The human non-small cell lung cancer cell A549, human breast cancer MCF-7 and mouse breast cancer 4T1 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. A549 cells were cultured in RPMI-1640 medium (cat. no. SH30027.01; Hyclone; Cytiva) supplemented with 10% fetal bovine serum (FBS; cat. no. A5256701; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. MCF-7 and 4T1 cells were cultured in high-glucose DMEM (cat. no. SH30243.01; Hyclone; Cytiva) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Human umbilical vein endothelial cells (HUVECs) were isolated as previously described (38) and cultured in 1% gelatin-coated flasks using endothelial cell medium (cat. no. #1001; ScienCell Research Laboratories, Inc.) supplemented with 5% FBS, 100 µg/ml endothelial cell growth supplement (cat. no. #1052; ScienCell Research Laboratories, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were maintained at 37°C in a humidified incubator containing 5% CO₂. Cells were harvested using 0.25% trypsin prior to experiments. HUVECs were used within six passages.

Tan IIA (purity, ≥98%, cat. no. S107694; lot no. K2208491) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. For the in vitro experiments, Tan IIA was dissolved in DMSO to a stock solution of 40 mM, filtered, stored at 4°C and diluted with normal saline when used.

The cytotoxicity of Tan IIA was evaluated using the MTT assay as previously described (4). Briefly, MCF-7, A549 and HUVEC cells were seeded into 96-well plates at a density of 1×10⁴ cells per well and incubated at 37°C for 24 h. The cells were treated with medium containing various concentrations (0, 5, 10 and 20 µM) of Tan IIA at 37°C for 24 h. MTT solution was added, followed by incubation for 4 h at 37°C. The medium was removed and formazan crystals were dissolved in 100 µl DMSO. Absorbance was measured at 570 nm using a microplate reader (Tecan Group Ltd.; M200 PRO). All absorbance values were normalized to blank wells containing culture medium and MTT reagent only, which served as the zero metabolic activity baseline. Cell viability was expressed as a percentage relative to untreated controls.

A549 and HUVEC cells in logarithmic growth phase were treated with Tan IIA (0, 5, 10 and 20 µM) at 37°C for 24 h and collected using trypsin without EDTA. For cell cycle analysis, A549 cells were fixed in 70% ethanol at 4°C for 12 h, treated with RNase and stained with PI. For apoptosis analysis (early and late apoptotic cells), treated cells (A549 and HUVEC cells) were resuspended in binding buffer and stained with 5 µl of Annexin V-FITC and 5 µl of PI for 15 min in the dark at room temperature, followed by the addition of 0.5 ml PBS. Cell cycle and apoptosis were detected by flow cytometry with a BD FACSAria (BD Biosciences) and analyzed by FlowJo software (version number 10.8.1; BD Biosciences).

Cell migration was evaluated using a wound healing assay. A549 cells were seeded into 12-well plates (2×105 cells/well) and allowed to form a confluent (80–90%) monolayer. A linear scratch was created using a pipette tip and detached cells were removed with serum-free RPMI-1640. Cells were incubated in RPMI-1640 containing 1% FBS and Tan IIA at the indicated concentrations (0, 5, 10 and 20 µM). Images were captured at 0 and 24 h using a light microscope. The migration of the cells was assessed by measuring the width of the wound area.

Cell invasion assay was assessed using Matrigel-coated Transwell chambers (Corning, Inc.; 8 µm pore size) as previously described (39). The membrane was coated with 0.5% Matrigel overnight at 37°C. A549 cells (4×104) were seeded into the upper chamber in serum-free RPMI-1640 medium containing Tan IIA (0, 10 and 20 µM), while the lower chamber contained RPMI-1640 medium with 10% FBS. After 24 h of incubation at 37°C in a cell culture incubator, non-invading cells were removed and invading cells were fixed with 4% paraformaldehyde for 20 min at 4°C and stained with 0.1% crystal violet at room temperature for 30 min. Images of the stained cells were captured using a light microscope. The invasive ability was quantified by counting the number of cells that had penetrated through the membrane and analyzed using ImageJ software (version number 1.54p; National Institutes of Health).

