SDC1 as a breast cancer diagnostic marker and its inhibitor in the preparation of breast cancer drugs

Abstract

This invention discloses the application of SDC1 in the diagnosis and treatment of breast cancer. Through bioinformatics analysis, cell function experiments, multi-omics analysis, and animal model validation, this invention reveals for the first time the high expression of SDC1 in breast cancer and its correlation with poor patient prognosis. It elucidates the molecular mechanism by which SDC1 promotes the malignant progression of breast cancer by regulating ECM remodeling, EMT, angiogenesis, and ferroptosis resistance, and verifies the therapeutic potential of MZ1 in inhibiting breast cancer growth and metastasis by downregulating SDC1 expression. This invention provides the application of reagents for detecting SDC1 expression levels in the preparation of diagnostic or prognostic kits for breast cancer, as well as the application of SDC1 inhibitors in the preparation of drugs for treating breast cancer. This invention provides new technical solutions for the diagnosis, prognostic assessment, and treatment of breast cancer.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and tumor diagnosis and treatment technology, specifically to the application of the SDC1 gene and its expression products as biomarkers for breast cancer diagnosis and prognostic assessment, and the application of SDC1 inhibitors in the preparation of drugs for treating breast cancer. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide and one of the leading causes of cancer death in women. Despite continuous advancements in clinical treatments such as surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, the incidence and mortality rates of breast cancer remain high.

[0003] Tumor metastasis is a leading cause of death in breast cancer patients. Tumor metastasis is a complex, multi-stage cascade process involving local invasion, intravascular infiltration, survival in the bloodstream, extravascular exudation, and colonization in distant organs. Epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) remodeling, and angiogenesis are key biological events in this process. Furthermore, disseminated tumor cells must evade multiple cell death pathways, including anoikis and the recently discovered iron-dependent cell death pathway—ferroptosis.

[0004] Syndecan-1 (SDC1) belongs to the transmembrane heparan sulfate proteoglycan family and plays an important role in regulating cell-microenvironment interactions and influencing cell proliferation, adhesion, and migration. Previous studies have shown that SDC1 expression is dysregulated in various malignant tumors, including pancreatic cancer, ovarian cancer, and colon cancer. However, the role of SDC1 in breast cancer remains controversial, with evidence suggesting it may have both tumor-promoting and tumor-suppressing functions. Therefore, elucidating the molecular mechanisms by which SDC1 plays a role in breast cancer pathogenesis is crucial to resolving this controversy.

[0005] Currently, there is a lack of specific biomarkers in clinical practice that can effectively predict the prognosis of breast cancer patients and guide treatment decisions, as well as effective treatment strategies for breast cancer metastasis. Therefore, developing new biomarkers for breast cancer diagnosis and prognostic assessment, and identifying effective therapeutic targets, has significant clinical importance and application value. Summary of the Invention

[0006] The technical problem that the invention aims to solve The technical problem to be solved by this invention is to provide a new use for SDC1 as a biomarker for the diagnosis and treatment of breast cancer, and the application of SDC1 inhibitors in the preparation of drugs for the treatment of breast cancer.

[0007] Specifically, this invention, through bioinformatics analysis, cell function experiments, multi-omics analysis, and animal model validation, reveals for the first time the high expression of SDC1 in breast cancer and its correlation with poor patient prognosis. It elucidates the molecular mechanism by which SDC1 promotes the malignant progression of breast cancer by regulating ECM remodeling, EMT, angiogenesis, and ferroptosis resistance, and verifies the therapeutic potential of MZ1 to inhibit breast cancer growth and metastasis by downregulating SDC1 expression.

[0008] Technical solution In a first aspect, the present invention provides the use of reagents for detecting SDC1 expression levels in the preparation of kits for breast cancer diagnosis or prognostic assessment.

[0009] Preferably, the reagent is an antibody that specifically binds to the SDC1 protein.

[0010] Preferably, the reagent is a primer for specifically amplifying the SDC1 gene.

[0011] Preferably, the kit is used for immunohistochemical detection, Western blotting detection, ELISA detection, or qRT-PCR detection.

[0012] Preferably, the kit further includes instructions describing judgment rules, wherein when the detected SDC1 expression level is higher than that of the normal control, it indicates that the subject has breast cancer or has a poor prognostic risk.

