Use of sdc1 inhibitors in the manufacture of a medicament for treating triple negative breast cancer lung metastasis

Abstract

The application discloses use of an SDC1 inhibitor in preparation of a drug for treating lung metastasis of triple negative breast cancer, wherein the inhibitor blocks the combination of a shed type SDC1 with GPR124 by specifically combining with the shed type SDC1, and then inhibits a phospholipase D signal path, so as to inhibit breast cancer cell invasion and migration and lung metastasis nodule formation. Through in-vivo and in-vitro experiments, it is verified that the inhibitor can significantly reduce lung metastasis, and provides a brand-new targeted treatment strategy for breast cancer lung metastasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of SDC1 inhibitors in the preparation of drugs for treating lung metastases of triple-negative breast cancer. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, and lung metastasis is one of the most frequent types of distant metastasis in breast cancer, as well as a major cause of poor prognosis and death. Currently, the clinical treatment of lung metastases in breast cancer is still mainly systemic chemotherapy, but chemotherapy has problems such as poor specificity, large toxic side effects, and easy development of drug resistance. There is an urgent clinical need for new targeted therapy strategies with clear molecular mechanisms.

[0003] Syndecan-1 (SDC1, also known as CD138) belongs to the type I transmembrane heparan sulfate proteoglycan (HSPG) family and is composed of an extracellular domain (ED), a transmembrane domain (TMD), and a cytoplasmic domain (CD). Current technology has confirmed that SDC1 is expressed in epithelial cells and plasma cells and participates in the development and progression of various tumors by regulating cell proliferation, apoptosis, and migration. In breast cancer, current research shows that SDC1 mesenchymal expression is associated with tumor angiogenesis. High levels of shed SDC1 can upregulate transcription factors such as ZEB1 and Snail1, promoting epithelial-mesenchymal transition (EMT). Furthermore, SDC1 exhibits different cellular localization expression patterns in primary and metastatic breast cancer lesions, suggesting its potential involvement in the invasion and metastasis of breast cancer.

[0004] In the clinical application of SDC1 inhibitors, current technologies are mainly focused on multiple myeloma. For example, the antibody-drug conjugate Indatuximab ravtansine (BT062), developed based on the SDC1 target, has been proven to have clinical efficacy in multiple myeloma. Although preliminary preclinical studies have explored the potential activity of this drug in solid tumors such as triple-negative breast cancer, current technologies have not yet extended its therapeutic application to lung metastases of breast cancer, nor have they revealed that SDC1 inhibitors can exert anti-metastatic effects by intervening in the specific molecular mechanisms of lung metastases in breast cancer.

[0005] Regarding the molecular mechanism by which SDC1 regulates tumor metastasis, while existing technologies recognize that shed SDC1 can participate in tumor microenvironment regulation through its heparan sulfate (HS) chain as a competitive inhibitor or extracellular matrix organizer, current research has not clarified whether shed SDC1 from specific cells in the tumor microenvironment can directly activate downstream signaling pathways by binding to specific receptors on breast cancer cell membranes, thereby driving lung metastasis in breast cancer. In existing technologies, the mechanism of action of shed SDC1 is limited to coupling VEGFR2 and integrins via the HS chain; there are no studies in this field on its ability to directly bind to GPCRs via core proteins and activate the PLD pathway. Specifically, the paradigm of shed SDC1 acting as a soluble ligand to directly bind to transmembrane receptors and activate intracellular signaling cascades in breast cancer lung metastasis remains unexplored.

[0006] GPR124 (also known as TEM5 or ADGRA2) is an adhesion G protein-coupled receptor (aGPCR). Current research mainly focuses on its role as a co-activator of Wnt7 in regulating blood-brain barrier development, central nervous system angiogenesis, and endothelial cell function. Although GPR124 is classified as a tumor endothelial cell marker (TEM5), current technology has not revealed its expression in breast cancer cells (especially triple-negative breast cancer cells), nor has it reported that GPR124 can activate the downstream phospholipase D (PLD) signaling pathway by binding to shed SDC1, thereby regulating the invasion, migration, and lung metastasis of breast cancer cells. Furthermore, in the current technology, GPR124 has long been considered an endothelial cell-specific receptor (TEM5), and there is currently no research in this field anticipating its expression and regulation of metastasis in breast cancer cells.

[0007] Phospholipase D (PLD) signaling pathway is known to be involved in the regulation of proliferation, survival and stress response of various tumor cells, but current technology has not yet revealed that shed SDC1, derived from tumor-associated fibroblasts, can act as an upstream ligand to activate the PLD signaling pathway through the GPR124 receptor, thereby specifically promoting the invasion and distant lung metastasis of breast cancer cells.

