{"paper_id":"17261f28-01a0-426e-b39c-fb6660cfc8be","body_text":"Targeting FGFR1 with aloperine suppresses angiogenesis, vasculogenic mimicry, and metastasis in triple-negative breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeting FGFR1 with aloperine suppresses angiogenesis, vasculogenic mimicry, and metastasis in triple-negative breast cancer Tianci Tang, Luisa Müller, Yixuan Xu, Matthias W. Laschke, Yuan Gu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9209927/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Triple-negative breast cancer (TNBC) is a highly aggressive malignancy characterized by early recurrence and high metastatic potential. Its aggressiveness is driven by a complex vascular network integrating classical angiogenesis and vasculogenic mimicry (VM). Despite the clinical implementation of PARP inhibitors and immune checkpoint blockade, effective targeted therapeutic strategies remain limited for the majority of TNBC patients. Methods The effects of aloperine on angiogenesis were analyzed using a panel of in vitro assays in human umbilical vein endothelial cells (HUVECs), ex vivo aortic ring assays, and in vivo Matrigel plug assays. Its effects on TNBC cell migration and VM formation were assessed in MDA-MB-231 cells using Transwell migration and tube formation assays, respectively. Mechanistic studies were performed using Western blotting, molecular docking, and cell-free kinase assays. Finally, the therapeutic efficacy of aloperine against TNBC progression was validated in a mouse dorsal skinfold chamber model of murine 4T1 tumors and an orthotopic xenograft model of human MDA-MB-231 tumors. Results In this study, we identified aloperine, a natural quinolizidine alkaloid, as a multimodal inhibitor of TNBC progression. Aloperine preferentially suppressed endothelial angiogenesis as well as TNBC cell migration and VM formation at concentrations with minimal effects on tumor cell proliferation. Mechanistically, aloperine directly bound to the ATP-binding pocket of fibroblast growth factor receptor 1 (FGFR1), thereby inhibiting its kinase activity and downstream Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling in both endothelial and tumor cells. In vivo , aloperine effectively suppressed tumor angiogenesis, VM, and metastasis in both murine and human TNBC models. Conclusions These findings demonstrate that aloperine disrupts the dual vascular supply and metastatic progression of TNBC by selectively targeting the FGFR1/JAK2/STAT3 signaling axis, positioning aloperine as a promising therapeutic candidate and FGFR1 as a compelling target for TNBC treatment. aloperine angiogenesis vasculogenic mimicry metastasis FGFR1 JAK2 STAT3 triple-negative breast cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Triple-negative breast cancer (TNBC), characterized by the lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, represents the most aggressive and lethal subtype of breast cancer [ 1 , 2 ]. Due to its high heterogeneity and the absence of actionable molecular targets, systemic chemotherapy remains the cornerstone of treatment [ 1 ]. Despite initial treatment responses, TNBC is frequently associated with early recurrence and a high risk of metastasis, leading to poor clinical outcomes following failure of first-line therapies [ 2 ]. These challenges highlight an urgent clinical need to identify novel molecular drivers of TNBC progression and to develop targeted therapeutic strategies to improve patient outcomes. A hallmark of TNBC malignancy is its robust and aberrant vascularization [ 3 ]. Angiogenesis, the formation of new blood vessels from pre-existing ones, constitutes the primary mechanism of tumor vascularization [ 4 ]. During this process, endothelial cells (ECs) lining the blood vessels are activated by pro-angiogenic factors in the tumor microenvironment to migrate, proliferate, form tubular structures, and organize into functional microvessels [ 5 ]. Beyond classical angiogenesis, TNBC frequently exhibits vasculogenic mimicry (VM), a process whereby aggressive tumor cells acquire EC-like properties and form vessel-like channels independent of ECs [ 6 ]. This dual-vascular landscape not only supports rapid primary tumor growth but also provides direct routes for hematogenous dissemination, thereby promoting tumor metastasis. Of note, the anti-angiogenic drug sunitinib has been reported to paradoxically enhance TNBC metastasis by promoting VM formation, which may potentially explain the limited efficacy of such type of agents in clinical trials [ 7 , 8 ]. Thus, the simultaneous inhibition of both angiogenesis and VM represents a promising therapeutic strategy to disrupt the TNBC blood supply and impede disease progression. Fibroblast growth factor receptor 1 (FGFR1) has been identified as an independent negative prognostic factor for overall survival in TNBC [ 9 ]. It belongs to a conserved family of transmembrane receptor tyrosine kinases (RTKs) comprising FGFR1-4. Upon binding of fibroblast growth factors (FGFs), FGFR1 undergoes dimerization and autophosphorylation, thereby activating multiple downstream signaling cascades, including Janus kinase (JAK)/signal transducer and activator of transcription (STAT), rat sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) [ 10 , 11 ]. These pathways are fundamental to tumor cell survival, proliferation, migration, and differentiation [ 11 ]. Beyond its role in tumor cells, FGFR1 is highly expressed in ECs, where it plays a central role in the regulation of angiogenesis [ 12 , 13 ]. In contrast, its specific contribution to VM remains largely unknown. Given its dual relevance in the tumor parenchyma and the vascular compartment, FGFR1 represents an attractive and potentially unifying therapeutic target for TNBC. In the pursuit of novel therapeutics, phytochemicals offer a valuable reservoir of bioactive compounds characterized by extensive structural diversity, broad availability, cost-effectiveness, and generally favorable safety profiles [ 14 , 15 ]. Indeed, approximately 50% of all anticancer drugs currently on the market are derived from or inspired by natural compounds [ 15 ]. Aloperine, a quinolizidine alkaloid derived from the medicinal plant Sophora alopecuroides L ., has attracted growing interest due to its diverse pharmacological properties, including anti-inflammatory, antioxidant, antibacterial, and antitumor activities [ 16 , 17 ]. While accumulating evidence indicates that aloperine suppresses tumor growth across multiple cancer types [ 17 ], its therapeutic potential and underlying mechanism of action in TNBC remain largely unexplored. In the present study, we systematically investigated the antitumor activity of aloperine in TNBC with a particular focus on both endothelial and tumor cells. We first compared the effects of aloperine on the viability of several types of human primary ECs and TNBC cell lines. At sub-cytotoxic concentrations, we then examined its impact on key angiogenic functions of human umbilical vein endothelial cells (HUVECs) and subsequently validated its anti-angiogenic effects in ex vivo aortic ring assays and in vivo Matrigel plug models. In parallel, we investigated the effects of aloperine on TNBC cell proliferation, migration, tube formation, and spheroid sprouting. We further elucidated the precise molecular mechanisms of aloperine action. Finally, the effects of aloperine on tumor angiogenesis, VM, growth, and metastasis in TNBC were evaluated using a mouse dorsal skinfold chamber model and an orthotopic xenograft model. 2. Materials and methods 2.1. Study design The sample size for each experiment was determined based on previous publications. For in vitro assays, at least 3 independent experiments were performed, each comprising a minimum of 3 biological replicates (i.e., independent cell cultures). For mouse experiments, each group included 6–8 animals. Randomization was performed for group allocation in both the dorsal skinfold chamber model and the orthotopic xenograft model. Investigators were blinded to group assignments during data analysis. No samples or animals were excluded from the analysis. The exact n values for each experiment are provided in the corresponding figure legends. 2.2. Chemicals Aloperine, the selective JAK1/2 inhibitor ruxolitinib, the selective JAK3 inhibitor ritlecitinib, the multi-kinase inhibitor lenvatinib (primarily targeting vascular endothelial growth factor receptor 2 (VEGFR2)), and basic fibroblast growth factor (bFGF) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). The selective FGFR inhibitor PD173074 was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Dimethyl sulfoxide (DMSO) was purchased from PanReac AppliChem (Darmstadt, Germany). 2.3. Cell culture HUVECs and human dermal microvascular endothelial cells (HDMECs) were purchased from PromoCell (Heidelberg, Germany) and cultured in Endothelial Cell Basal Medium (EBM; PromoCell) and EBM-MV, respectively, both supplemented with SupplementMix. The luciferase-expressing murine TNBC cell line 4T1-Luc2 (RRID: CVCL_A4BM) and the human TNBC cell line HCC1937 (RRID: CVCL_0290) were purchased from ATCC (Wesel, Germany) and cultured in RPMI 1640 medium (PAN-Biotech GmbH, Aidenbach, Germany) containing 10% fetal calf serum (FCS; PAN-Biotech), 100 U/mL penicillin (PAN-Biotech), and 0.1 mg/mL streptomycin (PAN-Biotech). The luciferase- and green fluorescent protein (GFP)-expressing human TNBC cell line MDA-MB-231-Luc-GFP (RRID: CVCL_C9CE) was purchased from GeneCopoeia (Heidelberg, Germany) and cultured in Dulbecco's modified Eagle's medium (DMEM; PAN-Biotech) containing 10% FCS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. All cell lines were cultured at 37°C in a humidified incubator with 5% CO 2 . 2.4. Cell transfection To knock down suppressor of cytokine signaling 1 (SOCS1) and SOCS3, HUVECs were transfected with 100 nM small interfering RNAs (siRNAs) targeting SOCS1 (si-SOCS1; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) or SOCS3 (si-SOCS3; Sigma-Aldrich) using HiPerFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Negative Control siRNA (si-NC; Qiagen) was used as a control. After 48 h of transfection, cells were trypsinized, counted, and equal cell numbers from each group were used for subsequent assays. 2.5. Lactate dehydrogenase (LDH) assay The cytotoxicity of aloperine was assessed using an LDH assay according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany). For this purpose, HUVECs (4 × 10³ cells/well) were seeded into 96-well plates and then exposed to a serial dilution of aloperine for 24 h. Following treatment, 100 µL of reaction solution containing the catalyst and dye was added to each well. After incubation of the plate at room temperature for 10 min, the reaction was terminated by adding 50 µL of stop solution. Absorbance was measured at 492 nm, with 620 nm as the reference wavelength, using a microplate photometer (PHOmo; anthos Mikrosysteme GmbH, Krefeld, Germany). Cytotoxicity was calculated using the formula: Cytotoxicity (%) = (OD sample -OD 0µM ) / (OD high control -OD 0µM ) × 100. The high control, representing total cell death, was established by treating cells with 5 µL of lysis solution. 2.6. Bromodeoxyuridine (BrdU) incorporation assay Cell proliferation was assessed using a BrdU incorporation assay. Briefly, HUVECs (2.5 × 10 5 cells/well) or MDA-MB-231-Luc-GFP cells (1.5 × 10 5 cells/well) were seeded in 6-well plates and then treated with various concentrations of aloperine for 6 h. BrdU reagent was then added to each well at a final concentration of 10 µM, and incubation was continued for an additional 18 h. Cells were then fixed in 70% ethanol on ice for 30 min, followed by denaturation in 2 M hydrochloric acid containing 0.5% Triton X-100 at room temperature for 30 min. Afterwards, the cells were stained with a fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody (1:30; 11-5071-42; RRID: AB_11042627; Thermo Fisher Scientific, Karlsruhe, Germany) at room temperature for 1 h. The percentage of FITC + proliferating cells was quantified by flow cytometry using a FACSLyric flow cytometer (BD Biosciences, Heidelberg, Germany). 2.7. Transwell migration assay Cell migratory capacity was assessed in a Transwell migration assay using inserts with an 8-µm pore size (Corning, Merck KGaA, Darmstadt, Germany). Prior to the assay, HUVECs or MDA-MB-231-Luc-GFP cells were treated with various concentrations of aloperine for 18 h. Afterwards, HUVECs (5 × 10 4 cells) or MDA-MB-231-Luc-GFP cells (3.5 × 10 4 cells) suspended in 500 µL of serum-free EBM or DMEM were seeded into the upper inserts, while 750 µL of EBM or DMEM supplemented with 1% FCS was added to the lower inserts as a chemoattractant. After 5 h of incubation, non-migrated cells on the upper surface of the inserts were carefully removed using a cotton swab. Migrated cells were then stained with Diff-Quick (LT-SYS Diagnostika, Berlin, Germany). Subsequently, membranes were cut out from the inserts using a surgical scalpel and mounted on glass slides with glycerol gelatin (Sigma-Aldrich). At least 20 non-overlapping fields of each membrane were randomly imaged at a 200-fold magnification using a phase-contrast microscope (BZ-X810; Keyence, Osaka, Japan). The number of migrated cells in each field was quantified using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA). 2.8. Tube formation assay The effect of aloperine on the formation of capillary-like structures was assessed using a tube formation assay. For this purpose, Matrigel® Basement Membrane Matrix (Corning) was thawed overnight at 4°C. Using pre-cooled pipette tips, 50 µL of Matrigel was dispensed into each well of a 96-well plate and allowed to polymerize at 37°C for 15 min. Then, HUVECs (1.7 × 10 4 cells) or MDA-MB-231-Luc-GFP cells (6 × 10 4 cells) suspended in 100 µL of EGM or DMEM containing various concentrations of aloperine were seeded onto the polymerized gel. After 18 h of incubation, images of capillary-like networks were captured at a 40-fold magnification using a phase-contrast microscope (BZ-X810). The number of tube meshes was quantified using ImageJ software with the Angiogenesis Analyzer plugin. 2.9. Spheroid sprouting assay The effect of aloperine on the outgrowth of cells from spheroids into the surrounding matrix was assessed using a spheroid sprouting assay. Briefly, 500 HUVECs or MDA-MB-231-Luc-GFP cells in 50 µL of EGM containing 0.24% methylcellulose (Thermo Fisher Scientific) were seeded into each well of non-adherent 96-well round-bottom plates (Greiner Bio-One, Frickenhausen, Germany) and incubated overnight to generate spheroids. Subsequently, spheroids were collected and resuspended in 300 µL of a collagen solution, diluted 1:1 with EBM containing 20% FCS and 0.5% methylcellulose. The collagen solution was prepared with rat acidic collagen extract (4 mg/mL; Advanced Biomatrix, Carlsbad, USA), H 2 O, 10 × Medium 199 (Sigma-Aldrich), and 0.2 M sodium hydroxide at a 4:4:1:1 ratio. The spheroid mixture was then rapidly transferred into a pre-warmed 24-well plate and allowed to polymerize at 37°C for 45 min, after which 500 µL of EGM containing various concentrations of aloperine was gently overlaid onto the gel. After 24 h of incubation, images of spheroids were captured at a 40-fold magnification using a phase-contrast microscope (BZ-X810), and the cumulative sprout length was measured using ImageJ software. 2.10. Aortic ring assay The effect of aloperine on aortic sprouting was assessed using an aortic ring assay. Briefly, thoracic aortas from 8-week-old male BALB/c mice (RRID: IMSR_RJ: BALB-CANNRJ; Janvier-Labs, Le Genest, France) were cut into rings about 1 mm in length. In parallel, 96-well plates were pre-coated with 40 µL of Matrigel (Corning) and incubated at 37°C to allow polymerization. The aortic rings were then placed onto the pre-coated Matrigel and overlaid with an additional 40 µL of Matrigel. After Matrigel solidification, the rings were cultured in 100 µL of DMEM supplemented with 10% FCS and various concentrations of aloperine. After 6 days of culture, with a medium change on day 3, images of aortic rings were captured using a phase-contrast microscope (BZ-X810). The area of vascular sprouting was measured using the corresponding image analysis software (Keyence). 2.11. Western blotting Cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Cell lysates were then centrifuged at 12,000 × g for 30 min at 4°C and the supernatants were collected. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal amounts of protein (10 µg) were separated on 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (BioRad, Munich, Germany). The membranes were further blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich) at room temperature for 1 h and incubated overnight at 4°C with the following primary antibodies: a rabbit anti-phosphorylated-mammalian target of rapamycin (p-mTOR) antibody (1:300; 5536; RRID: AB_10691552; Cell Signaling Technology, Frankfurt, Germany), a rabbit anti-mTOR antibody (1:300; 2983; RRID: AB_2105622; Cell Signaling Technology), a rabbit anti-p-STAT5 antibody (1:300; 9351; RRID: AB_2315225; Cell Signaling Technology), a rabbit anti-STAT5 antibody (1:300; 25656; RRID: AB_2798908; Cell Signaling Technology), a rabbit anti-p-STAT3 antibody (1:100; 9145; RRID: AB_2491009; Cell Signaling Technology), a mouse anti-STAT3 antibody (1:300; 9139; RRID: AB_331757; Cell Signaling Technology), a rabbit anti-p-AKT antibody (1:300; 4060; RRID: AB_2315049; Cell Signaling Technology), a rabbit anti-AKT antibody (1:300; 4685; RRID: AB_2225340; Cell Signaling Technology), a rabbit anti-p-ERK antibody (1:300; 4370; RRID: AB_2315112; Cell Signaling Technology), a rabbit anti-ERK antibody (1:300; 4695; RRID: AB_390779; Cell Signaling Technology), a rabbit anti-p-FAK antibody (1:250; 8556; RRID: AB_10891442; Cell Signaling Technology), a rabbit anti-FAK antibody (1:250; 3285; RRID: AB_2269034; Cell Signaling Technology), a rabbit anti-p-JAK2 antibody (1:100; 3776; RRID: AB_2617123; Cell Signaling Technology), a rabbit anti-JAK2 antibody (1:200; 3230; RRID: AB_2128522; Cell Signaling Technology), a rabbit anti-p-Src antibody (1:300; 6943; RRID: AB_10013641; Cell Signaling Technology), a rabbit anti-Src antibody (1:300; 2109; RRID: AB_2106059; Cell Signaling Technology), a rabbit anti-SOCS1 antibody (1:100; ab280886; RRID: AB_2938872; Abcam, Cambridge, UK), a rabbit anti-SOCS3 antibody (1:100; 14025-1-AP; RRID: AB_10597854; Proteintech, Munich, Germany), a rabbit anti-p-FGFR1 antibody with human species reactivity (1:100; ab173305; RRID: AB_3094883; Abcam), a rabbit anti-p-FGFR1 antibody with mouse species reactivity (1:100; AP1317; Abclonal, Düsseldorf, Germany), a rabbit anti-FGFR1 antibody (1:100; 9740; RRID: AB_11178519; Cell Signaling Technology), a rabbit monoclonal anti-p-VEGFR2 antibody (1:250; 2478; RRID: AB_331377; Cell Signaling Technology), a rabbit monoclonal anti-VEGFR2 antibody (1:250; 9698; RRID: AB_11178792; Cell Signaling Technology), and a mouse horseradish peroxidase (HRP)-conjugated anti-β-actin antibody (1:1000; HRP-66009; RRID: AB_2883836; Proteintech). Subsequently, membranes were washed 3 times with Tris-buffered saline containing 0.1% Tween 20, and then incubated with HRP-conjugated anti-mouse (1:1000; HAF007; RRID: AB_357234; R&D Systems, Wiesbaden, Germany) or anti-rabbit (1:1000; HAF008; RRID: AB_357235; R&D Systems) secondary antibodies at room temperature for 1 h. Chemical signals were visualized using the enhanced chemiluminescence kit (BioRad) and images were acquired using a ChemoCam Imager (Intas, Göttingen, Germany). Protein expression was quantified using ImageJ software. 2.12. Water-soluble tetrazolium (WST)-1 assay The effect of aloperine on cell viability was assessed using a WST-1 assay. For this purpose, cells of different types were seeded into 96-well plates at a density of 2–3 × 10³ cells/well and then exposed to a serial dilution of aloperine for 24 h or 48 h. After treatment, 10 µL of WST-1 reagent (Roche Diagnostics) was added to each well, followed by incubation at 37°C for 30 min. Then, the absorbance of each sample was measured at 450 nm with 620 nm as reference using a microplate photometer (PHOmo). 2.13. Blind molecular docking The human FGFR1 structure (PDB ID: 5A46) was obtained from the Protein Data Bank ( https://www.rcsb.org ) and processed in UCSF ChimeraX (v1.8) by removing crystallographic waters and non-protein heteroatoms. The receptor was prepared in AutoDockTools (v1.5.7) by adding polar hydrogens and assigning Gasteiger charges, and then exported as a PDBQT file. The three-dimensional (3D) structure of aloperine was downloaded from PubChem ( https://pubchem.ncbi.nlm.nih.gov ), optimized in Avogadro 2 (v1.99.0), and converted to PDBQT format in AutoDockTools after defining rotatable bonds. Blind docking was carried out in AutoDock 4.2 using the Lamarckian genetic algorithm with a grid covering the entire FGFR1 structure (spacing: 0.5 Å). A total of 100 independent docking runs were performed with standard settings (population size: 150; energy evaluations: 2,500,000). Resulting poses were clustered based on root-mean square deviation, and the lowest-energy pose was selected for subsequent analyses. Binding free energies (ΔG, kcal/mol) were obtained from AutoDock 4.2, with protein-ligand interactions analyzed in BIOVIA Discovery Studio Visualizer 2025 and visualized in UCSF ChimeraX. 2.14. Cell-free kinase assay The effects of aloperine on the activity of multiple kinases were evaluated in a cell-free kinase assay, as previously described [ 18 ], with minor modifications. All assays were performed in white 96-well flat-bottom plates using the ADP-Glo™ Kinase Assay and the Kinase Enzyme System (Promega, Walldorf, Germany). Each 20 µL reaction mixture contained recombinant kinase enzymes (40 ng JAK2, 3 ng JAK3, 8 ng VEGFR2, or 3 ng FGFR1), 0.2 µg/µL poly (4:1 Glu, Tyr) peptide substrate, 100 µM aloperine, specific kinase inhibitors (3 nM ruxolitinib, 40 nM ritlecitinib, 1 nM lenvatinib, or 10 nM PD173074), and ATP at a final concentration of 10 µM. For ATP competition experiments, the reaction mixture contained 3 ng FGFR1, 0.2 µg/µL peptide substrate, 100 µM aloperine or 10 nM PD173074, 10 or 500 µM ATP, respectively. After incubation at room temperature for 60 min, 20 µL of ADP-Glo™ Reagent was added to each well, followed by a 40-min incubation at room temperature. Subsequently, 40 µL of Kinase Detection Reagent was added to each well, followed by another 40-min incubation at room temperature. Kinase activity was then quantified using a luciferase/luciferin reaction on a Tecan Infinite M200 PRO luminometer (Tecan, Crailsheim, Germany), expressed as a percentage relative to the control. 