[0013] Secondly, the present invention provides the use of SDC1 inhibitors in the preparation of medicaments for the treatment of breast cancer.

[0014] Preferably, the SDC1 inhibitor is MZ1.

[0015] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0016] Preferably, the dosage form of the drug is an injection, tablet, capsule, or powder for injection.

[0017] Preferably, the drug is administered via intratumoral injection, intravenous injection, or oral administration.

[0018] Thirdly, the present invention provides a kit for the diagnosis or prognostic assessment of breast cancer, which includes reagents for detecting SDC1 expression levels.

[0019] Fourthly, the present invention provides a pharmaceutical composition comprising an SDC1 inhibitor and a pharmaceutically acceptable carrier.

[0020] Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: 1. Revealing the multifunctional regulatory role of SDC1 in breast cancer: This invention, through multi-omics functional experiments, systematically elucidates for the first time the molecular mechanism by which SDC1, as a core signaling hub, simultaneously regulates ECM remodeling, EMT, angiogenesis, and ferroptosis resistance, thereby promoting the malignant progression of breast cancer.

[0021] 2. Validation of SDC1 as an independent prognostic biomarker for breast cancer: This invention, through TCGA database analysis and tissue microarray validation, is the first to demonstrate that high expression of SDC1 is significantly associated with poor prognosis in breast cancer patients, and that SDC1 can serve as an independent prognostic predictor.

[0022] 3. A novel mechanism by which SDC1 inhibits ferroptosis in breast cancer is discovered for the first time: This invention is the first to discover that SDC1 enhances the resistance of breast cancer cells to ferroptosis by upregulating the xCT-GPX4 antioxidant pathway and downregulating the HMOX1 / ACSL4 pro-ferroptosis pathway, revealing a novel mechanism by which SDC1 promotes tumor progression.

[0023] 4. First validation of the potential of MZ1 to treat breast cancer by downregulating SDC1: This invention is the first to validate the therapeutic effect of MZ1 in a breast cancer model by downregulating SDC1 expression, reversing its downstream pro-tumor effects, and inhibiting tumor growth and lung metastasis.

[0024] 5. Good safety profile and promising clinical application prospects Animal experiments showed that MZ1 was well tolerated, with no obvious tissue toxicity observed, and it has good prospects for clinical translation. Attached Figure Description

[0025] Figure 1 Analysis of SDC1 expression in breast cancer and its correlation with prognosis.

[0026] Figure 1 a: SDC1 expression radar map of 33 tumor types in the TCGA database.

[0027] Figure 1 bc: Comparison of SDC1 expression in tumor and normal tissues in the TCGA-BRCA dataset.

[0028] Figure 1 d: ROC curve of SDC1 in diagnosing breast cancer.

[0029] Figure 1 For example, high expression of SDC1 is associated with overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI) in breast cancer patients.

[0030] Figure 1hi: qRT-PCR and Western Blot were used to verify the high expression of SDC1 in breast cancer tissues.

[0031] Figure 1 j: SDC1 diagnostic ROC curve based on tissue chip.

[0032] Figure 1 k: Staining score analysis of SDC1 in breast cancer tissue and benign tissue.

[0033] Figure 1 l: SDC1 co-expression heatmap.

[0034] Figure 1 m: SDC1 expression based on tissue chip and Kaplan-Meier survival curve of OS.

[0035] Figure 2 The effect of SDC1 on the malignant phenotype of breast cancer cells.

[0036] Figure 2 a: Western blot validation of SDC1 knockdown and overexpression cell lines.

[0037] Figure 2 bd: CCK-8 and EdU assays were used to detect the effect of SDC1 on cell proliferation.

[0038] Figure 2 eh: Scratch assay and Transwell assay were used to detect the effects of SDC1 on cell migration and invasion.

[0039] Figure 3 : The mechanism by which SDC1 regulates ECM remodeling and EMT.

[0040] Figure 3 ab: Volcano plot analysis of transcriptome and proteome after SDC1 knockdown.

[0041] Figure 3 c: GO enrichment analysis results.

[0042] Figure 3 dg: Western blot analysis of the effects of SDC1 on the expression of FN1, ITGB3, N-cadherin, E-cadherin, and VIM.