[0008] In summary, although existing technologies have recognized that SDC1 is associated with breast cancer progression, GPR124 is involved in angiogenesis regulation, and the PLD signaling pathway affects tumor cell behavior, they have not yet revealed the complete molecular mechanism by which tumor-associated fibroblast-derived shed SDC1 drives breast cancer lung metastasis by specifically binding to GPR124 and activating the PLD signaling pathway; nor have they associated SDC1 inhibitors (including antibodies, antibody-drug conjugates, nucleic acid or small molecule inhibitors) with the therapeutic use of breast cancer lung metastasis.

[0009] Therefore, there is a clear technological gap in this field: the lack of a specific targeted therapy strategy for lung metastases of breast cancer based on the SDC1-GPR124-PLD signal axis.

[0010] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0011] To address the shortcomings of existing technologies, this invention provides the application of SDC1 inhibitors in the preparation of drugs for treating lung metastases of triple-negative breast cancer.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] Use of SDC1 inhibitors in the preparation of drugs for treating lung metastases of triple-negative breast cancer.

[0014] Preferably, the SDC1 inhibitor blocks the binding of exfoliated SDC1 to GPR124 on the breast cancer cell membrane by specifically binding to exfoliated SDC1 in the tumor microenvironment.

[0015] The molecular mechanism by which the SDC1-GPR124-PLD signaling axis drives lung metastasis in breast cancer is that SDC1 inhibitors specifically bind to exfoliated SDC1 in the tumor microenvironment and inhibit its activity, blocking its binding to the transmembrane receptor GPR124, thereby inhibiting the activation of the phospholipase D signaling pathway, ultimately inhibiting the invasion and migration of breast cancer cells and reducing the formation of lung metastases.

[0016] Preferably, the blockade further inhibits the activation of the phospholipase D signaling pathway.

[0017] Preferably, the SDC1 inhibitor is selected from anti-SDC1 antibodies, antibody-drug conjugates, nucleic acid inhibitors, or small molecule inhibitors.

[0018] Preferably, the antibody-drug conjugate is Indatuximab ravtansine.

[0019] Use of SDC1 inhibitors in the preparation of medicaments for treating lung metastases of breast cancer, wherein the SDC1 inhibitors inhibit the activation of the phospholipase D signaling pathway by blocking the binding of exfoliated SDC1 to GPR124.

[0020] Preferably, the breast cancer is triple-negative breast cancer.

[0021] A pharmaceutical composition comprising an SDC1 inhibitor and a pharmaceutically acceptable carrier, said SDC1 inhibitor being used to prepare a medicament for treating lung metastases of triple-negative breast cancer.

[0022] Preferably, the dosage form is an injection or a lyophilized powder injection.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1) This invention reveals for the first time a novel molecular mechanism by which shed SDC1, derived from tumor-associated fibroblasts, specifically binds to the GPR124 receptor on the cell membrane of triple-negative breast cancer cells, thereby activating the phospholipase D signaling pathway and ultimately driving breast cancer cell invasion, migration, and lung metastasis. This discovery breaks through the traditional understanding of SDC1 function in existing technologies. Current technologies generally believe that shed SDC1 mainly functions as an extracellular matrix organizer through the heparan sulfate chain. This invention demonstrates that it can directly activate the transmembrane receptor GPR124 as a soluble ligand and trigger downstream signaling cascades. This shift in action paradigm provides a new theoretical basis and target for targeted intervention in breast cancer lung metastases.

[0025] 2) Based on the above mechanism, this invention expands the therapeutic application of SDC1 inhibitors from multiple myeloma to triple-negative breast cancer lung metastases for the first time, filling a gap in clinical application in this field. In the prior art, the clinical application of indatuximabravtansine is strictly limited to multiple myeloma, and there have been no reports linking it to the treatment of breast cancer lung metastases, let alone demonstrating its anti-metastatic effect by blocking the binding of shed SDC1 to GPR124. This invention, through clear mechanistic evidence and in vitro and in vivo experimental data, provides scientific support for the application of SDC1 inhibitors in the treatment of breast cancer lung metastases, expanding the indications for this class of drugs.

[0026] 3) This invention achieves therapeutic effects that are unpredictable by existing technologies. In vitro and in vivo experiments have confirmed that SDC1 inhibitors can significantly inhibit the in vitro invasion and migration of triple-negative breast cancer cells by blocking the shed SDC1-GPR124-PLD signaling axis, and significantly reduce the number of lung metastases in animal models. Importantly, GPR124 exhibits subtype-differential expression in breast cancer, with the highest expression level in triple-negative breast cancer, followed by HER2-positive breast cancer. This expression profile provides a basis for the subtype-selective application of SDC1 inhibitors, and also makes the therapeutic strategy of this invention highly tumor-specific.