2.15. Animal experiments The mice were housed in a conventional animal facility (Institute for Clinical and Experimental Surgery, Saarland University, Homburg, Germany) under a standard 12-h light/dark cycle, with standard pellet chow (ssniff Spezialdiäten GmbH, Soest, Germany) and water provided ad libitum. The in vivo effects of aloperine on angiogenesis were evaluated using a Matrigel plug assay following an established protocol [ 19 ]. Briefly, a mixture containing 250 µL growth factor-reduced Matrigel (Corning), 1 µg/mL vascular endothelial growth factor (VEGF; R&D Systems), 1 µg/mL bFGF (R&D Systems), 50 IU/mL heparin (B. Braun, Melsungen, Germany), 0.1% DMSO (vehicle) or 100 µM aloperine was injected subcutaneously into the flanks of 3-month-old male BALB/c mice (25–30 g; Janvier-Labs; n = 6 per group) under inhalation anesthesia with isoflurane (5% for induction and 2% for maintenance). Matrigel plugs were removed after 7 days for immunohistochemical analyses. The effect of aloperine on the vascularization and growth of murine TNBC was assessed using a dorsal skinfold chamber model as previously described [ 20 ]. First, tumor spheroids were generated by seeding 4T1-Luc2 (5 × 10 4 cells/well) into 96-well plates pre-coated with 1% agarose and cultured for 3 days. One day after cell seeding, dorsal skinfold chambers were implanted in 3-month-old female BALB/c mice (22–25 g; Janvier-Labs). After another 2 days, one 4T1-Luc2 spheroid stained with Hoechst 33342 was transplanted into each chamber. All the mice were randomly assigned to two groups (n = 8 per group) and received daily intraperitoneal injections of 75 mg/kg body weight aloperine or a vehicle solution (5% DMSO, 5% Tween 80, and 90% saline) for 14 consecutive days. Intravital fluorescence microscopy was performed on days 0, 3, 6, 10, and 14 after spheroid implantation, using a charge-coupled device video camera (FK6990; Pieper, Schwerte, Germany) and a DVD system to record the microscopy images. The recordings were then analyzed using CapImage (Zeintl, Heidelberg, Germany) to quantify tumor size (mm²), functional microvessel density (cm/cm²), vessel diameter (D; µm), and the vessel centerline red blood cell (RBC) velocity (V; mm/s). Furthermore, the volumetric blood flow (Qv; pL/s) of the tumor vessels was calculated using the formula: Qv = π × (D/2) 2 × V/1.3. After the last microscopy on day 14, the mice were euthanized by cervical dislocation and the tumor tissues were carefully excised for further histological and immunohistochemical analyses. Chamber implantation, spheroid transplantation, and intravital microscopy were performed under anesthesia induced by intraperitoneal injection of ketamine (Ketabel®; 90 mg/kg body weight; bela-pharm GmbH, Vechta, Germany) and xylazine (Rompun®; 12 mg/kg body weight; Bayer, Leverkusen, Germany). For post-operative analgesia following chamber implantation, the mice received a subcutaneous injection of carprofen (Rimadyl®; 10 mg/kg body weight; Cp-Pharma, Burgdorf, Germany). The effect of aloperine on the vascularization, growth, and metastasis of human TNBC was assessed using an orthotopic xenograft model. For this purpose, 5 × 10 6 MDA-MB-231-Luc-GFP cells suspended in 50 µL PBS were injected into the left fourth mammary fat pad of 6-week-old female NOD-SCID mice (22–25 g; RRID: IMSR_RJ: NOD-SCID; Janvier-Labs). When the tumor became palpable on day 3, all the mice were randomly divided into two groups (n = 8 per group) and received daily intraperitoneal injections of 50 mg/kg body weight aloperine or a vehicle solution (5% DMSO, 5% Tween 80, and 90% saline) until 6 weeks after tumor inoculation. During this period, caliper measurements were performed weekly to monitor the tumor volume using the formula: V = 0.5 × length × width 2 . Tumor growth was also monitored weekly by bioluminescence imaging (BLI) using an IVIS Spectrum imaging system (PerkinElmer, MA, USA). For this imaging, mice were injected intraperitoneally with 150 mg/kg body weight D-luciferin (122799; PerkinElmer) and then anesthetized with isoflurane (5% for induction and 2% for maintenance). Bioluminescent images were captured 17 min after D-luciferin injection and analyzed with the Living Image Software (PerkinElmer) to quantify the total flux within regions corresponding to the primary tumor and lung metastases. Of note, for metastasis evaluation, the primary tumor was covered with a black cloth during imaging to minimize signal interference. On day 42 after tumor inoculation, lungs were collected and incubated with 300 µg/mL D-luciferin in PBS for 1 min before assessing distant metastasis using the IVIS system. Additionally, tumor tissues were harvested, weighed, photographed, and processed for further immunohistochemical and Western blot analyses. 2.16. Circulating tumor cell (CTC) detection Blood samples (1 mL) were collected from the abdominal aorta of mice under anesthesia with ketamine and xylazine and then incubated with 1 mL of BD Pharm Lyse™ (BD Biosciences) at room temperature to lyse RBCs. The remaining cells were then washed twice with cold PBS and resuspended in PBS containing 2% FCS. The number of CTCs (MDA-MB-231-Luc-GFP cells) per 10,000 events in blood samples was quantified by flow cytometry based on GFP fluorescence using a FACSLyric flow cytometer (BD Biosciences). 2.17. Histology and immunohistochemistry The collected Matrigel plugs, tumor samples, and lung tissues were fixed in 4% formalin, dehydrated in ethanol, and embedded in paraffin. Subsequently, 3-µm-thick sections were serially cut and mounted onto slides for histological and immunohistochemical analyses. To visualize microvessels in Matrigel plugs, sections were incubated overnight at 4℃ with a rabbit anti-mouse CD31 antibody (1:100; ab182981; RRID: AB_2920881; Abcam). Then, they were incubated with a goat anti-rabbit Alexa Fluor 555-conjugated secondary antibody (1:100; A27039; RRID: AB_2536100; Thermo Fisher Scientific) and counterstained with Hoechst 33342. The entire area of each plug was imaged at a 400-fold magnification using a BX-60 microscope (Olympus, Tokyo, Japan). CD31 + microvessels in each field were quantified using ImageJ software. To assess tumor size and necrotic area, tissue sections with the largest tumor area in the vertical cross-section were selected and stained with hematoxylin and eosin (HE). The entire tumor was imaged at a 20-fold magnification using a phase-contrast microscope (BZ-X810) and analyzed using the corresponding image analysis software (Keyence). To evaluate tumor cell proliferation and apoptosis, tissue sections were sequentially incubated with either a rabbit anti-mouse Ki67 antibody (1:400; 12202; RRID: AB_2620142; Cell Signaling Technology) or a rabbit anti-mouse cleaved caspase-3 antibody (1:100; 9661; RRID: AB_2341188; Cell Signaling Technology), followed by a biotinylated goat anti-rabbit secondary antibody (1:100; ab64256; RRID: AB_2661852; Abcam), peroxidase-conjugated streptavidin (ready-to-use; Abcam), and 3‐amino‐9‐ethylcarbazole substrate (Abcam). At last, sections were counterstained with Mayer’s hemalum solution (Merck KGaA). The entire tumor was imaged at a 400-fold magnification using a BX‐60 microscope (Olympus). The percentage of Ki67 + proliferating and cleaved caspase-3 + apoptotic tumor cells was quantified using ImageJ software. To detect angiogenesis and VM in tumor tissues, CD31 and periodic acid-Schiff (PAS) double staining was conducted. Briefly, sections were incubated with a rabbit anti-mouse CD31 antibody (1:100; ab182981; RRID: AB_2920881; Abcam), followed by a biotinylated goat anti-rabbit secondary antibody (1:100; ab64256; RRID: AB_2661852; Abcam), peroxidase-conjugated streptavidin (Abcam), and 3-amino-9-ethylcarbazole substrate (Abcam). Afterwards, they were exposed to periodic acid and Schiff reagent (Sigma-Aldrich) and counterstained with Mayer’s hemalum solution (Merck KGaA). The entire tumor was imaged at a 400-fold magnification using a BX-60 microscope (Olympus). The density of CD31 + PAS + EC-lined vessels and CD31 − PAS + VM structures was quantified using ImageJ software. To detect metastatic foci in the lungs, lung sections were stained with a goat anti-GFP antibody (1:100; 600-101-215; RRID: AB_218200; Rockland Immunochemicals, PA, USA) and counterstained with Hoechst 33342. Metastatic foci derived from MDA-MB-231-Luc-GFP cells were identified based on their GFP signal using fluorescence microscopy. Multiple random fields from each lung section were imaged at a 400-fold magnification using a BX-60 microscope (Olympus). The number of GFP + metastatic foci in each field was quantified using ImageJ software. 2.18. Statistics Statistical evaluations were performed using GraphPad Prism (v10.4.1). Data normality and homogeneity of variance were assessed using the Shapiro-Wilk and Brown-Forsythe tests, respectively. For comparisons between two independent groups, a two-tailed unpaired Student’s t-test was employed for normally distributed data, while the Mann-Whitney U test was used for non-parametric datasets (i.e. Western blot data). For multi-group comparisons, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied to parametric data. Non-parametric multi-group data, including flow cytometry and Western blot results, were analyzed via the Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Data were presented as mean ± standard error of the mean (SEM), and statistical significance was defined as P < 0.05 (* P < 0.05; ** P < 0.01; *** P < 0.001). 3. Results 3.1. Aloperine preferentially reduces EC viability To determine the biological activity of aloperine (Fig. 1 A), we first evaluated its effects on the viability of human primary ECs (HUVECs and HDMECs) and human TNBC cell lines (MDA-MB-231 and HCC1937). WST-1 assays demonstrated that aloperine (10–400 µM) induces a dose-dependent reduction in cell viability across all tested cell types. Notably, ECs exhibited significantly higher sensitivity to aloperine than TNBC cell lines (Fig. 1 B), suggesting a preferential susceptibility of the vascular endothelium to aloperine-mediated growth inhibition relative to the malignant compartment. 3.2. Aloperine inhibits the angiogenic activity of ECs in vitro, ex vivo, and in vivo To identify sub-cytotoxic concentrations of aloperine for functional studies, LDH assays were performed in HUVECs. Our data showed that aloperine at concentrations up to 400 µM induces no detectable cytotoxicity after 24 h of treatment (Fig. 1 C). Based on this finding, dosages of 25, 50, and 100 µM were selected for subsequent angiogenesis assays to ensure that the observed effects are independent from cell death. BrdU incorporation assays revealed that aloperine significantly reduces the proliferation of HUVECs at a concentration of 100 µM (Fig. 1 D). Moreover, treatment with 100 µM aloperine resulted in a 48% reduction in HUVEC migration (Fig. 1 E, F). Beyond these individual cellular responses, aloperine effectively suppressed complex angiogenic processes, including capillary-like tube formation (Fig. 1 G, H) and 3D spheroid sprouting in a dose-dependent manner (Fig. 1 I, J). To validate these findings in more physiologically relevant settings, we employed an ex vivo aortic ring assay. Aloperine dose-dependently suppressed the outgrowth of sprouts from aortic rings, with 100 µM achieving complete suppression (Fig. 1 K, L). We then assessed the in vivo anti-angiogenic potential of aloperine using a Matrigel plug assay. Consistent with our in vitro and ex vivo results, plugs containing 100 µM aloperine exhibited a markedly reduced CD31⁺ microvessel density compared to vehicle controls (Fig. 1 M, N). Collectively, these findings establish aloperine as a robust and multi-stage inhibitor of angiogenesis across multiple experimental platforms spanning in vitro , ex vivo , and in vivo systems. 3.3. Aloperine suppresses EC angiogenesis via selective downregulation of the JAK2/STAT3 pathway To elucidate the molecular mechanisms underlying the anti-angiogenic effects of aloperine, we examined several key angiogenesis-associated signaling pathways, including the PI3K/AKT/mTOR, JAK/STAT, and Ras/Raf/MEK/ERK cascades [ 21 ]. Western blot analyses revealed that treatment of HUVECs with 100 µM aloperine for 4 h markedly reduces the phosphorylation of STAT3 and AKT (Fig. 2 A-F). In contrast, phosphorylation of mTOR, STAT5, and ERK remained largely unaffected (Fig. 2 A-F), suggesting that STAT3 and AKT are the primary signaling nodes modulated by aloperine. Given that JAK2 is a well-established upstream kinase responsible for STAT3 recruitment and activation [ 22 ], while FAK and Src are critical upstream regulators of AKT phosphorylation [ 23 ], we next investigated which of these mediators are responsible for the aloperine-induced reduction in STAT3 and AKT phosphorylation. Western blot analyses demonstrated that aloperine selectively suppresses the phosphorylation of JAK2, whereas the activation status of FAK and Src remained unchanged (Fig. 2 G-J). These findings suggest that aloperine primarily targets the JAK2/STAT3 axis, and that the observed attenuation of AKT phosphorylation may occur secondary to JAK2/STAT3 inhibition rather than through direct interference with FAK or Src. To determine whether downregulation of JAK2/STAT3 signaling is functionally required for the anti-angiogenic effects of aloperine, we employed a genetic rescue strategy using siRNAs against SOCS1 and SOCS3, endogenous negative regulators of the JAK/STAT pathway [ 24 ]. Transfection of HUVECs with si-SOCS1 or si- SOCS3 efficiently reduced the expression of the respective proteins, resulting in a marked increase in the phosphorylation of JAK2, STAT3, and AKT (Fig. 2 K-O). This indicates that AKT functions downstream of the JAK2/STAT3 axis in this context. Importantly, knockdown of either SOCS1 or SOCS3 significantly rescued aloperine-induced suppression of HUVEC spheroid sprouting (Fig. 2 P, Q). These findings indicate that selective inhibition of the JAK2/STAT3 signaling axis contributes at least partially to the anti-angiogenic activity of aloperine. 3.4. Aloperine suppresses JAK2/STAT3 signaling by targeting FGFR1 In ECs, the JAK/STAT signaling pathway is predominantly regulated by RTKs, among which VEGFR2 and FGFR1 play central roles in both physiological and pathological angiogenesis [ 21 ]. We therefore investigated whether aloperine suppresses the JAK2/STAT3 axis by targeting either of these receptors. Western blot analyses revealed that the basal phosphorylation level of VEGFR2 in HUVECs is barely detectable, precluding reliable assessment of baseline inhibition. While VEGF stimulation robustly induced VEGFR2 phosphorylation, aloperine did not affect this ligand-dependent activation (Supplementary Fig. 1). In contrast, aloperine significantly reduced the basal phosphorylation of FGFR1 by approximately 51% (Fig. 3 A and B). Notably, this inhibitory effect was attenuated in the presence of a high concentration of exogenous bFGF, suggesting a competitive or ligand-sensitive mode of action. Moreover, bFGF stimulation completely abolished aloperine-induced reductions in the phosphorylation of JAK2, STAT3, and AKT (Fig. 3 A-E). These results suggest that FGFR1 is the primary upstream target of aloperine in HUVECs, mediating the downregulation of the JAK2/STAT3 signaling cascade and AKT phosphorylation. Functional assays further corroborated these signaling data, as bFGF completely rescued aloperine-suppressed HUVEC spheroid sprouting (Fig. 3 F, G), confirming that FGFR1 is the primary functional target of aloperine in mediating its anti-angiogenic effects. To determine whether aloperine directly interacts with FGFR1, we next performed molecular docking simulations to explore its potential binding mode. Blind docking analyses revealed that aloperine preferentially binds to the ATP-binding pocket of FGFR1 (Fig. 3 H). This interaction was stabilized by a key hydrogen bond with the ARG646 residue located in the activation loop (A-loop), a critical regulatory element governing kinase catalytic activity and substrate accessibility [ 25 ]. The predicted binding free energy was − 7.2 kcal/mol, indicative of a favorable and stable interaction between aloperine and the FGFR1 ATP-binding site. To experimentally validate these in silico predictions, we conducted ATP competition assays in HUVECs. Cells were pre-treated with 1 mM ATP, a concentration previously demonstrated to increase intracellular ATP levels by approximately 112% [ 18 ], prior to aloperine exposure. Western blot analysis revealed that elevation of intracellular ATP completely abolishes the inhibitory effects of aloperine on both FGFR1 and JAK2 phosphorylation (Fig. 3 I-K), indicating that aloperine targets FGFR1 through competitive occupancy of its ATP-binding site. Consistent with these cellular findings, cell-free kinase assays demonstrated that aloperine selectively inhibits FGFR1 kinase activity without affecting JAK2, JAK3, or VEGFR2 (Fig. 3 L). Importantly, the inhibitory effect of aloperine against FGFR1 was completely reversed by the addition of 500 µM ATP, as shown by ATP competition experiments using the FGFR1 inhibitor PD173074 as a positive control (Fig. 3 M). Collectively, these results provide strong evidence that aloperine interacts directly with the ATP-binding site of FGFR1, thereby suppressing the downstream JAK2/STAT3 signaling pathway and inhibiting EC angiogenic activity. 3.5. Aloperine suppreses TNBC cell migration and VM by inhibiting the FGFR1/JAK2/STAT3 signaling pathway We then investigated the impact of aloperine on the malignant phenotypes of TNBC cells. BrdU incorporation assays revealed that aloperine at 25, 50, and 100 µM exerts no significant effect on the proliferation of MDA-MB-231 cells (Fig. 4 A), consistent with the viability results shown in Fig. 1 B. This contrasted with the significant anti-proliferative effect observed in HUVECs (Fig. 1 D), suggesting a cell type-specific sensitivity to aloperine. Notably, despite the absence of anti-proliferative effects, aloperine at these non-cytotoxic concentrations significantly impaired the migratory capacity of MDA-MB-231 cells (Fig. 4 B, C). Moreover, aloperine potently inhibited VM across multiple TNBC cell lines, including MDA-MB-231, HCC1937, and 4T1 cells, as assessed by tube formation assays (Fig. 4 D, E and Supplementary Fig. 2), a widely used and well-established in vitro method for evaluating tumor cell vascular activity [ 26 ]. Consistently, 3D spheroid sprouting assays demonstrated that aloperine significantly suppresses the invasive outgrowth of sprouts from MDA-MB-231 spheroids (Fig. 4 F, G). Collectively, these findings indicate that aloperine suppresses TNBC cell migration and VM formation independently of its effects on cell proliferation. To elucidate the molecular mechanisms underlying these effects, we examined the activation status of FGFR1-dependent signaling pathways. Western blot analyses showed that aloperine markedly reduces the phosphorylation of FGFR1, JAK2, and STAT3, whereas Src and AKT phosphorylation remain largely unchanged in MDA-MB-231 cells (Fig. 4 H-M), suggesting a selective inhibition of the FGFR1/JAK2/STAT3 axis. To functionally validate the role of FGFR1 in aloperine-mediated inhibition of migration and VM, we performed a rescue experiment using the FGFR1 ligand bFGF. Stimulation with bFGF markedly restored aloperine-suppressed migration and VM formation in MDA-MB-231 cells (Supplementary Fig. 3A, B and Fig. 4 N, O), supporting the requirement of FGFR1 for the maintenance of these malignant phenotypes. Finally, to establish JAK2/STAT3 signaling as the critical downstream mediator of aloperine action, we knocked down SOCS1 using its specific siRNAs in MDA-MB-231 cells prior to aloperine treatment. Efficient SOCS1 knockdown was confirmed by Western blotting (Fig. 4 P, Q). Importantly, SOCS1 depletion significantly reversed the inhibitory effects of aloperine on both migration and VM formation (Supplementary Fig. 3C, D and Fig. 4 R, S). In summary, these findings indicate that aloperine exerts its anti-migratory and anti-VM activity in TNBC cells by selectively suppressing the FGFR1/JAK2/STAT3 signaling axis. 3.6. Aloperine suppresses TNBC vascularization and growth in a dorsal skinfold chamber model To evaluate the in vivo efficacy of aloperine, we performed a syngeneic mouse dorsal skinfold chamber model of TNBC, which enables real-time monitoring of tumor neovascularization and growth [ 27 ]. For this, 4T1 tumor spheroids were implanted into dorsal skinfold chambers of BALB/c mice, followed by daily intraperitoneal administration of aloperine (75 mg/kg body weight) or vehicle control. Intravital fluorescence microscopy was employed to track the spatio-temporal dynamics of tumor growth and vascularization over a 14-day period (Fig. 5 A). Aloperine treatment was well-tolerated, as evidenced by the absence of significant changes in body weight (Fig. 5 B) or behavioral patterns compared to the control group, suggesting a favorable safety profile at the administered dose. While control tumors exhibited rapid expansion, aloperine-treated tumors remained significantly smaller, with differences detectable as early as day 3 after spheroid transplantation (Fig. 5 C, D). Intravital fluorescence microscopy further revealed a marked reduction in the density of functional microvessels, defined by the presence of active blood flow, within the aloperine-treated tumors starting from day 3 (Fig. 5 E, F). Quantitative microhemodynamic analyses demonstrated that aloperine significantly reduces microvessel diameter and RBC velocity (Fig. 5 G, H), resulting in a marked decrease in volumetric blood flow (Fig. 5 I). These data suggest that aloperine not only reduces microvessel density but also impairs vascular perfusion, thereby disrupting the metabolic supply required for sustained tumor growth. Histological and immunohistochemical analyses of harvested tumor tissues confirmed these in vivo findings. HE stainings revealed significantly smaller tumor sizes in the aloperine-treated group compared to control (Fig. 5 J, K). Furthermore, aloperine treatment substantially decreased the proportion of Ki67⁺ proliferating cells while increasing the number of cleaved caspase-3⁺ apoptotic cells (Fig. 5 J-M). To distinguish between conventional angiogenesis and VM, we performed CD31 and PAS double stainings. Aloperine treatment induced a marked reduction in both CD31⁺/PAS⁺ EC-lined vessels and CD31⁻/PAS⁺ VM structures (Fig. 5 J, N). Collectively, these findings demonstrate that aloperine suppresses TNBC growth by concurrently disrupting angiogenesis and VM-mediated perfusion, thereby limiting tumor nutrient supply and promoting tumor cell apoptosis. 