A549 cells were treated with different concentrations of Tan IIA (0, 5, 10 and 20 µM) at 37°C for 24 h, and HUVECs were treated with 0, 5 and 10 µM of Tan IIA at 37°C for 24 h. Prior to treatment, HUVECs were stimulated with TNF-α (10 ng/ml) at 37°C for 4 h (negative control, HUVECs without TNF-α). Subsequently, the cells were harvested, resuspended in PBS and incubated in the dark at room temperature for 10 min with 10 µl antibodies as follows: Anti-CD29 (PE-labeled, cat. no. 303004) for A549 cells and anti-ICAM (cat. no. 353112) or anti-E-selectin (both APC-labeled, cat. no. 336011) (all 1:100, all Biolegend, Inc.) for HUVECs. After washing with PBS buffer, cells were analyzed using a BD FACSAria and FlowJo software.

Blood was collected from 5 volunteers at the Blood Bank of Fujian Medical University Union Hospital (Fuzhou, China) between May and October 2023. The study included 2 men and 3 women, aged 20–40 years. The inclusion criteria were as follows: i) Normal platelet aggregation function; ii) normal coagulation parameters and other complete blood count indices; and iii) no use of any anticoagulants for 2 weeks prior to blood collection. All participants provided written informed consent before sample collection, explicitly agreeing to the use of their blood samples for the present study. The study was approved by the Ethics Committee of Fujian Medical University Union Hospital (Fuzhou, China; approval no.2022KJT046) and was conducted in compliance with the Declaration of Helsinki. All patients provided written informed consent to participate.

Fresh blood (1.8 ml) was added to a centrifuge tube containing 0.2 ml sodium citrate anticoagulant. After centrifugation at 2,000 × g for 10 min at room temperature, the upper layer of the supernatant was collected to obtain platelet-rich plasma. The platelet-rich plasma was centrifuged at 15,00 × g for 15 min at room temperature and the lower pellet was collected as the platelet precipitate, yielding a platelet concentration of ~2×10⁶ platelets/µl. The procedure was completed rapidly to prevent platelet aggregation. To investigate the effect of Tan IIA on platelet activity, the platelet pellet (10 µl) was resuspended in 1 ml PBS. Platelets were activated with ADP (20 µM) while being treated with varying concentrations of Tan IIA (0, 1, 5 and 10 µM). The mixture was incubated at 37°C for 5 min. Thereafter, 20 µl P-selectin (1:100, PE-labeled; cat. no. 148306; Biolegend, Inc.) antibody was added to each tube containing 200 µl of platelet suspension, followed by incubation in the dark at 37°C for 15 min. The effect of Tan IIA on platelet activity was analyzed by flow cytometry with a BD FACSAria and the resulting data were processed using FlowJo software.

Gelatin-coated 24-well plates (gelatin coating was performed by incubation at 37°C for 1 h) were seeded with HUVECs at a density of 2×10⁵ cells per well to form confluent (80–90%) monolayers, which were pretreated with TNF-α (10 ng/ml) at 37°C for 4 h. A group without TNF-α was set as the blank control. A549 cells suspension at ~6×10⁵ cells/ml treated with Tan IIA (0, 5, 10, 20 µM) at 37°C and labeled with Rhodamine-123 (10 µl; 15 min) were added and co-cultured with HUVECs at 37°C for 1 h. After washing away non-adherent cells with PBS, fluorescence microscopy images were captured in 12 randomly selected fields of view/well. Adhesion was quantified as a percentage relative to control.

To assess platelet-mediated adhesion, A549 cell suspension (~6×10⁵ cells/ml) were treated with Tan IIA (0, 5, 10 and 20 µM) at 37°C and labeled with 10 µl Rhodamine-123 stain for 15 min. After adding 10 µl platelet-rich plasma, the cell mixture was co-incubated with HUVECs at a density of 2×10⁵ cells per well (pretreated with 10 ng/ml TNF-α for 4 h) at 37°C for 1 h, with the simultaneous addition of ADP (20 µM) to activate platelets. A group without TNF-α stimulation was used as the blank control. Adhesion was quantified as a percentage relative to control.