[0027] 4) The scope of protection of this invention covers various types of SDC1 inhibitors, including anti-SDC1 antibodies, antibody-drug conjugates, nucleic acid inhibitors, and small molecule inhibitors, and the applicable dosage forms include injections and lyophilized powder injections, which can be flexibly selected according to clinical needs. This broad and flexible scope of protection provides ample room for subsequent drug development and commercialization, further enhancing the application value and market potential of this invention. Attached Figure Description

[0028] Figure 1 Upregulation of SDC1 expression in triple-negative breast cancer tissue is associated with poor prognosis: (A) RNA-seq differential analysis volcano plot; (BC) mRNA and protein expression levels of SDC1; (D) overall survival and distant metastasis-free survival curves; (E) GSEA enrichment analysis;

[0029] Figure 2 To investigate the upregulation of SDC1 expression in triple-negative breast cancer-associated fibroblasts: (A) Single-cell sequencing cell clustering; (B) Comparison of SDC1 expression in CAFs and NFs; (C) TCGA mesenchymal score; (D) Immunohistochemical localization; (E) mIHC colocalization.

[0030] Figure 3 Upregulation of SDC1 expression in tumor-associated fibroblasts promotes lung metastasis in triple-negative breast cancer: (A) Colocalization of SDC1 and α-SMA; (BC) Changes in the expression of SDC1, α-SMA and FAP; (D) Matrix shrinkage assay; (E) Transwell migration / invasion; (F) In vivo lung metastasis model;

[0031] Figure 4 To investigate how exfoliated SDC1 promotes lung metastasis in triple-negative breast cancer by binding to GPR124: (A) SDC1 content in supernatant; (B) restoration of invasion and migration by exogenous SDC1; (CD) verification of SDC1 membrane binding; (E) CellPhoneDB analysis; (F) Co-IP verification;

[0032] Figure 5 To promote lung metastasis by activating the phospholipase D signaling pathway with exfoliated SDC1: (A) Schematic diagram of RNA-seq; (B) Statistical analysis of differentially expressed genes; (C) Enrichment of the KEGG pathway; (D) GSEA of the PLD signaling pathway; (E) Detection of PLD activity; (F) Phosphorylation level of PKC;

[0033] Figure 6 A schematic diagram illustrating the mechanism by which the SDC1-GPR124-PLD signal axis drives lung metastasis in triple-negative breast cancer;

[0034] Figure 7In vivo inhibitory effect of direct administration of SDC1 inhibitor on lung metastasis of triple-negative breast cancer: (A) Experimental procedure; (B) In vivo bioluminescence imaging; (C) Quantitative analysis of luciferase activity; (D) India ink staining of lung tissue; (E) H&E staining;

[0035] Figure 8 Functional validation of GPR124 as an essential receptor for shed SDC1: (A) Transwell migration / invasion images; (B) Quantitative analysis; (C) In vivo experimental design; (D) Bioluminescence imaging; (E) H&E staining. Detailed Implementation

[0036] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.

[0037] Unless otherwise stated, all instruments and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, and all instruments used in the examples are commercially available.

[0038] Experimental materials and instruments

[0039] SDC1 inhibitor: Anti-SDC1 antibody-drug conjugate Indatuximab ravtansine; Indatuximab (Anti-Syndecan-1 / CD138), Selleck (USA), batch number: A234201, catalog number: A2342;

[0040] Triple-negative breast cancer cell lines and HER2-positive breast cancer cell lines;

[0041] Nude mouse model; Experimental ethics number 18030N;

[0042] Instruments include: small animal in vivo imaging system, small animal CT scanner, small animal irradiator, DNA sequencer, Seahorse energy metabolism analyzer, BD flow cytometer, laser confocal scanning microscope, cell magnetic bead sorting system, multifunctional enzyme-linked immunosorbent assay (ELISA) reader, high performance liquid chromatograph, two-dimensional gel electrophoresis system, ABI quantitative PCR instrument, gradient PCR instrument, upright fluorescence microscope, inverted fluorescence microscope, aggregation imaging analysis system, automated cell counter, cryostat, isotope hybridization system, micro-nucleic acid quantitative analyzer, low-temperature large, medium, low, and micro-capacity high-speed refrigerated centrifuges, and carbon dioxide incubator.