[0043] Figure 4 SDC1 promotes angiogenesis.

[0044] Figure 4 ab: Transwell assay to detect the effect of conditioned medium on HUVEC migration.

[0045] Figure 4 cf: CCK-8 assay to detect the effect of conditioned medium on HUVEC proliferation.

[0046] Figure 4 gj: Matrigel tube formation assay to detect the effect of conditioned medium on the angiogenesis capacity of HUVECs.

[0047] Figure 4 k: Correlation analysis of SDC1 expression and MVD in tissue microarrays.

[0048] Figure 5 The role of SDC1 in ferroptosis resistance.

[0049] Figure 5 ab: Results of KEGG pathway enrichment analysis.

[0050] Figure 5 cd: Western blotting to detect the effect of SDC1 on the expression of GPX4, xCT, HMOX1, and ACSL4.

[0051] Figure 5 ef: Detection of lipid peroxidation levels using confocal microscopy and flow cytometry.

[0052] Figure 6 SDC1 knockdown induces ferroptosis-related mitochondrial alterations.

[0053] Figure 6 a: Mito-FerroGreen staining to detect mitochondrial ferrous ion accumulation.

[0054] Figure 6 b: Transmission electron microscopy observation of mitochondrial morphological changes.

[0055] Figure 7 MZ1 inhibits the pro-tumor effect of SDC1 in vitro and in vivo.

[0056] Figure 7 a: IC50 determination of MZ1 in different breast cancer cell lines.

[0057] Figure 7 b: The inhibitory effect of MZ1 on the proliferation of SDC1-overexpressing cells.

[0058] Figure 7 c: MZ1 dose-dependently downregulates SDC1 protein expression.

[0059] Figure 7 df: The inhibitory effect of MZ1 on tumor growth in an orthotopic mouse model.

[0060] Figure 7gh: Effects of MZ1 on the expression of ECM, EMT and ferroptosis-related proteins.

[0061] Figure 8 MZ1 inhibits SDC1 overexpression-driven lung metastasis in breast cancer.

[0062] Figure 8 ab: Representative photographs and quantitative analysis of lung metastatic nodules.

[0063] Figure 8 cd: H&E and Masson staining of lung, vertebrae and femur tissues.

[0064] Figure 8 e: Dynamic changes in mouse body weight during treatment.

[0065] Figure 8 f: H&E and Masson staining of major organs to assess the organ toxicity of MZ1. Detailed Implementation

[0066] The present invention will be described in detail below through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All technical solutions implemented based on the above content of the present invention fall within the scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0067] Example 1: Bioinformatics Analysis 1.1 Data Acquisition and Processing

[0068] Transcriptome sequencing data for 33 tumor types were obtained from the TCGA database, totaling 1,226 samples (1,113 breast cancer tissues and 113 adjacent normal tissues). The raw data were normalized to TPM (transcripts per million) format. 1.2 SDC1 Expression Analysis and Prognostic Analysis

[0069] Pan-cancer analysis was performed using the ggplot2 package in R software to visualize the expression levels of SDC1 in different tumor types. The Wilcoxon rank-sum test was used to compare the differences in SDC1 expression between tumor and normal tissues. Kaplan-Meier survival analysis was used to assess the correlation between SDC1 expression levels and overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI).

[0070] Results: Radar plot analysis showed that SDC1 was significantly upregulated in various malignant tumors, including breast cancer. Figure 1a). Analysis of the TCGA-BRCA dataset showed that SDC1 mRNA levels were significantly higher in tumor tissues (n=1113) than in paired adjacent normal tissues (n=113), a result confirmed by both unpaired and paired analyses. Figure 1 bc). ROC curve analysis showed that SDC1 had good diagnostic performance (AUC=0.846). Figure 1 d). Kaplan-Meier survival analysis showed that patients with high SDC1 expression had a significantly longer overall survival ( Figure 1 e) Disease-specific survival ( Figure 1 f) and the progression-free interval ( Figure 1 g) All were significantly shortened.