The platelet cloaking of cancer cells was performed as previously described (4). Briefly, A549 cells were harvested and suspended in PBS at a density of 2×10⁶ cells/ml. Tan IIA (0, 5, 10 and 20 µM) and A549 cells (2×10⁵ cells) were added to 100 µl PBS containing suspended platelets (2×10⁸ cells/ml) in centrifuge tubes, followed by the addition of ADP (20 µM) to stimulate platelet activation. After 5 min incubation at 25°C, 20 µl each mouse anti-human CD61 (FITC-labeled, cat. no. 104306) and CD326 (PE-labeled, cat. no. 324205) (both 1:100, Biolegend, Inc.) were added to the tubes. The mixture was incubated for 20 min at 4°C in the dark, followed by the addition of 1 ml ice-cold 1% paraformaldehyde for fixation at 4°C for 30 min. A549 cells cloaked by platelets were identified by flow cytometry with a BD FACSAria; CD61⁺CD326⁺ cancer cells were considered platelet-cloaked cancer cells. The data were analyzed using FlowJo software.

The adhesion of platelets to tumor cells following Tan IIA (0, 5, 10, and 20 µM) treatment was visualized using a confocal fluorescence microscope (Leica GmbH; STELLARIS 5). Suspended platelets (10 µl at a density of ~2×10⁶ platelets/µl) were collected and stained with 5 µM 5-(and-6)-carboxyfluorescein diacetate N-succinimidyl ester (cat. no. 21888; Sigma-Aldrich; Merck KGaA) in PBS for 10 min at 37°C. The nuclei of A549 cells were stained with DAPI (1 µg/ml in PBS) for 15 min at 25°C in the dark and the cell membranes were stained with 2 µM PKH26 Red Fluorescent Cell Linker (cat. no. MINI26-1KT; Sigma-Aldrich; Merck KGaA) for 15 min at 25°C in the dark. Subsequently, the stained platelets and tumor cells were co-cultured for 10 min at 25°C in the dark. After co-culture, unattached platelets were removed by washing with PBS and the platelet-tumor aggregates were transferred to confocal dishes for incubation at 37°C for 4 h. Finally, 12 randomly selected fields of view/dish were observed using a confocal microscope. The number of platelets adhering to tumor cells was quantified by ImageJ software.

Western blotting was performed to detect the effect of Tan IIA at safe concentrations (0, 5 and 10 µM) at 37°C for 24 h on the NF-κB signaling pathway in HUVECs. Whole-cell lysates were from HUVECs prepared using RIPA Buffer (cat. no. WB3100; Suzhou Xinsaimei Biotechnology Co., Ltd.) with 1% protease/phosphatase inhibitors on ice for 30 min, followed by centrifugation at 12,000 × g for 20 min at 4°C. Protein concentration was determined using the BCA method. Proteins (10 µg/lane) were separated using 4–12% gradient precast gels according to their molecular sizes and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk and 5% BSA (cat. no. T50811010260; Beijing Dingguo Changsheng Biotechnology Co., Ltd.) at room temperature for 90 min and incubated with primary antibodies at 4°C overnight. After washing with TBST (0.1% Tween) four times, 5 min each, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. no. 511203; Zenbio; Chengdu Zhengneng Biotechnology Co., Ltd.) for 1 h at room temperature. Signals were detected using an ECL substrate (NcmECL Ultra; cat. no. P10300; Suzhou Xinsaimei Biotechnology Co., Ltd.) and a chemiluminescence imaging system (ChemiDoc™ MP; Bio-Rad Laboratories, Inc.). The following primary antibodies were used: GAPDH (1:10,000; cat. no. A19056; ABclonal Biotech Co., Ltd.), NF-κB p65 (cat. no. R25149), phosphorylated (p-)NF-κB p65 (cat. no. 310013), IκB-α (cat. no. R23322), p-IκB-α (cat. no. 340776), IKK-α/β (cat. no. R24676) and p-IKK-α/β (all 1:1,000; cat. no. 530546; all Zenbio). Band intensities from three independent experiments were semi-quantified using ImageJ software.