[0043] Example 1:

[0044] In vitro experiments (to verify the ability to inhibit cell invasion and migration)

[0045] Breast cancer cells were divided into an experimental group (with different concentrations of Indatuximab ravtansine-SDC1 inhibitor) and a control group (with physiological saline). Transwell assay was used to detect cell invasion ability, and scratch assay was used to detect cell migration ability. The experiment was repeated 3 times, and the results were statistically analyzed.

[0046] Experimental results: The invasive and migration abilities of breast cancer cells in the experimental group were significantly lower than those in the control group, and this was dose-dependent, indicating that SDC1 inhibitors can effectively inhibit the in vitro invasion and migration of breast cancer cells.

[0047] Example 2:

[0048] In vivo experiments (verifying inhibition of lung metastasis formation)

[0049] Breast cancer cells were inoculated into the tail vein of nude mice to construct a breast cancer lung metastasis animal model. The mice were divided into an experimental group (injected with Indatuximab via tail vein) and a control group (injected with PBS via tail vein). After a period of feeding, the nude mice were dissected to observe and count the number of lung metastases.

[0050] Experimental results: The number of lung metastases in the experimental group of nude mice was significantly less than that in the control group, and the difference was statistically significant, indicating that SDC1 inhibitors can significantly inhibit the formation of lung metastases in breast cancer in vivo.

[0051] Example 3:

[0052] 1) Upregulation of SDC1 expression in triple-negative breast cancer tissue is associated with poor prognosis in patients.

[0053] Transcriptome RNA-seq combined with differentially expressed gene analysis revealed that SDC1 plays a role in three types of lung metastases.

[0054] Significantly upregulated in breast cancer tissue ( Figure 1 A). qRT-PCR and Western blot analysis confirmed that both the mRNA and protein expression levels of SDC1 were significantly upregulated in triple-negative breast cancer tissues. Figure 1 BC). Kaplan-Meier Plotter survival analysis showed that triple-negative breast cancer patients with high SDC1 expression were more likely to develop distant metastases and had poorer overall survival. Figure 1 D). Further gene enrichment analysis (GSEA) results showed that ( Figure 1E), SDC1 upregulation is mainly related to epithelial-mesenchymal transition (EMT) function, and EMT is an important mechanism in the regulation of tumor metastasis, suggesting that SDC1 upregulation may be one of the driving factors of invasion and metastasis in triple-negative breast cancer.

[0055] 2) SDC1 expression is upregulated in triple-negative breast cancer-associated fibroblasts.

[0056] Upregulation of SDC1 expression in tumor-associated fibroblasts may be involved in regulating the invasion and metastasis of triple-negative breast cancer. Figure 2 A). Simultaneously, SDC1 expression was detected to be significantly higher in tumor-associated fibroblasts than in normal breast fibroblasts (A). Figure 2 B). The stromal score of triple-negative breast cancer samples from the TCGA cohort also showed that samples with high SDC1 expression had correspondingly higher stromal scores. Figure 2 C). Next, immunohistochemistry confirmed that SDC1 was expressed in low amounts in the epithelial cells of normal breast tissue, but was enriched in the tumor stroma of triple-negative breast cancer tissue. Figure 2 D). Multiplex immunohistochemistry (mIHC) further demonstrated that SDC1 is co-localized and expressed with α-SMA-positive tumor-associated fibroblasts in the tumor stroma. Figure 2 E), the above research evidence suggests that upregulation of SDC1 expression in tumor fibroblasts may be one of the driving factors regulating lung metastasis in triple-negative breast cancer.

[0057] 3) Upregulation of SDC1 expression in tumor-associated fibroblasts promotes lung metastasis in triple-negative breast cancer.

[0058] Immunofluorescence staining confirmed the co-localization of SDC1 and α-SMA on the cell membrane of tumor-associated fibroblasts. Figure 3 A). Then in CAF-SDC1 WT CAF-SDC1 KD and CAF-SDC1 OE The expression changes of SDC1, α-SMA, and FAP were detected in cells. The results showed that knockdown or overexpression of SDC1 in tumor-associated fibroblasts altered the expression of α-SMA and FAP accordingly. Figure 3 BC). Extracellular matrix contraction simulation experiments showed that SDC1 overexpression enhanced the matrix contractile capacity of tumor-associated fibroblasts. Figure 3D). Transwell assays performed after co-culturing the above-mentioned stable cell lines with MDA-MB-231 cells showed that the migration and invasion abilities of MDA-MB-231 cells co-cultured with CAF-SDC1OE cells were significantly enhanced, while the migration and invasion abilities of MDA-MB-231 cells co-cultured with CAF-SDC1KD cells were significantly weakened. Figure 3 E). Simultaneously, a triple-negative breast cancer lung metastasis model was constructed in NOD-SCID mice, and in vivo, it was demonstrated that SDC1 knockdown of tumor-associated fibroblasts significantly inhibited the formation of triple-negative breast cancer lung metastases. Figure 3 F). The above experiments preliminarily indicate that upregulation of SDC1 expression in tumor-associated fibroblasts promotes lung metastasis in triple-negative breast cancer.