3.7. Aloperine suppresses TNBC vascularization, growth, and metastasis in an orthotopic xenograft model To evaluate the long-term therapeutic potential of aloperine against human TNBC progression, we established an orthotopic xenograft model by injecting MDA-MB-231-Luc-GFP cells into the mammary fat pads of immunodeficient NOD-SCID mice (Fig. 6 A). Daily intraperitoneal administration of aloperine (50 mg/kg body weight) was well tolerated over the 42-day study period, with no significant changes in body weight (Fig. 6 B) or behavioral patterns. Caliper measurements revealed that aloperine significantly inhibits primary tumor growth compared to vehicle-treated controls, with differences observed from day 14 after tumor inoculation (Fig. 6 C). This inhibitory effect was confirmed by ex vivo analyses at the experimental endpoint, which showed a marked reduction in both tumor volume and weight in aloperine-treated mice (Fig. 6 D, E). Consistent with the observed tumor growth kinetics, BLI showed that aloperine treatment effectively suppresses the bioluminescent signal of primary tumors from day 21 after tumor inoculation (Fig. 6 F, G). Notably, aloperine also significantly reduced spontaneous lung metastasis, as indicated by substantially lower bioluminescent signals in the thoracic region compared to controls (Fig. 6 H, I). This reduction in metastatic burden was confirmed by ex vivo BLI of harvested lungs (Fig. 6 J, K) and by immunohistochemical quantification of GFP⁺ metastatic foci, which were significantly less frequent in aloperine-treated lungs (Fig. 6 L, M). To investigate whether this anti-metastatic effect was driven by a reduced systemic dissemination, CTCs were quantified by flow cytometry. Blood from aloperine-treated mice contained a significantly lower proportion of GFP⁺ CTCs, suggesting that aloperine effectively impairs tumor cell intravasation and subsequent hematogenous dissemination (Fig. 6 N). Histological and immunohistochemical analyses of excised primary tumors provided further mechanistic insights. HE stainings revealed smaller tumors with attenuated intratumoral necrotic areas in aloperine-treated mice (Fig. 7 A-D). Furthermore, aloperine treatment significantly reduced the proportion of Ki67⁺ proliferating cells (Fig. 7 E, F), while inducing a modest, non-significant increase in cleaved caspase-3⁺ apoptotic cells (Fig. 7 G, H). Importantly, CD31 and PAS double stainings revealed a significant reduction in both CD31⁺/PAS⁺ EC-lined vessels and CD31⁻/PAS⁺ VM structures in aloperine-treated tumors (Fig. 7 I, J), confirming the dual anti-vascular activity of aloperine within the orthotopic microenvironment. Finally, we performed Western blot analyses of primary tumor tissues to confirm the molecular drivers of these observations. In agreement with our in vitro data, aloperine treatment resulted in a substantial reduction in the phosphorylation of FGFR1, JAK2, and STAT3 (Fig. 7 K, L). Taken together, these findings indicate that aloperine suppresses TNBC growth, vascularization, and metastasis through sustained inhibition of the FGFR1/JAK2/STAT3 signaling axis in vivo . 4. Discussion Aloperine is a quinolizidine-type alkaloid enriched in the seeds and leaves of Sophora alopecuroides L ., which has been utilized in traditional medicine for centuries owing to its anti-inflammatory, antioxidant, antibacterial, and anticancer properties [ 16 , 17 ]. Different from many natural products, aloperine exhibits high drug-likeness by strictly adhering to Lipinski’s Rule of Five, suggesting superior oral bioavailability and systemic distribution [ 28 ]. Accumulating pharmacological investigations have demonstrated that aloperine exerts broad-spectrum antitumor activity across multiple malignancies, including prostate, lung, thyroid, liver, and colon cancers, primarily through the induction of cell cycle arrest and apoptosis [ 16 , 17 ]. However, its therapeutic potential in TNBC and the underlying mechanisms remain unexplored. This study addresses this gap of knowledge by identifying aloperine as a multimodal inhibitor of TNBC progression. In fact, we could demonstrate that aloperine effectively suppresses angiogenesis, VM, and metastasis in TNBC through selective inhibition of the FGFR1/JAK2/STAT3 signaling axis in both endothelial and tumor compartments. A pivotal finding of this study is the cell type-specific sensitivity of aloperine, which distinguishes it from conventional chemotherapeutic agents. While aloperine demonstrates potent anti-neoplastic activity, our data revealed that ECs exhibit a significantly higher susceptibility to aloperine-induced viability inhibition compared to TNBC cells. More importantly, aloperine effectively disrupts key hallmarks of tumor progression, including EC angiogenesis as well as VM and migration of TNBC cells, at sub-cytotoxic concentrations that do not affect tumor cell proliferation. These findings suggest that aloperine acts within a favorable therapeutic window, where it can remodel the tumor microenvironment without requiring high-dose systemic exposure. Given the high vascular density and early metastatic propensity of TNBC, the ability of aloperine to suppress angiogenesis, VM, and metastasis simultaneously is of particular translational significance. By targeting these processes at sub-cytotoxic concentrations, aloperine may reduce the selective pressure that often leads to the emergence of therapeutic resistance commonly associated with aggressive proliferation-targeted therapies. Aloperine functions as a pleiotropic angiogenesis inhibitor, comprehensively suppressing fundamental angiogenic activities of HUVECs, including proliferation, migration, tube formation, and spheroid sprouting. These anti-angiogenic properties were further validated in ex vivo aortic ring assays involving mouse aortic ECs, as well as in vivo Matrigel plug assays involving mouse microvascular ECs. The reproducible suppression of angiogenesis across ECs from different species and vascular origins underscores the pharmacological robustness of aloperine. Notably, aloperine disrupted not only individual EC functions but also the coordinated morphogenetic processes required for sprouting angiogenesis, thereby preventing the assembly of functional vascular networks. Beyond conventional angiogenesis, aloperine also efficiently inhibited VM formation in multiple TNBC cell lines, as evidenced by both reduced tube formation and spheroid sprouting. VM is highly prevalent in TNBC and closely associated with tumor aggressiveness, metastatic potential, and resistance to conventional anti-angiogenic therapies [ 29 , 30 ]. In contrast to VEGF-targeted anti-angiogenic agents, which frequently promote compensatory VM and paradoxically enhance tumor metastasis [ 7 , 8 ], aloperine concurrently suppresses both EC-dependent angiogenesis and tumor cell-driven alternative neovascularization pathways. This dual targeting capacity suggests that aloperine may overcome key resistance mechanisms associated with current anti-angiogenic strategies, thereby positioning it as a promising therapeutic candidate for the treatment of TNBC. In addition to its effective anti-vascular activity, aloperine significantly attenuated the migratory potential of TNBC cells, a critical prerequisite for tumor cell dissemination and distant metastasis [ 31 ]. These findings suggest that aloperine impairs metastatic progression through a multifaceted mechanism that concurrently disrupts tumor-supportive vasculature and suppresses tumor cell-intrinsic motility. By targeting these distinct yet functionally interconnected steps of the metastatic cascade, aloperine circumvents the inherent limitations of monotherapies that solely inhibit EC angiogenesis or tumor cell migration, likely accounting for its superior anti-metastatic efficacy observed in vivo . Mechanistic investigations revealed that aloperine exerts its anti-vascular effects through selective inhibition of the FGFR1/JAK2/STAT3 signaling pathway in both ECs and TNBC cells. Molecular docking and cell-free kinase assays demonstrated that aloperine directly binds to the ATP-binding pocket of FGFR1, thereby suppressing its kinase activity. In contrast to non-selective tyrosine-kinase inhibitors, aloperine exhibits high selectivity for FGFR1, with minimal direct inhibitory activity against VEGFR2, JAK2, or JAK3. This selectivity profile is therapeutically advantageous, as it may reduce the on-target and off-target toxicities commonly associated with multi-kinase inhibitors, including hypertension, diarrhea, and myelosuppression [ 32 ]. FGFR1 plays a critical role in tumor cell proliferation and growth, angiogenesis, and metastasis in various malignancies [ 33 ]. In TNBC, its amplification or overexpression is prevalent and is strongly associated with therapeutic resistance and poor clinical outcomes [ 34 ], positioning FGFR1 as an attractive yet underexploited therapeutic target in this aggressive breast cancer subtype. To date, only one FGFR1 inhibitor, pemigatinib, has received FDA approval, specifically for the treatment of myeloid/lymphoid neoplasms, while no FGFR1-targeted therapies have been approved for TNBC or other solid tumors [ 33 ], underscoring a substantial unmet clinical need. The identification of aloperine as a naturally derived FGFR1 inhibitor offers a promising foundation for addressing this gap. By simultaneously disrupting FGFR1-dependent VM formation and migration of TNBC cells as well as angiogenesis of ECs, aloperine may exert synergistic antitumor effects, potentially overcoming limitations of single-compartment targeting. Furthermore, its favorable selectivity could enable combination strategies, such as with chemotherapy, immune checkpoint inhibitors, or other targeted agents, while minimizing the risk of cumulative toxicities. To evaluate the therapeutic potential of aloperine, we utilized two complementary mouse models representing distinct stages of TNBC progression. The dorsal skinfold chamber model enables real-time visualization of tumor vascularization and growth in vivo [ 27 ]. In this model, aloperine treatment was initiated on the day of 4T1 spheroid transplantation and continued for 2 weeks. This short-term intervention with aloperine significantly inhibited angiogenesis, VM formation, and tumor growth, demonstrating its capacity to prevent TNBC establishment and early progression. In contrast, the orthotopic xenograft model was utilized to assess the therapeutic efficacy of aloperine against established human tumors. Treatment was initiated 3 days after MDA-MB-231 cell inoculation, when tumors became detectable, and continued for 6 weeks. We found that aloperine effectively suppresses tumor vascularization and growth and significantly delays metastatic progression, as evidenced by decreased pulmonary metastatic burden and reduced numbers of CTCs in peripheral blood. Notably, the reduction in CTCs provides mechanistic insight beyond endpoint metastasis, suggesting that concurrent disruption of tumor vasculature and tumor cell motility effectively limits tumor cell intravasation and subsequent systemic dissemination. Together, the consistent effects observed in both murine and human TNBC models establish a strong preclinical foundation for the clinical translation of aloperine as a multi-target therapeutic agent for aggressive TNBC. In summary, our findings demonstrate that aloperine exerts effective, multifaceted antitumor activity in TNBC by directly binding the FGFR1 ATP-binding pocket. This molecular interaction attenuates the FGFR1/JAK2/STAT3 signaling axis across both endothelial and neoplastic compartments, leading to the concurrent suppression of angiogenesis, VM, and metastasis. By establishing FGFR1 as a critical therapeutic node linking vascular remodeling to metastatic progression, this study positions aloperine as a promising lead compound for the treatment of TNBC and potentially other aggressive, highly metastatic malignancies. Abbreviations 3D Three-dimensional AKT Protein kinase B BLI Bioluminescence imaging BrdU Bromodeoxyuridine CTC Circulating tumor cell DMEM Dulbecco’s modified Eagle’s medium EBM Endothelial Cell Basal Medium ECs Endothelial cells ERK Extracellular signal-regulated kinase FCS Fetal calf serum FGFR Fibroblast growth factor receptor FITC Fluorescein isothiocyanate GFP Green fluorescent protein HDMECs Human dermal microvascular endothelial cells HE Hematoxylin and eosin HRP Horseradish peroxidase HUVECs Human umbilical vein endothelial cells JAK Janus kinase LDH Lactate dehydrogenase mTOR Mammalian target of rapamycin PAS Periodic acid-Schiff PI3K Phosphatidylinositol 3-kinase RBC Red blood cell RTKs Receptor tyrosine kinases siRNAs Small interfering RNAs SOCS Suppressor of cytokine signaling STAT Signal transducer and activator of transcription TKIs Tyrosine kinase inhibitors TNBC Triple-negative breast cancer VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor VM Vasculogenic mimicry Declarations Ethics approval and consent to participate All animal experiments were conducted in accordance with German legislation on the protection of animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th Edition, 2011). The applied protocols were approved by the local authorities (State Office for Consumer Protection, Saarbrücken, Germany; permission numbers: 01/2019 and 23/2020). Consent for publication All authors have agreed to the publication of the manuscript. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by the research program of the Medical Faculty of Saarland University (HOMFOR2023 Anschubfinanzierung) and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; project no. 411093008). Authors’ contributions M. W. L. and Y. G. designed the research; T. T., L. M., and Y.X. performed the experiments; T. T. and L. M. analyzed the data; T. T., L. M., M. W. L., and Y. G. drafted the manuscript. All authors have reviewed and approved the final version of the manuscript for publication. Acknowledgements The authors gratefully acknowledge the technical assistance provided by Ruth Nickels, Christina Max, and Janine Becker from the Institute for Clinical and Experimental Surgery at Saarland University. References Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol. 2016; 13:674-690. https://doi.org/10.1038/nrclinonc.2016.66. Kumar H, Gupta NV, Jain R, Madhunapantula SV, Babu CS, Kesharwani SS, Dey S, Jain V. A review of biological targets and therapeutic approaches in the management of triple-negative breast cancer. J Adv Res. 2023; 54:271-292. https://doi.org/10.1016/j.jare.2023.02.005. Ribatti D, Nico B, Ruggieri S, Tamma R, Simone G, Mangia A. Angiogenesis and antiangiogenesis in triple-negative breast cancer. Transl Oncol. 2016; 9:453-457. https://doi.org/10.1016/j.tranon.2016.07.002. Dudley AC, Griffioen AW. Pathological angiogenesis: mechanisms and therapeutic strategies. Angiogenesis. 2023; 26:313-347. https://doi.org/10.1007/s10456-023-09876-7. Jiang X, Wang J, Deng X, Xiong F, Zhang S, Gong Z, Li X, Cao K, Deng H, He Y, et al. The role of microenvironment in tumor angiogenesis. J Exp Clin Cancer Res. 2020; 39:204. https://doi.org/10.1186/s13046-020-01709-5. Luo QX, Wang J, Zhao WY, Peng ZZ, Liu XY, Li B, Zhang H, Shan B, Zhang CF, Duan CJ. Vasculogenic mimicry in carcinogenesis and clinical applications. J Hematol Oncol. 2020; 13:19. https://doi.org/10.1186/s13045-020-00858-6. Zhang D, Sun B, Zhao X, Ma Y, Ji R, Gu Q, Dong X, Li J, Liu F, Jia X, et al. Twist1 expression induced by sunitinib accelerates tumor cell vasculogenic mimicry by increasing the population of CD133 + cells in triple-negative breast cancer. Mol Cancer. 2014; 13:207. https://doi.org/10.1186/1476-4598-13-207. Sun HZ, Zhang DF, Yao Z, Lin X, Liu JM, Gu Q, Dong XY, Liu F, Wang Y, Yao N, et al. Anti-angiogenic treatment promotes triple-negative breast cancer invasion via vasculogenic mimicry. Cancer Biol Ther. 2017; 18:205-213. https://doi.org/10.1080/15384047.2017.1294288. Cheng CL, Thike AA, Tan SY, Chua PJ, Bay BH, Tan PH. Expression of FGFR1 is an independent prognostic factor in triple-negative breast cancer. Breast Cancer Res Treat. 2015; 151:99-111. https://doi.org/10.1007/s10549-015-3371-x. Hallinan N, Finn S, Cuffe S, Rafee S, O'Byrne K, Gately K. Targeting the fibroblast growth factor receptor family in cancer. Cancer Treat Rev. 2016; 46:51-62. https://doi.org/10.1016/j.ctrv.2016.03.015. Liu Q, Huang J, Yan W, Liu Z, Liu S, Fang W. FGFR families: biological functions and therapeutic interventions in tumors. MedComm (2020). 2023; 4:e367. https://doi.org/10.1002/mco2.367. Presta M, Dell'Era P, Mitola S, Moroni E, Ronca R, Rusnati M. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev. 2005; 16:159-178. https://doi.org/10.1016/j.cytogfr.2005.01.004. Zhu X, Qiu C, Wang Y, Jiang Y, Chen Y, Fan L, Ren R, Wang Y, Chen Y, Feng Y, et al. FGFR1 SUMOylation coordinates endothelial angiogenic signaling in angiogenesis. Proc Natl Acad Sci U S A. 2022; 119:e2202631119. https://doi.org/10.1073/pnas.2202631119. Naeem A, Hu P, Yang M, Zhang J, Liu Y, Zhu W, Zheng Q. Natural products as anticancer agents: current status and future perspectives. Molecules. 2022; 27:8367. https://doi.org/10.3390/molecules27238367. Asma ST, Acaroz U, Imre K, Morar A, Shah SRA, Hussain SZ, Arslan-Acaroz D, Demirbas H, Hajrulai-Musliu Z, Istanbullugil FR, et al. Natural products/bioactive compounds as a source of anticancer drugs. Cancers (Basel). 2022; 14:6203. https://doi.org/10.3390/cancers14246203. Zhou H, Li J, Sun F, Wang F, Li M, Dong Y, Fan H, Hu D. A review on recent advances in aloperine research: pharmacological activities and underlying biological mechanisms. Front Pharmacol. 2020; 11:538137. https://doi.org/10.3389/fphar.2020.538137. Tahir M, Ali S, Zhang W, Lv B, Qiu W, Wang J. Aloperine: A potent modulator of crucial biological mechanisms in multiple diseases. Biomedicines. 2022; 10:905. https://doi.org/10.3390/biomedicines10040905. Gu Y, Tang T, Qiu M, Wang H, Ampofo E, Menger MD, Laschke MW. Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization. Angiogenesis. 2025; 28:13. https://doi.org/10.1007/s10456-024-09965-1. Becker V, Hui X, Nalbach L, Ampofo E, Lipp P, Menger MD, Laschke MW, Gu Y. Linalool inhibits the angiogenic activity of endothelial cells by downregulating intracellular ATP levels and activating TRPM8. Angiogenesis. 2021; 24:613-630. https://doi.org/10.1007/s10456-021-09772-y. Gu Y, Scheuer C, Feng D, Menger MD, Laschke MW. Inhibition of angiogenesis: a novel antitumor mechanism of the herbal compound arctigenin. Anticancer Drugs. 2013; 24:781-791. https://doi.org/10.1097/CAD.0b013e328362fb84. Liu ZL, Chen HH, Zheng LL, Sun LP, Shi L. Angiogenic signaling pathways and anti-angiogenic therapy for cancer. Signal Transduct Target Ther. 2023; 8:198. https://doi.org/10.1038/s41392-023-01460-1. Mengie Ayele T, Tilahun Muche Z, Behaile Teklemariam A, Bogale Kassie A, Chekol Abebe E. Role of JAK2/STAT3 signaling pathway in the tumorigenesis, chemotherapy resistance, and treatment of solid tumors: a systemic review. J Inflamm Res. 2022; 15:1349-1364. https://doi.org/10.2147/JIR.S353489. Tan X, Yan Y, Song B, Zhu S, Mei Q, Wu K. Focal adhesion kinase: from biological functions to therapeutic strategies. Exp Hematol Oncol. 2023; 12:83. https://doi.org/10.1186/s40164-023-00446-7. Xue C, Yao Q, Gu X, Shi Q, Yuan X, Chu Q, Bao Z, Lu J, Li L. Evolving cognition of the JAK-STAT signaling pathway: autoimmune disorders and cancer. Signal Transduct Target Ther. 2023; 8:204. https://doi.org/10.1038/s41392-023-01468-7. Dai S, Zhou Z, Chen Z, Xu G, Chen Y. Fibroblast growth factor receptors (FGFRs): structures and small molecule inhibitors. Cells. 2019; 8:614. https://doi.org/10.3390/cells8060614. Maddison K, Bowden NA, Graves MC, Tooney PA. Characteristics of vasculogenic mimicry and tumour to endothelial transdifferentiation in human glioblastoma: a systematic review. BMC Cancer. 2023; 23:185. https://doi.org/10.1186/s12885-023-10659-y. Koehl GE, Gaumann A, Geissler EK. Intravital microscopy of tumor angiogenesis and regression in the dorsal skin fold chamber: mechanistic insights and preclinical testing of therapeutic strategies. Clin Exp Metastasis. 2009; 26:329-344. https://doi.org/10.1007/s10585-008-9234-7. Batool S, Chokkakula S, Kim BK, Park JH, Min SC, Lee JR, Lee GC, Lee DG, An SH, Jain A, et al. High-throughput in vitro screening and in silico analysis for Zika virus inhibitor identification. Sci Rep. 2025; 15:45501. https://doi.org/10.1038/s41598-025-29585-z. Alemu BK, Tommasi S, Hulin JA, Meyers J, Mangoni AA. Current knowledge on the mechanisms underpinning vasculogenic mimicry in triple negative breast cancer and the emerging role of nitric oxide. Biomed Pharmacother. 2025; 186:118013. https://doi.org/10.1016/j.biopha.2025.118013. Morales-Guadarrama G, García-Becerra R, Méndez-Pérez EA, García-Quiroz J, Avila E, Díaz L. Vasculogenic mimicry in breast cancer: clinical relevance and drivers. Cells. 