[0071] Example 2: Validation using clinical tissue samples 2.1 Tissue Sample Collection

[0072] We collected 165 breast cancer tissue specimens and 70 paired adjacent normal tissue specimens from the Affiliated Hospital of Nantong University between 2012 and 2017. We also collected 8 pairs of freshly surgically removed breast cancer tissues and their paired adjacent normal tissues; these tissues remained after pathological sampling and were stored at -80°C. None of the patients received any preoperative treatment. This study was approved by the Medical Ethics Committee of the Affiliated Hospital of Nantong University (No.: 2024-L201), and all patients signed informed consent forms. 2.2 Immunohistochemical staining

[0073] Tissue sections were dewaxed with xylene, rehydrated with graded ethanol, and then subjected to antigen retrieval in a microwave oven using 1× retrieval solution. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and nonspecific sites were blocked with 5% BSA. Sections were incubated overnight with primary antibody at 4°C, followed by incubation with enhancement reagent and secondary antibody. DAB staining was performed, and hematoxylin counterstaining was applied. Staining results were independently evaluated by two senior pathologists using a double-blind method, scoring based on the percentage of positive cells and staining intensity.

[0074] Results: Tissue microarray IHC staining showed that SDC1 expression was significantly stronger in tumor tissues than in benign tissues. Figure 1 k). ROC curves based on tissue microarrays further confirmed the diagnostic value of SDC1 (k). Figure 1 j). High SDC1 expression and poor overall survival (OS) Figure 1 m) and invasive clinicopathological features (including advanced T stage, distant metastasis, high TNM stage, high grade histological grade and high microvessel density) were significantly correlated (Table 1). 2.3 qRT-PCR and Western Blot Validation

[0075] Total RNA was extracted using an RNA extraction kit, and cDNA was synthesized by reverse transcription. qRT-PCR was performed using SYBR Green qPCR MasterMix on a QuantStudio 5 Real-Time PCR system. Total protein was extracted using RIPA lysis buffer, and protein concentration was determined by the BCA method. After separation by SDS-PAGE, the protein was transferred to a PVDF membrane, incubated overnight at 4°C with primary antibody, and incubated at room temperature for 1 hour with secondary antibody. ECL staining was performed, and grayscale values ​​were analyzed using ImageJ software.

[0076] Results: Both qRT-PCR and Western Blot showed that SDC1 expression in tumor tissues was significantly higher than that in paired adjacent normal tissues. Figure 1 hi).

[0077] Example 3: Cell Function Experiment 3.1 Cell Culture

[0078] Breast cancer cell lines MDA-MB-231, BT-549, SUM159PT, MCF-7, 4T1, and SK-BR3, normal breast epithelial cells MCF-10A, HEK-293T cells, and human umbilical vein endothelial cells (HUVECs) were all purchased from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231, SUM159PT, MCF-7, 4T1, and HEK-293T cells were cultured in DMEM medium, BT-549 cells in RPMI-1640 medium, and HUVECs in DMEM / F12 medium, all supplemented with 10% fetal bovine serum and 100 U / mL penicillin-streptomycin. MCF-10A cells were cultured in Prologis complete medium. All cells were cultured in a 37°C, 5% CO2 humidified incubator. 3.2 Construction of stable cell lines

[0079] SDC1-specific siRNA fragments and shRNA constructs were designed, along with an SDC1 overexpression vector (OE-SDC1) and a negative control plasmid. The target plasmid, along with helper vectors pMD2.G and psPAX2, was co-transfected into HEK-293T cells to package lentivirus. Viral supernatant was collected after 48-72 hours, filtered, and used to infect target cells. Stable cell lines were established by selection with puromycin for 2-3 weeks.

[0080] The SDC1-specific siRNA and shRNA sequences, as well as the non-targeted control sequences (scramble and siNC), are as follows: Scramble: 5′-GGGTGAACTCACGTCAGAA-3′ Human-shSDC1 1 #:5′-GGTGCTTTTGCAAGATATCACC-3′ Human-shSDC1 2 # :5′-GGCCTCCTGGACAGGAAAGA-3′ Mouse-shSDC1 1 # :5′-CCGCAAATTGTGGCTGTAAAT-3′ Mouse-shSDC1 2 # :5′-GAACAAGACTTCACCTTTGAA-3′ siNC: Forward: 5′-UUCUCCGAACGUGUCACGUTT-3′ siNC: Direction: 5′-ACGUGACACGUUCGGAGAATT-3 Human-shSDC1 1 # Forward: 5′-CCACCAAACAGGAGGAAUUTT-3′′ Direction: 5′-AAUUCCUCCUGUUUGGUGGTT-3′ Human-shSDC1 2 # Forward: 5′-CGAGAGGGCUGCUGAGGAUTT-3′ Direction: 5′-AUCCUCAGCAGCCCUCUCGTT-3′ 3.3 CCK-8 Proliferation Experiment