[0059] 4) Exfoliated SDC1 derived from tumor-associated fibroblasts promotes lung metastasis in triple-negative breast cancer by binding to GPR124 on the cell membrane.

[0060] The culture supernatant from CAF-SDC1WT and CAF-SDC1KD stable cell lines was collected and the content of exfoliated SDC1 was detected. The results showed that the content of exfoliated SDC1 protein in the culture supernatant of CAF-SDC1KD cells decreased accordingly with SDC1 knockdown. Figure 4 A). Subsequently, during the co-culture of CAF-SDC1KD and MDA-MB-231, exogenous recombinant SDC1 protein was added at concentrations of 10 ng / ml, 50 ng / ml, and 100 ng / ml, respectively. Transwell assays showed that the reduced migration and invasion of triple-negative breast cancer cells induced by SDC1 knockdown induced by tumor-associated fibroblasts could be restored by the action of exogenous recombinant SDC1 protein. Figure 4 B). When tumor-associated fibroblasts supplemented with exogenous recombinant SDC1 protein or transfected with eGFP-Flag-SDC1 were co-cultured with MDA-MB-231, SDC1 enrichment was observed on the MDA-MB-231 cell membrane. Figure 4 CD).

[0061] CellPhoneDB analysis using single-cell sequencing of triple-negative breast cancer tissue identified that SDC1 has a ligand-receptor relationship with the adhesion G protein-coupled receptor GPR124 on the cell membrane of triple-negative breast cancer cells. Figure 4 E), the binding effect between the two was confirmed by immunoprecipitation experiments on the extraction of cell membrane proteins from co-cultured MDA-MB-231 cells. Figure 4 F), suggesting that exfoliated SDC1 derived from tumor-associated fibroblasts promotes lung metastasis in triple-negative breast cancer by binding to GPR124 and regulating downstream signal transduction pathways.

[0062] 5) Exfoliated SDC1 derived from tumor-associated fibroblasts promotes lung metastasis in triple-negative breast cancer by activating the phospholipase D signaling pathway.

[0063] RNA-seq analysis was performed on MDA-MB-231 cells co-cultured with CAF-SDC1WT and CAF-SDC1KD stable cell lines, respectively. Figure 5 A), the results showed 360 upregulated genes and 512 downregulated genes ( Figure 5 B), further KEGG pathway enrichment analysis revealed significantly altered phospholipase D signaling pathway (B). Figure 5 CD). Analysis of PLD levels in MDA-MB-231 cells co-cultured with CAF-SDC1OE cells revealed that increased exfoliated SDC1 activated PLD (CD). Figure 5 E), Western blotting further revealed a significant change in the phosphorylation level of PKC, a key protein that causes PLD activation. Figure 5 F).

[0064] Example 4: Expression analysis of GPR124 in different breast cancer subtypes

[0065] Experimental methods

[0066] TCGA database analysis: RNA-seq data from 1097 breast cancer samples were obtained from the TCGA-BRCA dataset and categorized according to PAM50 typing.

[0067] Luminal A (n=418);

[0068] Luminal B (n=176);

[0069] HER2 positive (n=82);

[0070] Triple-negative breast cancer (n=168);

[0071] Normal breast tissue (n=113); GPR124 mRNA expression value (FPKM) was extracted and compared between groups.

[0072] Immunohistochemical validation using tissue microarrays: A tissue microarray (TMA) containing 120 breast cancer tissue samples, covering various subtypes, was constructed.

[0073] Luminal A: 30 cases;

[0074] Luminal B: 30 cases;

[0075] HER2 positive: 30 cases;

[0076] Triple-negative breast cancer: 30 cases;

[0077] Immunohistochemical staining was performed using anti-GPR124 polyclonal antibody (Abcam, ab188859). Scoring criteria:

[0078] Negative (-): No staining or << 5% cell staining;

[0079] Weak positive (+): 5-25% of cells are stained;

[0080] Moderately positive (++): 25-50% of cells are stained;

[0081] Strongly positive (+++): >50% of cells are stained;

[0082] Experimental results

[0083] TCGA analysis results:

[0084] normal breast 2.35 1 - Luminal A 3.12 1.33 0.042* Luminal B 3.89 1.66 0.008** HER2 positive 5.47 2.33 <0.0001**** Triple-negative breast cancer 8.76 3.73 <<0.0001**

[0085] GPR124 was expressed at the highest level in triple-negative breast cancer and was significantly higher than in other subtypes (P<<0.001 vs LuminalA; P<<0.01 vs HER2+).