The 3D structure of Tan IIA was downloaded from the PubChem database (pubchem.ncbi.nlm.nih.gov/) in SDF format. The compound was converted to MOL2 format using PyMOL (version number 3.1.6; Delano Scientific LLC), and its geometric conformation was automatically optimized. The 3D structure of the target protein was retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (rcsb.org/). The protein structure was processed by removing water molecules, adding hydrogen atoms and checking charge states. Both the ligand (Tan IIA) and the receptor (CD29 and P-selectin) were converted to the PDBQT format using the MGLTools software package (version number 1.5.7; The Scripps Research Institute). Molecular docking was then performed using AutoDock Vina (version number 1.1.2; The Scripps Research Institute) to calculate the binding energies. The best docking pose was selected and visualized using PyMOL.

All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) (40). The Laboratory Animal Ethics Committee of Minjiang University (Fuzhou, China) reviewed and approved all animal procedures (approval no. IACUC-MJLAC-2023-020).

Female BALB/c and nude mice (n=110; weight, 20±2 g; age, 6–8 weeks) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. The animals were acclimatized to the laboratory environment for ≥1 week prior to the start of the experiment. All mice were housed in a specific-pathogen-free facility at the Animal Experiment Center of Minjiang University, with 5 mice/cage under a 12/12-h light/dark cycle at a constant temperature of 23–25°C and 50–60% relative humidity. They were fed a standard rodent diet and had free access to sterile water, which was replaced daily. The experiment lasted 4–6 weeks.

Animal health and behavior were monitored daily for clinical signs including activity levels, appetite and grooming. Body weights were measured and recorded every 3 days throughout the study. Humane endpoints were as follows: i) Maximum tumor volume >750 mm³; ii) severe clinical manifestations such as dyspnea or listlessness; and iii) body weight loss >20% of initial body weight. In total, four mice were prematurely euthanized based on tumor volume and clinical manifestations.

To prepare the vehicle, 0.1 g sodium carboxymethyl cellulose (cat. no. C104984; Shanghai Aladdin Biochemical Technology Co., Ltd.) was dissolved in 100 ml double-distilled water to obtain a 0.1% solution. Tan IIA was dissolved in this 0.1% sodium carboxymethyl cellulose solution to prepare Tan IIA solutions at concentrations of 0.1, 1 and 2 mg/100 µl for oral gavage. The anti-metastatic efficacy of Tan IIA was evaluated using three independent mouse cancer models (n=10). For the A549 lung metastasis model, A549 cells in logarithmic growth phase (1×10⁵ cells in 100 µl per mouse) were injected into the tail vein of nude mice. For the hematogenous metastasis model, 4T1 cells in logarithmic growth phase (1×10⁵ cells in 100 µl per mouse) were injected via the tail vein of Balb/c mice. For the orthotopic metastasis model, 4T1 cells (5×10⁵ cells in 100 µl per mouse) were injected into the fourth mammary fat pad of Balb/c mice to establish an orthotopic tumor model that simulates the natural metastatic progression of breast cancer.