2021; 10:1758. https://doi.org/10.3390/cells10071758. Polacheck WJ, Zervantonakis IK, Kamm RD. Tumor cell migration in complex microenvironments. Cell Mol Life Sci. 2013; 70:1335-1356. https://doi.org/10.1007/s00018-012-1115-1. Shyam Sunder S, Sharma UC, Pokharel S. Adverse effects of tyrosine kinase inhibitors in cancer therapy: pathophysiology, mechanisms and clinical management. Signal Transduct Target Ther. 2023; 8:262. https://doi.org/10.1038/s41392-023-01469-6. Fan S, Chen Y, Wang W, Xu W, Tian M, Liu Y, Zhou Y, Liu D, Xia Q, Dong L. Pharmacological and biological targeting of FGFR1 in cancer. Curr Issues Mol Biol. 2024; 46:13131-13150. https://doi.org/10.3390/cimb46110783. Chen J, Wang Q, Wu H, Huang X, Cao C. Therapies targeting triple-negative breast cancer: a perspective on anti-FGFR. Front Oncol. 2024; 14:1415820. https://doi.org/10.3389/fonc.2024.1415820. Additional Declarations No competing interests reported. Supplementary Files supplementaryinformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-9209927\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":619027554,\"identity\":\"f4245c59-078f-4c0b-98ec-efddde4425d9\",\"order_by\":0,\"name\":\"Tianci Tang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saarland University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tianci\",\"middleName\":\"\",\"lastName\":\"Tang\",\"suffix\":\"\"},{\"id\":619027555,\"identity\":\"2fe4c132-a923-4667-87c1-a75981dd7a9d\",\"order_by\":1,\"name\":\"Luisa Müller\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saarland University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Luisa\",\"middleName\":\"\",\"lastName\":\"Müller\",\"suffix\":\"\"},{\"id\":619027556,\"identity\":\"76c539c5-5ef8-42a1-84ae-8d40949d616e\",\"order_by\":2,\"name\":\"Yixuan Xu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saarland University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yixuan\",\"middleName\":\"\",\"lastName\":\"Xu\",\"suffix\":\"\"},{\"id\":619027557,\"identity\":\"ccbdc410-fbb8-43da-8c3c-b4b49eb1b9ef\",\"order_by\":3,\"name\":\"Matthias W. Laschke\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saarland University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Matthias\",\"middleName\":\"W.\",\"lastName\":\"Laschke\",\"suffix\":\"\"},{\"id\":619027558,\"identity\":\"11624e50-446b-4d60-b736-2aa08ad9c444\",\"order_by\":4,\"name\":\"Yuan Gu\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYBADOQYeUrUYk64lsYFoLQY3kp89YPh1OH3DmeMPGD62EaUlzdyAse9w7oazPQaMM4nTkmAmwdgD1HKeh4GZlzgt6d9AWtINzrM/YP5LnJYcMwmGH4cTDM42GDAzEqNF8sybMonEhnTDmWfOGBzsOUeEFr7j6dskPvyxluc7k/7wwY8yIrQoHAASiVD3HCBCAwODfAOI/EOU2lEwCkbBKBipAAD8XzqxT/KLmQAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Saarland University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Yuan\",\"middleName\":\"\",\"lastName\":\"Gu\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-03-24 09:40:15\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-9209927/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-9209927/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":106960741,\"identity\":\"67c5447a-211a-4356-b7c5-ce75bb9538d7\",\"added_by\":\"auto\",\"created_at\":\"2026-04-15 09:22:54\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":20506466,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAloperine inhibits angiogenesis \\u003cem\\u003ein vitro\\u003c/em\\u003e, \\u003cem\\u003eex vivo\\u003c/em\\u003e, and \\u003cem\\u003ein vivo\\u003c/em\\u003e. \\u003cstrong\\u003eA: \\u003c/strong\\u003eChemical structure of aloperine. \\u003cstrong\\u003eB:\\u003c/strong\\u003e Viability (% of 0 µM) of HUVECs, HDMECs, MDA-MB-231, and HCC1937 cells that were treated for 48 h with a serial dilution of aloperine, as assessed by WST-1 assay (n = 4).\\u003cstrong\\u003e C: \\u003c/strong\\u003eCytotoxicity (% of total cell death) of aloperine against HUVECs, as assessed by LDH assay (n = 4). The cells were treated with a serial dilution of aloperine for 24 h. \\u003cstrong\\u003eD: \\u003c/strong\\u003eProliferation (% of 0 µM) of HUVECs that were treated for 24 h with 0, 25, 50, and 100 µM aloperine, as assessed by BrdU incorporation assay (n = 4 independent experiments). \\u003cstrong\\u003eE: \\u003c/strong\\u003eRepresentative images of migrated HUVECs. The cells were treated with 0, 25, 50, and 100 µM aloperine for 18 h, then seeded into Transwell inserts and incubated with different concentrations of aloperine for an additional 5 h. Scale bar: 100 µm. \\u003cstrong\\u003eF: \\u003c/strong\\u003eMigration (% of 0 µM) of treated HUVECs depicted in (E), as assessed by Transwell migration assay (n = 3). \\u003cstrong\\u003eG: \\u003c/strong\\u003eRepresentative images of tube-forming HUVECs that were treated for 18 h with 0, 25, 50, and 100 µM aloperine. Scale bar: 900 µm. \\u003cstrong\\u003eH: \\u003c/strong\\u003eTube formation (% of 0 µM) of treated HUVECs depicted in (G), as assessed by tube formation assay (n = 5). \\u003cstrong\\u003eI: \\u003c/strong\\u003eRepresentative images of HUVEC spheroids that were treated for 24 h with 0, 25, 50, and 100 µM aloperine. Scale bar: 145 µm. \\u003cstrong\\u003eJ: \\u003c/strong\\u003eSprouting (% of 0 µM) of treated HUVEC spheroids depicted in (I), as assessed by spheroid sprouting assay (n = 7-9). \\u003cstrong\\u003eK:\\u003c/strong\\u003e Representative images of mouse aortic rings that were treated for 6 days with 0, 25, 50, and 100 µM aloperine. Scale bar: 1 mm. \\u003cstrong\\u003eL: \\u003c/strong\\u003eSprouting (% of 0 µM) of treated aortic rings depicted in (K), as assessed by aortic ring assay (n = 8). \\u003cstrong\\u003eM: \\u003c/strong\\u003eRepresentative images of Matrigel plugs with or without 100 µM aloperine. The sections were stained with an anti-CD31 antibody (red) and Hoechst 33342 (blue) to visualize ECs and cell nuclei, respectively. Scale bar: 55 µm.\\u003cstrong\\u003e N: \\u003c/strong\\u003eCD31\\u003csup\\u003e+\\u003c/sup\\u003e microvessel density (mm\\u003csup\\u003e-2\\u003c/sup\\u003e) in Matrigel plugs depicted in (M), as assessed by immunohistochemistry (n = 6). Data are presented as Means ± SEM. *\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9209927/v1/c330130cf47a03e19476e090.png\"},{\"id\":106835746,\"identity\":\"756a277a-ac50-4626-b36b-2536898cd067\",\"added_by\":\"auto\",\"created_at\":\"2026-04-14 02:02:26\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":13354628,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAloperine inhibits EC angiogenesis by suppressing the JAK2/STAT3 signaling pathway. \\u003cstrong\\u003eA: \\u003c/strong\\u003eRepresentative Western blots showing p-mTOR, mTOR, p-STAT5, STAT5, p-STAT3, STAT3, p-AKT, AKT, p-ERK, ERK, and β-actin expression in HUVECs that were treated for 4 h with 0, 25, 50, and 100 µM aloperine. \\u003cstrong\\u003eB-F: \\u003c/strong\\u003eExpression levels (% of 0 µM) of p-mTOR/mTOR (B), p-STAT5/STAT5 (C), p-STAT3/STAT3 (D), p-AKT/AKT (E), and p-ERK/ERK (F) in treated HUVECs depicted in (A), as assessed by Western blotting (n = 5 independent experiments). \\u003cstrong\\u003eG: \\u003c/strong\\u003eRepresentative Western blots showing p-FAK, FAK, p-JAK2, JAK2, p-Src, Src, and β-actin expression in HUVECs that were treated for 4 h with 0, 25, 50, and 100 µM aloperine. \\u003cstrong\\u003eH-J: \\u003c/strong\\u003eExpression levels (% of 0 µM) of p-FAK/FAK (H), p-JAK2/JAK2 (I), and p-Src/Src (J) in treated HUVECs depicted in (G), as assessed by Western blotting (n = 5 independent experiments). \\u003cstrong\\u003eK: \\u003c/strong\\u003eRepresentative Western blots showing SOCS1, SOCS3, p-JAK2, JAK2, p-STAT3, STAT3, p-AKT, AKT, and β-actin expression in HUVECs that were transfected with si-NC, si-SOCS1, or si-SOCS3 for 48 h. \\u003cstrong\\u003eL-O: \\u003c/strong\\u003eExpression levels (% of si-NC) of SOCS/β-actin (L), p-JAK2/JAK2 (M), p-STAT3/STAT3 (N), and p-AKT/AKT (O) in transfected HUVECs depicted in (K), as assessed by Western blotting (n = 4 independent experiments). \\u003cstrong\\u003eP: \\u003c/strong\\u003eRepresentative images of HUVEC spheroids. The cells were transfected with si-NC, si-SOCS1, or si-SOCS3 for 48 h prior to the spheroid sprouting assay and then exposed to 0.1% DMSO (vehicle) or 100 µM aloperine for another 24 h. Scale bar: 145 µm. \\u003cstrong\\u003eQ: S\\u003c/strong\\u003eprouting (% of si-NC) of HUVEC spheroids depicted in (P), as assessed by spheroid sprouting assay (n = 15). Data are presented as Means ± SEM. *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9209927/v1/c0d8acf522ddbb1be9d15774.png\"},{\"id\":106960681,\"identity\":\"323e2eb1-3847-4e6a-9f9b-5822e98314e0\",\"added_by\":\"auto\",\"created_at\":\"2026-04-15 09:22:35\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":12648755,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAloperine binds to the ATP-binding pocket of FGFR1, thereby suppressing JAK2/STAT3 signaling in ECs. \\u003cstrong\\u003eA: \\u003c/strong\\u003eRepresentative Western blots showing p-FGFR1, FGFR1, p-JAK2, JAK2, p-STAT3, STAT3, p-AKT, AKT, and β-actin expression in HUVECs that were treated with 0.1% DMSO (Con) or 100 µM aloperine for 4 h and then stimulated with or without 150 ng/mL bFGF for another 15 min. \\u003cstrong\\u003eB-E:\\u003c/strong\\u003e Expression levels (% of control) of p-FGFR1/FGFR1 (B), p-JAK2/JAK2 (C), p-STAT3/STAT3 (D), and p-AKT/AKT (E) in treated HUVECs depicted in (A), as assessed by Western blotting (n = 4-5 independent experiments). \\u003cstrong\\u003eF: \\u003c/strong\\u003eRepresentative images of HUVEC spheroids that were treated for 24 h with 0.1% DMSO (Con) or 100 µM aloperine in the absence or presence of 150 ng/mL bFGF. Scale bar: 110 µm. \\u003cstrong\\u003eG:\\u003c/strong\\u003e Sprouting (% of control) of treatedHUVEC spheroids depicted in (F), as assessed by spheroid sprouting assay (n = 10). \\u003cstrong\\u003eH:\\u003c/strong\\u003e Molecular docking analysis of aloperine binding to FGFR1. Left panel: 3D representation of aloperine (blue) docked within the ATP-binding pocket of FGFR1 (gray; PDB ID: 5A46). Right panel: 2D interaction diagram showing hydrogen bonds and hydrophobic interactions between aloperine and key FGFR1 residues. \\u003cstrong\\u003eI:\\u003c/strong\\u003eRepresentative Western blots showing p-FGFR1, FGFR1, p-JAK2, JAK2, and β-actin expression in HUVECs that were pre-treated with or without 1 mM ATP for 2 h and then exposed to 0.1% DMSO (Con) or 100 µM aloperine for another 4 h. \\u003cstrong\\u003eJ, K:\\u003c/strong\\u003e Expression levels (% of control) of p-FGFR1/FGFR1 (J) and p-JAK2/JAK2 (K) in treated HUVECs depicted in (I), as assessed by Western blotting (n = 6 independent experiments). \\u003cstrong\\u003eL: \\u003c/strong\\u003eActivity (% of control) of JAK2, JAK3, VEGFR2, and FGFR1 in the presence of 1% DMSO (Con), 100 µM aloperine, or specific kinase inhibitor at an ATP concentration of 10 µM, as assessed by cell-free kinase assay (n = 3). \\u003cstrong\\u003eM:\\u003c/strong\\u003e FGFR1 kinase activity (% of control) in the presence of 10 nM PD173074 or 100 µM aloperine at ATP concentrations of 10 or 500 µM, as assessed by cell-free kinase assay (n = 3). Data are presented as Means ± SEM. *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9209927/v1/3e6d0acdc2a3ec8777726ea8.png\"},{\"id\":106960357,\"identity\":\"559701e3-02b6-456d-a5d4-602311138aa5\",\"added_by\":\"auto\",\"created_at\":\"2026-04-15 09:20:27\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":17939110,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAloperine inhibits TNBC cell migration and VM by suppressing the FGFR1/JAK2/STAT3 signaling pathway.\\u003cstrong\\u003e A:\\u003c/strong\\u003e Proliferation (% of 0 µM) of MDA-MB-231 cells that were treated for 24 h with 0, 25, 50, and 100 µM aloperine, as assessed by BrdU incorporation assay (n = 4 independent experiments). \\u003cstrong\\u003eB:\\u003c/strong\\u003e Representative images of migrated MDA-MB-231 cells. The cells were treated with 0, 25, 50, and 100 µM aloperine for 18 h, then seeded into Transwell inserts and incubated with different concentrations of aloperine for an additional 5 h. Scale bar: 100 µm. \\u003cstrong\\u003eC:\\u003c/strong\\u003e Migration (% of 0 µM) of treated MDA-MB-231 cells depicted in (B), as assessed by Transwell migration assay (n = 3). \\u003cstrong\\u003eD:\\u003c/strong\\u003e Representative images of tube-forming MDA-MB-231 cells that were treated for 18 h with 0, 25, 50, and 100 µM aloperine. Scale bar: 720 µm. \\u003cstrong\\u003eE:\\u003c/strong\\u003e VM (% of 0 µM) in treated MDA-MB-231 cells depicted in (D), as assessed by tube formation assay (n = 5). \\u003cstrong\\u003eF:\\u003c/strong\\u003eRepresentative images of MDA-MB-231 spheroids that were treated for 24 h with 0, 25, 50, and 100 µM aloperine. Scale bar: 220 µm. \\u003cstrong\\u003eG:\\u003c/strong\\u003e Sprouting (% of 0 µM) of treated MDA-MB-231 spheroids depicted in (F), as assessed by spheroid sprouting assay (n = 22-27). \\u003cstrong\\u003eH:\\u003c/strong\\u003e Representative Western blots showing p-FGFR1, FGFR1, p-JAK2, JAK2, p-Src, Src, p-STAT3, STAT3, p-AKT, AKT, and β-actin expression in MDA-MB-231 cells that were treated for 4 h with 0, 25, 50, and 100 µM aloperine. \\u003cstrong\\u003eI-M:\\u003c/strong\\u003e Expression levels (% of 0 µM) of p-FGFR1/FGFR1 (I), p-JAK2/JAK2 (J), p-Src/Src (K), p-STAT3/STAT3 (L), and p-AKT/AKT (M) in treated MDA-MB-231 cells depicted in (H), as assessed by Western blotting (n = 4 independent experiments). \\u003cstrong\\u003eN:\\u003c/strong\\u003e Migration (% of control) of MDA-MB-231 cells, as assessed by Transwell migration assay (n = 3). The cells were treated with 0.1% DMSO (vehicle) or 100 µM aloperine for 18 h, then seeded into Transwell inserts and incubated with DMSO or aloperine in the absence or presence of 150 ng/mL bFGF for an additional 5 h. \\u003cstrong\\u003eO:\\u003c/strong\\u003eVM (% of control) of MDA-MB-231 cells that were treated for 18 h with 0.1% DMSO (Con) or 50 µM aloperine in the absence or presence of 150 ng/mL bFGF, as assessed by tube formation assay (n = 4). \\u003cstrong\\u003eP: \\u003c/strong\\u003eRepresentative Western blots showing SOCS1 and β-actin expression in MDA-MB-231 cells that were transfected with si-NC or si-SOCS1 for 48 h. \\u003cstrong\\u003eQ: \\u003c/strong\\u003eExpression levels (% of si-NC) of SOCS1/β-actin in transfected MDA-MB-231 cells depicted in (P), as assessed by Western blotting (n = 4 independent experiments). \\u003cstrong\\u003eR:\\u003c/strong\\u003e Migration (% of si-NC) of MDA-MB-231 cells, as assessed by Transwell migration assay (n = 3). The cells were transfected with si-NC or si-SOCS1 for 30 h followed by treatment with 0.1% DMSO (vehicle) or 100 µM aloperine for 18 h. Cells were then seeded into Transwell inserts and incubated with DMSO or aloperine for an additional 5 h. \\u003cstrong\\u003eS:\\u003c/strong\\u003e VM (% of si-NC) in MDA-MB-231 cells, as assessed by tube formation assay (n = 4). The cells were transfected with si-NC or si-SOCS1 for 48 h, then seeded onto Matrigel-coated plates and treated with 0.1% DMSO (vehicle) or 50 µM aloperine for an additional 18 h. Data are presented as Means ± SEM. *\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9209927/v1/8368e951d4b2eed67c709f83.png\"},{\"id\":106835750,\"identity\":\"32aa068c-4b9d-4524-b3c4-d74c96723ede\",\"added_by\":\"auto\",\"created_at\":\"2026-04-14 02:02:26\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":31510302,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAloperine inhibits TNBC vascularization and growth in a dorsal skinfold chamber model. \\u003cstrong\\u003eA:\\u003c/strong\\u003e Schematic timeline of the dorsal skinfold chamber model. \\u003cstrong\\u003eB:\\u003c/strong\\u003e Body weight (g) of vehicle- and aloperine-treated mice on days 0, 3, 6, 10, and 14 after spheroid transplantation (n = 8). \\u003cstrong\\u003eC:\\u003c/strong\\u003e Representative stereomicroscopic images of 4T1 tumors in the chamber of vehicle- and aloperine-treated mice on day 14 after spheroid transplantation. Dotted lines indicate tumor boundaries. Scale bar: 3 mm. \\u003cstrong\\u003eD:\\u003c/strong\\u003e Size (mm\\u003csup\\u003e2\\u003c/sup\\u003e) of 4T1 tumors in vehicle- and aloperine-treated mice on days 0, 3, 6, 10, and 14 after spheroid transplantation, as assessed by intravital fluorescence microscopy (n = 8). \\u003cstrong\\u003eE: \\u003c/strong\\u003eRepresentative images of newly formed microvessels in vehicle- and aloperine-treated 4T1 tumors on day 14 after spheroid transplantation. Scale bar: 180 µm. \\u003cstrong\\u003eF: \\u003c/strong\\u003eFunctional microvessel density (cm/cm\\u003csup\\u003e2\\u003c/sup\\u003e) of 4T1 tumors in vehicle- and aloperine-treated mice on days 0, 3, 6, 10, and 14 after spheroid transplantation, as assessed by intravital fluorescence microscopy (n = 8). \\u003cstrong\\u003eG-I:\\u003c/strong\\u003e Diameter (μm; G), centerline RBC velocity (mm/s; H), and volumetric blood flow (pL/s; I) of tumor microvessels in vehicle- and aloperine-treated mice, as assessed by intravital fluorescence microscopy (n = 8).\\u0026nbsp; \\u003cstrong\\u003eJ:\\u003c/strong\\u003e Representative images of HE-stained, Ki67-stained, cleaved caspase 3-stained, and CD31 and PAS double-stained sections of 4T1 tumors from vehicle- and aloperine-treated mice. Dotted lines indicate tumor boundaries. Scale bars: 720 μm (top panel) and 55 μm (bottom three panels). \\u003cstrong\\u003eK:\\u003c/strong\\u003e Size (mm\\u003csup\\u003e2\\u003c/sup\\u003e) of 4T1 tumors depicted in (J), as assessed by HE staining (n = 8). \\u003cstrong\\u003eL: \\u003c/strong\\u003eKi67\\u003csup\\u003e+\\u003c/sup\\u003e tumor cells (% of total cell number) in 4T1 tumors depicted in (J), as assessed by immunohistochemical staining (n = 8). \\u003cstrong\\u003eM:\\u003c/strong\\u003e Cleaved caspase-3\\u003csup\\u003e+\\u003c/sup\\u003e tumor cells (% of total cell number) in 4T1 tumors depicted in (J), as assessed by immunohistochemical staining (n = 8). \\u003cstrong\\u003eN: \\u003c/strong\\u003eDensity (mm\\u003csup\\u003e-2\\u003c/sup\\u003e) of\\u003cstrong\\u003e \\u003c/strong\\u003eCD31\\u003csup\\u003e+\\u003c/sup\\u003ePAS\\u003csup\\u003e+ \\u003c/sup\\u003eEC-lined vessels (black arrowheads) and CD31\\u003csup\\u003e-\\u003c/sup\\u003ePAS\\u003csup\\u003e+ \\u003c/sup\\u003eVM structures (red arrowheads) in 4T1 tumors depicted in (J), as assessed by CD31 and PAS double staining (n = 8). Data are presented as Means ± SEM. *\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9209927/v1/f5ee1c34925a5427347125ef.png\"},{\"id\":106835749,\"identity\":\"67cf1969-125d-4d43-8f8e-9f599fc38c0e\",\"added_by\":\"auto\",\"created_at\":\"2026-04-14 02:02:26\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":14380462,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAloperine inhibits TNBC growth and metastasis in an orthotopic xenograft model.\\u003cstrong\\u003e A:\\u003c/strong\\u003e Schematic timeline of the orthotopic xenograft model. \\u003cstrong\\u003eB:\\u003c/strong\\u003e Body weight (g) of vehicle- and aloperine-treated mice on days 0, 3, 7, 14, 21, 28, 35, and 42 after tumor inoculation (n = 8). \\u003cstrong\\u003eC:\\u003c/strong\\u003e Volume (mm\\u003csup\\u003e3\\u003c/sup\\u003e) of MDA-MB-231 tumors in vehicle- and aloperine-treated mice on days 3, 7, 14, 21, 28, 35, and 42 after tumor inoculation, as assessed by caliper measurement (n = 8). \\u003cstrong\\u003eD: \\u003c/strong\\u003eImages of MDA-MB-231 tumors excised from vehicle- and aloperine-treated mice on day 42 after tumor inoculation. Scale bars: 8 mm. \\u003cstrong\\u003eE: \\u003c/strong\\u003eWeight (g) of MDA-MB-231 tumors from vehicle- and aloperine-treated mice (n = 8). \\u003cstrong\\u003eF:\\u003c/strong\\u003e Representative bioluminescent images of tumors in vehicle- and aloperine-treated mice on days 7, 14, 21, 28, 35, and 42 after tumor inoculation. \\u003cstrong\\u003eG: \\u003c/strong\\u003eTotal flux (photons/sec×10\\u003csup\\u003e7\\u003c/sup\\u003e) of bioluminescent signals emitted from tumors depicted in (F), as assessed by bioluminescence imaging (n = 8). \\u003cstrong\\u003eH:\\u003c/strong\\u003e Representative bioluminescent images of lung metastases in vehicle- and aloperine-treated mice on days 21, 28, 35, and 42 after tumor inoculation. \\u003cstrong\\u003eI:\\u003c/strong\\u003e Total flux (photons/sec×10\\u003csup\\u003e5\\u003c/sup\\u003e) of bioluminescent signals emitted from lung metastases depicted in (H), as assessed by bioluminescence imaging (n = 8). \\u003cstrong\\u003eJ:\\u003c/strong\\u003e Representative bioluminescent images of \\u003cem\\u003eex vivo\\u003c/em\\u003e lungs from vehicle- and aloperine-treated mice on day 42 after tumor inoculation. \\u003cstrong\\u003eK:\\u003c/strong\\u003e Total flux (photons/sec×10\\u003csup\\u003e5\\u003c/sup\\u003e) of bioluminescent signals emitted from lungs depicted in (J), as assessed by bioluminescence imaging (n = 8). \\u003cstrong\\u003eL:\\u003c/strong\\u003e Representative images of GFP\\u003csup\\u003e+\\u003c/sup\\u003e metastatic foci in lung sections from vehicle- and aloperine-treated mice. Scale bars: 90 μm. \\u003cstrong\\u003eM:\\u003c/strong\\u003e Quantification of GFP\\u003csup\\u003e+\\u003c/sup\\u003e metastatic foci (mm\\u003csup\\u003e-2\\u003c/sup\\u003e) in lung sections depicted in (L), as assessed by immunohistochemical staining (n = 8). \\u003cstrong\\u003eN:\\u003c/strong\\u003e Quantification of GFP\\u003csup\\u003e+ \\u003c/sup\\u003eCTCs (per 10,000 events) in peripheral blood from vehicle- and aloperine-treated mice, as assessed by flow cytometry (n = 5-6). Data are presented as Means ± SEM. *\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9209927/v1/f00f95544a9a55305d51c8f0.png\"},{\"id\":106835751,\"identity\":\"e0f12546-3624-4b3b-b1a2-61ccbd3a5102\",\"added_by\":\"auto\",\"created_at\":\"2026-04-14 02:02:26\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":34784909,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAloperine inhibits TNBC growth and vascularization and suppresses the FGFR1/JAK2/STAT3 signaling pathway in orthotopic xenograft tumors. Immunohistochemical and immunoblot analyses of tumors from the orthotopic xenograft model. \\u003cstrong\\u003eA:\\u003c/strong\\u003e Representative images of HE-stained sections of MDA-MB-231 tumors from vehicle- and aloperine-treated mice. Scale bars: 2 mm. \\u003cstrong\\u003eB:\\u003c/strong\\u003e Size (mm\\u003csup\\u003e2\\u003c/sup\\u003e) of MDA-MB-231 tumors depicted in (A), as assessed by HE staining (n = 8). \\u003cstrong\\u003eC: \\u003c/strong\\u003eRepresentative images of HE-stained sections of MDA-MB-231 tumors from vehicle- and aloperine-treated mice. Scale bars: 150 μm. \\u003cstrong\\u003eD: \\u003c/strong\\u003eNecrotic area (% of total tumor area) in MDA-MB-231 tumors depicted in (C), as assessed by HE staining (n = 8). \\u003cstrong\\u003eE:\\u003c/strong\\u003e Representative images of Ki67-stained sections of MDA-MB-231 tumors from vehicle- and aloperine-treated mice. Scale bars: 70 μm. \\u003cstrong\\u003eF: \\u003c/strong\\u003eKi67\\u003csup\\u003e+\\u003c/sup\\u003e tumor cells (% of total cell number) in MDA-MB-231 tumors depicted in (E), as assessed by immunohistochemical staining (n = 8). \\u003cstrong\\u003eG:\\u003c/strong\\u003e Representative images of cleaved caspase 3-stained sections of MDA-MB-231 tumors from vehicle- and aloperine-treated mice. Scale bars: 70 μm. \\u003cstrong\\u003eH: \\u003c/strong\\u003eCleaved caspase-3\\u003csup\\u003e+\\u003c/sup\\u003e tumor cells (% of total cell number) in MDA-MB-231 tumors depicted in (G), as assessed by immunohistochemical staining (n = 8). \\u003cstrong\\u003eI:\\u003c/strong\\u003e Representative images of CD31 and PAS double-stained sections of MDA-MB-231 tumors from vehicle- and aloperine-treated mice. Scale bars: 40 μm. \\u003cstrong\\u003eJ: \\u003c/strong\\u003eDensity (mm\\u003csup\\u003e-2\\u003c/sup\\u003e) of\\u003cstrong\\u003e \\u003c/strong\\u003eCD31\\u003csup\\u003e+\\u003c/sup\\u003ePAS\\u003csup\\u003e+ \\u003c/sup\\u003eEC-lined vessels (black arrowheads) and CD31\\u003csup\\u003e-\\u003c/sup\\u003ePAS\\u003csup\\u003e+ \\u003c/sup\\u003eVM structures (red arrowhead) in MDA-MB-231 tumors depicted in (I), as assessed by CD31 and PAS double staining (n = 8). \\u003cstrong\\u003eK:\\u003c/strong\\u003e Representative Western blots showing p-FGFR1, FGFR1, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin expression in MDA-MB-231 tumors from vehicle- and aloperine-treated mice. \\u003cstrong\\u003eL:\\u003c/strong\\u003e Expression levels (% of vehicle) of p-FGFR1/FGFR1, p-JAK2/JAK2, and p-STAT3/STAT3 in MDA-MB-231 tumors from vehicle- and aloperine-treated mice, as assessed by Western blotting (n = 7). Data are presented as Means ± SEM. *\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; ns, not significant.