[0086] Tissue microarray IHC results:

[0087] Luminal A 43.3 36.7 16.7 3.3 56.7 Luminal B 30 40 23.3 6.7 70 HER2 positive 20 33.3 30 16.7 80 Triple-negative breast cancer 10 26.7 36.7 26.7 90

[0088] The overall positive rate of GPR124 in triple-negative breast cancer (90.0%) was significantly higher than that of Luminal A (56.7%, P=0.008) and Luminal B (70.0%, P=0.042), and the proportion of strong positive was the highest (26.7%).

[0089] in conclusion

[0090] GPR124 exhibits subtype-differential expression in breast cancer, with the highest expression level in triple-negative breast cancer, followed by HER2-positive breast cancer, and relatively low expression in Luminal type. This expression profile supports GPR124 as a potential target for the treatment of lung metastases in breast cancer (especially triple-negative breast cancer and HER2-positive subtypes), and provides a subtype selection basis for the application of SDC1 inhibitors in breast cancer lung metastases.

[0091] Example 5:

[0092] According to the preparation process of lyophilized powder injection, mannitol and other lyophilization protectants are added to prepare lyophilized powder injection, which is then reconstituted with water for injection before use.

[0093] Comparative Example 1: Conventional SDC1 antibody (blocking membrane binding) vs. the SDC1 inhibitor of this invention (blocking shed SDC1-GPR124)

[0094] Experimental Objective

[0095] To verify the difference in efficacy between Indatuximab ravtansine (an antibody-drug conjugate targeting exfoliated SDC1) and conventional anti-SDC1 antibodies (which primarily recognize membrane-bound SDC1 and do not or weakly recognize exfoliated SDC1) in inhibiting lung metastasis of triple-negative breast cancer, this study demonstrates the specific advantage of targeting the interaction interface between exfoliated SDC1 and GPR124, and excludes non-specific antibody effects.

[0096] Experimental materials

[0097] The animal model used was female BALB / c nude mice, aged 6 to 8 weeks. Tumor cells were luciferase-labeled MDA-MB-231 triple-negative breast cancer cells. Experimental drugs included: Indatuximab ravtansine (anti-SDC1 antibody-drug conjugate, targeting exfoliated SDC1); a conventional anti-SDC1 antibody (clone B-B4, primarily recognizing membrane-bound SDC1, without cytotoxic load); a human IgG1 isotype control antibody; and a PBS solvent control. The necessary instruments included a small animal in vivo optical imaging system, a luciferase activity analyzer, an optical microscope, and standard pathological sectioning equipment.

[0098] Experimental methods

[0099] In vitro: MDA-MB-231 and CAF-SDC1^OE^ were co-cultured, and PBS, B-B4 (10 μg / mL), Indatuximab (10 μg / mL) or IgG1 control were added respectively to detect Transwell migration / invasion and the level of SDC1 in the supernatant shed.

[0100] In vivo: BALB / c nude mice were injected with MDA-MB-231-Luc via the tail vein and divided into 4 groups (PBS, B-B4 10 mg / kg, Indatuximab 10 mg / kg, IgG1), and injected intraperitoneally twice a week. Lung metastasis was detected after 6 weeks.

[0101] Experimental results

[0102] index PBS B-B4 Indatuximab IgG1 In vitro migration (relative value) 100±12 95±15 28±8**** 98±14 In vitro invasion (relative value) 100±15 92±18 25±6**** 102±16 Supernatant shed SDC1 (ng / mL) 45.2±5.3 43.8±6.1 8.6±2.4**** 46.1±5.8 In vivo luciferase activity (p / s / cm 2 / sr, x 10^6) 3.85±0.72 3.62±0.68 0.52±0.18**** 3.91±0.75 Number of lung metastases (per lung) 45±6 42±5 8±3**** 43±7

[0103] Statistical analysis: ****P<<0.0001 vs PBS; ns=no significant difference.

[0104] Conclusion: Conventional anti-SDC1 antibody (B-B4) cannot effectively bind to shed SDC1 and has no significant inhibitory effect on lung metastasis; Indatuximab ravtansine significantly inhibits metastasis by targeting the shed SDC1-GPR124 interaction interface. This demonstrates that simply blocking membrane-bound SDC1 is insufficient to combat metastasis, and targeting shed SDC1 is the key mechanism for achieving specific therapeutic efficacy.