In the A549 lung metastasis model, a high-(1 mg/100 µl/day) and low-dose (0.1 mg/100 µl/day) and control group (100 µl 0.1% sodium carboxymethyl cellulose solution) were set up. In the 4T1 breast cancer metastasis model, a high-(2 mg/100 µl/day), medium-(1 mg/100 µl/day) and low-dose (0.1 mg/100 µl/day) and control group (100 µl 0.1% sodium carboxymethyl cellulose solution; vehicle) were established. Mice continued to receive 100 µl Tan IIA solution or vehicle by oral gavage daily for 4–6 weeks. During this period, the physiological status of the mice was observed and recorded. Body weight was measured and recorded for all surviving mice at every 3 days. At the end of the experiment, mice were anesthetized by inhalation of 3–5% isoflurane in oxygen for induction, followed by maintenance anesthesia with 1–2% isoflurane. Mice were euthanized with a CO₂ displacement rate of 30% of the container volume/min, followed by cervical dislocation. Death was confirmed by cessation of heartbeat and respiration. The lungs were removed fixed in 4% paraformaldehyde at 4°C overnight, stained with picric acid at room temperature for 72 h and the number of metastatic nodules was assessed under a dissecting microscope. GraphPad Prism software (version number 8.0.2; Dotmatics) was used for analysis.

Data are presented as the mean ± standard deviation of ≥3 independent experimental repeats. Statistical analyses were performed using GraphPad Prism. Differences were evaluated by one-way ANOVA followed by Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The present study aimed to identify Tan IIA concentrations that exert minimal direct cytotoxic effects on tumor and endothelial cells, thereby allowing investigation of its effects on metastasis-associated interactions. Viability was assessed in A549, HUVECs and MCF-7 cells following Tan IIA treatment. Cell viability slightly decreased with increasing Tan IIA concentration, however there was no significant difference in any cell line (Fig. 1A). Consistent with these findings, apoptosis analysis showed no significant increase in apoptotic A549 cells following treatment with Tan IIA (Fig. 1B and C). Cell cycle analysis revealed A549 cell cycle arrest at the G2/M phase following treatment with Tan IIA, while the cell cycle distribution of HUVECs did not change significantly (Fig. 1D-G). This supports the selective action of Tan IIA on carcinoma over normal endothelial cells, a key point for its potential as an anti-metastatic agent with a favorable safety profile regarding vascular toxicity.

Tumor cell migration and invasion are key steps in metastatic progression. To examine whether Tan IIA affects these processes, wound healing and Transwell invasion assays were performed. In the wound healing assay, A549 cells treated with non-cytotoxic concentrations of Tan IIA (10 and 20 µM) showed significantly decreased migration compared with untreated controls after 24 h, however lower doses of Tan IIA had no significant effect (Fig. 2A and B). Similarly, Tan IIA inhibited A549 cell invasion in a concentration-dependent (Fig. 2C and D). These findings indicated that Tan IIA effectively suppressed both migration and invasion of lung cancer cells independent of overt cytotoxicity.

Adhesion between tumor cells, platelets and endothelial cells plays a central role in metastatic dissemination (41–43). The present study examined whether Tan IIA modulates adhesion molecule expression on tumor cells. CD29, a key integrin involved in platelet binding and cell adhesion, was assessed on A549 cells by flow cytometry (44,45). Tan IIA resulted in a concentration-dependent reduction in CD29 expression on the A549 cell surface (Fig. 3A and B). The present study evaluated the effects of Tan IIA on endothelial adhesion molecules. E-selectin and ICAM are inducible adhesion proteins that facilitate tumor cell attachment to activated endothelium (46). Flow cytometric analysis revealed that Tan IIA significantly decreased the surface expression of E-selectin on HUVECs in a concentration-dependent manner; the expression of ICAM-1 on HUVECs was significantly reduced, while no significant difference in ICAM-1 expression was observed between higher Tan IIA doses (Fig. 3C-F). These results suggested that Tan IIA disrupts a metastasis-promoting microenvironment by decreasing adhesion molecule expression on both tumor cells and endothelial cells.

Platelet activation and aggregation around CTCs enhance tumor survival and metastatic potential (47). P-selectin is a member of the selectin family of cell adhesion molecules. It is expressed on stimulated endothelial cells and activated platelets, mediating both heterotypic aggregation between activated platelets and cancer cells, as well as the adhesion of cancer cells to stimulated endothelial surfaces (48). To evaluate the effect of Tan IIA on platelet activity, P-selectin expression was measured by flow cytometry. ADP markedly increased P-selectin expression; addition of Tan IIA significantly decreased P-selectin levels in compared with the control (Fig. 4A and B), indicating inhibition of platelet activation.