[0081] 2,000 cells / well were seeded into 96-well plates. After approximately 8 hours, the cells adhered (set as 0 hours). CCK-8 reagent (90 μL basal medium + 10 μL CCK-8 reagent) was added, and the plates were incubated at 37°C for 2 hours. The absorbance was measured at 450 nm. The above procedure was repeated at 24, 48, 72, and 96 hours.

[0082] Results: CCK-8 assays showed that SDC1 knockdown significantly inhibited the proliferation of BT-549 and SUM159PT cells, while SDC1 overexpression promoted the proliferation of MCF-7 and MDA-MB-231 cells. Figure 2 bc). 3.4 EdU Proliferation Experiment

[0083] 5,000 cells / well were seeded into 96-well plates. After 24 hours, 50 μM EdU reagent was added and incubated for 2 hours. Cells were fixed with 4% PFA for 30 minutes, permeabilized with 0.5% Triton X-100 for 15 minutes, incubated in Azide Alexa Fluor 594 for 30 minutes in the dark, and then stained with Hoechst 33342 for 10 minutes. The proportion of EdU-positive cells was observed and calculated under a fluorescence microscope.

[0084] Results: EdU assays showed that SDC1 knockdown significantly reduced the DNA synthesis capacity of BT-549 cells. Figure 2 d). 3.5 Scratch Healing Test

[0085] Cells were seeded in 6-well plates and cultured until complete confluence. A straight line was drawn on the cell layer using a sterile pipette tip, and the cells were washed with PBS to remove suspension. Serum-free medium was then added. Images were taken under a microscope at 0 and 24 hours, and the scratch healing rate was analyzed using ImageJ software.

[0086] Results: Scratch assays showed that SDC1 knockdown reduced cell migration, while SDC1 overexpression enhanced cell migration. Figure 2 ef). 3.6 Transwell migration and invasion experiments

[0087] Migration assay: Cells were resuspended in serum-free medium and the upper chamber of an 8 μm pore size Transwell chamber (MDA-MB-231, BT-549, SUM159PT: 3 × 10⁻⁶) was added. 4 Cells / well; MCF-7: 10 × 10 4 Cells / well). Add 600 μL of medium containing 10% FBS to the lower chamber. After incubation at 37°C for 24 hours, wipe away unmigrated cells from the upper chamber, fix with 4% PFA for 30 minutes, stain with 0.1% crystal violet for 15 minutes, and photograph and count under a microscope.

[0088] Invasion test: Transwell chambers were pre-coated with Matrigel (37°C, 1 hour), and the remaining steps were the same as for the migration test.

[0089] Results: Transwell assays showed that SDC1 knockdown reduced cell migration and invasion abilities, while SDC1 overexpression enhanced cell migration and invasion abilities. Figure 2 gh).

[0090] Example 4: Multi-omics analysis 4.1 Transcriptome and proteome sequencing

[0091] BT-549 cell samples were prepared for the control group and the SDC1 knockdown group, with three biological replicates for each group. The samples were sent to Shanghai Meiji Biotechnology Co., Ltd. for transcriptome and proteome sequencing. 4.2 Differential Expression and Enrichment Analysis

[0092] Gene and protein expression changes were analyzed using volcano plots. GO and KEGG pathway enrichment analyses were used to identify biological pathways with differentially expressed genes and proteins.

[0093] Results: Transcriptomic and proteomic sequencing showed that SDC1 knockdown resulted in the gene ( Figure 3 a) and protein ( Figure 3 b) Expression underwent extensive changes. GO enrichment analysis showed that differentially expressed genes and proteins were significantly enriched in pathways such as "extracellular matrix tissue organization," "cell-matrix adhesion," "cell migration regulation," and "epithelial-mesenchymal transition." Figure 3 c). KEGG enrichment analysis showed that differentially expressed genes and proteins were enriched in programmed cell death pathways such as ferroptosis, apoptosis, and necroptosis. Figure 5 ab).