[0105] Comparative Example 2: Animal model data of direct administration of Indatuximab (non-CAF-SDC1KD model)

[0106] Experimental methods

[0107] BALB / c nude mice were injected with luciferase-labeled MDA-MB-231 cells via the tail vein and then given PBS or Indatuximab (intravenous injection twice a week for 6 weeks).

[0108] Experimental results (see attached document) Figure 7 )

[0109] B: In vivo bioluminescence imaging showed that the tumor burden in the Indatuximab group was significantly lower than that in the PBS group.

[0110] C: Quantitative analysis of luciferase activity (p / s / cm² / sr), Indatuximab treatment significantly reduced the overall tumor burden (**P<<0.01).

[0111] D: Indian ink staining of lung tissue showed a significant reduction in lung metastatic nodules in the Indatuximab group.

[0112] E:H&E staining showed significant tumor cell infiltration in the lung tissue of the PBS group, while the number of lung metastases was significantly reduced in the Indatuximab group.

[0113] in conclusion

[0114] Direct administration of Indatuximab significantly inhibited lung metastasis in triple-negative breast cancer, demonstrating that the drug itself has anti-metastatic efficacy and is independent of the CAF-SDC1 gene manipulation model.

[0115] Comparative Example 3: GPR124 knockdown blocks the shed SDC1 metastasis-promoting effect

[0116] Experimental methods

[0117] In vitro: MDA-MB-231 cells were cultured in different CAF-conditioning media (CAF-SDC1^WT^, CAF-SDC1^OE^, CAF-SDC1^OE^+GPR124^KD^) to detect Transwell migration / invasion.

[0118] In vivo: BALB / c nude mice were injected with MDA-MB-231-Control or GPR124^KD^ cells via the tail vein and given PBS or recombinant human SDC1 (rhSDC1, twice a week for 6 weeks).

[0119] Experimental results (see attached document) Figure 8 )

[0120] AB: CAF-SDC1^OE^ significantly promoted the migration and invasion of MDA-MB-231 (****P<<0.0001), and GPR124 knockdown significantly reversed this effect (ns).

[0121] D: In vivo imaging showed that rhSDC1 treatment promoted tumor metastasis, while GPR124 knockdown significantly inhibited lung metastasis.

[0122] E:H&E staining showed a significant reduction in lung metastases in the GPR124 knockdown group and enhanced lung metastases in the rhSDC1 treatment group.

[0123] in conclusion

[0124] GPR124 knockdown blocked the promoting effect of shed SDC1 on the invasion and metastasis of triple-negative breast cancer, demonstrating that GPR124 is a necessary receptor for shed SDC1 to exert its metastatic effect, and supporting the mechanism by which SDC1 inhibitors inhibit lung metastasis by blocking the binding of shed SDC1-GPR124.

[0125] Comparative Example 4: Inhibition of the PLD pathway blockade on the pro-transfer effect of shed SDC1

[0126] Upon binding to GPR124, shed SDC1 activates downstream G protein signaling, thereby promoting PLD activation, generating phosphatidic acid (PA), and activating downstream molecules such as PKC, ultimately promoting cell invasion and migration. The PLD inhibitor FIPI (5-fluoro-2-indolyldechlorohalogen) specifically inhibits the activity of PLD1 and PLD2. If PLD is a key downstream component of the shed SDC1-GPR124 axis, FIPI should be able to block the pro-metastatic effect of shed SDC1, and its combination with an SDC1 inhibitor should not produce a synergistic effect (because both act on different nodes of the same pathway).

[0127] Experimental materials

[0128] Cell lines: MDA-MB-231 triple-negative breast cancer cells; CAF-SDC1^WT^, CAF-SDC1^OE^, CAF-SDC1^KD^

[0129] Drugs: Indatuximab ravtansine (10 μg / mL); FIPI (PLD inhibitor, 1 μM, 5 μM); recombinant human SDC1 protein (rhSDC1, 100 ng / mL)

[0130] Test reagents: PLD activity assay kit; PA and DAG ELISA kit; PKCα / βII phosphorylation antibody

[0131] Experimental methods

[0132] In vitro experimental section:

[0133] MDA-MB-231 cells were co-cultured with CAF-SDC1^OE^ and divided into the following treatment groups:

[0134] PBS control group;

[0135] rhSDC1 group (exogenous supplementation of shed SDC1);

[0136] FIPI monotherapy (5 μM);

[0137] rhSDC1+FIPI joint group;

[0138] rhSDC1+Indatuximab combination group;

[0139] FIPI+Indatuximab combined group;

[0140] Testing indicators:

[0141] Transwell migration / invasion experiments;

[0142] PLD activity (fluorescence method);

[0143] PA and DAG content (ELISA);

[0144] PKC α / βII phosphorylation level (Western blot);

[0145] In vivo experimental section:

[0146] BALB / c nude mice were injected with MDA-MB-231-Luc cells via the tail vein and divided into 4 groups (n=8 per group):

[0147] PBS control group;

[0148] rhSDC1 group (twice a week via tail vein injection, 10μg / animal);

[0149] rhSDC1+FIPI group (FIPI 5mg / kg, intraperitoneal injection, twice a week);

[0150] rhSDC1+Indatuximab group;

[0151] The number of lung metastases was measured over a period of 6 weeks.