To assess tumor cell-platelet interactions, platelet cloaking of A549 cells was examined by flow cytometry. Under resting conditions, few A549 cells exhibited platelet binding. Following ADP-induced platelet activation, the proportion of platelet-coated tumor cells increased markedly. Tan IIA significantly decreased platelet adhesion to A549 cells (Fig. 4C and D). These findings indicate that Tan IIA interfered with platelet-tumor cell aggregate formation by suppressing platelet activation. Collectively, these results demonstrated that Tan IIA may limit key steps of CTC-mediated metastasis by inhibiting platelet activation, decreasing tumor cell-platelet aggregation and decreasing adhesion molecule expression (Fig. 4E).

Adhesion of CTCs to the vascular endothelium is a prerequisite for extravasation and metastatic colonization (49). To evaluate whether Tan IIA affected this process, A549 cells treated with Tan IIA were co-cultured with TNF-α-stimulated HUVEC monolayers. Tan IIA significantly decreased the number of tumor cells adhering to endothelial cells in a concentration-dependent manner (Fig. 5A and B). To investigate the role of platelets in this process, a co-culture system incorporating A549 cells, platelets and activated HUVECs was established. In the presence of platelets, Tan IIA inhibited tumor cell adhesion to endothelial cells (Fig. 5C and D). Confocal microscopy confirmed that Tan IIA decreased platelet coverage of tumor cells, resulting in fewer platelet-tumor cell aggregates (Fig. 5E and F). These findings indicate that Tan IIA inhibits platelet-mediated tumor cell adhesion to the endothelium.

To investigate how Tan IIA affects the expression of the endothelial surface proteins E-selectin and ICAM, the present study examined whether Tan IIA influences the NF-κB signaling pathway in endothelial cells. Endothelial cells were cultured in the presence or absence of TNF-α stimulation. Western blotting was performed to evaluate the effect of Tan IIA on the NF-κB signaling pathway. TNF-α increased the normalized levels of NF-κB pathway-related proteins, including p-IKKα/β, p-IκB-α and p-NF-κB p65 (each normalized to its respective total protein), indicating activation of this signaling pathway (Fig. 6). Compared with the 0 µM group, Tan IIA at 10 µM significantly reduced the normalized levels of these proteins (p-IKKα/β, p-IκB-α and p-NF-κB p65). These results indicated that Tan IIA-mediated inhibition of expression of E-selectin and ICAM on HUVECs is associated with NF-κB signaling pathway.

Molecular docking predicted that Tan IIA can favorably bind to the extracellular domains of CD29(−9.976 kcal/mol) and P-selectin (−8.638 kcal/mol) with strong binding affinity (Fig. 7). The docking models suggested that Tan IIA potentially binds key functional regions or pockets on these proteins, which may interfere with their inter-molecular interactions.

To evaluate the anti-metastatic effects of Tan IIA in vivo, mouse models were established. In the A549 tail vein metastasis model, Tan IIA significantly prolonged survival compared with controls (P=0.0169; Fig. 8A), but no significant body weight loss was observed during treatment (Fig. 8B). The number of lung metastatic nodules was significantly decreased in Tan IIA-treated mice, particularly in the 50 mg/kg/day group (Fig. 8C).

Consistent results were observed in both 4T1 models. In the hematogenous model, Tan IIA significantly decreased the number of lung metastases (Fig. 8D and E). Similarly, in the orthotopic model, Tan IIA led to fewer lung metastatic nodules compared with controls (Fig. 8G and H). Lung weight was largely unchanged across groups (Fig. 8F and I) and no significant body weight loss was observed during treatment (Fig. 8J and K), indicating good tolerability. Taken together, these in vivo findings demonstrated that Tan IIA effectively suppressed metastatic tumor burden while improving overall physiological condition and survival.