metastatic breast cancer.

[0024] Figure 10 This study investigated the binding and expression of the FABP5 / S100A9 complex in patients with metastatic breast cancer. Detailed Implementation

[0025] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. The specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0026] Example 1

[0027] (1) Collection and processing of serum samples:

[0028] Serum exosomes were extracted from patient serum for the detection of metastatic breast cancer. Serum exosomes were extracted using an exosome extraction and purification kit (blood) (Umibio, UR52136). The specific steps are as follows:

[0029] First, remove the serum sample frozen at -80℃ from the freezer and thaw it in a 25℃ water bath. Place the completely thawed sample on ice. Transfer 200 μL of sample to a centrifuge tube and centrifuge at 10,000g for 10 min at 4℃ to remove impurities. Transfer the supernatant to a new centrifuge tube. Dilute the supernatant with 1 ml of ice-cold 1×PBS, then add Blood PureExo Solution (BPS). Immediately cap the centrifuge tube and vortex for 1 min. Let it stand at 4℃ for 2 h. Then centrifuge at 10,000g for 60 min at 4℃, aspirating as much supernatant as possible. The precipitate should be rich in exosome particles. Take 100 μL of 1×PBS and gently pipette the centrifuged precipitate until dissolved. Transfer the resuspended solution to a new 1.5 mL centrifuge tube and centrifuge at 12000 g for 2 min at 4 °C. Transfer the supernatant to the upper chamber of an Exosome Purification Filter (EPF column) and centrifuge at 3000 g for 10 min at 4 °C. Collect the liquid at the bottom of the EPF column after centrifugation; this liquid is the purified exosome particles. If the purified exosomes are not used immediately, they can be stored at -80 °C for subsequent experiments.

[0030] (2) Mass spectrometry detection

[0031] First, serum exosomes were extracted from the serum of healthy individuals, patients with non-metastatic breast cancer, and patients with metastatic breast cancer, and then mass spectrometry was performed. Each group of samples contained blood samples from 10 individuals. Figure 1-3 and Figure 4-7These are mass spectra of FABP5 and S100A9, used to confirm the accuracy of these two proteins. The full-length amino acid sequence of FABP5 is: MATVQQLEGRWRLVDSKGFDEYMK EL GVGIALR KMGAMAKPDCIITCDGKNLTIKTESTLKTTQFSCTLGEK FEETTADGRK TQTVCNNFTDGALVQHQEWDGKESTITRKLKDGKLVVECVMNNVTCTRIYEKVE, Figure 1 , Figure 2 and Figure 3 The intermediate peptides are ELGVGIALR, FEETTADGR, and FEETTADGRK, corresponding to the underlined positions in the full-length amino acid sequence of FABP5. Therefore, this protein is identified as FABP5. The full-length amino acid sequence of S100A9 is: MTCKMSQLERNIETIINTFHQYSVK LGHPDTLNQGEFK ELVRK DLQNFL K KENKNEKVIEHIMEDLDTNADKQLSFEEFIMLMAR LTWASHEKMHEGDEGPGHKP

[0032] GLGEGTP , Figures 4-7 The four peptide segments DLQNFLK, LGHPDTLNQGEFK, LTWASHEK, and MHEGDEGPGHHHKPGLGEGTP correspond to the underlined positions in the full-length amino acid sequence of S100A9, thus identifying this protein as S100A9. Table 1 shows that the expression levels of FABP5 and S100A9 proteins in the serum of patients with metastatic breast cancer are significantly higher than those in healthy individuals and patients with non-metastatic breast cancer. This indicates that FABP5 and S100A9 play important roles in the metastasis process of breast cancer, and that the two may bind to each other.

[0033] The expression levels of FABP5 / S100A9 protein in serum exosomes from healthy individuals, patients with non-metastatic breast cancer, and patients with metastatic breast cancer were validated by Western blotting. Extracted serum exosomes were mixed 1:1 with exosome protein lysis buffer (Umibio, UR333101), lysed on ice for 10 min, centrifuged at 12000g for 5 min, and the supernatant was collected. BCA quantification was performed before Western blotting. First, 30 μg of sample from each group was placed in a new EP tube, and an appropriate amount of 5× protein loading buffer was added to a final concentration of 1×. The tube was incubated at 95℃ for 5 min to ensure complete protein denaturation, and then cooled to room temperature. After loading, SDS-PAGE gel electrophoresis was performed. Electrophoresis was first run at 60V until the stacking gel was completed, then at 120V until the separating gel was completed. Then, wet transfer was performed. PVDF membranes were activated with methanol before use. Transfer conditions were ice bath, 100V, 35 min. After transfer, wash the membrane with TBS for 10 min, block with TBST containing 5% skim milk powder for 1.5 h, and wash with TBST 3 times for 10 min each time. Place the membrane in FABP5 (1:500) and S100A9 (1:500) primary antibody working solution and incubate overnight at 4°C. Wash the membrane 3 times with TBST for 10 min each time. Place the membrane in the corresponding secondary antibody working solution and incubate at room temperature for 1.5 h. Wash the membrane 3 times with TBST for 10 min each time. Wash the membrane with TBS for 10 min. Place the membrane in a 1:1 ECL chemiluminescence colorimetric solution and incubate at room temperature in the dark for 2 min. Preheat the chemiluminescence analyzer, place the membrane on the chemiluminescence analyzer panel, and observe the color development.