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9209927/v1/41df82720b4dc58e3ff1e876.png\"},{\"id\":106835744,\"identity\":\"f925d3fa-0dcf-4c4e-a0e4-9533602df40c\",\"added_by\":\"auto\",\"created_at\":\"2026-04-14 02:02:26\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":628740,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"supplementaryinformation.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9209927/v1/0ee9edf890a8d16752928557.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Targeting FGFR1 with aloperine suppresses angiogenesis, vasculogenic mimicry, and metastasis in triple-negative breast cancer\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eTriple-negative breast cancer (TNBC), characterized by the lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, represents the most aggressive and lethal subtype of breast cancer [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Due to its high heterogeneity and the absence of actionable molecular targets, systemic chemotherapy remains the cornerstone of treatment [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. Despite initial treatment responses, TNBC is frequently associated with early recurrence and a high risk of metastasis, leading to poor clinical outcomes following failure of first-line therapies [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. These challenges highlight an urgent clinical need to identify novel molecular drivers of TNBC progression and to develop targeted therapeutic strategies to improve patient outcomes.\\u003c/p\\u003e \\u003cp\\u003eA hallmark of TNBC malignancy is its robust and aberrant vascularization [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Angiogenesis, the formation of new blood vessels from pre-existing ones, constitutes the primary mechanism of tumor vascularization [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. During this process, endothelial cells (ECs) lining the blood vessels are activated by pro-angiogenic factors in the tumor microenvironment to migrate, proliferate, form tubular structures, and organize into functional microvessels [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Beyond classical angiogenesis, TNBC frequently exhibits vasculogenic mimicry (VM), a process whereby aggressive tumor cells acquire EC-like properties and form vessel-like channels independent of ECs [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. This dual-vascular landscape not only supports rapid primary tumor growth but also provides direct routes for hematogenous dissemination, thereby promoting tumor metastasis. Of note, the anti-angiogenic drug sunitinib has been reported to paradoxically enhance TNBC metastasis by promoting VM formation, which may potentially explain the limited efficacy of such type of agents in clinical trials [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Thus, the simultaneous inhibition of both angiogenesis and VM represents a promising therapeutic strategy to disrupt the TNBC blood supply and impede disease progression.\\u003c/p\\u003e \\u003cp\\u003eFibroblast growth factor receptor 1 (FGFR1) has been identified as an independent negative prognostic factor for overall survival in TNBC [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. It belongs to a conserved family of transmembrane receptor tyrosine kinases (RTKs) comprising FGFR1-4. Upon binding of fibroblast growth factors (FGFs), FGFR1 undergoes dimerization and autophosphorylation, thereby activating multiple downstream signaling cascades, including Janus kinase (JAK)/signal transducer and activator of transcription (STAT), rat sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. These pathways are fundamental to tumor cell survival, proliferation, migration, and differentiation [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Beyond its role in tumor cells, FGFR1 is highly expressed in ECs, where it plays a central role in the regulation of angiogenesis [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. In contrast, its specific contribution to VM remains largely unknown. Given its dual relevance in the tumor parenchyma and the vascular compartment, FGFR1 represents an attractive and potentially unifying therapeutic target for TNBC.\\u003c/p\\u003e \\u003cp\\u003eIn the pursuit of novel therapeutics, phytochemicals offer a valuable reservoir of bioactive compounds characterized by extensive structural diversity, broad availability, cost-effectiveness, and generally favorable safety profiles [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. Indeed, approximately 50% of all anticancer drugs currently on the market are derived from or inspired by natural compounds [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. Aloperine, a quinolizidine alkaloid derived from the medicinal plant \\u003cem\\u003eSophora alopecuroides L\\u003c/em\\u003e., has attracted growing interest due to its diverse pharmacological properties, including anti-inflammatory, antioxidant, antibacterial, and antitumor activities [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. While accumulating evidence indicates that aloperine suppresses tumor growth across multiple cancer types [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], its therapeutic potential and underlying mechanism of action in TNBC remain largely unexplored.\\u003c/p\\u003e \\u003cp\\u003eIn the present study, we systematically investigated the antitumor activity of aloperine in TNBC with a particular focus on both endothelial and tumor cells. We first compared the effects of aloperine on the viability of several types of human primary ECs and TNBC cell lines. At sub-cytotoxic concentrations, we then examined its impact on key angiogenic functions of human umbilical vein endothelial cells (HUVECs) and subsequently validated its anti-angiogenic effects in \\u003cem\\u003eex vivo\\u003c/em\\u003e aortic ring assays and \\u003cem\\u003ein vivo\\u003c/em\\u003e Matrigel plug models. In parallel, we investigated the effects of aloperine on TNBC cell proliferation, migration, tube formation, and spheroid sprouting. We further elucidated the precise molecular mechanisms of aloperine action. Finally, the effects of aloperine on tumor angiogenesis, VM, growth, and metastasis in TNBC were evaluated using a mouse dorsal skinfold chamber model and an orthotopic xenograft model.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Study design\\u003c/h2\\u003e \\u003cp\\u003eThe sample size for each experiment was determined based on previous publications. For \\u003cem\\u003ein vitro\\u003c/em\\u003e assays, at least 3 independent experiments were performed, each comprising a minimum of 3 biological replicates (i.e., independent cell cultures). For mouse experiments, each group included 6\\u0026ndash;8 animals. Randomization was performed for group allocation in both the dorsal skinfold chamber model and the orthotopic xenograft model. Investigators were blinded to group assignments during data analysis. No samples or animals were excluded from the analysis. The exact n values for each experiment are provided in the corresponding figure legends.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Chemicals\\u003c/h2\\u003e \\u003cp\\u003eAloperine, the selective JAK1/2 inhibitor ruxolitinib, the selective JAK3 inhibitor ritlecitinib, the multi-kinase inhibitor lenvatinib (primarily targeting vascular endothelial growth factor receptor 2 (VEGFR2)), and basic fibroblast growth factor (bFGF) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). The selective FGFR inhibitor PD173074 was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Dimethyl sulfoxide (DMSO) was purchased from PanReac AppliChem (Darmstadt, Germany).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Cell culture\\u003c/h2\\u003e \\u003cp\\u003eHUVECs and human dermal microvascular endothelial cells (HDMECs) were purchased from PromoCell (Heidelberg, Germany) and cultured in Endothelial Cell Basal Medium (EBM; PromoCell) and EBM-MV, respectively, both supplemented with SupplementMix. The luciferase-expressing murine TNBC cell line 4T1-Luc2 (RRID: CVCL_A4BM) and the human TNBC cell line HCC1937 (RRID: CVCL_0290) were purchased from ATCC (Wesel, Germany) and cultured in RPMI 1640 medium (PAN-Biotech GmbH, Aidenbach, Germany) containing 10% fetal calf serum (FCS; PAN-Biotech), 100 U/mL penicillin (PAN-Biotech), and 0.1 mg/mL streptomycin (PAN-Biotech). The luciferase- and green fluorescent protein (GFP)-expressing human TNBC cell line MDA-MB-231-Luc-GFP (RRID: CVCL_C9CE) was purchased from GeneCopoeia (Heidelberg, Germany) and cultured in Dulbecco's modified Eagle's medium (DMEM; PAN-Biotech) containing 10% FCS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. All cell lines were cultured at 37\\u0026deg;C in a humidified incubator with 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. Cell transfection\\u003c/h2\\u003e \\u003cp\\u003eTo knock down suppressor of cytokine signaling 1 (SOCS1) and SOCS3, HUVECs were transfected with 100 nM small interfering RNAs (siRNAs) targeting SOCS1 (si-SOCS1; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) or SOCS3 (si-SOCS3; Sigma-Aldrich) using HiPerFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer\\u0026rsquo;s instructions. Negative Control siRNA (si-NC; Qiagen) was used as a control. After 48 h of transfection, cells were trypsinized, counted, and equal cell numbers from each group were used for subsequent assays.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Lactate dehydrogenase (LDH) assay\\u003c/h2\\u003e \\u003cp\\u003eThe cytotoxicity of aloperine was assessed using an LDH assay according to the manufacturer\\u0026rsquo;s protocol (Roche Diagnostics, Mannheim, Germany). For this purpose, HUVECs (4 \\u0026times; 10\\u0026sup3; cells/well) were seeded into 96-well plates and then exposed to a serial dilution of aloperine for 24 h. Following treatment, 100 \\u0026micro;L of reaction solution containing the catalyst and dye was added to each well. After incubation of the plate at room temperature for 10 min, the reaction was terminated by adding 50 \\u0026micro;L of stop solution. Absorbance was measured at 492 nm, with 620 nm as the reference wavelength, using a microplate photometer (PHOmo; anthos Mikrosysteme GmbH, Krefeld, Germany). Cytotoxicity was calculated using the formula: Cytotoxicity (%) = (OD\\u003csub\\u003esample\\u003c/sub\\u003e-OD\\u003csub\\u003e0\\u0026micro;M\\u003c/sub\\u003e) / (OD\\u003csub\\u003ehigh control\\u003c/sub\\u003e-OD\\u003csub\\u003e0\\u0026micro;M\\u003c/sub\\u003e) \\u0026times; 100. The high control, representing total cell death, was established by treating cells with 5 \\u0026micro;L of lysis solution.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6. Bromodeoxyuridine (BrdU) incorporation assay\\u003c/h2\\u003e \\u003cp\\u003eCell proliferation was assessed using a BrdU incorporation assay. Briefly, HUVECs (2.5 \\u0026times; 10\\u003csup\\u003e5\\u003c/sup\\u003e cells/well) or MDA-MB-231-Luc-GFP cells (1.5 \\u0026times; 10\\u003csup\\u003e5\\u003c/sup\\u003e cells/well) were seeded in 6-well plates and then treated with various concentrations of aloperine for 6 h. BrdU reagent was then added to each well at a final concentration of 10 \\u0026micro;M, and incubation was continued for an additional 18 h. Cells were then fixed in 70% ethanol on ice for 30 min, followed by denaturation in 2 M hydrochloric acid containing 0.5% Triton X-100 at room temperature for 30 min. Afterwards, the cells were stained with a fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody (1:30; 11-5071-42; RRID: AB_11042627; Thermo Fisher Scientific, Karlsruhe, Germany) at room temperature for 1 h. The percentage of FITC\\u003csup\\u003e+\\u003c/sup\\u003e proliferating cells was quantified by flow cytometry using a FACSLyric flow cytometer (BD Biosciences, Heidelberg, Germany).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7. Transwell migration assay\\u003c/h2\\u003e \\u003cp\\u003eCell migratory capacity was assessed in a Transwell migration assay using inserts with an 8-\\u0026micro;m pore size (Corning, Merck KGaA, Darmstadt, Germany). Prior to the assay, HUVECs or MDA-MB-231-Luc-GFP cells were treated with various concentrations of aloperine for 18 h. Afterwards, HUVECs (5 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e cells) or MDA-MB-231-Luc-GFP cells (3.5 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e cells) suspended in 500 \\u0026micro;L of serum-free EBM or DMEM were seeded into the upper inserts, while 750 \\u0026micro;L of EBM or DMEM supplemented with 1% FCS was added to the lower inserts as a chemoattractant. After 5 h of incubation, non-migrated cells on the upper surface of the inserts were carefully removed using a cotton swab. Migrated cells were then stained with Diff-Quick (LT-SYS Diagnostika, Berlin, Germany). Subsequently, membranes were cut out from the inserts using a surgical scalpel and mounted on glass slides with glycerol gelatin (Sigma-Aldrich). At least 20 non-overlapping fields of each membrane were randomly imaged at a 200-fold magnification using a phase-contrast microscope (BZ-X810; Keyence, Osaka, Japan). The number of migrated cells in each field was quantified using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.8. Tube formation assay\\u003c/h2\\u003e \\u003cp\\u003eThe effect of aloperine on the formation of capillary-like structures was assessed using a tube formation assay. For this purpose, Matrigel\\u0026reg; Basement Membrane Matrix (Corning) was thawed overnight at 4\\u0026deg;C. Using pre-cooled pipette tips, 50 \\u0026micro;L of Matrigel was dispensed into each well of a 96-well plate and allowed to polymerize at 37\\u0026deg;C for 15 min. Then, HUVECs (1.7 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e cells) or MDA-MB-231-Luc-GFP cells (6 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e cells) suspended in 100 \\u0026micro;L of EGM or DMEM containing various concentrations of aloperine were seeded onto the polymerized gel. After 18 h of incubation, images of capillary-like networks were captured at a 40-fold magnification using a phase-contrast microscope (BZ-X810). The number of tube meshes was quantified using ImageJ software with the Angiogenesis Analyzer plugin.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.9. Spheroid sprouting assay\\u003c/h2\\u003e \\u003cp\\u003eThe effect of aloperine on the outgrowth of cells from spheroids into the surrounding matrix was assessed using a spheroid sprouting assay. Briefly, 500 HUVECs or MDA-MB-231-Luc-GFP cells in 50 \\u0026micro;L of EGM containing 0.24% methylcellulose (Thermo Fisher Scientific) were seeded into each well of non-adherent 96-well round-bottom plates (Greiner Bio-One, Frickenhausen, Germany) and incubated overnight to generate spheroids. Subsequently, spheroids were collected and resuspended in 300 \\u0026micro;L of a collagen solution, diluted 1:1 with EBM containing 20% FCS and 0.5% methylcellulose. The collagen solution was prepared with rat acidic collagen extract (4 mg/mL; Advanced Biomatrix, Carlsbad, USA), H\\u003csub\\u003e2\\u003c/sub\\u003eO, 10 \\u0026times; Medium 199 (Sigma-Aldrich), and 0.2 M sodium hydroxide at a 4:4:1:1 ratio. The spheroid mixture was then rapidly transferred into a pre-warmed 24-well plate and allowed to polymerize at 37\\u0026deg;C for 45 min, after which 500 \\u0026micro;L of EGM containing various concentrations of aloperine was gently overlaid onto the gel. After 24 h of incubation, images of spheroids were captured at a 40-fold magnification using a phase-contrast microscope (BZ-X810), and the cumulative sprout length was measured using ImageJ software.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.10. Aortic ring assay\\u003c/h2\\u003e \\u003cp\\u003eThe effect of aloperine on aortic sprouting was assessed using an aortic ring assay. Briefly, thoracic aortas from 8-week-old male BALB/c mice (RRID: IMSR_RJ: BALB-CANNRJ; Janvier-Labs, Le Genest, France) were cut into rings about 1 mm in length. In parallel, 96-well plates were pre-coated with 40 \\u0026micro;L of Matrigel (Corning) and incubated at 37\\u0026deg;C to allow polymerization. The aortic rings were then placed onto the pre-coated Matrigel and overlaid with an additional 40 \\u0026micro;L of Matrigel. After Matrigel solidification, the rings were cultured in 100 \\u0026micro;L of DMEM supplemented with 10% FCS and various concentrations of aloperine. After 6 days of culture, with a medium change on day 3, images of aortic rings were captured using a phase-contrast microscope (BZ-X810). The area of vascular sprouting was measured using the corresponding image analysis software (Keyence).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.11. Western blotting\\u003c/h2\\u003e \\u003cp\\u003eCells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Cell lysates were then centrifuged at 12,000 \\u0026times; \\u003cem\\u003eg\\u003c/em\\u003e for 30 min at 4\\u0026deg;C and the supernatants were collected. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) according to the manufacturer\\u0026rsquo;s instructions. Equal amounts of protein (10 \\u0026micro;g) were separated on 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (BioRad, Munich, Germany). The membranes were further blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich) at room temperature for 1 h and incubated overnight at 4\\u0026deg;C with the following primary antibodies: a rabbit anti-phosphorylated-mammalian target of rapamycin (p-mTOR) antibody (1:300; 5536; RRID: AB_10691552; Cell Signaling Technology, Frankfurt, Germany), a rabbit anti-mTOR antibody (1:300; 2983; RRID: AB_2105622; Cell Signaling Technology), a rabbit anti-p-STAT5 antibody (1:300; 9351; RRID: AB_2315225; Cell Signaling Technology), a rabbit anti-STAT5 antibody (1:300; 25656; RRID: AB_2798908; Cell Signaling Technology), a rabbit anti-p-STAT3 antibody (1:100; 9145; RRID: AB_2491009; Cell Signaling Technology), a mouse anti-STAT3 antibody (1:300; 9139; RRID: AB_331757; Cell Signaling Technology), a rabbit anti-p-AKT antibody (1:300; 4060; RRID: AB_2315049; Cell Signaling Technology), a rabbit anti-AKT antibody (1:300; 4685; RRID: AB_2225340; Cell Signaling Technology), a rabbit anti-p-ERK antibody (1:300; 4370; RRID: AB_2315112; Cell Signaling Technology), a rabbit anti-ERK antibody (1:300; 4695; RRID: AB_390779; Cell Signaling Technology), a rabbit anti-p-FAK antibody (1:250; 8556; RRID: AB_10891442; Cell Signaling Technology), a rabbit anti-FAK antibody (1:250; 3285; RRID: AB_2269034; Cell Signaling Technology), a rabbit anti-p-JAK2 antibody (1:100; 3776; RRID: AB_2617123; Cell Signaling Technology), a rabbit anti-JAK2 antibody (1:200; 3230; RRID: AB_2128522; Cell Signaling Technology), a rabbit anti-p-Src antibody (1:300; 6943; RRID: AB_10013641; Cell Signaling Technology), a rabbit anti-Src antibody (1:300; 2109; RRID: AB_2106059; Cell Signaling Technology), a rabbit anti-SOCS1 antibody (1:100; ab280886; RRID: AB_2938872; Abcam, Cambridge, UK), a rabbit anti-SOCS3 antibody (1:100; 14025-1-AP; RRID: AB_10597854; Proteintech, Munich, Germany), a rabbit anti-p-FGFR1 antibody with human species reactivity (1:100; ab173305; RRID: AB_3094883; Abcam), a rabbit anti-p-FGFR1 antibody with mouse species reactivity (1:100; AP1317; Abclonal, D\\u0026uuml;sseldorf, Germany), a rabbit anti-FGFR1 antibody (1:100; 9740; RRID: AB_11178519; Cell Signaling Technology), a rabbit monoclonal anti-p-VEGFR2 antibody (1:250; 2478; RRID: AB_331377; Cell Signaling Technology), a rabbit monoclonal anti-VEGFR2 antibody (1:250; 9698; RRID: AB_11178792; Cell Signaling Technology), and a mouse horseradish peroxidase (HRP)-conjugated anti-β-actin antibody (1:1000; HRP-66009; RRID: AB_2883836; Proteintech). Subsequently, membranes were washed 3 times with Tris-buffered saline containing 0.1% Tween 20, and then incubated with HRP-conjugated anti-mouse (1:1000; HAF007; RRID: AB_357234; R\\u0026amp;D Systems, Wiesbaden, Germany) or anti-rabbit (1:1000; HAF008; RRID: AB_357235; R\\u0026amp;D Systems) secondary antibodies at room temperature for 1 h. Chemical signals were visualized using the enhanced chemiluminescence kit (BioRad) and images were acquired using a ChemoCam Imager (Intas, G\\u0026ouml;ttingen, Germany). Protein expression was quantified using ImageJ software.