[0094] Example 5: Study on ECM remodeling and EMT mechanisms The expression changes of ECM-related proteins (FN1, ITGB3) and EMT markers (N-cadherin, E-cadherin, VIM) after SDC1 knockdown or overexpression were detected by Western blotting.

[0095] Results: After SDC1 knockdown, the expression of FN1, ITGB3, N-cadherin, and VIM decreased in BT-549 and SUM159PT cells, while the expression of E-cadherin increased. Figure 3 d, f). Conversely, after SDC1 overexpression, the expression of FN1, ITGB3, N-cadherin, and VIM increased in MCF-7 and MDA-MB-231 cells, while the expression of E-cadherin decreased ( Figure 3 e, g).

[0096] Example 6: Angiogenesis Study 6.1 Collection of conditioned media

[0097] Conditioned culture medium from SDC1-regulated cells was collected and applied to HUVECs to evaluate the paracrine effect of SDC1 on angiogenesis. 6.2 HUVEC migration, proliferation and tube formation experiments

[0098] The migration assay used Transwell chambers, the proliferation assay used CCK-8, and the tube formation assay used Matrigel.

[0099] Results: Conditioned culture medium with SDC1 knockdown significantly inhibited HUVEC migration. Figure 4 a) Proliferation ( Figure 4 cd) and tube-forming ability ( Figure 4 g, i); Conditioned culture medium for SDC1-overexpressing cells significantly promoted HUVEC migration (g, i); Figure 4 b) Proliferation ( Figure 4 ef) and tube forming ability ( Figure 4 h, j). Tissue microarray CD34 / PAS double staining showed that SDC1 expression was strongly positively correlated with microvessel density (h, j). Figure 4 k).

[0100] Example 7: Study on the mechanism of ferroptosis 7.1 Western Blot Detection of Ferroptosis-Related Proteins

[0101] The expression changes of GPX4, xCT, HMOX1, and ACSL4 after SDC1 regulation were detected.

[0102] Results: SDC1 knockdown downregulated GPX4 and xCT expression, and upregulated HMOX1 and ACSL4 expression; SDC1 overexpression had the opposite effect. Figure 5 cd). 7.2 Detection of lipid peroxidation

[0103] Lipid peroxidation levels were detected by confocal microscopy and flow cytometry using BODIPY™ 581 / 591 C11 staining.

[0104] Results: SDC1 knockdown led to a significant increase in lipid peroxidation levels, an effect that could be reversed by the ferroptosis inhibitor Fer-1. Figure 5 ef). 7.3 Detection of mitochondrial ferrous ions

[0105] Mito-FerroGreen and MitoBright Deep Red co-staining was used to detect mitochondrial ferrous ion accumulation.

[0106] Results: SDC1 knockdown increased mitochondrial ferrous ion accumulation, which was exacerbated by the ferroptosis inducer RSL3 and blocked by the iron chelator DFO. Figure 6 a). 7.4 Observation of mitochondrial morphology using transmission electron microscopy

[0107] Cells from different treatment groups were collected, fixed with 2.5% glutaraldehyde for 2 hours, embedded in 2% low-melting-point agarose, post-fixed with 1% osmium tetroxide, dehydrated with graded ethanol, embedded in epoxy resin, ultrathin sections (50-70 nm), stained with uranium acetate and lead citrate, and observed under a transmission electron microscope.

[0108] Results: SDC1 knockdown combined with RSL3 treatment induced typical ferroptosis-related mitochondrial changes, manifested as mitochondrial shrinkage and increased membrane density, an effect that could be reversed by Fer-1. Figure 6 b).

[0109] Example 8: In vivo animal experiments 8.1 Establishment of an in situ breast cancer model

[0110] Six-week-old female BALB / c mice were housed in an SPF (Special Purpose Facility) environment. 2.5 × 10⁻⁶ mice were placed in a 2.5 × 10⁻⁶ pyrrolizumab (SPF) container. 5 A lentivirus-modified 4T1 cell was injected into the right fourth mammary fat pad of mice to establish an in situ breast cancer model. 8.2 SDC1 knockdown therapy trial