[0034] The results are as follows Figure 8 As shown, the levels of FABP5 and S100A9 proteins in the serum exosomes of patients with metastatic breast cancer were significantly higher than those in healthy individuals and patients with non-metastatic breast cancer, validating the accuracy of the mass spectrometry results.

[0035] Example 2

[0036] The kit used in this embodiment to detect the expression levels of FABP5 and S100A9 proteins in serum includes rabbit-derived FABP5 and S100A9 polyclonal antibodies and an immunoprecipitation assay reagent. The ordering companies and catalog numbers for the antibodies used to detect the two proteins are: FABP5 (Proteintech Group, 12348-1-AP) and S100A9 (Proteintech Group, 26992-1-AP).

[0037] The immunoprecipitation assay reagents included: PBS (pH=7.2-7.4), Protein A / G magnetic beads (Medchemexpress, HY-K020), IP Lysis / Wash Buffer (Biolinkedin, L-7101), PMSF (Beyotime, ST507-10ml), Phosphatase inhibitor cocktail II (Medchemexpress, HY-K0022), and SDS-PAGE protein loading buffer (5×) (Beyotime, P0015).

[0038] The specific testing steps are as follows:

[0039] (1) Take 200 µL of patient serum, add 300 µL of PBS to make up to 500 µL, then add an equal volume of IP Lysis / Wash Buffer, 5 µL of PMSF, and 10 µL of Phosphatase inhibitor cocktail II. Mix well and let stand on ice for 20 min. Centrifuge at 12000g for 10 min at 4℃ to collect the supernatant. Keep 200 µL of the supernatant as the Input group.

[0040] (2) Add 4 μL of FABP5 antibody and incubate overnight at 4°C to form an antigen sample / antibody mixture.

[0041] (3) Repeatedly invert and mix Protein A / G magnetic beads. Take 30 µL of magnetic beads into a new 1.5 mL centrifuge tube, add 600 µL of pre-chilled PBS on ice, mix gently, collect the magnetic beads with a magnetic rack, remove the supernatant, add 400 µL of IPLysis / Wash Buffer to the centrifuge tube, gently invert the centrifuge tube for 1 min, collect the magnetic beads with a magnetic rack, and remove the supernatant.

[0042] (4) Add the antigen sample / antibody mixture to the centrifuge tube containing the magnetic beads, place it on a rotary mixer, and incubate at room temperature for 2 h.

[0043] (5) Collect the magnetic beads with a magnetic rack, remove the unbound sample, add 1000µL IP Lysis / Wash Buffer to the centrifuge tube, gently mix the magnetic beads for 10 min, collect the magnetic beads with a magnetic rack, remove the supernatant, and repeat the operation twice.

[0044] (6) Take 20 µL of SDS-PAGE protein loading buffer (5×) and add 80 µL of PBS to dilute the concentration of SDS-PAGE protein loading buffer to 1×.

[0045] (7) Add the diluted 1× SDS-PAGE protein loading buffer to the precipitate obtained in step (5), heat it in a 100℃ metal bath for 10 min, separate the magnetic beads with a magnetic rack, and retain the supernatant containing the target antigen, which is the IP group sample.

[0046] (8) Perform Western Blot analysis on the obtained samples. Simultaneously incubate FABP5 antibody (1:500) and S100A9 antibody (1:500) with the primary antibody. If bands of FABP5 and S100A9 are present in both the Input group and the IP group during the final color development, it indicates that the two have bound together. If no S100A9 band is found in the IP group when performing immunoprecipitation with FABP5 antibody, it indicates that the two have not bound together. Similarly, if no FABP5 band is found in the IP group when performing immunoprecipitation with S100A9 antibody, it also indicates that the two have not bound together.

[0047] Figure 9 The binding of FABP5 and S100A9 in the serum of patients with metastatic breast cancer was investigated, and the results showed that the two do bind. Metastatic breast cancer can be screened by observing the high expression of FABP5 and S100A9 in the serum of breast cancer patients and the co-precipitation phenomenon between them.

[0048] After testing samples from 10 randomly selected patients using this immunoprecipitation kit, the presence of the FABP5 / S100A9 complex was found in 8 patients. Some results are as follows: Figure 10 As shown, this result is consistent with the clinical diagnosis.

[0049] In summary, the immunoprecipitation kit for detecting the FABP5 / S100A9 complex of the present invention utilizes the characteristic that FABP5 and S100A9 are simultaneously highly expressed and form a complex, and can be used for the identification of metastatic breast cancer.

[0050] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

[0051] Table 1 shows the mass spectrometry information of the protein complex (FABP5 / S100A9) in serum exosomal tissues of healthy individuals, patients with non-metastatic breast cancer, and patients with metastatic breast cancer.

[0052]

[0053] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. Application of FABP5 / S100A9 protein complex in the preparation of rapid detection kit for breast cancer metastasis.

2. The application according to claim 1, characterized in that: The rapid detection kit for breast cancer metastasis uses an immunoprecipitation method to detect the expression level and co-precipitation of the FABP5 / S100A9 protein complex in the serum of patients with breast cancer metastasis.

3. The application according to claim 1, characterized in that: The rapid detection kit for breast cancer metastasis includes rabbit-derived FABP5 polyclonal antibody, rabbit-derived S100A9 polyclonal antibody, and immunoprecipitation assay reagent; the immunoprecipitation assay reagent includes Protein A / G magnetic beads, IP Lysis / Wash Buffer, PBS buffer with pH 7.2-7.4, and 5× SDS-PAGE protein loading buffer.

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