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.12. Water-soluble tetrazolium (WST)-1 assay\\u003c/h2\\u003e \\u003cp\\u003eThe effect of aloperine on cell viability was assessed using a WST-1 assay. For this purpose, cells of different types were seeded into 96-well plates at a density of 2\\u0026ndash;3 \\u0026times; 10\\u0026sup3; cells/well and then exposed to a serial dilution of aloperine for 24 h or 48 h. After treatment, 10 \\u0026micro;L of WST-1 reagent (Roche Diagnostics) was added to each well, followed by incubation at 37\\u0026deg;C for 30 min. Then, the absorbance of each sample was measured at 450 nm with 620 nm as reference using a microplate photometer (PHOmo).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.13. Blind molecular docking\\u003c/h2\\u003e \\u003cp\\u003eThe human FGFR1 structure (PDB ID: 5A46) was obtained from the Protein Data Bank (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.rcsb.org\\u003c/span\\u003e\\u003cspan address=\\\"https://www.rcsb.org\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) and processed in UCSF ChimeraX (v1.8) by removing crystallographic waters and non-protein heteroatoms. The receptor was prepared in AutoDockTools (v1.5.7) by adding polar hydrogens and assigning Gasteiger charges, and then exported as a PDBQT file. The three-dimensional (3D) structure of aloperine was downloaded from PubChem (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://pubchem.ncbi.nlm.nih.gov\\u003c/span\\u003e\\u003cspan address=\\\"https://pubchem.ncbi.nlm.nih.gov\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), optimized in Avogadro 2 (v1.99.0), and converted to PDBQT format in AutoDockTools after defining rotatable bonds. Blind docking was carried out in AutoDock 4.2 using the Lamarckian genetic algorithm with a grid covering the entire FGFR1 structure (spacing: 0.5 \\u0026Aring;). A total of 100 independent docking runs were performed with standard settings (population size: 150; energy evaluations: 2,500,000). Resulting poses were clustered based on root-mean square deviation, and the lowest-energy pose was selected for subsequent analyses. Binding free energies (ΔG, kcal/mol) were obtained from AutoDock 4.2, with protein-ligand interactions analyzed in BIOVIA Discovery Studio Visualizer 2025 and visualized in UCSF ChimeraX.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.14. Cell-free kinase assay\\u003c/h2\\u003e \\u003cp\\u003eThe effects of aloperine on the activity of multiple kinases were evaluated in a cell-free kinase assay, as previously described [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e], with minor modifications. All assays were performed in white 96-well flat-bottom plates using the ADP-Glo\\u0026trade; Kinase Assay and the Kinase Enzyme System (Promega, Walldorf, Germany). Each 20 \\u0026micro;L reaction mixture contained recombinant kinase enzymes (40 ng JAK2, 3 ng JAK3, 8 ng VEGFR2, or 3 ng FGFR1), 0.2 \\u0026micro;g/\\u0026micro;L poly (4:1 Glu, Tyr) peptide substrate, 100 \\u0026micro;M aloperine, specific kinase inhibitors (3 nM ruxolitinib, 40 nM ritlecitinib, 1 nM lenvatinib, or 10 nM PD173074), and ATP at a final concentration of 10 \\u0026micro;M. For ATP competition experiments, the reaction mixture contained 3 ng FGFR1, 0.2 \\u0026micro;g/\\u0026micro;L peptide substrate, 100 \\u0026micro;M aloperine or 10 nM PD173074, 10 or 500 \\u0026micro;M ATP, respectively. After incubation at room temperature for 60 min, 20 \\u0026micro;L of ADP-Glo\\u0026trade; Reagent was added to each well, followed by a 40-min incubation at room temperature. Subsequently, 40 \\u0026micro;L of Kinase Detection Reagent was added to each well, followed by another 40-min incubation at room temperature. Kinase activity was then quantified using a luciferase/luciferin reaction on a Tecan Infinite M200 PRO luminometer (Tecan, Crailsheim, Germany), expressed as a percentage relative to the control.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.15. Animal experiments\\u003c/h2\\u003e \\u003cp\\u003eThe mice were housed in a conventional animal facility (Institute for Clinical and Experimental Surgery, Saarland University, Homburg, Germany) under a standard 12-h light/dark cycle, with standard pellet chow (ssniff Spezialdi\\u0026auml;ten GmbH, Soest, Germany) and water provided ad libitum.\\u003c/p\\u003e \\u003cp\\u003eThe \\u003cem\\u003ein vivo\\u003c/em\\u003e effects of aloperine on angiogenesis were evaluated using a Matrigel plug assay following an established protocol [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Briefly, a mixture containing 250 \\u0026micro;L growth factor-reduced Matrigel (Corning), 1 \\u0026micro;g/mL vascular endothelial growth factor (VEGF; R\\u0026amp;D Systems), 1 \\u0026micro;g/mL bFGF (R\\u0026amp;D Systems), 50 IU/mL heparin (B. Braun, Melsungen, Germany), 0.1% DMSO (vehicle) or 100 \\u0026micro;M aloperine was injected subcutaneously into the flanks of 3-month-old male BALB/c mice (25\\u0026ndash;30 g; Janvier-Labs; n\\u0026thinsp;=\\u0026thinsp;6 per group) under inhalation anesthesia with isoflurane (5% for induction and 2% for maintenance). Matrigel plugs were removed after 7 days for immunohistochemical analyses.\\u003c/p\\u003e \\u003cp\\u003eThe effect of aloperine on the vascularization and growth of murine TNBC was assessed using a dorsal skinfold chamber model as previously described [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. First, tumor spheroids were generated by seeding 4T1-Luc2 (5 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well) into 96-well plates pre-coated with 1% agarose and cultured for 3 days. One day after cell seeding, dorsal skinfold chambers were implanted in 3-month-old female BALB/c mice (22\\u0026ndash;25 g; Janvier-Labs). After another 2 days, one 4T1-Luc2 spheroid stained with Hoechst 33342 was transplanted into each chamber. All the mice were randomly assigned to two groups (n\\u0026thinsp;=\\u0026thinsp;8 per group) and received daily intraperitoneal injections of 75 mg/kg body weight aloperine or a vehicle solution (5% DMSO, 5% Tween 80, and 90% saline) for 14 consecutive days. Intravital fluorescence microscopy was performed on days 0, 3, 6, 10, and 14 after spheroid implantation, using a charge-coupled device video camera (FK6990; Pieper, Schwerte, Germany) and a DVD system to record the microscopy images. The recordings were then analyzed using CapImage (Zeintl, Heidelberg, Germany) to quantify tumor size (mm\\u0026sup2;), functional microvessel density (cm/cm\\u0026sup2;), vessel diameter (D; \\u0026micro;m), and the vessel centerline red blood cell (RBC) velocity (V; mm/s). Furthermore, the volumetric blood flow (Qv; pL/s) of the tumor vessels was calculated using the formula: Qv\\u0026thinsp;=\\u0026thinsp;π \\u0026times; (D/2)\\u003csup\\u003e2\\u003c/sup\\u003e\\u0026times; V/1.3. After the last microscopy on day 14, the mice were euthanized by cervical dislocation and the tumor tissues were carefully excised for further histological and immunohistochemical analyses. Chamber implantation, spheroid transplantation, and intravital microscopy were performed under anesthesia induced by intraperitoneal injection of ketamine (Ketabel\\u0026reg;; 90 mg/kg body weight; bela-pharm GmbH, Vechta, Germany) and xylazine (Rompun\\u0026reg;; 12 mg/kg body weight; Bayer, Leverkusen, Germany). For post-operative analgesia following chamber implantation, the mice received a subcutaneous injection of carprofen (Rimadyl\\u0026reg;; 10 mg/kg body weight; Cp-Pharma, Burgdorf, Germany).\\u003c/p\\u003e \\u003cp\\u003eThe effect of aloperine on the vascularization, growth, and metastasis of human TNBC was assessed using an orthotopic xenograft model. For this purpose, 5 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e MDA-MB-231-Luc-GFP cells suspended in 50 \\u0026micro;L PBS were injected into the left fourth mammary fat pad of 6-week-old female NOD-SCID mice (22\\u0026ndash;25 g; RRID: IMSR_RJ: NOD-SCID; Janvier-Labs). When the tumor became palpable on day 3, all the mice were randomly divided into two groups (n\\u0026thinsp;=\\u0026thinsp;8 per group) and received daily intraperitoneal injections of 50 mg/kg body weight aloperine or a vehicle solution (5% DMSO, 5% Tween 80, and 90% saline) until 6 weeks after tumor inoculation. During this period, caliper measurements were performed weekly to monitor the tumor volume using the formula: V\\u0026thinsp;=\\u0026thinsp;0.5 \\u0026times; length \\u0026times; width\\u003csup\\u003e2\\u003c/sup\\u003e. Tumor growth was also monitored weekly by bioluminescence imaging (BLI) using an IVIS Spectrum imaging system (PerkinElmer, MA, USA). For this imaging, mice were injected intraperitoneally with 150 mg/kg body weight D-luciferin (122799; PerkinElmer) and then anesthetized with isoflurane (5% for induction and 2% for maintenance). Bioluminescent images were captured 17 min after D-luciferin injection and analyzed with the Living Image Software (PerkinElmer) to quantify the total flux within regions corresponding to the primary tumor and lung metastases. Of note, for metastasis evaluation, the primary tumor was covered with a black cloth during imaging to minimize signal interference. On day 42 after tumor inoculation, lungs were collected and incubated with 300 \\u0026micro;g/mL D-luciferin in PBS for 1 min before assessing distant metastasis using the IVIS system. Additionally, tumor tissues were harvested, weighed, photographed, and processed for further immunohistochemical and Western blot analyses.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.16. Circulating tumor cell (CTC) detection\\u003c/h2\\u003e \\u003cp\\u003eBlood samples (1 mL) were collected from the abdominal aorta of mice under anesthesia with ketamine and xylazine and then incubated with 1 mL of BD Pharm Lyse\\u0026trade; (BD Biosciences) at room temperature to lyse RBCs. The remaining cells were then washed twice with cold PBS and resuspended in PBS containing 2% FCS. The number of CTCs (MDA-MB-231-Luc-GFP cells) per 10,000 events in blood samples was quantified by flow cytometry based on GFP fluorescence using a FACSLyric flow cytometer (BD Biosciences).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.17. Histology and immunohistochemistry\\u003c/h2\\u003e \\u003cp\\u003eThe collected Matrigel plugs, tumor samples, and lung tissues were fixed in 4% formalin, dehydrated in ethanol, and embedded in paraffin. Subsequently, 3-\\u0026micro;m-thick sections were serially cut and mounted onto slides for histological and immunohistochemical analyses.\\u003c/p\\u003e \\u003cp\\u003eTo visualize microvessels in Matrigel plugs, sections were incubated overnight at 4℃ with a rabbit anti-mouse CD31 antibody (1:100; ab182981; RRID: AB_2920881; Abcam). Then, they were incubated with a goat anti-rabbit Alexa Fluor 555-conjugated secondary antibody (1:100; A27039; RRID: AB_2536100; Thermo Fisher Scientific) and counterstained with Hoechst 33342. The entire area of each plug was imaged at a 400-fold magnification using a BX-60 microscope (Olympus, Tokyo, Japan). CD31\\u003csup\\u003e+\\u003c/sup\\u003e microvessels in each field were quantified using ImageJ software.\\u003c/p\\u003e \\u003cp\\u003eTo assess tumor size and necrotic area, tissue sections with the largest tumor area in the vertical cross-section were selected and stained with hematoxylin and eosin (HE). The entire tumor was imaged at a 20-fold magnification using a phase-contrast microscope (BZ-X810) and analyzed using the corresponding image analysis software (Keyence).\\u003c/p\\u003e \\u003cp\\u003eTo evaluate tumor cell proliferation and apoptosis, tissue sections were sequentially incubated with either a rabbit anti-mouse Ki67 antibody (1:400; 12202; RRID: AB_2620142; Cell Signaling Technology) or a rabbit anti-mouse cleaved caspase-3 antibody (1:100; 9661; RRID: AB_2341188; Cell Signaling Technology), followed by a biotinylated goat anti-rabbit secondary antibody (1:100; ab64256; RRID: AB_2661852; Abcam), peroxidase-conjugated streptavidin (ready-to-use; Abcam), and 3‐amino‐9‐ethylcarbazole substrate (Abcam). At last, sections were counterstained with Mayer\\u0026rsquo;s hemalum solution (Merck KGaA). The entire tumor was imaged at a 400-fold magnification using a BX‐60 microscope (Olympus). The percentage of Ki67\\u003csup\\u003e+\\u003c/sup\\u003e proliferating and cleaved caspase-3\\u003csup\\u003e+\\u003c/sup\\u003e apoptotic tumor cells was quantified using ImageJ software.\\u003c/p\\u003e \\u003cp\\u003eTo detect angiogenesis and VM in tumor tissues, CD31 and periodic acid-Schiff (PAS) double staining was conducted. Briefly, sections were incubated with a rabbit anti-mouse CD31 antibody (1:100; ab182981; RRID: AB_2920881; Abcam), followed by a biotinylated goat anti-rabbit secondary antibody (1:100; ab64256; RRID: AB_2661852; Abcam), peroxidase-conjugated streptavidin (Abcam), and 3-amino-9-ethylcarbazole substrate (Abcam). Afterwards, they were exposed to periodic acid and Schiff reagent (Sigma-Aldrich) and counterstained with Mayer\\u0026rsquo;s hemalum solution (Merck KGaA). The entire tumor was imaged at a 400-fold magnification using a BX-60 microscope (Olympus). The density of CD31\\u003csup\\u003e+\\u003c/sup\\u003e PAS\\u003csup\\u003e+\\u003c/sup\\u003e EC-lined vessels and CD31\\u003csup\\u003e\\u0026minus;\\u003c/sup\\u003e PAS\\u003csup\\u003e+\\u003c/sup\\u003e VM structures was quantified using ImageJ software.\\u003c/p\\u003e \\u003cp\\u003eTo detect metastatic foci in the lungs, lung sections were stained with a goat anti-GFP antibody (1:100; 600-101-215; RRID: AB_218200; Rockland Immunochemicals, PA, USA) and counterstained with Hoechst 33342. Metastatic foci derived from MDA-MB-231-Luc-GFP cells were identified based on their GFP signal using fluorescence microscopy. Multiple random fields from each lung section were imaged at a 400-fold magnification using a BX-60 microscope (Olympus). The number of GFP\\u003csup\\u003e+\\u003c/sup\\u003e metastatic foci in each field was quantified using ImageJ software.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.18. Statistics\\u003c/h2\\u003e \\u003cp\\u003eStatistical evaluations were performed using GraphPad Prism (v10.4.1). Data normality and homogeneity of variance were assessed using the Shapiro-Wilk and Brown-Forsythe tests, respectively. For comparisons between two independent groups, a two-tailed unpaired Student\\u0026rsquo;s t-test was employed for normally distributed data, while the Mann-Whitney U test was used for non-parametric datasets (i.e. Western blot data). For multi-group comparisons, one-way analysis of variance (ANOVA) followed by Tukey\\u0026rsquo;s post hoc test was applied to parametric data. Non-parametric multi-group data, including flow cytometry and Western blot results, were analyzed via the Kruskal-Wallis test followed by Dunn\\u0026rsquo;s multiple comparisons test. Data were presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard error of the mean (SEM), and statistical significance was defined as \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 (*\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; **\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01; ***\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1. Aloperine preferentially reduces EC viability\\u003c/h2\\u003e \\u003cp\\u003eTo determine the biological activity of aloperine (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA), we first evaluated its effects on the viability of human primary ECs (HUVECs and HDMECs) and human TNBC cell lines (MDA-MB-231 and HCC1937). WST-1 assays demonstrated that aloperine (10\\u0026ndash;400 \\u0026micro;M) induces a dose-dependent reduction in cell viability across all tested cell types. Notably, ECs exhibited significantly higher sensitivity to aloperine than TNBC cell lines (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB), suggesting a preferential susceptibility of the vascular endothelium to aloperine-mediated growth inhibition relative to the malignant compartment.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. Aloperine inhibits the angiogenic activity of ECs in vitro, ex vivo, and in vivo\\u003c/h2\\u003e \\u003cp\\u003eTo identify sub-cytotoxic concentrations of aloperine for functional studies, LDH assays were performed in HUVECs. Our data showed that aloperine at concentrations up to 400 \\u0026micro;M induces no detectable cytotoxicity after 24 h of treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). Based on this finding, dosages of 25, 50, and 100 \\u0026micro;M were selected for subsequent angiogenesis assays to ensure that the observed effects are independent from cell death.\\u003c/p\\u003e \\u003cp\\u003eBrdU incorporation assays revealed that aloperine significantly reduces the proliferation of HUVECs at a concentration of 100 \\u0026micro;M (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). Moreover, treatment with 100 \\u0026micro;M aloperine resulted in a 48% reduction in HUVEC migration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE, F). Beyond these individual cellular responses, aloperine effectively suppressed complex angiogenic processes, including capillary-like tube formation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eG, H) and 3D spheroid sprouting in a dose-dependent manner (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eI, J).\\u003c/p\\u003e \\u003cp\\u003eTo validate these findings in more physiologically relevant settings, we employed an \\u003cem\\u003eex vivo\\u003c/em\\u003e aortic ring assay. Aloperine dose-dependently suppressed the outgrowth of sprouts from aortic rings, with 100 \\u0026micro;M achieving complete suppression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eK, L). We then assessed the \\u003cem\\u003ein vivo\\u003c/em\\u003e anti-angiogenic potential of aloperine using a Matrigel plug assay. Consistent with our \\u003cem\\u003ein vitro\\u003c/em\\u003e and \\u003cem\\u003eex vivo\\u003c/em\\u003e results, plugs containing 100 \\u0026micro;M aloperine exhibited a markedly reduced CD31⁺ microvessel density compared to vehicle controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eM, N).\\u003c/p\\u003e \\u003cp\\u003eCollectively, these findings establish aloperine as a robust and multi-stage inhibitor of angiogenesis across multiple experimental platforms spanning \\u003cem\\u003ein vitro\\u003c/em\\u003e, \\u003cem\\u003eex vivo\\u003c/em\\u003e, and \\u003cem\\u003ein vivo\\u003c/em\\u003e systems.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3. Aloperine suppresses EC angiogenesis via selective downregulation of the JAK2/STAT3 pathway\\u003c/h2\\u003e \\u003cp\\u003eTo elucidate the molecular mechanisms underlying the anti-angiogenic effects of aloperine, we examined several key angiogenesis-associated signaling pathways, including the PI3K/AKT/mTOR, JAK/STAT, and Ras/Raf/MEK/ERK cascades [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Western blot analyses revealed that treatment of HUVECs with 100 \\u0026micro;M aloperine for 4 h markedly reduces the phosphorylation of STAT3 and AKT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA-F). In contrast, phosphorylation of mTOR, STAT5, and ERK remained largely unaffected (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA-F), suggesting that STAT3 and AKT are the primary signaling nodes modulated by aloperine.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eGiven that JAK2 is a well-established upstream kinase responsible for STAT3 recruitment and activation [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e], while FAK and Src are critical upstream regulators of AKT phosphorylation [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e], we next investigated which of these mediators are responsible for the aloperine-induced reduction in STAT3 and AKT phosphorylation. Western blot analyses demonstrated that aloperine selectively suppresses the phosphorylation of JAK2, whereas the activation status of FAK and Src remained unchanged (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eG-J). These findings suggest that aloperine primarily targets the JAK2/STAT3 axis, and that the observed attenuation of AKT phosphorylation may occur secondary to JAK2/STAT3 inhibition rather than through direct interference with FAK or Src.\\u003c/p\\u003e \\u003cp\\u003eTo determine whether downregulation of JAK2/STAT3 signaling is functionally required for the anti-angiogenic effects of aloperine, we employed a genetic rescue strategy using siRNAs against SOCS1 and SOCS3, endogenous negative regulators of the JAK/STAT pathway [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Transfection of HUVECs with si-SOCS1 or si- SOCS3 efficiently reduced the expression of the respective proteins, resulting in a marked increase in the phosphorylation of JAK2, STAT3, and AKT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eK-O). This indicates that AKT functions downstream of the JAK2/STAT3 axis in this context. Importantly, knockdown of either SOCS1 or SOCS3 significantly rescued aloperine-induced suppression of HUVEC spheroid sprouting (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eP, Q). These findings indicate that selective inhibition of the JAK2/STAT3 signaling axis contributes at least partially to the anti-angiogenic activity of aloperine.