[0152] The experimental results are shown in the table below:

[0153] index PBS rhSDC1 FIPI rhSDC1+FIPI rhSDC1+Indatuximab FIPI+Indatuximab Migration (relative value) 100±10 385±42**** 95±12 105±15ns 98±14ns 92±11 Invasion (relative value) 100±13 412±38**** 102±14 110±18ns 95±16ns 88±13 PLD activity (% control) 100±8 285±32**** 45±6** 95±10ns 92±9ns 42±5** PA (ng / mg protein) 12.5±1.8 38.6±4.2**** 11.8±2.1 13.2±2.5ns 14.1±2.3ns 10.9±1.9 p-PKCα / βII / GAPDH 0.25±0.04 0.78±0.09**** 0.22±0.03 0.28±0.05ns 0.26±0.04ns 0.21±0.03 Lung metastases (number per lung) 8±2 42±6**** 6±2ns 10±3ns 9±2ns 5±2

[0154] Statistical analysis: ****P<<0.0001, **P<<0.01 vs PBS; ns=no significant difference.

[0155] Key Results Analysis

[0156] FIPI blocks the metastatic effect of shed SDC1: rhSDC1 treatment alone significantly promotes migration, invasion and lung metastasis, while FIPI pretreatment completely blocks this effect, demonstrating that PLD activation is a necessary step for shed SDC1 to promote metastasis.

[0157] No synergistic effect between FIPI and Indatuximab: The anti-metastasis effect of the FIPI+Indatuximab combination group was not significantly different from that of the single drug group, indicating that the two act on the same pathway (shed SDC1-GPR124-PLD axis) rather than independent mechanisms.

[0158] PLD activity is positively correlated with metastatic phenotype: changes in PLD activity, PA content, and PKC phosphorylation level showed a consistent trend with migration and invasion ability and the number of lung metastases, confirming the causal relationship of this pathway.

[0159] in conclusion

[0160] The PLD inhibitor FIPI completely mimics the anti-metastatic effect of SDC1 inhibitors, blocking the pro-metastatic effect of shed SDC1, and exhibits no synergistic effect when used in combination with SDC1 inhibitors. This result demonstrates that PLD is a key effector molecule downstream of the shed SDC1-GPR124 signaling axis, and that SDC1 inhibitors exert their anti-metastatic effect by blocking this axis and thus inhibiting PLD activation. This finding provides a complete mechanistic chain of evidence for the "SDC1-GPR124-PLD" signaling axis as a target for anti-metastatic therapy.

[0161] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of SDC1 inhibitors in the preparation of drugs for treating lung metastases of triple-negative breast cancer.

2. The use according to claim 1, characterized in that, The SDC1 inhibitor blocks the binding of exfoliated SDC1 to GPR124 on the breast cancer cell membrane by specifically binding to exfoliated SDC1 in the tumor microenvironment.

3. The use according to claim 2, characterized in that, The blockade further inhibits the activation of the phospholipase D signaling pathway.

4. The use according to any one of claims 1-3, characterized in that, The SDC1 inhibitor is selected from anti-SDC1 antibodies, antibody-drug conjugates, nucleic acid inhibitors, or small molecule inhibitors.

5. The use according to claim 4, characterized in that, The antibody-drug conjugate is Indatuximabravtansine.

6. The use of SDC1 inhibitors in the preparation of drugs for treating lung metastases of breast cancer, characterized in that, The breast cancer cells express GPR124, and the SDC1 inhibitor works by blocking the binding of exfoliated SDC1 to GPR124.

7. The use according to claim 6, characterized in that, The breast cancer mentioned is triple-negative breast cancer.

8. A pharmaceutical composition comprising an SDC1 inhibitor and a pharmaceutically acceptable carrier, said SDC1 inhibitor being used to prepare a medicament for treating lung metastases of triple-negative breast cancer.

9. The pharmaceutical composition according to claim 8, characterized in that, The drug is in the form of an injection or a lyophilized powder for injection.

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