[0111] Thirty mice were randomly divided into a non-targeted scramble group (n=12) and a shSDC1 knockdown group (n=18). When the tumor volume reached approximately 100 mm³, the mice were further divided into five treatment groups (n=6): scramble + PBS, shSDC1 + PBS, scramble + RSL3 (1.0 mM, 30 μL, intratumoral injection every 3 days), shSDC1 + RSL3, and shSDC1 + Fer-1 + RSL3 (Fer-1: 5 mg / kg intraperitoneal injection; RSL3: 1.0 mM, 30 μL intratumoral injection every 3 days; Fer-1 was administered 2 hours before RSL3). 8.3 SDC1 overexpression therapy trial

[0112] Fifteen mice were randomly divided into an empty vector control group (n=5) and an OE-SDC1 overexpression group (n=10). When the tumor volume reached approximately 100 mm³, the OE-SDC1 group was further divided into three treatment subgroups (n=5): vector + PBS, OE-SDC1 + PBS, and OE-SDC1 + MZ1 (15 mg / kg, daily peritumoral and intratumoral injections). MZ1 was purchased from MCE (HY-107425).

[0113] Results: MZ1 treatment significantly inhibited the growth of SDC1-overexpressing tumors, as evidenced by a reduction in tumor volume and weight. Figure 7 df). MZ1 reversed the regulation of ECM, EMT and ferroptosis-related protein expression by SDC1 overexpression in vivo. Figure 7g). IHC staining showed that MZ1 significantly reduced the expression of the proliferation marker Ki67 in tumor cells ( Figure 7 Masson staining showed that MZ1 reversed the increase in collagen fiber deposition induced by SDC1 overexpression (h). Figure 7 h).

[0114] Example 9: Lung Metastasis Assessment Distant metastasis was assessed in an orthotopic mouse model. Metastasis to the lung, vertebrae, and femur was evaluated using H&E staining and Masson staining.

[0115] Results: MZ1 intervention significantly inhibited lung metastasis, as evidenced by a significant reduction in the number and size of lung metastatic lesions. Figure 8 ab). H&E staining confirmed the metastatic effect of SDC1, and MZ1 treatment significantly reduced metastatic lesions ( Figure 8 c). Masson staining showed that MZ1 reversed SDC1-driven collagen fiber deposition within metastatic lesions ( Figure 8 c). No obvious metastatic lesions were detected in the vertebrae and femur of any group. Figure 8 d). During MZ1 treatment, the mice maintained stable body weight, and no necrosis, inflammation, or other abnormalities were observed in the major organs by H&E and Masson staining. Figure 8 f).

[0116] Example 10: Statistical Analysis The Pearson chi-square test was used to assess the correlation between SDC1 protein levels and clinicopathological features. Cox regression models were used to analyze survival data, and the results are presented as Kaplan-Meier curves. Mann-Whitney U tests were used to assess differences between groups for continuous variables, and chi-square tests were used for categorical variables. Student's t-test was used for comparisons between groups, and two-way ANOVA was used for multivariate experiments. SPSS software (version 22.0) was used for some statistical calculations, and GraphPad Prism (version 10.0) was used for data visualization and main statistical tests. A p-value <0.05 was considered statistically significant.

Claims

1. Application of reagents for detecting SDC1 expression levels in the preparation of kits for breast cancer diagnosis or prognostic assessment.

2. Use according to claim 1, characterized in that, The reagent is an antibody that specifically binds to the SDC1 protein.

3. Use according to claim 1, characterized in that, The reagents are primers that specifically amplify the SDC1 gene.

4. Use according to claim 1, characterized in that, The kit is used for immunohistochemical detection, Western blotting detection, ELISA detection, or qRT-PCR detection.

5. The use according to claim 1, characterized in that, The kit also includes instructions describing the judgment rules, which state that when the detected SDC1 expression level is higher than that of the normal control, it indicates that the subject has breast cancer or has a poor prognostic risk.

6. Application of SDC1 inhibitors in the preparation of drugs for the treatment of breast cancer.

7. The application according to claim 6, characterized in that, The SDC1 inhibitor is MZ1.

8. The application according to claim 6, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

9. The application according to claim 6, characterized in that, The dosage form of the drug is injection, tablet, capsule, or powder for injection.

10. The application according to claim 6, characterized in that, The drug can be administered via intratumoral injection, intravenous injection, or oral administration.

Images