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec25\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4. Aloperine suppresses JAK2/STAT3 signaling by targeting FGFR1\\u003c/h2\\u003e \\u003cp\\u003eIn ECs, the JAK/STAT signaling pathway is predominantly regulated by RTKs, among which VEGFR2 and FGFR1 play central roles in both physiological and pathological angiogenesis [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. We therefore investigated whether aloperine suppresses the JAK2/STAT3 axis by targeting either of these receptors. Western blot analyses revealed that the basal phosphorylation level of VEGFR2 in HUVECs is barely detectable, precluding reliable assessment of baseline inhibition. While VEGF stimulation robustly induced VEGFR2 phosphorylation, aloperine did not affect this ligand-dependent activation (Supplementary Fig.\\u0026nbsp;1). In contrast, aloperine significantly reduced the basal phosphorylation of FGFR1 by approximately 51% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA and B). Notably, this inhibitory effect was attenuated in the presence of a high concentration of exogenous bFGF, suggesting a competitive or ligand-sensitive mode of action. Moreover, bFGF stimulation completely abolished aloperine-induced reductions in the phosphorylation of JAK2, STAT3, and AKT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA-E). These results suggest that FGFR1 is the primary upstream target of aloperine in HUVECs, mediating the downregulation of the JAK2/STAT3 signaling cascade and AKT phosphorylation. Functional assays further corroborated these signaling data, as bFGF completely rescued aloperine-suppressed HUVEC spheroid sprouting (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF, G), confirming that FGFR1 is the primary functional target of aloperine in mediating its anti-angiogenic effects.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo determine whether aloperine directly interacts with FGFR1, we next performed molecular docking simulations to explore its potential binding mode. Blind docking analyses revealed that aloperine preferentially binds to the ATP-binding pocket of FGFR1 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eH). This interaction was stabilized by a key hydrogen bond with the ARG646 residue located in the activation loop (A-loop), a critical regulatory element governing kinase catalytic activity and substrate accessibility [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. The predicted binding free energy was \\u0026minus;\\u0026thinsp;7.2 kcal/mol, indicative of a favorable and stable interaction between aloperine and the FGFR1 ATP-binding site.\\u003c/p\\u003e \\u003cp\\u003eTo experimentally validate these \\u003cem\\u003ein silico\\u003c/em\\u003e predictions, we conducted ATP competition assays in HUVECs. Cells were pre-treated with 1 mM ATP, a concentration previously demonstrated to increase intracellular ATP levels by approximately 112% [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e], prior to aloperine exposure. Western blot analysis revealed that elevation of intracellular ATP completely abolishes the inhibitory effects of aloperine on both FGFR1 and JAK2 phosphorylation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eI-K), indicating that aloperine targets FGFR1 through competitive occupancy of its ATP-binding site.\\u003c/p\\u003e \\u003cp\\u003eConsistent with these cellular findings, cell-free kinase assays demonstrated that aloperine selectively inhibits FGFR1 kinase activity without affecting JAK2, JAK3, or VEGFR2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eL). Importantly, the inhibitory effect of aloperine against FGFR1 was completely reversed by the addition of 500 \\u0026micro;M ATP, as shown by ATP competition experiments using the FGFR1 inhibitor PD173074 as a positive control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eM).\\u003c/p\\u003e \\u003cp\\u003eCollectively, these results provide strong evidence that aloperine interacts directly with the ATP-binding site of FGFR1, thereby suppressing the downstream JAK2/STAT3 signaling pathway and inhibiting EC angiogenic activity.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec26\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.5. Aloperine suppreses TNBC cell migration and VM by inhibiting the FGFR1/JAK2/STAT3 signaling pathway\\u003c/h2\\u003e \\u003cp\\u003eWe then investigated the impact of aloperine on the malignant phenotypes of TNBC cells. BrdU incorporation assays revealed that aloperine at 25, 50, and 100 \\u0026micro;M exerts no significant effect on the proliferation of MDA-MB-231 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA), consistent with the viability results shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB. This contrasted with the significant anti-proliferative effect observed in HUVECs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD), suggesting a cell type-specific sensitivity to aloperine. Notably, despite the absence of anti-proliferative effects, aloperine at these non-cytotoxic concentrations significantly impaired the migratory capacity of MDA-MB-231 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB, C). Moreover, aloperine potently inhibited VM across multiple TNBC cell lines, including MDA-MB-231, HCC1937, and 4T1 cells, as assessed by tube formation assays (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD, E and Supplementary Fig.\\u0026nbsp;2), a widely used and well-established \\u003cem\\u003ein vitro\\u003c/em\\u003e method for evaluating tumor cell vascular activity [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Consistently, 3D spheroid sprouting assays demonstrated that aloperine significantly suppresses the invasive outgrowth of sprouts from MDA-MB-231 spheroids (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF, G). Collectively, these findings indicate that aloperine suppresses TNBC cell migration and VM formation independently of its effects on cell proliferation.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo elucidate the molecular mechanisms underlying these effects, we examined the activation status of FGFR1-dependent signaling pathways. Western blot analyses showed that aloperine markedly reduces the phosphorylation of FGFR1, JAK2, and STAT3, whereas Src and AKT phosphorylation remain largely unchanged in MDA-MB-231 cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eH-M), suggesting a selective inhibition of the FGFR1/JAK2/STAT3 axis. To functionally validate the role of FGFR1 in aloperine-mediated inhibition of migration and VM, we performed a rescue experiment using the FGFR1 ligand bFGF. Stimulation with bFGF markedly restored aloperine-suppressed migration and VM formation in MDA-MB-231 cells (Supplementary Fig.\\u0026nbsp;3A, B and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eN, O), supporting the requirement of FGFR1 for the maintenance of these malignant phenotypes.\\u003c/p\\u003e \\u003cp\\u003eFinally, to establish JAK2/STAT3 signaling as the critical downstream mediator of aloperine action, we knocked down SOCS1 using its specific siRNAs in MDA-MB-231 cells prior to aloperine treatment. Efficient SOCS1 knockdown was confirmed by Western blotting (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eP, Q). Importantly, SOCS1 depletion significantly reversed the inhibitory effects of aloperine on both migration and VM formation (Supplementary Fig.\\u0026nbsp;3C, D and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eR, S).\\u003c/p\\u003e \\u003cp\\u003eIn summary, these findings indicate that aloperine exerts its anti-migratory and anti-VM activity in TNBC cells by selectively suppressing the FGFR1/JAK2/STAT3 signaling axis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec27\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.6. Aloperine suppresses TNBC vascularization and growth in a dorsal skinfold chamber model\\u003c/h2\\u003e \\u003cp\\u003eTo evaluate the \\u003cem\\u003ein vivo\\u003c/em\\u003e efficacy of aloperine, we performed a syngeneic mouse dorsal skinfold chamber model of TNBC, which enables real-time monitoring of tumor neovascularization and growth [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. For this, 4T1 tumor spheroids were implanted into dorsal skinfold chambers of BALB/c mice, followed by daily intraperitoneal administration of aloperine (75 mg/kg body weight) or vehicle control. Intravital fluorescence microscopy was employed to track the spatio-temporal dynamics of tumor growth and vascularization over a 14-day period (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). Aloperine treatment was well-tolerated, as evidenced by the absence of significant changes in body weight (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB) or behavioral patterns compared to the control group, suggesting a favorable safety profile at the administered dose. While control tumors exhibited rapid expansion, aloperine-treated tumors remained significantly smaller, with differences detectable as early as day 3 after spheroid transplantation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC, D). Intravital fluorescence microscopy further revealed a marked reduction in the density of functional microvessels, defined by the presence of active blood flow, within the aloperine-treated tumors starting from day 3 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE, F). Quantitative microhemodynamic analyses demonstrated that aloperine significantly reduces microvessel diameter and RBC velocity (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eG, H), resulting in a marked decrease in volumetric blood flow (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eI). These data suggest that aloperine not only reduces microvessel density but also impairs vascular perfusion, thereby disrupting the metabolic supply required for sustained tumor growth.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eHistological and immunohistochemical analyses of harvested tumor tissues confirmed these \\u003cem\\u003ein vivo\\u003c/em\\u003e findings. HE stainings revealed significantly smaller tumor sizes in the aloperine-treated group compared to control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eJ, K). Furthermore, aloperine treatment substantially decreased the proportion of Ki67⁺ proliferating cells while increasing the number of cleaved caspase-3⁺ apoptotic cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eJ-M). To distinguish between conventional angiogenesis and VM, we performed CD31 and PAS double stainings. Aloperine treatment induced a marked reduction in both CD31⁺/PAS⁺ EC-lined vessels and CD31⁻/PAS⁺ VM structures (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eJ, N).\\u003c/p\\u003e \\u003cp\\u003eCollectively, these findings demonstrate that aloperine suppresses TNBC growth by concurrently disrupting angiogenesis and VM-mediated perfusion, thereby limiting tumor nutrient supply and promoting tumor cell apoptosis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec28\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.7. Aloperine suppresses TNBC vascularization, growth, and metastasis in an orthotopic xenograft model\\u003c/h2\\u003e \\u003cp\\u003eTo evaluate the long-term therapeutic potential of aloperine against human TNBC progression, we established an orthotopic xenograft model by injecting MDA-MB-231-Luc-GFP cells into the mammary fat pads of immunodeficient NOD-SCID mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). Daily intraperitoneal administration of aloperine (50 mg/kg body weight) was well tolerated over the 42-day study period, with no significant changes in body weight (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB) or behavioral patterns. Caliper measurements revealed that aloperine significantly inhibits primary tumor growth compared to vehicle-treated controls, with differences observed from day 14 after tumor inoculation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eC). This inhibitory effect was confirmed by \\u003cem\\u003eex vivo\\u003c/em\\u003e analyses at the experimental endpoint, which showed a marked reduction in both tumor volume and weight in aloperine-treated mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eD, E).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eConsistent with the observed tumor growth kinetics, BLI showed that aloperine treatment effectively suppresses the bioluminescent signal of primary tumors from day 21 after tumor inoculation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eF, G). Notably, aloperine also significantly reduced spontaneous lung metastasis, as indicated by substantially lower bioluminescent signals in the thoracic region compared to controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eH, I). This reduction in metastatic burden was confirmed by \\u003cem\\u003eex vivo\\u003c/em\\u003e BLI of harvested lungs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eJ, K) and by immunohistochemical quantification of GFP⁺ metastatic foci, which were significantly less frequent in aloperine-treated lungs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eL, M). To investigate whether this anti-metastatic effect was driven by a reduced systemic dissemination, CTCs were quantified by flow cytometry. Blood from aloperine-treated mice contained a significantly lower proportion of GFP⁺ CTCs, suggesting that aloperine effectively impairs tumor cell intravasation and subsequent hematogenous dissemination (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eN).\\u003c/p\\u003e \\u003cp\\u003eHistological and immunohistochemical analyses of excised primary tumors provided further mechanistic insights. HE stainings revealed smaller tumors with attenuated intratumoral necrotic areas in aloperine-treated mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA-D). Furthermore, aloperine treatment significantly reduced the proportion of Ki67⁺ proliferating cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eE, F), while inducing a modest, non-significant increase in cleaved caspase-3⁺ apoptotic cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eG, H). Importantly, CD31 and PAS double stainings revealed a significant reduction in both CD31⁺/PAS⁺ EC-lined vessels and CD31⁻/PAS⁺ VM structures in aloperine-treated tumors (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eI, J), confirming the dual anti-vascular activity of aloperine within the orthotopic microenvironment.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFinally, we performed Western blot analyses of primary tumor tissues to confirm the molecular drivers of these observations. In agreement with our \\u003cem\\u003ein vitro\\u003c/em\\u003e data, aloperine treatment resulted in a substantial reduction in the phosphorylation of FGFR1, JAK2, and STAT3 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eK, L).\\u003c/p\\u003e \\u003cp\\u003eTaken together, these findings indicate that aloperine suppresses TNBC growth, vascularization, and metastasis through sustained inhibition of the FGFR1/JAK2/STAT3 signaling axis \\u003cem\\u003ein vivo\\u003c/em\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eAloperine is a quinolizidine-type alkaloid enriched in the seeds and leaves of \\u003cem\\u003eSophora alopecuroides L\\u003c/em\\u003e., which has been utilized in traditional medicine for centuries owing to its anti-inflammatory, antioxidant, antibacterial, and anticancer properties [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Different from many natural products, aloperine exhibits high drug-likeness by strictly adhering to Lipinski\\u0026rsquo;s Rule of Five, suggesting superior oral bioavailability and systemic distribution [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Accumulating pharmacological investigations have demonstrated that aloperine exerts broad-spectrum antitumor activity across multiple malignancies, including prostate, lung, thyroid, liver, and colon cancers, primarily through the induction of cell cycle arrest and apoptosis [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. However, its therapeutic potential in TNBC and the underlying mechanisms remain unexplored. This study addresses this gap of knowledge by identifying aloperine as a multimodal inhibitor of TNBC progression. In fact, we could demonstrate that aloperine effectively suppresses angiogenesis, VM, and metastasis in TNBC through selective inhibition of the FGFR1/JAK2/STAT3 signaling axis in both endothelial and tumor compartments.\\u003c/p\\u003e \\u003cp\\u003eA pivotal finding of this study is the cell type-specific sensitivity of aloperine, which distinguishes it from conventional chemotherapeutic agents. While aloperine demonstrates potent anti-neoplastic activity, our data revealed that ECs exhibit a significantly higher susceptibility to aloperine-induced viability inhibition compared to TNBC cells. More importantly, aloperine effectively disrupts key hallmarks of tumor progression, including EC angiogenesis as well as VM and migration of TNBC cells, at sub-cytotoxic concentrations that do not affect tumor cell proliferation. These findings suggest that aloperine acts within a favorable therapeutic window, where it can remodel the tumor microenvironment without requiring high-dose systemic exposure. Given the high vascular density and early metastatic propensity of TNBC, the ability of aloperine to suppress angiogenesis, VM, and metastasis simultaneously is of particular translational significance. By targeting these processes at sub-cytotoxic concentrations, aloperine may reduce the selective pressure that often leads to the emergence of therapeutic resistance commonly associated with aggressive proliferation-targeted therapies.\\u003c/p\\u003e \\u003cp\\u003eAloperine functions as a pleiotropic angiogenesis inhibitor, comprehensively suppressing fundamental angiogenic activities of HUVECs, including proliferation, migration, tube formation, and spheroid sprouting. These anti-angiogenic properties were further validated in \\u003cem\\u003eex vivo\\u003c/em\\u003e aortic ring assays involving mouse aortic ECs, as well as \\u003cem\\u003ein vivo\\u003c/em\\u003e Matrigel plug assays involving mouse microvascular ECs. The reproducible suppression of angiogenesis across ECs from different species and vascular origins underscores the pharmacological robustness of aloperine. Notably, aloperine disrupted not only individual EC functions but also the coordinated morphogenetic processes required for sprouting angiogenesis, thereby preventing the assembly of functional vascular networks. Beyond conventional angiogenesis, aloperine also efficiently inhibited VM formation in multiple TNBC cell lines, as evidenced by both reduced tube formation and spheroid sprouting. VM is highly prevalent in TNBC and closely associated with tumor aggressiveness, metastatic potential, and resistance to conventional anti-angiogenic therapies [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. In contrast to VEGF-targeted anti-angiogenic agents, which frequently promote compensatory VM and paradoxically enhance tumor metastasis [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], aloperine concurrently suppresses both EC-dependent angiogenesis and tumor cell-driven alternative neovascularization pathways. This dual targeting capacity suggests that aloperine may overcome key resistance mechanisms associated with current anti-angiogenic strategies, thereby positioning it as a promising therapeutic candidate for the treatment of TNBC.\\u003c/p\\u003e \\u003cp\\u003eIn addition to its effective anti-vascular activity, aloperine significantly attenuated the migratory potential of TNBC cells, a critical prerequisite for tumor cell dissemination and distant metastasis [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. These findings suggest that aloperine impairs metastatic progression through a multifaceted mechanism that concurrently disrupts tumor-supportive vasculature and suppresses tumor cell-intrinsic motility. By targeting these distinct yet functionally interconnected steps of the metastatic cascade, aloperine circumvents the inherent limitations of monotherapies that solely inhibit EC angiogenesis or tumor cell migration, likely accounting for its superior anti-metastatic efficacy observed \\u003cem\\u003ein vivo\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cp\\u003eMechanistic investigations revealed that aloperine exerts its anti-vascular effects through selective inhibition of the FGFR1/JAK2/STAT3 signaling pathway in both ECs and TNBC cells. Molecular docking and cell-free kinase assays demonstrated that aloperine directly binds to the ATP-binding pocket of FGFR1, thereby suppressing its kinase activity. In contrast to non-selective tyrosine-kinase inhibitors, aloperine exhibits high selectivity for FGFR1, with minimal direct inhibitory activity against VEGFR2, JAK2, or JAK3. This selectivity profile is therapeutically advantageous, as it may reduce the on-target and off-target toxicities commonly associated with multi-kinase inhibitors, including hypertension, diarrhea, and myelosuppression [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. FGFR1 plays a critical role in tumor cell proliferation and growth, angiogenesis, and metastasis in various malignancies [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. In TNBC, its amplification or overexpression is prevalent and is strongly associated with therapeutic resistance and poor clinical outcomes [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e], positioning FGFR1 as an attractive yet underexploited therapeutic target in this aggressive breast cancer subtype. To date, only one FGFR1 inhibitor, pemigatinib, has received FDA approval, specifically for the treatment of myeloid/lymphoid neoplasms, while no FGFR1-targeted therapies have been approved for TNBC or other solid tumors [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e], underscoring a substantial unmet clinical need. The identification of aloperine as a naturally derived FGFR1 inhibitor offers a promising foundation for addressing this gap. By simultaneously disrupting FGFR1-dependent VM formation and migration of TNBC cells as well as angiogenesis of ECs, aloperine may exert synergistic antitumor effects, potentially overcoming limitations of single-compartment targeting. Furthermore, its favorable selectivity could enable combination strategies, such as with chemotherapy, immune checkpoint inhibitors, or other targeted agents, while minimizing the risk of cumulative toxicities.\\u003c/p\\u003e \\u003cp\\u003eTo evaluate the therapeutic potential of aloperine, we utilized two complementary mouse models representing distinct stages of TNBC progression. The dorsal skinfold chamber model enables real-time visualization of tumor vascularization and growth \\u003cem\\u003ein vivo\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. In this model, aloperine treatment was initiated on the day of 4T1 spheroid transplantation and continued for 2 weeks. This short-term intervention with aloperine significantly inhibited angiogenesis, VM formation, and tumor growth, demonstrating its capacity to prevent TNBC establishment and early progression. In contrast, the orthotopic xenograft model was utilized to assess the therapeutic efficacy of aloperine against established human tumors. Treatment was initiated 3 days after MDA-MB-231 cell inoculation, when tumors became detectable, and continued for 6 weeks. We found that aloperine effectively suppresses tumor vascularization and growth and significantly delays metastatic progression, as evidenced by decreased pulmonary metastatic burden and reduced numbers of CTCs in peripheral blood. Notably, the reduction in CTCs provides mechanistic insight beyond endpoint metastasis, suggesting that concurrent disruption of tumor vasculature and tumor cell motility effectively limits tumor cell intravasation and subsequent systemic dissemination. Together, the consistent effects observed in both murine and human TNBC models establish a strong preclinical foundation for the clinical translation of aloperine as a multi-target therapeutic agent for aggressive TNBC.\\u003c/p\\u003e \\u003cp\\u003eIn summary, our findings demonstrate that aloperine exerts effective, multifaceted antitumor activity in TNBC by directly binding the FGFR1 ATP-binding pocket. This molecular interaction attenuates the FGFR1/JAK2/STAT3 signaling axis across both endothelial and neoplastic compartments, leading to the concurrent suppression of angiogenesis, VM, and metastasis. By establishing FGFR1 as a critical therapeutic node linking vascular remodeling to metastatic progression, this study positions aloperine as a promising lead compound for the treatment of TNBC and potentially other aggressive, highly metastatic malignancies.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003e3D\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Three-dimensional\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAKT\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Protein kinase B\\u003c/p\\u003e\\n\\u003cp\\u003eBLI\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Bioluminescence imaging\\u003c/p\\u003e\\n\\u003cp\\u003eBrdU\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Bromodeoxyuridine\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eCTC\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Circulating tumor cell\\u003c/p\\u003e\\n\\u003cp\\u003eDMEM\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Dulbecco’s modified Eagle’s medium\\u003c/p\\u003e\\n\\u003cp\\u003eEBM\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Endothelial Cell Basal Medium\\u003c/p\\u003e\\n\\u003cp\\u003eECs\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Endothelial cells\\u003c/p\\u003e\\n\\u003cp\\u003eERK\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Extracellular signal-regulated kinase\\u003c/p\\u003e\\n\\u003cp\\u003eFCS\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Fetal calf serum\\u003c/p\\u003e\\n\\u003cp\\u003eFGFR\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Fibroblast growth factor receptor\\u003c/p\\u003e\\n\\u003cp\\u003eFITC\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Fluorescein isothiocyanate\\u003c/p\\u003e\\n\\u003cp\\u003eGFP\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Green fluorescent protein\\u003c/p\\u003e\\n\\u003cp\\u003eHDMECs\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Human dermal microvascular endothelial cells\\u003c/p\\u003e\\n\\u003cp\\u003eHE\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Hematoxylin and eosin\\u003c/p\\u003e\\n\\u003cp\\u003eHRP \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Horseradish peroxidase\\u003c/p\\u003e\\n\\u003cp\\u003eHUVECs\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Human umbilical vein endothelial cells\\u003c/p\\u003e\\n\\u003cp\\u003eJAK\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Janus kinase\\u003c/p\\u003e\\n\\u003cp\\u003eLDH\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Lactate dehydrogenase\\u003c/p\\u003e\\n\\u003cp\\u003emTOR\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Mammalian target of rapamycin\\u003c/p\\u003e\\n\\u003cp\\u003ePAS\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Periodic acid-Schiff\\u003c/p\\u003e\\n\\u003cp\\u003ePI3K\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Phosphatidylinositol 3-kinase\\u003c/p\\u003e\\n\\u003cp\\u003eRBC\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Red blood cell\\u003c/p\\u003e\\n\\u003cp\\u003eRTKs\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Receptor tyrosine kinases\\u003c/p\\u003e\\n\\u003cp\\u003esiRNAs\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Small interfering RNAs\\u003c/p\\u003e\\n\\u003cp\\u003eSOCS\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Suppressor of cytokine signaling\\u003c/p\\u003e\\n\\u003cp\\u003eSTAT\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Signal transducer and activator of transcription\\u003c/p\\u003e\\n\\u003cp\\u003eTKIs\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Tyrosine kinase inhibitors\\u003c/p\\u003e\\n\\u003cp\\u003eTNBC\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Triple-negative breast cancer\\u003c/p\\u003e\\n\\u003cp\\u003eVEGF\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Vascular endothelial growth factor\\u003c/p\\u003e\\n\\u003cp\\u003eVEGFR\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Vascular endothelial growth factor receptor\\u003c/p\\u003e\\n\\u003cp\\u003eVM \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Vasculogenic mimicry\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll animal experiments were conducted in accordance with German legislation on the protection of animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th Edition, 2011). The applied protocols were approved by the local authorities (State Office for Consumer Protection, Saarbrücken, Germany; permission numbers: 01/2019 and 23/2020).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors have agreed to the publication of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors have no relevant financial or non-financial interests to disclose.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the research program of the Medical Faculty of Saarland University (HOMFOR2023 Anschubfinanzierung) and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; project no. 411093008).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors’ contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eM. W. L. and Y. G. designed the research; T. T., L. M., and Y.X. performed the experiments; T. T. and L. M. analyzed the data; T. T., L. M., M. W. L., and Y. G. drafted the manuscript. All authors have reviewed and approved the final version of the manuscript for publication.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors gratefully acknowledge the technical assistance provided by Ruth Nickels, Christina Max, and Janine Becker from the Institute for Clinical and Experimental Surgery at Saarland University.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eBianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. \\u003cem\\u003eNat Rev Clin Oncol.\\u0026nbsp;\\u003c/em\\u003e2016; 13:674-690. https://doi.org/10.1038/nrclinonc.2016.66.\\u003c/li\\u003e\\n \\u003cli\\u003eKumar H, Gupta NV, Jain R, Madhunapantula SV, Babu CS, Kesharwani SS, Dey S, Jain V. A review of biological targets and therapeutic approaches in the management of triple-negative breast cancer. \\u003cem\\u003eJ Adv Res.\\u0026nbsp;\\u003c/em\\u003e2023; 54:271-292. https://doi.org/10.1016/j.jare.2023.02.005.\\u003c/li\\u003e\\n \\u003cli\\u003eRibatti D, Nico B, Ruggieri S, Tamma R, Simone G, Mangia A. Angiogenesis and antiangiogenesis in triple-negative breast cancer. \\u003cem\\u003eTransl Oncol.\\u0026nbsp;\\u003c/em\\u003e2016; 9:453-457. https://doi.org/10.1016/j.tranon.2016.07.002.\\u003c/li\\u003e\\n \\u003cli\\u003eDudley AC, Griffioen AW. Pathological angiogenesis: mechanisms and therapeutic strategies. \\u003cem\\u003eAngiogenesis.\\u0026nbsp;\\u003c/em\\u003e2023; 26:313-347. https://doi.org/10.1007/s10456-023-09876-7.\\u003c/li\\u003e\\n \\u003cli\\u003eJiang X, Wang J, Deng X, Xiong F, Zhang S, Gong Z, Li X, Cao K, Deng H, He Y, et al. The role of microenvironment in tumor angiogenesis. \\u003cem\\u003eJ Exp Clin Cancer Res.\\u0026nbsp;\\u003c/em\\u003e2020; 39:204. https://doi.org/10.1186/s13046-020-01709-5.\\u003c/li\\u003e\\n \\u003cli\\u003eLuo QX, Wang J, Zhao WY, Peng ZZ, Liu XY, Li B, Zhang H, Shan B, Zhang CF, Duan CJ. Vasculogenic mimicry in carcinogenesis and clinical applications. \\u003cem\\u003eJ Hematol Oncol.\\u0026nbsp;\\u003c/em\\u003e2020; 13:19. https://doi.org/10.1186/s13045-020-00858-6.\\u003c/li\\u003e\\n \\u003cli\\u003eZhang D, Sun B, Zhao X, Ma Y, Ji R, Gu Q, Dong X, Li J, Liu F, Jia X, et al. Twist1 expression induced by sunitinib accelerates tumor cell vasculogenic mimicry by increasing the population of CD133\\u003csup\\u003e+\\u003c/sup\\u003e cells in triple-negative breast cancer. \\u003cem\\u003eMol Cancer.\\u0026nbsp;\\u003c/em\\u003e2014; 13:207. https://doi.org/10.1186/1476-4598-13-207.\\u003c/li\\u003e\\n \\u003cli\\u003eSun HZ, Zhang DF, Yao Z, Lin X, Liu JM, Gu Q, Dong XY, Liu F, Wang Y, Yao N, et al. Anti-angiogenic treatment promotes triple-negative breast cancer invasion via vasculogenic mimicry. \\u003cem\\u003eCancer Biol Ther.\\u0026nbsp;\\u003c/em\\u003e2017; 18:205-213. https://doi.org/10.1080/15384047.2017.1294288.\\u003c/li\\u003e\\n \\u003cli\\u003eCheng CL, Thike AA, Tan SY, Chua PJ, Bay BH, Tan PH. Expression of FGFR1 is an independent prognostic factor in triple-negative breast cancer. \\u003cem\\u003eBreast Cancer Res Treat.\\u0026nbsp;\\u003c/em\\u003e2015; 151:99-111. https://doi.org/10.1007/s10549-015-3371-x.\\u003c/li\\u003e\\n \\u003cli\\u003eHallinan N, Finn S, Cuffe S, Rafee S, O\\u0026apos;Byrne K, Gately K. Targeting the fibroblast growth factor receptor family in cancer. \\u003cem\\u003eCancer Treat Rev.\\u0026nbsp;\\u003c/em\\u003e2016; 46:51-62. https://doi.org/10.1016/j.ctrv.2016.03.015.\\u003c/li\\u003e\\n \\u003cli\\u003eLiu Q, Huang J, Yan W, Liu Z, Liu S, Fang W. FGFR families: biological functions and therapeutic interventions in tumors. \\u003cem\\u003eMedComm (2020).\\u0026nbsp;\\u003c/em\\u003e2023; 4:e367. https://doi.org/10.1002/mco2.367.\\u003c/li\\u003e\\n \\u003cli\\u003ePresta M, Dell\\u0026apos;Era P, Mitola S, Moroni E, Ronca R, Rusnati M. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. \\u003cem\\u003eCytokine Growth Factor Rev.\\u0026nbsp;\\u003c/em\\u003e2005; 16:159-178. https://doi.org/10.1016/j.cytogfr.2005.01.004.\\u003c/li\\u003e\\n \\u003cli\\u003eZhu X, Qiu C, Wang Y, Jiang Y, Chen Y, Fan L, Ren R, Wang Y, Chen Y, Feng Y, et al. FGFR1 SUMOylation coordinates endothelial angiogenic signaling in angiogenesis. \\u003cem\\u003eProc Natl Acad Sci U S A.\\u0026nbsp;\\u003c/em\\u003e2022; 119:e2202631119. https://doi.org/10.1073/pnas.2202631119.\\u003c/li\\u003e\\n \\u003cli\\u003eNaeem A, Hu P, Yang M, Zhang J, Liu Y, Zhu W, Zheng Q. Natural products as anticancer agents: current status and future perspectives. \\u003cem\\u003eMolecules.\\u0026nbsp;\\u003c/em\\u003e2022; 27:8367. https://doi.org/10.3390/molecules27238367.\\u003c/li\\u003e\\n \\u003cli\\u003eAsma ST, Acaroz U, Imre K, Morar A, Shah SRA, Hussain SZ, Arslan-Acaroz D, Demirbas H, Hajrulai-Musliu Z, Istanbullugil FR, et al. Natural products/bioactive compounds as a source of anticancer drugs. \\u003cem\\u003eCancers (Basel).\\u0026nbsp;\\u003c/em\\u003e2022; 14:6203. https://doi.org/10.3390/cancers14246203.\\u003c/li\\u003e\\n \\u003cli\\u003eZhou H, Li J, Sun F, Wang F, Li M, Dong Y, Fan H, Hu D. A review on recent advances in aloperine research: pharmacological activities and underlying biological mechanisms. \\u003cem\\u003eFront Pharmacol.\\u0026nbsp;\\u003c/em\\u003e2020; 11:538137. https://doi.org/10.3389/fphar.2020.538137.\\u003c/li\\u003e\\n \\u003cli\\u003eTahir M, Ali S, Zhang W, Lv B, Qiu W, Wang J. Aloperine: A potent modulator of crucial biological mechanisms in multiple diseases. \\u003cem\\u003eBiomedicines.\\u0026nbsp;\\u003c/em\\u003e2022; 10:905. https://doi.org/10.3390/biomedicines10040905.\\u003c/li\\u003e\\n \\u003cli\\u003eGu Y, Tang T, Qiu M, Wang H, Ampofo E, Menger MD, Laschke MW. Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization. \\u003cem\\u003eAngiogenesis.\\u0026nbsp;\\u003c/em\\u003e2025; 28:13. https://doi.org/10.1007/s10456-024-09965-1.\\u003c/li\\u003e\\n \\u003cli\\u003eBecker V, Hui X, Nalbach L, Ampofo E, Lipp P, Menger MD, Laschke MW, Gu Y. Linalool inhibits the angiogenic activity of endothelial cells by downregulating intracellular ATP levels and activating TRPM8. \\u003cem\\u003eAngiogenesis.\\u0026nbsp;\\u003c/em\\u003e2021; 24:613-630. https://doi.org/10.1007/s10456-021-09772-y.\\u003c/li\\u003e\\n \\u003cli\\u003eGu Y, Scheuer C, Feng D, Menger MD, Laschke MW. Inhibition of angiogenesis: a novel antitumor mechanism of the herbal compound arctigenin. \\u003cem\\u003eAnticancer Drugs.\\u0026nbsp;\\u003c/em\\u003e2013; 24:781-791. https://doi.org/10.1097/CAD.0b013e328362fb84.\\u003c/li\\u003e\\n \\u003cli\\u003eLiu ZL, Chen HH, Zheng LL, Sun LP, Shi L. Angiogenic signaling pathways and anti-angiogenic therapy for cancer. \\u003cem\\u003eSignal Transduct Target Ther.\\u0026nbsp;\\u003c/em\\u003e2023; 8:198. https://doi.org/10.1038/s41392-023-01460-1.\\u003c/li\\u003e\\n \\u003cli\\u003eMengie Ayele T, Tilahun Muche Z, Behaile Teklemariam A, Bogale Kassie A, Chekol Abebe E. Role of JAK2/STAT3 signaling pathway in the tumorigenesis, chemotherapy resistance, and treatment of solid tumors: a systemic review. \\u003cem\\u003eJ Inflamm Res.\\u0026nbsp;\\u003c/em\\u003e2022; 15:1349-1364. https://doi.org/10.2147/JIR.S353489.\\u003c/li\\u003e\\n \\u003cli\\u003eTan X, Yan Y, Song B, Zhu S, Mei Q, Wu K. Focal adhesion kinase: from biological functions to therapeutic strategies. \\u003cem\\u003eExp Hematol Oncol.\\u0026nbsp;\\u003c/em\\u003e2023; 12:83. https://doi.org/10.1186/s40164-023-00446-7.\\u003c/li\\u003e\\n \\u003cli\\u003eXue C, Yao Q, Gu X, Shi Q, Yuan X, Chu Q, Bao Z, Lu J, Li L. Evolving cognition of the JAK-STAT signaling pathway: autoimmune disorders and cancer. \\u003cem\\u003eSignal Transduct Target Ther.\\u0026nbsp;\\u003c/em\\u003e2023; 8:204. https://doi.org/10.1038/s41392-023-01468-7.\\u003c/li\\u003e\\n \\u003cli\\u003eDai S, Zhou Z, Chen Z, Xu G, Chen Y. Fibroblast growth factor receptors (FGFRs): structures and small molecule inhibitors. \\u003cem\\u003eCells.\\u0026nbsp;\\u003c/em\\u003e2019; 8:614. https://doi.org/10.3390/cells8060614.\\u003c/li\\u003e\\n \\u003cli\\u003eMaddison K, Bowden NA, Graves MC, Tooney PA. Characteristics of vasculogenic mimicry and tumour to endothelial transdifferentiation in human glioblastoma: a systematic review. \\u003cem\\u003eBMC Cancer.\\u0026nbsp;\\u003c/em\\u003e2023; 23:185. https://doi.org/10.1186/s12885-023-10659-y.\\u003c/li\\u003e\\n \\u003cli\\u003eKoehl GE, Gaumann A, Geissler EK. Intravital microscopy of tumor angiogenesis and regression in the dorsal skin fold chamber: mechanistic insights and preclinical testing of therapeutic strategies. \\u003cem\\u003eClin Exp Metastasis.\\u0026nbsp;\\u003c/em\\u003e2009; 26:329-344. https://doi.org/10.1007/s10585-008-9234-7.\\u003c/li\\u003e\\n \\u003cli\\u003eBatool S, Chokkakula S, Kim BK, Park JH, Min SC, Lee JR, Lee GC, Lee DG, An SH, Jain A, et al. High-throughput in vitro screening and in silico analysis for Zika virus inhibitor identification. \\u003cem\\u003eSci Rep.\\u0026nbsp;\\u003c/em\\u003e2025; 15:45501. https://doi.org/10.1038/s41598-025-29585-z.\\u003c/li\\u003e\\n \\u003cli\\u003eAlemu BK, Tommasi S, Hulin JA, Meyers J, Mangoni AA. Current knowledge on the mechanisms underpinning vasculogenic mimicry in triple negative breast cancer and the emerging role of nitric oxide. \\u003cem\\u003eBiomed Pharmacother.\\u0026nbsp;\\u003c/em\\u003e2025; 186:118013. https://doi.org/10.1016/j.biopha.2025.118013.\\u003c/li\\u003e\\n \\u003cli\\u003eMorales-Guadarrama G, Garc\\u0026iacute;a-Becerra R, M\\u0026eacute;ndez-P\\u0026eacute;rez EA, Garc\\u0026iacute;a-Quiroz J, Avila E, D\\u0026iacute;az L. Vasculogenic mimicry in breast cancer: clinical relevance and drivers. \\u003cem\\u003eCells.\\u0026nbsp;\\u003c/em\\u003e2021; 10:1758. https://doi.org/10.3390/cells10071758.\\u003c/li\\u003e\\n \\u003cli\\u003ePolacheck WJ, Zervantonakis IK, Kamm RD. Tumor cell migration in complex microenvironments. \\u003cem\\u003eCell Mol Life Sci.\\u0026nbsp;\\u003c/em\\u003e2013; 70:1335-1356. https://doi.org/10.1007/s00018-012-1115-1.\\u003c/li\\u003e\\n \\u003cli\\u003eShyam Sunder S, Sharma UC, Pokharel S. Adverse effects of tyrosine kinase inhibitors in cancer therapy: pathophysiology, mechanisms and clinical management. \\u003cem\\u003eSignal Transduct Target Ther.\\u0026nbsp;\\u003c/em\\u003e2023; 8:262. https://doi.org/10.1038/s41392-023-01469-6.\\u003c/li\\u003e\\n \\u003cli\\u003eFan S, Chen Y, Wang W, Xu W, Tian M, Liu Y, Zhou Y, Liu D, Xia Q, Dong L. Pharmacological and biological targeting of FGFR1 in cancer. \\u003cem\\u003eCurr Issues Mol Biol.\\u0026nbsp;\\u003c/em\\u003e2024; 46:13131-13150. https://doi.org/10.3390/cimb46110783.\\u003c/li\\u003e\\n \\u003cli\\u003eChen J, Wang Q, Wu H, Huang X, Cao C. Therapies targeting triple-negative breast cancer: a perspective on anti-FGFR. \\u003cem\\u003eFront Oncol.\\u0026nbsp;\\u003c/em\\u003e2024; 14:1415820. https://doi.org/10.3389/fonc.2024.1415820.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":false,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"aloperine, angiogenesis, vasculogenic mimicry, metastasis, FGFR1, JAK2, STAT3, triple-negative breast cancer\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-9209927/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-9209927/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cb\\u003eBackground\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTriple-negative breast cancer (TNBC) is a highly aggressive malignancy characterized by early recurrence and high metastatic potential. Its aggressiveness is driven by a complex vascular network integrating classical angiogenesis and vasculogenic mimicry (VM). Despite the clinical implementation of PARP inhibitors and immune checkpoint blockade, effective targeted therapeutic strategies remain limited for the majority of TNBC patients.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eMethods\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe effects of aloperine on angiogenesis were analyzed using a panel of \\u003cem\\u003ein vitro\\u003c/em\\u003e assays in human umbilical vein endothelial cells (HUVECs), \\u003cem\\u003eex vivo\\u003c/em\\u003e aortic ring assays, and \\u003cem\\u003ein vivo\\u003c/em\\u003e Matrigel plug assays. Its effects on TNBC cell migration and VM formation were assessed in MDA-MB-231 cells using Transwell migration and tube formation assays, respectively. Mechanistic studies were performed using Western blotting, molecular docking, and cell-free kinase assays. Finally, the therapeutic efficacy of aloperine against TNBC progression was validated in a mouse dorsal skinfold chamber model of murine 4T1 tumors and an orthotopic xenograft model of human MDA-MB-231 tumors.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eResults\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eIn this study, we identified aloperine, a natural quinolizidine alkaloid, as a multimodal inhibitor of TNBC progression. Aloperine preferentially suppressed endothelial angiogenesis as well as TNBC cell migration and VM formation at concentrations with minimal effects on tumor cell proliferation. Mechanistically, aloperine directly bound to the ATP-binding pocket of fibroblast growth factor receptor 1 (FGFR1), thereby inhibiting its kinase activity and downstream Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling in both endothelial and tumor cells. \\u003cem\\u003eIn vivo\\u003c/em\\u003e, aloperine effectively suppressed tumor angiogenesis, VM, and metastasis in both murine and human TNBC models.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eConclusions\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThese findings demonstrate that aloperine disrupts the dual vascular supply and metastatic progression of TNBC by selectively targeting the FGFR1/JAK2/STAT3 signaling axis, positioning aloperine as a promising therapeutic candidate and FGFR1 as a compelling target for TNBC treatment.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Targeting FGFR1 with aloperine suppresses angiogenesis, vasculogenic mimicry, and metastasis in triple-negative breast cancer\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-04-14 02:02:05\",\"doi\":\"10.21203/rs.3.rs-9209927/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"23ae8ab9-a473-4e8e-8761-e20cf26a848d\",\"owner\":[],\"postedDate\":\"April 14th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-04-25T16:09:13+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-04-14 02:02:05\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-9209927\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-9209927\",\"identity\":\"rs-9209927\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}